bluuebunny/crossref_metadata_2025 huggingface dataset:
Angiotensin Converting Enzyme‐2 and Mas Receptor Are Hypoxia‐Regulated in Human CD34+ cells
Authors: Joshi Shrinidh , Jarajapu Yagna PR
Published: November 2023

Adult bone marrow‐derived CD34+ cells have the propensity of re‐endothelialization and vascular generation, and are currently being investigated for the potential benefit in the treatment of ischemic vascular diseases. Activation of the cardiovascular‐protective axis of renin angiotensin system, Angiotensin Converting Enzyme‐2 (ACE2)/Angiotensin‐(1–7)/Mas receptor (MasR) axis, stimulates vascular repair‐relevant functions in CD34+ cells. We have previously reported that exposure of CD34+ cells to hypoxia enhances vasoreparative functions. The current study tested the hypothesis that the beneficial effects of hypoxia are mediated by ACE2/MasR pathway. CD34+ cells were isolated from mononuclear cells (MNCs) derived from healthy volunteers (n=36). Exposure to normoxia (20% O2) and hypoxia (1% O2) was accomplished by using a hypoxia chamber. Proliferation and migration were evaluated by using BrdU‐ELISA or chemotaxis assay, respectively. Protein and mRNA expressions of ACE2 and MasR were determined in cells by western blotting or by flow cytometry and real‐time PCR, respectively. ACE2 activity was determined in cell‐lysates and cell supernatants by using an enzyme‐specific fluorogenic substrate, 7‐Mca‐YVADAPK(Dnp), and a selective inhibitor, MLN4760. Exposure to hypoxia increased proliferation and migration in basal conditions (n=8, P<0.05) or in response to stromal‐derived factor‐1a (SDF) (n=6, P<0.05) or vascular endothelial growth factor (VEGF) (n=6, P<0.05), compared to normoxic exposure. Hypoxia stimulated ACE2 activity in CD34+ cells (n=6, P<0.05 vs normoxia) but not in MNCs. ACE2 and MasR mRNA (n=5, P<0.05) and protein levels (n=5, P<0.01) were increased by hypoxia. ACE2 activity was increased (n=6, P<0.05) and immune‐reactive ACE2 fragments were observed in the cell‐supernatants of hypoxia‐exposed CD34+ cells compared to normoxic‐cell supernatants. Collectively, these results suggest that hypoxic potentiation of vascular‐repair relevant functions is associated with upregulation of ACE2 and MasR expression, which likely mediate the ischemic vascular repair by CD34+ cells.
Electrically‐Induced Cardioprotection via the Skin
Authors: Roessler Anne E , Luther Kristin , Ren Xiaoping , Wang Yang , Shen Zilong , Jones Walter Keith
Published: November 2023

Ischemic preconditioning (IPC) is a well‐characterized cardioprotective phenomenon involving multiple brief, non‐lethal ischemic episodes to the heart. Remote ischemic preconditioning (RIPC) elicits cardioprotection via repetitive ischemia and reperfusion to a distant vascular bed, organ, or limb. A non‐ischemic, nociceptive stimulus in distant tissues can result in the most powerful cardioprotection reported – an 80% decrease in infarct size – called remote nociceptor‐induced cardioprotection (NIC). Electrically‐induced cardioprotection via electroacupuncture with needles (EA) or electrical stimulation with patches (ES) on the skin of the abdomen may be a clinically feasible way to induce cardioprotection.

Our previous work shows that the transcription factor NF‐κB regulates many cardioprotective genes in the heart after IPC, including heat shock proteins (HSP), which act in unity to assist in protein folding, repair, and degradation following myocardial injury. We found that IPC requires NF‐κB‐dependent upregulation and post‐transcriptional regulation of HSP70.3. This study's objective is to determine the molecular mechanism of electrically‐induced NIC. Based upon our work in IPC, we hypothesized that electrical stimulation of the skin is cardioprotective via NF‐κB‐dependent upregulation of HSPs.

Our methodology uses a 5‐volt electrical stimulus applied to the abdomen via EA or ES. This reduces infarct size in a mouse surgical model of myocardial infarction when applied as either a preconditioning or postconditioning stimulus. Western blotting and PCR revealed only small non‐significant changes in HSP mRNA and protein relative to sham, inconsistent with the large significant increases in HSP seen in ischemic preconditioning. Whether these small increases in HSP mRNA and protein are functionally significant is under investigation using pharmacological and genetic inhibition of the NF‐κB‐dependent HSPs, and deep sequencing studies are planned to delineate additional potential genes that may be involved. Future investigations will also address the neurobiology of electrically‐induced cardioprotection via skin stimulation.

Support or Funding Information

grant number: R01HL091478
Validation of Click Chemistry with Mass Cytometry for Single Cell Analysis
Authors: Shaklee Jessica , Srivastava Krita , Grundhofer Heather , Pierre Valerie , van Berlo Jop
Published: November 2023

