bluuebunny/crossref_metadata_2025 huggingface dataset:
Electronic Cigarette Extracts Alter Endothelial Cell Inflammatory Responses
Authors: Barber Kirstin E. , Yin Wei , Rubenstein David Alan
Published: November 2023

Electronic cigarettes (e‐cigarettes) have recently become a popular alternative to traditional tobacco cigarettes. The effects of traditional cigarettes on endothelial cells (ECs) have been well‐documented and illustrate that tobacco products predispose ECs towards cardiovascular pathologies. However, the effects of e‐cigarettes on ECs have not been studied even though the composition of e‐vapor has been shown to resemble the composition of tobacco smoke in a number of important parameters that have been related to cardiovascular disease progression. We hypothesized that exposure to e‐cigarette extracts would elicit a pro‐inflammatory response through the activation of the complement cascade, a down‐regulation of the negative regulators of complement and an increase in the production/release of pro‐inflammatory cytokines. We also hypothesized that the increase in pro‐inflammatory state would be dependent on the dose of the e‐vapor exposure (e.g. nicotine concentration in the e‐cigarette) and that exposure to e‐vapor would induce an overall reduction in EC culture conditions. In order to test this hypothesis, human umbilical vein endothelial cells (HUVECs) were incubated with extracts of tobacco smoke, e‐cigarette vapors, and pure nicotine for 2–3 days. A solid‐phase ELISA technique was used to measure the deposition of various complement proteins, including C1q, iC3b, C4d and C5b‐9, onto the cell surface, along with the expression of the complement C1q receptor (gC1qR and cC1qR) and the negative regulators of complement. All conditions – tobacco smoke, e‐cigarette vapor, and pure nicotine – led to an increase in C1q receptor expression. In contrast to studies performed on other cell lines and results of tobacco smoke exposure, the deposition of complement proteins decreased after exposure to e‐cigarette vapor extracts. Interestingly, we also observed a decrease in the expression/release of negative regulators of complement for most of the e‐cigarette vapor extracts that were investigated here. After exposure to e‐vapor, ECs up‐regulated the production/release of pro‐inflammatory cytokines. To quantify changes in cell culture conditions, EC metabolic activity, cell viability and cell density were measured using a standard MTT assay and a live/dead cytotoxicity assay. After exposure to e‐cigarette vapor extracts, we observed a decrease in metabolic activity, in some cases to a further extent than that caused by mainstream tobacco smoke exposure. A slight decrease in cell viability was also observed, combined with a marked decrease in cell density. Thus, it is possible that the observed reductions in complement deposition (or complement inhibitor expression), were due to a decrease in the cell number as compared with the control conditions. Overall, these results indicate that e‐cigarettes present a significant risk to vascular endothelial cells and may alter the progression of cardiovascular diseases.
Pretreated with Evans blue can prevent mast cell degranulation and compound 48/80 induced anaphylaxis
Authors: Chen Yu‐Tong , Lin Shiuan‐Yea , Lue Sheng‐I , Lu Tzong‐Shi , Fu Yaw‐Syan
Published: November 2023

This study demonstrated that pretreated with Evans blue could prevent hypotension, plasma leakage, mesenteric mast cells degranulation induced by compound 48/80, but only prevent mast cells degranulation by substance P. Sprague‐Dawley rats were cannulated for monitoring arterial blood pressure, and femoral vein was cannulated for i.v. injected with substance P (3 μg/kg) and compound 48/80 (200 μg/kg) by syringe pump over 2 min to induced systemic inflammation. Half of animals were pretreated with i.v. injection of a dose of Evans blue (50 mg/kg) 20 sec prior to the drug infusion, and the remaining half of animals were post‐treated with same dose Evans blue after drug infusion immediately. The result of our study showed Evans blue pretreatment could complete inhibit compound 48/80‐induced hypotension, peripheral mast cell degranulation, and edema formation in various organs including the airways, esophagus, and skin. The pretreatment of Evans blue only had few effects on hypotension and plasma leakage induced by substance P, but could prevent the mesenteric mast cell degranulation. In conclusion, Evans blue pretreatment could inhibit the mast cells degranulation and that may be the main mechanism for prevent animals from C48/80‐triggered anaphylaxis and systemic inflammation.

Support or Funding Information

KMU‐TP104E34, KMU‐O105001
Validation of the doubly labeled water method using integrated cavity output spectroscopy against isotope ratio mass spectrometry and whole‐room indirect calorimetry
Authors: Melanson Edward L. , Plasqui Guy , Wouters Loek , Kohrt Wendy S. , Swibas Tracy , Catenacci Vicki A. , Speakman John R. , Berman Elena S.
Published: November 2023

Background

The doubly‐labeled water (DLW) method is the gold standard methodology for determination of free‐living energy expenditure. Isotope enrichments have traditionally been determined by isotope ratio mass spectrometry (IRMS), but new technologies are emerging, such as off‐axis integrated cavity output spectroscopy (OA‐ICOS). We have previously shown that compared to IRMS, OA‐ICOS provides comparable isotope enrichments of standard waters and of human urine samples, and to carbon dioxide (CO2) production measured using whole‐room indirect calorimetry (IC). In this study, we compared CO2 production in humans measured using OA‐ICOS and IRMS to simultaneous whole‐room indirect calorimetry (IC) measurements over a period of 7 days.

Methods

12 subjects (5 male, 7 female; age=24 to 63 yrs.; body mass index=19.4 to 46.4 kg/m2) were studied for 7 consecutive days in the IC. Prior to entry on the first day, subjects consumed an oral dose of 0.25 g H218O (95% APE) and 0.14 g 2H2O (99.8% APE) per kg of total body water (estimated as 73% of fat free mass, measured using Dual X‐ray Absorptiometry). Urine samples were obtained daily, and the samples on day 1 and 8 were used to measure average CO2 production over the 7 days using both OA‐ICOS and IRMS.

