bluuebunny/crossref_metadata_2025_split huggingface dataset:
Droplet Digital PCR Reveals Substantial Tissue‐Specific Differences in Estrogen Receptor Expression Profiles
Authors: Hutson Dillion D , Duong Jennifer L. , Sato Ryousuke , Lindsey Sarah H. 
Published: November 2023

Our previous work shows an important role for the novel G‐protein Coupled Estrogen Receptor (GPER) in female cardiovascular health. However, the relative importance of this receptor in the context of physiological estrogen signaling has thus far remained elusive, since direct comparison of transcript number has been unattainable due to the relative nature of existing PCR techniques. Droplet digital PCR (ddPCR) is a new technique that allows absolute quantification of nucleic acid concentration from experimental samples. We utilized this technology to compare absolute copy numbers for the three known estrogen receptors (GPER, ERα, ERβ) as well as aromatase, a key enzyme in the synthesis of estrogen. Quantitative ddPCR analysis of purified RNA gathered from cultured A7r5 embryonic rat aortic smooth muscle cells and rat kidney cortex was performed via the BioRad QX200 system for the genes of interest. Expression levels of GPER in A7r5 cells were 10.0 ± 1.4 copies per ng of RNA, while transcript levels for ERα, ERβ, and aromatase were significantly lower at 0.6 ± 0.1, 1.6 ± 0.2, and 2.9 ± 0.7 copies/ng of RNA, respectively (P<0.0001 vs. GPER). Interestingly, in kidney tissue ERα was the predominant transcript at 743.6 ± 4.4 copies in comparison with 30.8 ± 1.4 copies of GPER (P<0.0001). ERβ was expressed at very low levels in kidney (0.15 ± 0.05 copies) while aromatase was undetectable. This data suggests that GPER is expressed at significantly higher levels than nuclear estrogen receptors in A7r5 cells. In contrast, the profile of estrogen receptors in kidney tissue differs greatly, with a predominance of ERα. In conclusion, estrogen responses in different tissues may be modulated by the amounts of these receptors. Moreover, knowledge of these variable receptor profiles can be utilized to target estrogen's protective effects in a tissue‐specific manner.Support or Funding InformationNational Institute of General Medical Sciences IDeA Program (COBRE, P30GM103337)
Characterization of Acid Ceramidase and Its Regulation by HMGB1 in Mouse Coronary Arterial Myocytes
Authors: Lee RaMi , Meng Nan , Yuan Xinxu , Li Pin‐Lan 
Published: November 2023

Acid ceramidase (AC) has been reported to be a critical sphingolipid metabolizing enzyme in the regulation of cellular activity under physiological and pathological conditions. However, it remains unknown whether AC is functioning in coronary arterial myocytes (CAMs) to regulate vascular function. In the present study, we first characterized the expression and activity of AC in CAMs. By real time RT‐PCR, Western blot analysis and immunhistochemistry, AC was found highly expressed in the primary cultures of mouse CAMs and in the middle layer of mouse coronary arterial wall. Confocal microscopy showed that AC, in particular, its alpha subunit was mainly colocalized with lysosome marker, Lamp‐1, indicating a main location of this enzyme in lysosomes. Treatment of CAMs with AC inducer, genistein significantly increased AC expression in CAMs, while inhibition of AC gene expression by N‐oleoylethanolamine (NOE) markedly reduced the level of AC in CAMs. Using LC/MS analysis, we demonstrated that basal ceramide level in CAMs was 707.49 ± 103.94 pmol/1 million cells and that genistein mainly reduced the concentration of C‐14 ceramide (−35%) and C1–6 ceramide (−18%) and NOE increased both ceramide species by 2.4 and 3.4 folds, respectively. Given recent reports on the implication of a typical alarmin, high‐mobility‐group box 1 (HMGB1) in tissue injury and inflammation, we tested whether AC and related ceramide signaling is involved in the action of HMGB1. It was found that HMGB1 decreased AC mRNA and protein levels in a concentration‐dependent manner. Correspondingly, cellular total, C‐14‐, and C‐16 ceramide concentrations in HMGB1‐treated CAMs increased by 65%, 51% and 77%, respectively. This HMGB1‐indcued increase in ceramide accumulation in CAMs was blocked by AC inducer, genistein, but further enhanced by AC inhibitor, NOE. These results suggest that AC is enriched in CAMs and its activation or inhibition alters cellular ceramide levels and that AC may be a signaling enzyme mediating the action of HMGB1 in the regulation of CAM function in response to atherogenic or inflammatory stimuli.Support or Funding InformationSupported by NIH grants HL057244, HL075316 and HL122937
Oroxylin‐A suppresses adhesion molecules expression and endothelial barrier disruption in endotoxemic arteries
Authors: Tseng Tzu‐Ling , Chen Mei‐Fang , Liu Chin‐Hung , Hsu Yung‐Hsaiang , Lee Tony J. F. 
Published: November 2023