The field of molecular biology has rapidly advanced since the development of single cell characterization technologies, such as flow cytometry and monoclonal antibodies. More recently, novel single cell characterization methods have been developed to address the limitations of previous technologies. Mass cytometry detects the presence of antigen based on the molecular mass of a probe, sidestepping the problem of overlapping fluorescent signals in flow cytometry. Currently, mainly antibody‐based assays are possible for analysis on a mass cytometer. Here, we developed a novel probe that allows identification and detection of events using click chemistry. Click chemistry involves a method of labeling reagents in a stable, covalent way that circumvents the need for cumbersome antibody conjugations to metal probes and fastidious tittering and validation. Here we show that the thymidine analog 5‐ethynyl‐2′‐deoxyuridine (EdU) can be accurately detected via mass cytometry when labeled with a metal‐tagged azide in a click reaction. The level of detection is as expected based on comparative experiments analyzed using flow cytometry, showing that the combined use of click chemistry and mass cytometry is both feasible and valid. Furthermore, it paves the way for future experiments combining these techniques. Mass cytometry can theoretically measure up to about 100 parameters at once, many more than possible with flow cytometry, and without the need for laborious compensation. This feature makes it particularly attractive to immunologists and pharmacologists who are studying the intricate subtypes of immune cells and signaling cascades involving many parameters. In addition, click chemistry provides a very simple, easy staining procedure that is highly specific and compatible with many markers such as antibodies but could be especially valuable to detect non‐protein events. We believe the addition of click‐chemistry to the mass cytometry toolbox allows for expanded utility of these methods and will prove invaluable as molecular biologists probe deeper into our physiology.
Method to Assess Interactivity of Drugs with Nonparallel Concentration‐Effect Relationships
Authors: Zhao Liang , Au Jessie L.‐S. , Wientjes M. Guillaume
Published: November 2023

Commonly used methods for analyzing interactivity between drugs (e.g., synergy, antagonism) such as isobologram, combination index, and curve shift are based on the Loewe Additivity principle of dose equivalence and the inherent assumption of similar concentration‐effect (C‐E) including parallel curves and equal maximum effects (Emax), and therefore are not suitable for drugs with dissimilar C‐E. This study describes a new method that is without this limitation and has the additional advantage of enabling statistical analysis. The method comprises two steps. First, based on the dose equivalence principle, the experimentally obtained C‐E of one drug was used to calculate the equally effective C‐E of the other drug at no interactivity; the resulting two zero‐interactivity C‐E formed the upper and lower boundaries of Additivity Envelope. Next, 95% confidence intervals calculated from experimental data were added to Additivity Envelope to obtain Uncertainty Envelope (UE). Experimentally observed effects of drug combinations (C‐Ecomb,observed) located within UE indicate additivity whereas C‐Ecomb,observed located above or below UE indicate statistically significant (p<0.05) synergy or antagonism, respectively. Additional in silico studies demonstrated the shape and size of Additivity Envelope, which determine the ability to detect drug interactivity, depended on the Drug A‐to‐B concentration ratios and the ratios of their C‐E curve shape parameter. Analyses of experimental results of combinations of drugs with nonparallel C‐E and/or unequal Emax indicate UE was more versatile and provided more information, compared to earlier methods. UE is a broadly applicable method for analysis, including statistical significance assessment, of drug interactivity.

Support or Funding Information

This research is supported in part by the National Institutes of Health, National Institute of General Medical Sciences [Grant R01GM100487]; and National Cancer Institute [Grant R01CA163015].
Quantitative Pharmacology of Intercellular Drug Shuttle by Exosomes
Authors: Wang Jin , Cui Minjian , Yeung Bertrand Z. , Lu Ze , Wientjes Michael Guill , Woo Sukyung , Au Jessie L.‐S.
Published: November 2023

Background

Exosomes are extracellular vesicles (30–100 nm) that contain bioactive molecules, secreted by most cell types and play a role in intercellular communications. Tumor cells exosomes may affect tumor progression, metastasis, and therapeutic efficacy and resistance. The current study used in vitro experiments and in silico studies to investigate the roles of exosomes in intercellular drug transfer and the quantitative relationship between exosomes and pharmacodynamics of chemotherapy drugs in solid tumor cells.

Methods

Effects of paclitaxel (PTX) and doxorubicin (DOX) on exocytosis were studied in human breast (MCF7 and a lung metastatic subline of MDA‐MB‐231 cells, LM2) and ovarian (A2780 and OVCAR4) cancer cells. Inhibition of exocytosis was studied using omeprazole (proton pump inhibitor) and GW4869 (ceramide synthesis inhibitor). Time‐lapse laser confocal microscopy was used together with RFP‐transfected cells to monitor the release of exosomes into extracellular fluid. Extracellular exosomes were collected and quantified based on their acetylcholinesterase activity. Drug concentrations were measured using LC‐MS/MS. Pharmacodynamics of exosomes were measured in recipient cells, using the sulforhodamine B assay for anti‐proliferation and the wound‐healing assay for anti‐migration.

Results

All four cells in exponential growth phase maintained similar baseline exosome level (~220 exosomes/cell). PTX (2–1000 nM) or DOX (10–1000 nM) treatment for 24 or 48 h stimulated exosome production/secretion, in dose‐ and time‐dependent manner, by up to 10‐fold. After 24 h treatment, the amount of PTX in exosomes was 5–10% of that in donor cells, and these PTX‐containing exosomes inhibited cell viability and migration in vitro. Pretreatment for 24 h with omeprazole (10 μM) or GW4869 (10 μg/mL) reduced the production/secretion of, and the drug amount in, exosomes by >20%, and enhanced the exosome cytotoxicity in donor cells. Our quantitative pharmacology model, comprising 6 intracellular and intercellular compartments and 11 rate constants, captured and characterized the effect of exocytosis on intercellular transfer of drug‐containing exosomes and PTX pharmacodynamics in donor and recipient cells with good fitness (CV <19%).

Conclusion

Our results indicate (a) chemotherapeutic agents stimulate exosome production/secretion, (b) exocytosis is a mechanism of intercellular drug transfer responsible for conferring biological activities on neighboring recipient cells, and (c) successful application of quantitative pharmacology to depict the spatial and temporal effects of exosomes on chemotherapy pharmacodynamics.