Results

Average estimated CO2 production by IC, TIWA, and IRMS was 413.6±69.4, 400.6±74.5, and 385.5±71.1 L/day (P=0.03), respectively (mean±SD). Post‐hoc comparisons indicated significant differences in CO2 production between IRMS and IC (95% confidence interval of difference = +3.7 to +52.5 L/min), but not between TIWA and IC (−11.4 to 37.5 L/min) or TIWA and IRMS (−9.3 to 39.5 L/min). CO2 production measured with the different instruments were significantly correlated: TIWA vs. IC (r=0.89), IRMS vs. IC (r=0.93), IRMS vs. TIWA (r=0.86).

Conclusions

OA‐ICOS produces valid measurements of CO2 production compared to IC, and comparable results to IRMS. OA‐ICOS provides a valid and viable alternative to IRMS for DLW studies in humans.

Support or Funding Information

This work was supported by NIH SBIR 1R43DK093362‐01, The Colorado Nutrition and Obesity Research Center (P30 DK048520) and Colorado Clinical and Translational Science Institute (UL1 TR000154).
Experimental approaches for studying cell water and volume regulation in apoptotic cells
Authors: Model Michael A
Published: November 2023

Shrinkage and water loss are unique features of apoptosis. Despites decades of research, their exact roles, as well as the mechanisms of development, are poorly understood. Research in this area requires the ability to measure cell volume, intracellular water content, intracellular ion concentrations and the assessment of specific membrane permeability.

Cell volume

The volumes of adherent cells can be conveniently measured by transmission‐through‐dye (TTD) microscopy. This method is based on exclusion of a strongly absorbing extracellular dye by intact cells. The method can be realized on any transmission light microscope at nearly zero cost. TTD provides accurate measurements, even on severely deformed cells (which are common during apoptosis) and is compatible with fluorescence.

Cell water

Cell water is another essential parameter involved in apoptosis. Apoptotic shrinkage can occur by water expulsion as well as by cell fragmentation, and to discriminate between these two mechanisms, direct water measurements must complement measurements of the cell volume. Intracellular water is directly related to the average refractive index; the latter can be obtained from the the volume (which can be determined by the TTD) and from the delay of the light wave crossing the sample. A simple and effective way to measure the phase is based on the transport‐of‐intensity equation (TIE). Two transmission brightfield images are collected at different distances to the sample, and the phase is restored by solving the TIE. A Matlab‐based code for solving TIE is available.

Ion measurements

Monovalent ions play a major role in volume regulation. Because the properties of fluorescent ion probes depend on the environment, they have to be calibrated in situ. Ionophores are often used to control intracellular milieu; however, equalization of intracellular and extracellular ion concentrations would require the destruction of the membrane potential and a harsh treatment of cells. One way to avoid it is to measure the actual membrane potential y simultaneously with the ion of interest. The process can be presented by the following flow‐chart:

Membrane permeability

Apotosis can alter membrane permeability not only for inorganic ions, but for certain organic substances, such as choline. Some of the common cellular stains utilize same pathways and can be used to assess the cell permeability. More work is needed to characterize the channels involved in these processes.

Support or Funding Information

The work was partially funded by KSU Research Council grant
A Lymphedema‐Inducing Mutation of Cx47, R259C, Alters Growth Pattern and Cx43 Processing in Rat Insulinoma Cells
Authors: Kanady John , Pontifex Tasha , Taylor Samantha‐Su , Simon Alexander , Burt Janis
Published: November 2023

Connexins (Cx), proteins that mediate direct intercellular communication, are essential for normal vascular development and function. Mutations in Cx47 have been reported to underlie cases of lymphedema in humans, however the cellular and molecular mechanisms responsible for dysfunction have been largely unexplored. This study examines an orthologous mutation in mouse Cx47 to one of these lymphedema‐causing mutations, an arginine to cysteine substitution (R259C) in a highly‐conserved region of the second extracellular loop. We transfected connexin‐deficient rat insulinoma (Rin) cells with wild‐type (WT) Cx47 or Cx47R259C either alone or in combination with WT Cx43. Evaluation of cell growth was performed using light microscopy, protein expression through Western blot and immunocytochemistry, and electrical coupling via dual whole‐cell voltage clamp. Expression of Cx47 in Rin cells resulted in plexiform cell growth. Cx47R259C and Cx43/Cx47R259C coexpressing Rin cells also displayed plexiform growth patterns, but exhibited more elongated gaps between cell groups. Surprisingly, in Cx43/Cx47R259C cells a multiple banding pattern for Cx43 was detected via Western blot and full length Cx43 was conspicuously absent. Immunocytochemistry of Cx43/Cx47R259C cells revealed a diffuse cytoplasmic staining pattern for Cx43 and an absence of readily identifiable gap junction plaques. Based on initial electrophysiological recordings, Cx47‐expressing Rin cells have a low incidence of electrical coupling. Together, these preliminary data suggest that Cx47 influences cell growth, and subtle alterations in pattern occur with mutation of Cx47 at R259C. Furthermore, Cx47R259C affects cellular processing of Cx43, which may be a potential molecular mechanism by which this orthologous mutation leads to lymphatic defects in humans.