Vascular inflammation plays a critical role in the pathogenesis of sepsis, which is characterized by disruption of endothelial junctions, increasing vascular permeability and leakage, and promoting leukocytes and inflammatory exudates to enter the injured area. Here we assess the beneficial effects of post‐treatment of Oroxylin‐A (OroA), a flavonoid, in ameliorating lipopolysaccharide (LPS)‐induced adhesion molecules expression, endothelial barrier dysfunction, and vascular inflammation. Rats were injected with LPS (10 mg/kg, ip) to induce systemic inflammation, and OroA (15 mg/kg, iv) was given 6 hours after LPS treatment. Biochemical changes in the circulation, and morphological/histological alternations associated with inflammation in the aorta were investigated. LPS significantly enhanced proinflammatory cytokines release, increased vascular cell adhesion molecule (VCAM)‐1 and intercellular adhesion molecule (ICAM)‐1 expressions, disrupted endothelial tight junction zona occludens (ZO)‐1 surface expression, reduced stabilization of adherens junction vascular endothelial cadherin (VE‐cadherin) of endothelial barrier, and increased macrophage infiltration and accumulation in aortic arteries. All these changes were reversed by OroA (15 mg/kg, iv) post‐treatment. This post‐treatment also blocked the LPS‐activated aortic NF‐κB. Moreover, this post‐treatment suppressed interleukin‐1‐receptor‐associated‐kinase (IRAK)‐4‐targeted IKKα/β phosphorylation without directly affecting interaction between LPS and toll‐like receptor (TLR)‐4 receptor. These results obtained from studying OroA suggest that beside anti‐inflammation via inhibiting IRAK‐4‐mediated NF‐κB activation, reversal of adhesion molecules expression and endothelial barrier dysfunction may be another useful strategies in managing endotoxemic shock.Support or Funding Informationsupported by the MOST, Tzu Chi Foundation, Tzu Chi University and Tzu Chi Medical Center
O‐GlcNAc‐eNOS is decreased in blood vessels from pregnant Wistar rats, but not in vessels from pregnant SHR
Authors: Troiano Jessica Antonini , Potje Simone Regina , Graton Murilo Eduardo , Silva Daniela Souza , Antoniali Cristina 
Published: November 2023