Support or Funding Information

Supported in part by RO1CA163015 (GW, JA) from National Cancer Institute and RO1EB015253 (JA, GW) from National Institute of Biomedical Imaging and Bioengineering, DHHS and RSG‐16‐006‐01‐CCE (SW) from American Cancer Society.
Influence of fan induced photoperiod on selected blood physiological variables of male broilers reared to 63 days of age
Authors: Olanrewaju Hammed A , Purswell Joseph L , Collier Stephanie D , Branton Scott L
Published: November 2023

Increasing broiler house size and ventilation capacity have resulted in increased light ingress through ventilation system components. Effectively of this may alter the photoperiod for broiler chickens and create local increases in light intensity, which may also impact broiler body homeostasis. The objective of this study was to determine the influence of fan‐induced photoperiod on selected blood plasma biochemistry, enzyme activities and electrolyte levels of male broilers reared to 63 day of age. A total of 960 male broiler chicks were randomly distributed to 16 environmentally‐controlled rooms at 60 chicks/room. Similar feed and water were provided ad libitum to all chickens. The lighting program to day 35 was identical for both treatments and treatments were initiated at day 36. The two treatments consisted of constant (C) light intensity at 2.5 lx at 16L: 8D based on prescriptive intensities in typical heavy broiler lighting programs and a variable (V) light intensity and photoperiod program that was based on field measurements near tunnel fans in a solid‐walled broiler house. The study was a completely randomized design with room serving as the experimental unit. Blood samples were collected from the wing brachial vein of 6 birds per room on day 35 (before initiation of the treatments) and also on day 63 and analyzed immediately. The results showed broilers exposed to a constant light intensity of 2.5 lx as compared with broilers exposed to variable lighting to mimic areas near tunnel fans had significantly lower level of pH, Ca2+, K+, and higher levels of angap. However, all these changes were still within the acid‐base homeostasis and physiological ranges. Also, blood glucose and plasma corticosterone concentrations were not affected by treatments, suggesting an absence of physiological stress. It was concluded that the data herein illustrate the need to mitigate light ingress through ventilation system components to improve live performance in broilers without compromising welfare.

Support or Funding Information

USDA/ARS
Laminar Shear Stress Reduces NADPH Oxidase II Expression but Does Not Influence Superoxide Dismutase Expression in Human Adipose Microvascular Endothelial Cells
Authors: Robinson Austin T , Cook Marc D , Brown Michael D , Phillips Shane A
Published: November 2023

We have shown regular aerobic exercise reduces the expression of the pro‐oxidant enzyme NADPH Oxidase II (NOX II) and increases the expression of the anti‐oxidant enzyme superoxide dismutase (SOD) in ex vivo adipose resistance arteries. These adaptations contributed to preserved endothelial function following high intraluminal pressure‐ which elicits excess oxidative stress via the local renin angiotensin system. The purpose of this study was to determine if the primary mediator of exercise‐induced adaptations in NOX II and SOD in the adipose microcirculation is high physiological laminar shear stress (LSS).

Methods

Human adipose microvascular endothelial cells (HAMECs) were grown to 80–90% confluence and either 1) kept in the static condition; or 2) subjected to LSS (20 dyn/cm2) for 24 hours before cell harvesting. LSS was generated using a modified cone and plate design. Western blot was used to measure NOX II subunit and SOD isoform expression. In order to elucidate the role of Angiotensin II induced oxidative stress, HAMECs were either 1) untreated (control); or 2) treated with Ang II (400nm), and incubated with the superoxide (O2−) indicator DHE (5μM)‐with or without the Angiotensin Type I receptor blocker Losartan (50μM), the NOX II inhibitors VAS2870 (2μM) and NSC23766 (10 μM), or the SOD mimetic Tiron (100 mM).

Results

HAMECs exposed to LSS expressed significantly less of the NOX II subunits gp91phox and p47phox than static HAMECs. In contrast, there was no difference between static and LSS treated HAMECs in regards to SOD isoform expression. HAMECs exposed to Ang II produced significantly more O2− compared to control HAMECs. Pharmacological treatment with Losartan, VAS2870, NSC23766, or Tiron prevented the increased O2− production indicating NOX II and SOD play a role in mediating Ang II induced oxidative stress.

Conclusion

Laminar shear stress results in decreased NOX II, but not increased SOD expression in the adipose microvasculature. Both of these enzymes play a role in regulating Ang II induced O2− generation, thus shear stress may play a role in reducing Ang II mediated oxidative stress.

Support or Funding Information

K23HL085614 (SAP), R01HL095701 (SAP), R01HL095701‐03S1 (support for ATR)
Spironolactone can act on pannexin1 channels in smooth muscle cells to regulate vasoconstriction and blood pressure
Authors: Good Miranda Elizabeth , Poon Ivan KH , Chiu Yu‐Hsin , Butcher Joshua T , DeLalio Leon J , Mendu Suresh , Jaffe Iris Z , Leitinger Norbert , Desai Bimal N , Bayliss Douglas A , Ravichandran Kodi S , Isakson Brant E
Published: November 2023

Using an unbiased screen for potential inhibitors of pannexin1 (Panx1) channels, we identified spironolactone, a potent anti‐hypertensive, as being able to block Panx1 channels. This was especially interesting considering spironolactone has been considered a mineralcorticoid receptor (MR) antagonist. Initial confirmation of this result was obtained using electrophysiology, which showed that spironolactone could directly and effectively inhibit Panx1 channel activity without inhibiting other putative ATP release channels. Next, because we have previously demonstrated that Panx1 channels on smooth muscle cells (SMCs) can regulate α‐adrenergic vasoconstriction and blood pressure, we hypothesized that spironolactone would also inhibit α‐adrenergic vasoconstriction. Indeed, we found that spironolactone inhibited α‐adrenergic vasoconstriction from resistance arteries isolated from hypertensive humans. This effect was also seen in C57Bl6 mice, mice lacking SMC MR, or mice lacking Panx1 in endothelial cells. However, mice with SMC specific deletion of Panx1 (SMC‐Panx1 deficient) did not show any change in α‐adrenergic vasoconstriction when treated with spironolactone. TO‐PRO dye uptake on intact arteries further confirmed these functional results. Using radiotelemetry, we found that C57Bl6 mice and SMC MR deficient mice injected with spironolactone had in a significant drop in blood pressure acutely. In line with the vasoreactivity experiments, SMC‐Panx1 deficient mice did not have any change in blood pressure. Together these data suggest that Panx1 is a novel target of spironolactone and inhibition of Panx1 by spironolactone may contribute to this drug's anti‐hypertensive effects.