Support or Funding Information

This work was supported by the following grants: National Institutes of Health (R21HL122443) and Heart, Lung, and Blood Training Grant (T32HL007249)
Exercise Training Attenuates Vascular Smooth Muscle Remodeling of Coronary Microvasculature in Aged Heart
Authors: Hotta Kazuki , Chen Bei , Behnke Bradley J , Ghosh Payal , Alarcon David , Morgan Hannah , Cowan Morgan , Lucero Tiffani , Maraj Joshua , Haynes Anthony , Stabley John N , Delp Michael D , Muller‐Delp Judy M
Published: November 2023

Late life exercise training reverses age‐induced coronary microvascular dysfunction; however, the cellular mechanisms that underlie this reversal have not been identified. We tested the hypothesis that exercise training attenuates age‐induced vascular smooth muscle remodeling and proliferation, thus contributing to an improvement of microvascular function in coronary arterioles from old rats. Young and old Fischer 344 rats underwent daily exercise training (15 m/min, 15° incline, 1 hr/d, 5 d/wk for 10 wks) or served as sedentary controls. At the end of training, coronary arterioles were isolated, pressurized and fixed, and subsequently cryosectioned to assess vascular smooth muscle cell number and size, and vascular smooth muscle proliferation via immunohistochemistry. Serum samples were obtained to measure circulating adiponectin, an adipokine that promotes vascular smooth muscle differentiation. Immunoblot analysis was performed on pooled arteriolar samples to assess arteriolar protein levels of adenosine monophosphate‐activated kinase (AMPK), a downstream signaling protein of adiponectin receptors, smooth muscle myosin heavy chain isoform I (SMM I), and the synthetic protein, ribosomal protein s6 (rpS6). Aging increased vascular smooth muscle cell size and correspondingly reduced the number of vascular smooth muscle cells per cross‐sectional unit of vascular smooth muscle. Aging increased vascular smooth muscle cell proliferation. Aging decreased circulating adiponectin and reduced the levels of AMPK and SMM I in coronary arterioles; whereas the level of rpS6 increased in arterioles from old rats. After exercise training, the age‐related difference in the number and size of smooth muscle cells was no longer detectable. Exercise training also decreased vascular smooth muscle cell proliferation in arterioles from old rats, and increased circulating adiponectin in both young and old rats. Exercise training increased the level of SMM I in arterioles from old rats. Age‐related differences in the arteriolar levels of AMPK and rpS6 were no longer detectable in coronary arterioles after exercise training. These results indicate that exercise training‐induced reversal of coronary microvascular dysfunction in the aged heart is linked to attenuation of smooth muscle proliferation and hypertrophy. Furthermore, the restorative effect of exercise training on coronary microvascular function may result from adiponectin‐driven changes in the vascular smooth muscle cell phenotype characterized by increased expression of contractile proteins and reduced expression of synthetic proteins.

Support or Funding Information

NIH R01 HL077224 and NIH R01 HL90937
Hypertension‐induced impairment in synaptic plasticity and dysregulation of genes involved in synaptic function in the mouse hippocampus: implications for the pathogenesis of vascular cognitive impairment in aging
Authors: Valcarcel‐Ares Marta Noa , Tucsek Zsuzsanna , Tarantini Stefano , Hertelendy Peter , Giles Cory B. , Orock Albert , Sonntag William E , Wren Jonathan D , Csiszar Anna , Deak Ferenc , Ungvari Zoltan
Published: November 2023

Strong epidemiological and experimental evidence indicate that hypertension predisposes to cognitive decline in the elderly, but the underlying mechanisms remain elusive. Long‐term potentiation (LTP), defined as a long‐lasting increase in synaptic efficacy following high‐frequency stimulation of afferent fibers, is presumed to play an important role in the establishment and storage of memories in the hippocampus. The present study was designed to characterize the effects of hypertension on synaptic plasticity and the expression of genes involved in regulation of synaptic function in the hippocampus. Hypertension was induced in 3 and 24 month old C57BL/6 mice by chronic (4 weeks) infusion of angiotensin II. To determine whether hypertension compromises neuronal mechanisms of learning and memory, extracellular recordings were performed from acute hippocampal slices using multi‐electrode arrays. Field excitatory post‐synaptic potentials (fEPSPs) were invoked through stimulation of the performant path collaterals and obtained from the dentate gyrus area. We found that hypertension was associated with significant decreases in LTP, mimicking the aging phenotype. Synaptic density was decreased by hypertension, an effect that was exacerbated by aging. In aged mice hypertension decreased protein expression of NMDAR1 and NMDAR2A, whereas expression of GLUR1 and GLUR2 were unaffected. Hippocampal mRNA expression of genes involved in synaptic function was assessed using a targeted qPCR array. The hippocampal gene expression signature observed in hypertensive mice (including altered expression of Nptx2, Bdnf, Homer1, Stxbp1 (Munc‐18), Dlg4) provides important clues for subsequent studies to elucidate the mechanisms by which hypertension may contribute to the pathogenesis and clinical manifestation of both vascular cognitive impairment (VCI) and Alzheimer's disease.

Support or Funding Information

This work was supported by grants from the American Heart Association, the National Center for Complementary and Alternative Medicine, and the National Institute on Aging
Enterococcus Faecium Strain R30 Prevents Capillary Regression through Increasing Red Blood Cell Velocity in Rat Soleus Muscle under Disuse Condition
Authors: Hirayama Yusuke , Nakanishi Ryosuke , Ueno Mizuki , Yoshikawa Madoka , Tategaki Airo , Maeshige Noriaki , Fujino Hidemi
Published: November 2023