IntroductionLate pregnancy in spontaneously hypertensive rats (SHR) is related to low blood pressure levels and decreased vasoconstrictors responses to alpha 1‐adrenergic drugs. In blood vessels, increased endothelial nitric oxide synthase (eNOS) phosphorylation and nitric oxide (NO) production are associated to pregnancy effects. Some post‐translational modifications, such as O‐glycosylation by N‐acetyl‐glucosamine (O‐GlcNAc) decreases eNOS activity leading to a lower NO bioavailability. O‐GlcNAc is involved on the OGT (O‐GlcNAc transferase) activity, which binds O‐GlcNAc on serine and threonine residues of target proteins and OGA (O‐GlcNAcase), which removes O‐GlcNAc from these proteins. We hypothesized that O‐GlcNAc‐eNOS is decreased in blood vessels of pregnant SHR. We evaluate the effect of PugNAc, OGA inhibitor, on vascular reactivity to phenylephrine (PE) in aortas of pregnant Wistar rats and in mesenteric artery of pregnant SHR.MethodsAortic rings (2 mm) of non‐pregnant (NP) and pregnant (P) Wistar rats (n= 5–7) and rings of the 2nd and 3rd branches of the mesenteric artery of NP and P SHR (n = 4–6) were incubated in DMEM in the presence of PugNAc (100 μM) or methanol (vehicle) for 24 hours. These rings, with and without endothelium, or with endothelium in the presence of L‐NAME (100 μM, 30 min incubation), were stimulated with increasing and cumulative concentrations of phenylephrine (PE, 1 nM to 10 μM). The results were compared between the groups (ANOVA, p <0.05).ResultsPregnancy modulates the vascular reactivity of the blood vessels, reducing the maximum effect of PE curves in aortas from P Wistar rats (22.96 ± 1.36 mN) when compared to the NP Wistar (27.01 ± 0.68), and in mesenteric arteries from P SHR (12.04 ± 0.53 mN) when compared to NP SHR (24.12 ± 0.99 mN). Incubation with PugNAc did not alter the reactivity of NP Wistar aortas to PE (Emax: 26.16 ± 0.43 mN). However, aortas from P Wistar rats incubated with PugNAc showed greater maximum contractile effect to PE (32.75 ± 1.17 mN) when compared to non‐incubated aorta or to NP Wistar aortas. The increased endothelium modulation on PE vasoconstriction observed in aortas from P Wistar rats was reduced by PugNAc when compared to non‐incubated aortas. PugNAc did not alter mesenteric artery rings reactivity to PE in NP SHR (24.66 ± 0.69 mN) or P SHR (13.49 ± 0.71). PugNAc reversed the low reactivity to PE, which is dependent of eNOS activity, in aortas from pregnant Wistar rats, but not in mesenteric arteries from pregnant SHR.ConclusionThese results suggest that O‐GlcNAc‐eNOS is decreased in blood vessels from pregnant Wistar rats, but not in vessels from pregnant SHR.Support or Funding InformationFinancial support: Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) [Grant 2015/08188‐4].
Mechanisms Linking Chronic Obstructive Pulmonary Disease and Stroke
Authors: Bernard Ashton Juan , Ku Jacqueline M , Bozinovski Steven , Vlahos Ross , Miller Alyson A 
Published: November 2023

Recent studies have shown that chronic obstructive pulmonary disease (COPD) is associated with an increased stroke risk2 and mortality3. Cigarette smoke is a shared risk factor for COPD and stroke. Therefore it is conceivable that the link between COPD and stroke may relate, at least in part, to cigarette smoke. During stroke, excess reactive oxygen species (ROS) or oxidative stress disrupts the blood‐brain barrier (BBB), which in turn can accentuate brain injury. Importantly, cigarette smoke increases ROS production by endothelial cells. Therefore, we tested the hypothesis that COPD/smoking worsens stroke severity by exacerbating oxidative‐induced BBB disruption after stroke. Monolayers of mouse cerebral microvascular endothelial cells (bEnd.3 cells) were pre‐treated with either vehicle, 5, 10, 20 or 40% CSE (1 h) prior to exposure to oxygen glucose deprivation (OGD) (4 h) and reoxygenation (RO) (23 h). Cells cultured under normoxic conditions (27 h) were used as controls. We assessed BBB disruption by measuring paracellular permeability (FITC‐dextran), tight junction protein expression (Western blotting), and cell viability (MTS assay). Furthermore, we measured superoxide (L‐012‐enhanced chemiluminescence) and hydrogen peroxide (H2O2) production (Amplex Red fluorescence). 10% CSE increased the paracellular permeability of bEnd.3 cells cultured under normoxic conditions, indicating BBB disruption. Furthermore, 10% CSE exacerbated (~2‐fold) the hyperpermeability of bEnd.3 cells exposed to OGD + RO (10% CSE, 5±0.9 vs. vehicle, 2.8±0.7; fold‐change relative to normoxic controls, n=8, P<0.05), indicating a worsening of BBB disruption after ischaemic‐like conditions. However, this was found to be independent of increased tight junction protein degradation (occludin or ZO‐1) or cell death (n=6, P>0.05). Contrary to our hypothesis, CSE (10% or 20%) significantly attenuated OGD/RO‐induced elevations in superoxide production (10% CSE, 97±6.3, 20% CSE 75±5 vs. vehicle, 147±16; % normoxic, n=5, P<0.05), but had no significant effect on H2O2 production (n=5, P>0.05). Thus, CSE worsens BBB disruption in an in vitro model of stroke; however, this appears to be unrelated to increased oxidative stress. Although, much more research is needed, these tantalizing findings raise the possibility that such an effect may play a role in the link between COPD and stroke.Support or Funding InformationSupported by NHMRC funding
Do Non‐C1 Cells In The Rostral Ventrolateral Medulla Increase Blood Pressure?
Authors: Dash Kiana Elizabeth , Stornetta Ruth L. , Holloway Benjamin B. , Guyenet Patrice G. 
Published: November 2023