Support or Funding Information

Funding: HL088554, HL120840, HL007284, HL131399.
Determining eNOS Binding and Circulation Modulating Effects of an Alpha Globin Mimetic Peptide (HbαX)
Authors: Keller TC Stevenson , Butcher Joshua T , Swope Nicole K , Columbus Linda , Isakson Brant E
Published: November 2023

The ability of hemoglobin to scavenge the potent vasodilator nitric oxide (NO) in the blood has been well established as a mechanism of vascular tone homeostasis. In endothelial cells, the alpha chain of hemoglobin (hereafter, alpha globin) and endothelial NO synthase (eNOS) form a macromolecular complex, providing a sink for NO directly adjacent to the production source. We have developed an alpha globin mimetic peptide (named HbαX) that displaces endogenous alpha globin and increases bioavailable NO for vasodilation. Initially we performed structural analysis of HbαX via solution NMR in order to better understand the interaction of alpha globin and eNOS. Next we show that, in vivo, HbαX administration increases capillary oxygenation and blood flow in arterioles acutely and produces a sustained decrease in systolic blood pressure in normal and angiotensin II–induced hypertensive states. HbαX acts with high specificity and affinity to endothelial NO synthase, without toxicity to liver and kidney and no effect on p50 of O2 binding in red blood cells. In human vasculature, HbαX blunts vasoconstrictive response to cumulative doses of phenylephrine, a potent constricting agent. By binding to endothelial NO synthase and displacing endogenous alpha globin, HbαX modulates important metrics of vascular function, increasing vasodilation and flow in the resistance vasculature.

Support or Funding Information

Funding: HL088554, HL120840.
Adipose Stromal Vascular Fraction Cells Improve Coronary and Cardiac Physiology in the Aged Female Heart
Authors: Beare Jason , Shofner Charles , Dale Jacob , Tinney Joseph , Keller Brad , Hoying James , LeBlanc Amanda
Published: November 2023

Over 50% of adult women presenting with symptoms of ischemic heart disease have “clear” coronary artery angiograms. Additional testing indicates coronary microvascular dysfunction, contributing to the coronary perfusion deficits in these women. Current therapies for this condition, called microvascular angina, are relatively ineffective. Our previous research demonstrated that tissue‐resident macrophages within the therapeutic cell preparation derived from the stromal vascular fraction (SVF) of adipose tissue improved peripheral small artery function. Delivered intravascularly, these SVF‐derived macrophages migrate to the walls of small vessels and reset vasomotor tone. Therefore, we hypothesized that i.v. delivery of SVF cells can reverse coronary microvascular dysfunction. Using the aged female rat heart (22 mos. of age) as a model of coronary microvascular dysfunction without coronary artery involvement, combined with high‐resolution ultrasound and telemetry, we evaluated cardiac function and coronary flow reserve (CFR) in aged animals receiving fluorescently tagged GFP+ SVF cells from syngeneic young rats (6 × 106 cells/rat) via the tail vein at one‐week and four‐weeks post‐injection. At the time of explant, the location of injected SVF cells was determined via confocal microscopy, and isolated coronary arteriole preparations were used to evaluate vasoactivity. Injected GFP+ SVF cells had incorporated into the coronary vasculature one‐week post‐injection and remained there at 4 weeks. While coronary blood flow was improved in animals receiving the cell therapy, vasoreactivity of isolated arterioles to flow, pressure, bradykinin, and endothelin was unaffected. Measures of heart function such as maximal cardiac output, percent heart rate increase, and left ventricular diameter and volume during diastole ‐ which are all compromised in aged animals ‐ were significantly improved in response to a dobutamine challenge compared to pre‐SVF measurements. The cell therapy did not lead to arrhythmias or increased mortality. Our findings indicate that intravenously‐delivered adipose SVF cells disseminate to the aging heart and incorporate into the microvascular wall and perivascular spaces of the coronary vasculature. The presence of the therapeutic cells in the heart is associated with improved coronary perfusion and concomitant cardiac function.

Support or Funding Information

Jewish Heritage Fund for Excellence
Hypoxia Disturbs the Metformin‐Evoked Curtailment of Chemokine CX3CL1 (Fractalkine) Signaling in Human Umbilical Vein Endothelial Cells (HUVECs)
Authors: Szukiewicz Dariusz , Pyzlak Michal , Szewczyk Grzegorz , Wejman Jaroslaw , Kochanowski Jan
Published: November 2023