Inactivity leads to capillary regression in skeletal muscle. Angiogenesis is promoted by shear stress via endothelial nitric oxide synthase (eNOS)‐vascular endothelial growth factor (VEGF) pathway, and the shear stress correlates to blood flow velocity. Enterococcus faecium strain R30 (R30) has been shown to increase exercise tolerance and muscle sympathetic activity. Therefore, the aim of this study was to determine the effect of administration of R30 on hemodynamics and preventive effects via angiogenesis pathway on hindlimb unloading (HU) induced capillary regression in rat soleus muscle. Firstly, the red cells velocity in the soleus muscle was measured for assessment of the hemodynamics by single administration of R30 in rats. Secondly, chronic effects of R30 supplementation on HU‐induced capillary regression in skeletal muscle of rats were determined. Twenty‐five male SD rats were divided and administrated either saline or R30; control+saline, control+R30, HU+saline, and HU+R30. HU groups were unloaded for 2 weeks. The red cells velocity was increased by single administration of R30 compared to saline and the response was inhibited by beta‐blocker; propranolol (2 mg/kg i.v.). In addition, HU resulted in capillary regression compared to control, indicated by decreases in capillary‐to‐fiber ratio and capillary volume. However, the capillary‐to‐fiber ratio and the capillary volume in HU+R30 were higher than those in HU+saline. In addition, both the levels of eNOS and VEGF protein expression in HU+R30 were higher than those in HU+saline. These results indicated that R30 administration attenuated the capillary regression via eNOS/VEGF pathway by increasing red blood cell velocity in atrophied muscle.
Coronary Microvascular Smooth Muscle‐Endothelial Cell Communication and Notch 3 Signaling in Type 2 Diabetes
Authors: McCallinhart Patricia E. , Clark Olivia E. , Kanai Mariko , Lilly Brenda , Trask Aaron J.
Published: November 2023

The Notch signaling pathway, via cell‐cell communication, is critical during normal vascular development and homeostasis, and it is known to regulate vascular smooth muscle (VSMC) phenotype, including proliferation. The current study tested the hypothesis that VSMC‐endothelial cell (EC) Notch 3/Jagged 1 signaling is aberrant to account for adverse remodeling and stiffness observed in type 2 diabetic coronary resistance microvessels (CRMs). Low‐passage primary coronary VMSCs were isolated from normal and type 2 diabetic db/db mice and were either mono‐ or co‐cultured with normal or type 2 diabetic human coronary microvascular endothelial cells (hCMECs), respectively (n=4–6 per group). In separate experiments, normal (n=3) and diabetic db/db CRMs (n=3) were isolated, mounted, and perfusion pressure fixed on a pressure myograph. The size and frequency of fenestrae in the internal elastic laminas of intact CRMs were assessed using Alexa Fluor 633 hydrazide on a confocal microscope. Coronary VSMCs isolated from db/db mice expressed higher Notch 3 mRNA (2.12±0.10 vs. 1.00±0.20; p<0.01) compared to normal. Co‐culture of coronary VSCMs with hCMECs caused a robust induction of Notch 3 expression in normal VSMCs (3.67±0.60 vs. 1.00, p<0.001), which was exacerbated in coronary VSMCs isolated from db/db mice (5.52±0.51 vs. 3.67±0.60, p<0.05). Alpha‐smooth muscle actin (α‐SMA) trended lower in db/db coronary VSMCs (p=0.07), while co‐culture with hCMECs did not alter α‐SMA expression. Diabetic hCMECs expressed lower Jagged 1 mRNA compared to normal (0.58±0.04 vs. 1.00±0.07; p<0.01); co‐culture of hCMECs and coronary VSMCs caused a robust reduction in Jagged 1 expression in normal cells (0.63±0.06 vs. 1.00±0.07, p<0.001), while no further reduction was observed in co‐cultured diabetic cells. The number of fenestrae in the internal elastic lamina was reduced in diabetic CRMs (0.028±0.005 vs. 0.040±0.001 fen/μm2, p<0.05), while the size of the fenestrae remained unchanged. These data show that Notch3/Jagged 1 expression and VSMC differentiation are altered in diabetic coronary resistance microvessel cells, and VSMC‐EC contact via co‐culture induces differential dysregulation of Notch3/Jagged1 signaling. The data further show a reduction in the number of fenestrae in db/db CRMs, suggesting that the opportunity for Notch3/Jagged1 signaling to occur in vivo may be reduced to contribute to adverse remodeling and stiffness observed in those blood vessels.

Support or Funding Information

AHA 15UFEL22760002 to MK; NIH K99HL116769 and NCH to AJT.
Myocardium From the Left and Right Ventricles of Human Hearts Have Similar Mechanical Properties
Authors: Blair Cheavar A , Guglin Maya , Stromberg Arnold , Campbell Kenneth S
Published: November 2023

The left and right ventricles (LV and RV) of human hearts generate different pressures. It is not clear if this difference in pressure reflects the arrangement of cells in each ventricle, or the force generating capacity of individual myocytes. Thus, the aim of this study is to test the hypothesis that cells from the RV and LV of human hearts have different mechanical properties. Chemically permeabilized multicellular preparations from both ventricles of explanted hearts from patients with heart failure (n=12) and patients without a history of heart failure (non‐failing samples) (n=6) were attached between a force transducer and a motor to determine mechanical properties. Similar to prior data from our laboratory, heart failure reduced isometric force and maximum power by ~40% (p<0.001) and ~30% (p=0.004) respectively. Heart failure also increased pCa50 from ~5.41 to ~5.55 (p=0.006). None of these properties differed between the LV and the RV samples (all p‐values > 0.05). There were no significant interactions between heart failure status and region. These data imply that myocardium from the left and right ventricles has comparable mechanical properties and that heart failure impacts both ventricles in a similar way. Interventricular differences in chamber function are most likely to reflect structural effects as opposed to the intrinsic contractile properties of the cells.