Metformin (dimethylbiguanid) is an antihyperglycemic agent widely used for treating non‐insulin‐dependent diabetes mellitus (NIDDM). Moreover, it was reported that metformin may prevent nuclear factor kappa‐beta (NF‐κβ) activation in endothelial cells exposed to inflammatory cytokines. These anti‐inflammatory properties resulting from activation of AMP‐activated protein kinase (AMPK). Inflammation and hypoxia activate chemokine CX3CL1 receptor (CX3CR1)‐related signaling pathway. Thus, we hypothesized that lowering of cellular AMP levels during hypoxia may reduce ability of metformin to counteract the CX3CXL1‐CX3CR1‐axis‐dependent mechanism of endothelial dysfunction. In this study we examined comparatively the effect of metformin on lipopolysaccharide (LPS)‐induced CX3CL1 production as well as CX3CR1 expression in HUVECs cultured under normoxic vs. hypoxic conditions. Immediately after childbirth, HUVECs were isolated from a 12‐cm umbilical cord segment via collagenase digestion. The material obtained from primigravidas after term, normal‐course pregnancies was equally divided into two groups: group I (N = 24) – kept under hypoxia (5% O2), and the control normoxic (20% O2) group II (N = 24). After achieving the confluence, HUVECs were exposed to metformin (10 mmol/L; the subgroups IA and IIA) or cultured without metformin (the subgroups IB and IIB, respectively). Following pretreatment with LPS (1 μg/mL), the mean levels of CX3CL1 in the culture media supernatants were determined at predefined time points by ELISA. At the end of the culture period, HUVECs were formalin fixed, paraffin embedded, immunostained for CX3CR1 and cut into 5 μm sections. The expression of CX3CR1 was estimated by quantitative immunohistochemistry. The mean levels of CX3CL1 in group I were 5.9‐fold and 2.7‐fold higher than observed in normoxic group II (IA vs. IIA and IB vs. IIB, respectively). Initially, the mean levels of CX3CL1 were significantly (p < 0.05) lower in IA compared to IB, but these differences gradually disappeared, being non‐significant over the time. The mean expression of CX3CR1 in hypoxia did not differ statistically between IA and IB, but was significantly (p < 0.05) upregulated compared to the both normoxic subgroups IIA and IIB. In normoxia, exposition to metformin produced significant and stable decrease in CX3CL1 production in HUVECs, compared to metformin‐free subgroup IIB. A significant increase in CX3CR1 expression was observed in IIB compared to IIA. In conclusion, upregulation of CX3CR1 in response to hypoxia and/or metformin may serve as a compensatory mechanism to preserve or augment the level of CX3CL1/CX3CR1 signaling. Ineffectiveness of metformin in lowering of CX3CL1 production under hypoxia may suggest insufficient activation of AMPK in HUVECs.

Support or Funding Information

WUM grant: 2M2‐W1‐16
ADAM15‐mediated glycocalyx injury contributes to vascular barrier dysfunction during inflammation
Authors: Yang Xiaoyuan , Meegan Jamie E. , Jannaway Melanie , Yuan Sarah Y.
Published: November 2023

Endothelial surface glycocalyx composed of glycosaminoglycans, proteoglycans and glycoproteins plays an important role in regulating vascular barrier integrity. A disintegrin metalloprotease 15 (ADAM15) is a membrane‐associated sheddase which has recently been characterized as a mediator of endothelial hyperpermeability during inflammation. This study tested the hypothesis that ADAM15 contributes to microvascular barrier dysfunction by damaging glycocalyx integrity. Intravital microscopic analysis of mouse mesenteric microcirculation showed a significant reduction in the thickness of glycocalyx layer on the endothelial surface following bacterial lipopolysaccharide (LPS) injection; this response was greatly attenuated in ADAM15−/− mice. In parallel, LPS treatment caused a higher normalized plasma level of low molecular weight hyaluronan, an important component of glycocalyx that can be degraded into small fragments, in wild type mice compared to ADAM15−/− mice. Functionally, inflammation‐induced plasma leakage in mesenteric microvessels was ameliorated in the absence of ADAM15. Further molecular and confocal imaging analyses of cultured endothelial cells revealed evidence of ADAM15 co‐localization with CD44, a transmembrane glycoprotein that binds hyaluronan and stabilizes its attachment to cell surface. LPS stimulation resulted in an increase in ADAM15 expression and a decrease in CD44 and hyaluronan expression on endothelial cell surface, an effect greatly inhibited during siRNA knockdown of ADAM15. Moreover, the LPS‐induced increase in CD44 release into conditioned media was attenuated during ADAM15 knockdown. Taken together, the data suggest the involvement of ADAM15 in glycocalyx injury and endothelial barrier leakage during inflammation.
Exercise‐like mechanical stimulation improves myogenic responses in aged skeletal muscle resistance arteries
Authors: Shin Song Yi , Seawright John , Trache Andreea , Woodman Christopher
Published: November 2023

Objective

We tested the hypothesis that exposure to a short‐duration (1 h) increase in intraluminal pressure, to mimic pressure associated with a bout of exercise, would attenuate or reverse the age‐related decline in myogenic constrictor responses in skeletal muscle resistance arteries.

Methods

Soleus muscle feed arteries (SFA) from young (4 mo) and old (24 mo) Fischer 344 rats were cannulated and pressurized to 90 (P90) or 130 cmH2O (P130) for 1 h. Following the 1 h treatment period, pressure in the P130 arteries was reduced to 90 cmH2O and SFA were required to develop stable spontaneous tone for 10 min. After stable tone was achieved, myogenic contractile responses were assessed using step‐increases in intraluminal pressure up to 135 cm H2O in 15 cm H2O increments followed by step‐decreases down to 60 cm H2O.

Results

Myogenic vasoconstrictor responses were significantly impaired in old SFA compared to young SFA. Mechanical stimulation with increased pressure for 1 h improved myogenic constriction in old SFA.

Conclusion

These results indicate that stimulation with a short‐duration increase in intraluminal pressure, within a range of pressure believed to be present in these arteries during exercise, improves myogenic constrictor responses in aged skeletal muscle resistance arteries.