Support or Funding Information

NIH
8UL1TR000117‐02

NIH
P30GM110787

AHA
15GRNT25460003

NSF 153854
Improvement of coronary flow and cardiac function, as a function of deletion/downregulation of soluble epoxide sydrolase
Authors: Qin Jun , Kandhi Sharath , Froogh Ghezal , Jiang Houli , Sun Dong , Huang An
Published: November 2023

To test the hypothesis that increased epoxyeicosatrienoic acids (EETs) bioavailability via the deletion of soluble epoxide hydrolase (sEH) gene improves coronary perfusion in a sex‐different manner, hearts were isolated from male (M) and female (F) wild type (WT) and sEH knockout (KO) mice and perfused with constant flow at different pre‐loads. Hearts isolated from F‐WT and F‐KO mice required significantly higher basal coronary flow to maintain the perfusion pressure at 100 mmHg than their male counterparts, suggesting a reduced basal coronary resistance in females. Increases in pre‐loads initiated a greater reduction in coronary resistance, associated with greater cardiac contraction (developed tension) in F‐WT compared to M‐WT mice. These changes became more dramatic in both sexes of sEH‐KO mice, with more predominant in F‐KO mice. Similarly, increases in cardiac work elicited greater reduction of coronary resistance in both sexes of sEH‐KO than their WT controls in a female‐favorable manner. Cardiac expression of sEH was dwonregulated in F‐WT mice, and coincided with higher cardiac EET levels in F‐WT, as well as M‐KO and F‐KO mice. Thus, the elevation of cardiac EETs, as a function of deletion or female‐specific downregulation of sEH gene, contributes significantly to increases in coronary flow and improvement of cardiac function.

Support or Funding Information

This work was supported by grant HL070653
Only Two Weeks of Physical Inactivity Induces Insulin Resistance and Impairs Glycemic Control in Older Adults
Authors: McGlory Chris , von Allmen Mark , Stokes Tanner , Phillips Stuart
Published: November 2023

Periods of physical inactivity are known to result in deleterious consequences for skeletal muscle mass, strength, and insulin sensitivity. We propose that even transient periods of reduced activity likely have a greater impact in older individuals who often fail to fully recover. We aimed to examine the impact of two weeks of step reduction (SR), as a model of physical inactivity akin to that occurring during illness, on indices of glycemic control and muscle strength in thirteen healthy older adults (7 men, 6 women, 68 ± 3yr [all data mean±SD]). Participants underwent 7 d of monitored normal activity (pre‐SR) immediately before reducing their daily step‐count from 7379 ± 3809 to 967 ± 79 steps.d−1 for 14 d (SR), and then returning to their habitual step count for a period of 14 d (post‐SR). The HOMA‐IR and MATSUDA models of insulin resistance, and insulin sensitivity as well as glucose and insulin area under the curve GAUC and IAUC in response to an oral glucose tolerance test were assessed pre‐SR, SR, and post‐SR. Peak isometric maximal voluntary contraction (ISO‐MVCpeak), peak rate of torque development (RTDpeak) number of sit‐to‐stand cycles in 30 s, and total distance walked in 6 min were also assessed at these time points. HOMA‐IR increased from pre‐SR to SR (2.8 ± 0.3 to 3.6 ± 0.6, p<0.05) and MATSUDA decreased (3.7 ± 0.5 to 3.0 ± 0.1, p < 0.05), neither recovered at post‐SR. GAUC was not significantly different between pre‐SR (435 ± 121) and SR (467 ± 136) but was marginally lower at post‐SR (386 ± 140, p=0.05). IAUC increased from pre‐SR to SR (3796 ± 579 to 4329 ± 833, p<0.05) and, was still elevated at post‐SR (4051 ± 768, p<0.05). There was no change from pre‐SR to SR in either ISO‐MVCpeak (146 ± 34 to 148 ± 41 N.m, p>0.05), RTDpeak (2142 ± 1527 to 1956 ± 894 N.m.s−1, p>0.05), sit‐to‐stand cycles in 30 s (17 ± 5 to 16 ± 5, p>0.05) or total distance walked in 6 min (561 ± 108 to 563 ± 110 m, p>0.05). These data indicate that two weeks of SR negatively impacts indices of glycemic control in older adults that are not fully recovered after 14 d of return to habitual step count. However, indices of muscle function remained unaltered.

Support or Funding Information

Supported by the Canadian Diabetes Association
Influence of Aerobic Fitness, Contraceptive Use, and Height on Cardiac Augmentation Index in Young Healthy Females
Authors: Smith Michael , Devor Steven
Published: November 2023

PURPOSE

While female hormones and high levels of aerobic fitness may offer a protective advantage against arterial stiffness in young women, short stature may negate this benefit. Our goals were to identify the key determinates of augmentation index (AIx) and investigate the influence that cyclic fluctuations of estradiol and progesterone have on ventricular‐vascular coupling in young healthy women.

METHOD

We assessed the relationship between maximal aerobic capacity (VO2max) and AIx standardized to 75 beats per minute (AIx@75 BPM). Twenty two nulliparous women taking either Loestrin (n=5), Ortho‐cyclen (n=5), or no contraceptive (n=12) underwent graded exercise testing and applanation tonometry of the radial artery to measure VO2max and AIx, respectively, during low hormonal status (±48h menses). Subsequently, AIx@75 BPM was measured at days 7, 14, and 21 post‐menses. Saliva was collected for analysis of estradiol and progesterone at all 4 time points.

RESULTS

In all subjects, there was a statistical difference in AIx between mid‐follicular and early luteal phases (P <0.01), and between late luteal and early follicular phases (P<0.05). AIx was strongly and negatively associated with relative aerobic fitness (R2=.42, P<0.0001), and an additional 17% of this relationship was explained when corrected for height (R2=.69, P<0.0001). 14% of the variation in AIx@75 BPM was further explained by no contraceptive use, body composition, and peripheral diastolic blood pressure.