Support or Funding Information

Research support: AHA grant 0765043Y, AHA grant 4150031, The National Space Biomedical Research Institute through NCC 9–58 and a Sydney and J.L. Huffines Institute of Sports Medicine Graduate Student Research Grant.
Altered Basal Cerebral Perfusion in Adolescents with Insulin Resistance
Authors: Ward Aaron T. , Kellawan J. Mikhail , Peltonen Garrett L. , Carter Katrina J. , Rehm Jennifer L. , Oh Jennifer M. , Carrel Aaron L. , Bendlin Barbara B. , Schrage William G.
Published: November 2023

Insulin resistance (IR) is associated with reduced macrovascular cerebral blood flow (CBF) and microvascular cerebral perfusion (CP) in middle‐aged adults. Due to the obesity epidemic, more people are affected by IR; however, it is unknown if adolescents with IR have cerebral vascular dysfunction. This is surprising since obese adolescents are known to display abnormal executive function processes. Therefore, we tested the hypothesis that adolescents with IR will exhibit lower basal CBF, as well as CP, in neuronal regions known to be involved in executive function, learning, language, and memory. Five adolescents (15±3 yrs) were divided into groups based on Homeostatic Model of Assessment of Insulin Resistance (HOMA‐IR; Control, CTRL < 4.0, n=2 vs IR ≥ 4.0, n=3). Hemodynamics were measured (HR, SpO2, MAP, ETCO2) and all testing was performed in a fasted state. CBF was measured via 4D MRI utilizing phase contrast‐vastly undersampled isotropic projection reconstruction (PC VIPR). CP was measured by pseudo‐continuous arterial spin labelling (pcASL) MRI, normalized to global pcASL signal. The IR group exhibited higher HOMA‐IR (5.3±0.2 vs 3.3±0.3 p=0.006) and insulin compared to CTRL (28±2 vs 16±3 mU/L; p=0.027). Baseline HR (CTRL 75±6 vs IR 68±3.5 bpm; p=0.185), SpO2 (CTRL 99.0±0.0 vs 96.5±1.3%; p=0.113), MAP (CTRL 75±4.7 vs IR 81.9±5.4 mmHg; p=0.143), and ETCO2 (CTRL 35.5±0.4 vs 40.4±2.0 mmHg; p=0.07) was similar between groups. Total CBF was similar between groups (CTRL 735±79 vs IR 811±115 mL/min; p=0.64) as was CBF in each of the 11 regional arteries measured. Of 14 brain regions examined via pcASL, the IR group exhibited lower CP in the left frontal superior orbital gyrus (4% lower; p=0.002), left angular gyrus (10% lower; p=0.006), right frontal middle gyrus (11% lower; p=0.038), and right frontal superior medial gyrus (11% lower; p=0.017), and tended to be lower in the right frontal superior gyrus (10% lower; p=0.058). Collectively, these regions are notable for language, attention, memory retrieval and cognitive processing of decision making. Taken together, these data indicate preserved total and regional CBF in adolescents with IR. However, preserved bulk CBF appears to mask impairments in microvascular CP in key brain regions linked to learning and memory, which may have implications for long‐term brain health.

Support or Funding Information

University of Wisconsin‐Madison Graduate School Pilot Funds
Initial studies of cardiovascular monitoring feasibility using EndoGear 2, a fully implantable telemetry system in swine model
Authors: Goodwill Adam Glenn , Konecny Filip , Pitsillides Koullis , Sosa Margo , Barnard April P , Kiel Alex M , Tune Johnathan D
Published: November 2023

Introduction

Current methodological recommendations for directly translational and clinically relevant models of cardiovascular disease portray large animal models as a mainstay for drug, cell, and gene therapy and for device development and surgical procedure testing. The translational capacity of swine telemetry models has been plagued by an inability to longitudinally assess cardiovascular parameters due to its technological limitations using telemetric instrumentation. Historically, 24‐hour monitoring has required technical staff to physically connect externalized wires which are prone to damage, making the appeal of a fully enclosed automatable data collection system desirable.

Methods

We have performed initial studies to assess cardiovascular parameters employing Transonic EndoGear® 2 (EG2‐Q1S3T‐M25) biotelemetry system on 50kg male domestic swine. At telemeter implantation, a perivascular 2.5 QS flow probe was placed around the left anterior descending coronary artery, 2 ECG leads were attached directly to myocardium, a 3.5F systemic pressure transducer was inserted into the left internal mammary artery and a 7F left ventricular pressure transducer was inserted into the left ventricle via an apical transmural stab. All data were recorded using EMKA IOX software.

Results

Animals were monitored continuously for 24 hours following the procedure and then for 5 minutes each hour for 1 week subsequently prior to sacrifice. All measurements were within the expected range consistent with previous studies we have performed and animals were able to be reliably monitored for all indicated parameters in real time throughout the monitoring period.

Conclusion

These initial studies indicate the feasibility using EndoGear 2 in longitudinal studies monitoring major cardiovascular parameters in untethered and unrestrained swine. The platform enables, for the first time, telemetered monitoring of the coronary artery blood flow using transit time technology.

Support or Funding Information

Funded by: R01 HL117620 04
Treatment of long‐term coronary dysfunction in diabetes with mineralocorticoid receptor antagonism: vasomotor control and the MR‐dependent vascular transcriptome
Authors: Brown Scott M , Meuth Alex I , Meers Grace M , Davis J Wade , Rector R Scott , Bender Shawn B
Published: November 2023