CONCLUSIONS

AIx fluctuates inversely with estradiol concentrations throughout the menstrual cycle. High aerobic capacity is associated with favorable AIx and may offer a mechanism that protects ventricular‐vascular coupling efficiency from deleterious variables such as short stature, age, and impedance mismatch. These results underscore the importance of regular exercise for women for enhanced heart and vascular health, especially as they approach menopause.
Altered Ca2+ Dynamics and Vascular Function During Low‐flow Induced Vascular Remodeling
Authors: Weber David S , Choi Chung‐Sik , Bayazid Leith , Glosemeyer Katherine , Taylor Mark S
Published: November 2023

In light of the widely recognized fact that the integrated endothelial cell response to shear stress is pivotal in the control of VSMC‐mediated wall remodeling, it is striking that the underlying Ca2+ signaling remains poorly understood. We hypothesized that low flow and sustained oscillatory shear stress results in decreased number, size and range of functional Ca2+ events in endothelial cells facilitating changes in vascular function and neointima formation. To test this, we used a partial carotid ligation model in C57BL/6J mice to induce chronic in vivo low flow and oscillatory shear stress. After 14 days of ligation, both control and partially ligated carotid arteries were harvested from each animal. Partial ligation resulted in substantial vascular remodeling (intima/media ratio of .42±.13), while no remodeling occurred in non‐ligated contralateral control carotids. To measure Ca2+ dynamics, carotid artery segments were opened longitudinally and pinned, luminal‐side‐up on Sylgard blocks with tungsten micropins. Arteries were loaded with fluo‐4 AM and Ca2+‐dependent fluorescence measured with a spinning‐disk laser confocal microscope. Partial ligation led to basal reductions in distinct signaling sites as well as the magnitude and spread of endothelial Ca2+ events in the endothelium. Using a modified parallel‐plate flow chamber to induce acute changes in shear stress, our studies demonstrated that a chronic long‐term decrease in flow leads to decreased Ca2+signaling sites and a shift from wide‐scale predominant waves to frequent localized Ca2+ transients along the intima. Acetylcholine (1 uM) produced sustained Ca2+ events in control endothelium but these were reduced to small periodic transients in partially ligated carotids. This pattern corresponded with impaired acetylcholine‐mediated relaxation as determined using myography (Control 60±10% vs Partial ligation 25±7%) while maximal relaxation in response to sodium nitroprusside was similar. Phenylephrine‐induced vasoconstriction was enhanced 2‐fold in partially ligated carotid arteries compared to control while KCl induced contraction following partial ligation was decreased 50%. Our findings indicate that chronic low flow in vivo in the absence of other confounding factors leads to significant neointima formation and changes in Ca2+ dynamic patterning. Current studies are on‐going to identify unique pattern changes in endothelial Ca2+ events associated with altered vascular function and investigate the subsequent changes in Ca2+ mediated endothelial signaling underlying the development of vascular pathology.

Support or Funding Information

NIH HL084159 (DSW) and HL085887 (MST)
Altered vascular endothelial control of facial skin blood flow in rosacea
Authors: Metzler‐Wilson Kristen , Kube Megan , Mann Sarah , Demidova Olga , Sammons Dawn L , Wilson Thad E
Published: November 2023

Sympathetic outflow to the face may be augmented in rosacea, a disorder which clinically presents with flushing (Metzler‐Wilson & Toma, et al, J Neurophysiol, 2015). To determine whether regulatory mechanisms of skin blood flow (SkBF) are altered by these chronic outflow differences or associated inflammatory mediators, SkBF oscillations were assessed in 9 otherwise healthy subjects with rosacea and age‐sex matched controls during baseline and local heat stress (42°C, 30 min). Local heating is classified as a flushing trigger in rosacea and induces cutaneous vasodilation with an initial axon reflex‐mediated peak and a second nitric oxide‐mediated peak. We hypothesized that compared to controls, individuals with rosacea would demonstrate greater changes in spectral power in the vascular endothelial (very‐low frequency (VLF); 0.009–0.02 Hz) and sympathetic (low frequency (LF); 0.02–0.06 Hz) frequency ranges during heating of typically rosacea‐affected (forehead, cheek) but not rosacea‐unaffected (forearm, palm) skin areas. SkBF (laser‐Doppler flowmetry) was measured and changes in SkBF spectral power (fast Fourier transform) in VLF and LF ranges from baseline to heating were determined at each skin site. Individuals with rosacea had significantly greater changes in VLF SkBF spectral power in cheek (0.024±0.003 vs. 0.006±0.002 au; p=0.03) and forehead (0.030±0.003 vs. 0.011±0.001 au; p=0.04) but not forearm (0.015±0.002 vs. 0.013±0.001 au; p=0.76) or palm (−0.019±0.006 vs. −0.041±0.005 au, rosacea and control, respectively; p=0.38) sites. There were no significant differences in the change in LF SkBF spectral power between groups (cheek p=0.29, forehead p=0.35, forearm p=0.69, palm p=0.80). These data indicate that during local heat stress, individuals with rosacea have altered vascular endothelial control of facial SkBF. These vascular endothelial differences may be due to chronic neuroinflammatory processes.

Support or Funding Information

National Rosacea Society
Downregulation of the Nuclear Factor (Erythroid‐Derived 2)‐Like 2 (NRF2) Antioxidant Defense System Contributes to Endothelial Dysfunction and Microvascular Rarefaction with Elevated Dietary Salt Intake
Authors: Lombard Julian H. , Kautenburg Katie E. , Priestley Jessica R. C. , McCord Joe M. , Greene Andrew S. , Geurts Aron M.
Published: November 2023