Obesity and diabetes are associated with inappropriate activation of the renin‐angiotensin‐aldosterone system (RAAS) and impaired coronary microvascular function. We recently demonstrated that mineralocorticoid receptor (MR) antagonism with spironolactone (Spiro) treated coronary arteriolar endothelial dysfunction when administered during the development of dysfunction in obesity. Whether MR antagonism can treat long‐term established coronary arteriolar vasomotor dysfunction in diabetes and potential underlying molecular mechanisms have not been investigated. Here, we hypothesized that long‐term diabetes‐associated coronary arteriolar dysfunction will be abrogated by MR antagonism with Spiro involving alterations of the vascular transcriptome. Male Otsuka Long‐Evans Tokushima Fatty (OLETF) rats (29 weeks of age) were treated with Spiro (sc, 20 mg/kg/d) or placebo for 3 weeks. Control, lean Long‐Evans Tokushima Otsuka (LETO) rats were treated with placebo and the onset of coronary microvascular dysfunction was established in separate groups of 5–8, 20, and 32 week old OLETF and LETO rats. At 32 weeks of age, OLETF rats exhibited obesity, hyperglycemia, hypercholesterolemia, normal blood pressure, and reduced circulating aldosterone compared to LETO. Endothelium‐dependent vasodilation of isolated, pressurized coronary arterioles to acetylcholine (ACh) was impaired at 5–8 weeks of age and persisted to 32 weeks in OLETF rats with no change in endothelium‐independent vasodilation to sodium nitroprusside. Further, coronary arteriolar vasoconstriction to endothelin‐1 (ET‐1) and the thromboxane mimetic U‐46619 increased significantly in an age‐dependent manner in OLETF rats. OLETF rats treated with Spiro exhibited similar plasma glucose, insulin, cholesterol, blood pressure, and serum potassium, but elevated circulating aldosterone compared to placebo‐treated OLETF rats at 32 weeks of age. Spiro treatment restored endothelium‐dependent coronary arteriolar vasodilation to ACh and insulin and attenuated coronary vasoconstriction to the L‐type calcium channel agonist BayK‐8644, but not to ET‐1 or U46619 in OLETF rats. RNA sequencing and Ingenuity Pathway Analysis of aortic mRNA from Spiro and placebo‐treated OLETF rats revealed MR‐dependent alterations of gene networks associated with iNOS, ceramide, and TNFR2 signaling. These data support and extend our recently published work by demonstrating that, independent of blood pressure, MR antagonism treats established diabetes‐associated coronary arteriolar endothelial dysfunction, an independent predictor of future cardiovascular events in human patients. In addition, these data highlight novel MR‐dependent alterations of the vascular transcriptome in diabetes that warrant further investigation.

Support or Funding Information

Supported by VHA BLR&D BX002030 (SBB) and VHA BLR&D BX001299 (RSR)
No Evidence of Impaired Functional Sympatholysis in Young Obese Adults
Authors: Bunsawat Kanokwan , Grigoriadis Georgios , Wee Sang Ouk , Griffith Garett , Brown Michael D. , Phillips Shane A , Fadel Paul J , Clifford Philip S , Fernhall Bo , Baynard Tracy
Published: November 2023

In healthy individuals, sympathetic vasoconstriction is blunted in exercising muscles, contributing to the matching of blood flow with metabolic demand. This “functional sympatholysis” may be impaired in young obese adults due to greater sympathetic activation and reduced local vasodilatory capacity of both small and large arteries, but this remains poorly understood. We tested the hypothesis that functional sympatholysis is impaired in obese adults compared with lean counterparts. Participants were 13 lean (female=6; 26±1 yrs; 22.4±0.5 kg/m2) and 14 obese adults (female=7; 27±1 yrs; 32.6±0.6 kg/m2). Lower body negative pressure (LBNP −20 mmHg, 2 min), to evoke reflex‐mediated increases in sympathetic nerve activity, was performed at rest and during steady‐state dynamic handgrip exercise (HG, 30% maximal voluntary contraction; 50% duty cycle). Ultrasonography [brachial diameter, forearm blood flow (FBF), forearm vascular conductance (FVC)], and beat‐to‐beat cardiovascular variables [mean arterial pressure (MAP), heart rate (HR), and cardiac index (CI)] were obtained. FBF and FVC were normalized to lean forearm mass, and CI was cardiac output indexed to body surface area. No group differences were found at rest or during handgrip before LBNP for any variable. LBNP evoked similar decreases in FBF (−18.6±3.0% lean vs. −17.8±3.1% obese) and FVC (−16.6±3.1% lean vs. −14.9±3.1% obese) in the resting forearms of the lean and obese adults (P>0.05 for group). During HG, reductions in FBF (−1.7±3.8% lean vs. −2.0±2.3% obese) and FVC (1.2±4.3% lean vs. 0.1±2.5% obese) to LBNP were blunted in the exercising forearms of both groups (P>0.05 for group). In both lean and obese subjects, LBNP decreased MAP and CI at rest and during HG, but decreased brachial artery diameter and increased HR only during HG (all; P<0.05 for time effect). Our preliminary data suggests that functional sympatholysis is not impaired in young obese adults without comorbidities.

Support or Funding Information

This project is sponsored by the American Heart Association Predoctoral Fellowship (16PRE26430096).

Variable
Group
Rest
Rest with LBNP
30%MVC
30%MVC with LBNP

Brachial Artery Diameter (mm)
Lean
3.46±0.15
3.43±0.14
3.64±0.14
3.62±0.14*

Obese
3.62±0.14
3.60±0.14
3.76±0.14
3.73±0.13*

FBF (mL/min* 100g tissue)
Lean
11±1
8±1*
30±2
30±2

Obese
10±1
8±1*
27±2
27±2

FVC (mL/min* 100mmHg*100g tissue)
Lean
11±1
9±1*
30±2
31±2

Obese
10±1
9±1*
27±2
27±2

MAP (mmHg)
Lean
101±2
99±2*
102±3
100±3*

Obese
100±2
96±2*
103±3
101±3*

HR (bpm)
Lean
62±2
63±2
67±2
71±2*

Obese
64±2
64±2
67±2
72±2*

CI (L/min/m2)
Lean
3.3±0.2
3.0±0.2*
3.7±0.2
3.3±0.2*

Obese
3.0±0.2
2.7±0.2*
3.2±0.2
3.0±0.2*

Data are mean±SE.

P<0.05, time effect.
Brain Homogenates from Alzheimer's Patients Impact Vascular Function
Authors: Zhang Xiancong , Ma Jing , Hajjar Ihab , Sutliff Roy L.
Published: November 2023

Alzheimer's disease (AD) is characterized by a state of reduced cerebral blood flow, which may partly be secondary to cerebral artery vascular dysfunction. Recent studies have shown that systemic vascular dysfunction may precede Alzheimer's disease. We hypothesize that biochemical changes associated with AD progression can impact vascular function. Homogenates were prepared from temporal lobe samples from patients with or without tauopathies and were categorized into three groups: homogenates with amyloid Tau protein, homogenates with high concentrations of Tau protein but little amyloids, and control.

The chronic effects of brain homogenates on vascular function was characterized using isolated wild‐type rat aorta segments incubated with homogenates (0.1% or 1%) for 24 hours. Rat aortas were isometrically mounted and the vascular function was assessed by examining contractile responses to KCl and phenylephrine, and relaxation responses to methacholine and sodium nitroprusside. The acute effect of homogenates on vascular function was measured using wild‐type rat aorta segments after maximal relaxation by methacholine.

Preliminary data from chronic studies suggests that, amyloid group patient tissues are less sensitive to methacholine‐dependent relaxation compared to tissue from Tau and control groups(One‐way ANOVA; p<0.05). Preliminary data from acute studies suggests that tissue from the tau group results in more severe contractile effects on maximally relaxed aortas compared to amyloid and control groups. These results are significant because they suggest that biochemical changes associated with AD can impact vascular function. Therefore, these studies suggest that there is an association between AD and vascular dysfunction.

Support or Funding Information

Supported by grant AG051633 from NIA./NIH and SIRE to XZ
High Dietary Sodium Reduces Low‐Flow‐Mediated Constriction in Salt‐Resistant Adults
Authors: Ramick Meghan , Lennon‐Edwards Shannon , Rose William , Farquhar William , Edwards David
Published: November 2023

High dietary sodium has been shown to impair flow‐mediated dilation independent of blood pressure (BP). Low flow‐mediated (LFC) constriction has been proposed as a complementary measure of vascular function in humans. Therefore, the purpose of this study was to test the hypothesis that a high sodium diet would reduce brachial artery LFC in salt‐resistant adults. Additionally, we sought to determine if sex differences existed in the LFC response to high dietary sodium. Twenty healthy, normotensive adults (12 males, 28.8±1.8 yrs; 8 females, 29.8±2.2 yrs) participated in a controlled feeding study. Following a run‐in diet, participants completed 7 days of a low sodium (LS) diet (20mmol sodium/day) and 7 days of a high sodium (HS) diet (300mmol sodium/day) in random order. Diets were designed to be eucaloric and consistent in macronutrient distribution and potassium content. On the last day of the LS and HS diets, a 24‐hour urine was collected and 24‐hour ambulatory BP was assessed. Subjects were classified as salt‐resistant as defined by a change in 24‐hour ambulatory mean arterial pressure (MAP) of ≤5mmHg between the LS and HS diets. On day 7 of the LS and HS diet, LFC was assessed by ultrasound. LFC was calculated as the % change in brachial artery diameter following a 5‐minute distal cuff occlusion. By design, 24‐hour MAP was not different between LS and HS conditions (85±1 vs 84±1 mmHg, p>0.05) whereas urinary sodium excretion (34.8±6.0mmol/24hr vs 269.7±21.6 mmol/24hr, p<0.01) and serum sodium concentration (137.9±0.6mmol/L vs 140.2±0.3mmol/L, p<0.01) increased significantly on the HS diet. LFC was significantly attenuated on the HS diet compared to LS (2.2±0.3% vs 3.3±0.3%). LFC was reduced by HS in both men and women (men: LS 3.8±0.4% vs HS 2.6±0.4%; women: LS 2.6±0.5% vs HS 1.5±0.5%; p<0.05). These data demonstrate that high dietary sodium impairs LFC in salt‐resistant adults providing additional evidence for the adverse vascular effects of dietary sodium independent of BP.

Support or Funding Information

Research funded by NIH grant R01 HL104106
The Role of FGF2 and FGFBP1 in Cerebrovascular Response to Angiotensin II with Renal Ischemia‐Reperfusion Injury in Mice
Authors: Zhao Liang , Cao Xiaoyun , Zhou Suhan , Wang Qin , Li Lingli , Zhao Jingwei , Saavedra Juan , Wilcox Christopher S. , Wellstein Anton , Lai En Yin
Published: November 2023

Acute kidney injury (AKI) is defined as an abrupt deterioration in kidney function. Recent studies indicate that the prevalence of AKI is relatively high and associated with poor clinical outcomes including failure of other organs that include strokes. However, the effects of AKI on cerebrovascular function is poorly understood. Here we tested the hypothesis that AKI may promote strokes by cerebrovascular sensitization to angiotensin II (Ang II) in conjunction with FGF2 (fibroblast growth factor 2) plus the secreted FGF binding protein FGFBP1. Renal ischemia‐reperfusion (I/R) mice served as the AKI model and sham‐operated mice as controls. Brain medulla oblongata arterioles were dissected and microperfused. Also, brain basal and posterior communicating arteries were dissected and tested by vascular myography. We found that the effects of the vasoconstrictors endothelin 1 or norepinephrine were not impacted in the AKI model but observed that the effects of Ang II on cerebral arterioles and arteries were enhanced. Ang II effects were further amplified by treatment with FGF2 plus FGFBP1. Complementary to this, the FGF receptor (FGFR) kinase inhibitor PD173074 decreased the cerebrovascular response to Ang II indicating a crosstalk between Ang II and the FGFR pathway. Interestingly, Ang II type 2 receptor mRNA expression in isolated cerebral vessels was reduced in AKI model relative to controls. We conclude that synergism between the FGFR and Ang II pathway increases the brain vessel response to Ang II and this enhancement of the cerebral vascular responses may be due to the reduced expression of Ang II type 2 receptor.