A high salt diet leads to vascular oxidant stress, endothelial dysfunction, and microvascular rarefaction, which can be ameliorated by chronic infusion of a low dose of angiotensin II (ANG II) or angiotensin (1–7) [ANG(1–7]. The present study investigated the role of the NRF2 antioxidant defense system in mediating the protective effect of ANG II and ANG (1–7) to ameliorate endothelial dysfunction and microvascular rarefaction occurring with elevated dietary salt intake. In these experiments, short term (3 days) high salt diet (4% NaCl) eliminated endothelium‐dependent dilation to acetylcholine (ACh) in isolated middle cerebral arteries (MCA) and reduced microvessel density (assessed with rhodamine‐labeled GS1 lectin) in the cremaster muscle of Sprague‐Dawley (S‐D) parental strain rats. The NRF2 inducers Protandim and sulforaphane restored ACh‐induced dilation of MCA and increased microvessel density in S‐D rats fed HS diet for 2 weeks. Deletion mutation of the Nrf2 gene utilizing TALEN technology eliminated the protective effect of ANG II infusion and significantly reduced the protective effect of ANG (1–7) infusion to restore ACh‐induced dilation of the MCA. The Nrf2 (−/−) mutation also eliminated the ability of ANG II and ANG (1–7) to prevent microvascular rarefaction in HS fed rats. Protandim restored endothelium dependent dilation to ACh in MCA of HS fed wild type controls, but not MCA of HS‐fed Nrf2 (−/−) mutant rats. These findings indicate that down regulation of NRF2‐mediated antioxidant defenses due to salt‐induced suppression of the renin‐angiotensin system leads to vascular oxidant stress, endothelial dysfunction, and microvascular rarefaction; and suggest that dietary upregulation of the NRF2 antioxidant system may be a valuable therapeutic approach to ameliorate endothelial dysfunction with HS diet and in low renin forms of salt‐sensitive hypertension.

Support or Funding Information

NIH #R‐01 HL65289‐12; #R56 HL65289‐13A1, and #R21 OD018309‐01
Effects of acute TUDCA administration on postprandial hyperglycemia and endothelial function
Authors: Walsh Lauren Kay , Restaino Robert , Manrique Camila , Padilla Jaume
Published: November 2023

Previous studies have shown that postprandial hyperglycemia causes a transient impairment in endothelial function; however, the mechanisms remain largely unknown. Herein we tested the hypothesis that hyperglycemia‐induced endothelial dysfunction is attributed to endoplasmic reticulum (ER) stress. Specifically, we hypothesized that acute oral administration of tauroursodeoxycholic acid (TUDCA, 1500mg), a chemical chaperone known to alleviate ER stress, would prevent hyperglycemia‐induced endothelial dysfunction. In 10 young healthy subjects (five men, five women), brachial artery flow‐mediated dilation (FMD) was assessed at baseline, 1 hour, and 2 hours post oral administration of a 75g glucose load. Subjects were tested on two separate visits in a single‐blind randomized crossover design: after oral ingestion of TUDCA or placebo capsules. FMD was reduced during hyperglycemia under the placebo condition (baseline: 7.01± 1.00%, 1hr: 4.75± 0.68%, 2hr: 5.29± 0.55%) but not under the TUDCA condition (baseline: 5.62± 0.67%, 1hr: 5.43±0.84%, 2hr: 6.01±0.91%). These preliminary results suggest that hyperglycemia‐induced endothelial dysfunction can be mitigated with oral administration of TUDCA, thus supporting the hypothesis that ER stress may contribute to endothelial dysfunction during postprandial hyperglycemia.

Support or Funding Information

Support: NIH R21DK105368 (Jaume Padilla)
Decreased Systemic Microvascular Function May Contribute to Increased Cardiovascular Risk in Women with Asthma
Authors: Kapoor Karishma , Moore Linn E , Byers Bradley W , Stickland Michael K
Published: November 2023

INTRODUCTION

Individuals with asthma are at increased risk of developing cardiovascular disease (CVD), with the greatest risk being reported among asthmatic women. The mechanisms underlying differences in CVD risk between sexes are presently unknown. However, endothelial and microvascular dysfunction, and elevated arterial stiffness are early indicators of CVD development and have been shown to be predictive of adverse cardiovascular outcomes. The purpose of this study was to examine markers of vascular health and thus, CVD risk in men and women with asthma.

METHODS

Thirteen male (27.9±6.3 years) and eleven female asthmatics (24.7±4.7 years), matched for BMI, were recruited for the study and screened for known cardiovascular conditions. Participants were required to fast and refrain from drinking alcohol, exercising, and using a bronchodilator for 12 hours prior to testing. Endothelial function was assessed by flow‐mediated dilation normalized to shear stress (FMD/SS). Microvascular function was evaluated as hyperemic blood flow (Velocity Time Integral, VTI; cm/s) and arterial stiffness as carotid and radial pulse wave velocity (PWV; m/s). All measurements were done in the supine position following 10 minutes of rest. Two‐tailed Student's t‐tests were used to for statistical comparison between males and females.

RESULTS

No between‐group differences were seen in endothelial function (female: 0.054±0.009; male: 0.058±0.047, p=0.820), or arterial stiffness (7.8±1.5; 8.8±1.3 m/s, p=0.115). However, women had reduced microvascular function, evaluated as reactive hyperemic blood flow, in comparison to men (74.7±17.4; 96.7±28.2 cm/s, p=0.035).

CONCLUSIONS

Asthmatic women have reduced microvascular function compared to asthmatic men, which may be a contributing factor to the increased risk of CVD seen among women with asthma.

Support or Funding Information

The Lung Association, Alberta & North West Territories (NWT)
Development of a Novel Technique to Isolate and Characterize Mouse Podocytes
Authors: Kung Ling Hsuan , Wasti Ruby , Tibolla Annette , Gibson Glenn , Pullen Steven , MacDonnell Scott
Published: November 2023

INTRODUCTION

Podocytes play a key role in the selective filter that underlies kidney function. The loss of mature podocytes is involved in chronic kidney disease progression. The mechanisms of podocyte injury, leading to proteinuria are still unclear due to lack of consistent and validated methods to study primary podocytes. In vitro system, glomerular outgrowth and human immortalized cell line (HIC) have their merits; however, these cells fail to express podocyte specific markers and variations in culture protocols challenge their utility. Hence, an isolation method is required to define potential mechanism involved in disease progression.

METHODS

Mouse glomeruli were digested in Liberase (100 μg/ml, 40 min) followed by trypsin (0.075%, 20 min). Mixed cells were filtered (70μm) and a 15% BSA cushion was used to eliminate debris. Podocytes were then enriched using anti‐nephrin and magnetic beads via columns. Expression of selected genes was compared between glomerular (G), nephrin positive (N+) fraction, and HIC.

RESULTS

While podocyte selective markers were observed in the G fractions, significant expression can only be observed in N+. Podocyte specific genes are enriched in N+; ΔCT WT1 (N+ 3.6 vs. G 7.5), Podocin (N+ 0.8 vs. G 4.5), and TRPC6 (N+ 5.76 vs. G 12.4). Same genes also expressed higher in N+ than fully differentiated HIC; ΔCT WT1 (N+ 3.6 vs. HIC 12.2), podocin (N+ 1.9 vs. HIC 17.4), and TRPC6 (N+ 5.7 vs. HIC 16.6). Mesangial cell specific gene SerpinB7 was not observed in N+ (ΔCT14.8), but was expressed in HIC (ΔCT 8.8), confirmed the specificity of N+ cells.

CONCLUSION

A method has been developed to isolate and enrich mouse podocytes. It provides utility to study podocytes from healthy and diseased kidneys to identify druggable targets and pathways. However, additional studies are required to profile the function of N+ and possibility for long term culture.

Support or Funding Information

Supported by Cardio‐Metabolic Disease Research Department, Boehringer Ingelheim
Elucidating the Role of Arginases in Diabetic Nephropathy: Arginase 2‐dependent and ‐independent Effects
Authors: Awad Alaa S. , You Hanning , Gao Ting , Cooper Timothy K , Morris Sidney M
Published: November 2023

Diabetes is the leading cause of end stage renal disease, resulting in a significant health care burden and loss of economic productivity by affected individuals. As current therapies for progression of diabetic nephropathy are only moderately successful, identification of underlying mechanisms of disease process is essential in order to develop more effective therapies.

We showed previously that treatment with an arginase inhibitor or genetic deficiency of the arginase 2 isozyme was protective against several key features of nephropathy in diabetic mouse models. However, those studies did not determine whether all tested markers of diabetic nephropathy were dependent only on arginase 2 expression.

The objective of this study was to identify features of diabetic nephropathy that are associated specifically with arginase 2 expression.

The arginase inhibitor S‐(2‐boronoethyl)‐L‐cysteine or vehicle was continuously infused via minipump in male wild‐type and arginase 2‐deficient (Arg2−/−) mice for 12 weeks following induction of diabetes by streptozotocin injection.

Our results demonstrate that elevated urinary albumin excretion rate and plasma urea levels, and decreased renal medullary blood flow, were associated almost completely and specifically with arginase 2 expression. Furthermore, diabetes‐dependent increases in renal fibronectin mRNA levels were largely dependent on arginase 2 expression, with no indication of any significant contribution by arginase 1.

These findings indicate that arginase 2 selectively mediates major aspects of diabetic renal injury. However, increases in renal macrophage infiltration and renal TNF‐α mRNA levels occurred independently of arginase 2 expression but were almost entirely abolished by treatment with the arginase inhibitor, indicating that arginase 1 is required for increases in these markers of diabetic renal pathology.

Support or Funding Information

This study was supported by NIH Grants DK094930 and DK094930S1
ENaC activity in the collecting ducts of the STZ‐induced type 1 diabetic Dahl SS rats
Authors: Pavlov Tengis S. , Ilatovskaya Daria V. , Levchenko Vladislav , Brands Michael W. , Staruschenko Alexander
Published: November 2023

The epithelial sodium channel (ENaC) is expressed in the aldosterone sensitive distal nephron where it performs sodium reabsorption from the lumen. Previous studies indicate that high glucose upregulates ENaC expression and activity in cultured cortical collecting duct cells. However, activity of this channel has never been studied in a rodent models of diabetes. Streptozotocin (STZ) treatment is a widely accepted approach to study type 1 diabetes (T1D). However, while Sprague Dawley rats or other salt‐resistant rodent models develop hyperglycemia and T1D after STZ treatment, they do not develop essential features of diabetic nephropathy (DN). To address this limitation, we induced T1D in the Dahl Salt‐Sensitive (SS) rat, which is a model of salt‐sensitive hypertension. We and others reported that 11–12 weeks of STZ‐induced diabetes is sufficient for the animals to develop hyperglycemia, polyuria, weight loss, microalbuminuria, nephrinuria and display renal histological lesions typical for patients with DN. T1D was induced in 6 week old male SS rats with a single i.p. injection of 75 mg/kg STZ. An insulin pellet was implanted on day 7 after STZ treatment in order to keep blood glucose around 300 mg/dL. At the end of experiments the animals were sacrificed and the kidneys were flushed and collected for analysis. We did not measure any difference in plasma aldosterone levels between STZ‐treated and control animals. However, single channel patch‐clamp analysis of ENaC activity in isolated split‐opened tubules revealed that high blood glucose levels increased channel activity compared to normoglycemic animals (NPo were 1.57±0.20 and 0.59±0.19 in STZ‐treated and control animals, respectively). Western blotting and immunohistochemical analyses revealed greater abundance of beta‐ENaC subunits in the kidney cortex of hyperglycemic rats. We conclude that high blood glucose levels in T1D cause ENaC hyperactivity that can lead to excessive sodium and water reabsorption.

Support or Funding Information

Supported by NIH R00HL116603, R01HL056259.