C1 cells are a major group of neurons located in the rostral ventrolateral medulla (RVLM) that are glutamatergic and contain catecholamine synthetic enzymes such as tyrosine‐hydroxylase (TH), dopamine‐beta hydroxylase (DBH), and phenylethanolamine N‐methlytransferase (PNMT) (DePuy et al., 2013; Stornetta et al., 2002). Limited amount of information is available that suggests these neurons are the exclusive influence on blood pressure. Non‐C1 catechominergic cells also express vesicular glutamate transporter‐2 (VGLUT2) and are barosensitive and thus may have an impact on blood pressure (Stornetta et al., 2002). Optogenetic activation of C1 cells increases blood pressure specifically targeted in TH‐Cre rats by using a Cre‐dependent vector that drives channelrodopsin‐2 (ChR2) tagged with mCherry (Burke and Guyenet, 2014). However, in the mouse model when C1 cells are stimulated the results indicate a decrease in blood pressure. This difference is perhaps due to the close proximity of different types of C1 cells that are separated in the rat model. When C1 and non‐C1 neurons are stimulated in mice with the use of a CAMKII vector, there is a large increase in blood pressure. One unanswered question is whether CAMKII transduces glutamatergic neurons in the brainstem. This present study uses a CAMKII vector to express ChR2 in combination with mCherry. Oligonucleotide probes were used to label cells positive for VGLUT2. By using light microscopy we will ask if the non‐C1 cells transduced by CAMKII are glutamatergic. We found that on average, 90% (N=3) of CAMKII transduced cells within the RVLM contain VGLUT2.Support or Funding InformationUniversity of Virginia Summer Research Internship Program
Development and Characterization of a Fluorescent Protein‐Based Biosensor for the Detection of Extracellular ATP
Authors: Conley Jason M , Radhakrishnan Saranya , Valentino Stephen A , Tantama Mathew C 
Published: November 2023

Extracellular adenosine 5′‐triphosphate (ATP) is a fundamental component of purinergic signaling that contributes to wide ranging physiological processes and pathophysiological conditions. New methods that enable direct measurement of extracellular ATP dynamics with high spatial and temporal resolution are expected to advance our understanding of purinergic signaling mechanisms in healthy and pathological states. Several distinct classes of fluorescent protein‐based biosensors have emerged as powerful molecular tools that report real‐time intracellular ATP dynamics with single‐cell and subcellular resolution, but there are no reports of leveraging such technology for the detection of extracellular ATP. Therefore, the focus of this study was to develop and characterize a series of fluorescent protein‐based biosensors for the detection of extracellular ATP. Our strategy was to anchor several candidate ATP biosensors to the plasma membrane and to position the biosensor in the extracellular space, allowing for the detection of extracellular ATP. We present the extensive characterization of one such cell surface‐tethered candidate biosensor. Live‐cell fluorescence microscopy was used to verify biosensor expression and localization at the cell surface and to measure sensor response to exogenous application of ATP when expressed by cell types including Neuro2A and HEK293 cells. Furthermore, we present progress using this sensor to measure extracellular ATP dynamics that are modulated by cellular processes, including ligand‐stimulated cellular release of ATP and degradation of ATP by ectonucleotidase activity. In conclusion, we are developing a genetically‐encoded fluorescent protein‐based biosensor that currently demonstrates the ability to measure dynamic changes in extracellular ATP levels in live‐cell proof‐of‐concept studies.Support or Funding InformationWe acknowledge funding support from National Institutes of Health grant NS092010
Alternative Therapeutic Nanoparticle Approach For Treatment Of Invasive Streptococcal Infections
Authors: Escajadillo Tamara , Olson Joshua , Luk Bryan , Zhang Liangfang , Nizet Victor 
Published: November 2023

Bacterial infections continue to be a significant cause of morbidity and mortality worldwide. Streptococcus pyogenes, also known as group A Streptococcus (GAS), is an important human‐specific Gram‐positive bacterial pathogen, capable of causing a broad spectrum of disease from mild superficial infections such as pharyngitis and impetigo to serious invasive infections including necrotizing fasciitis and streptococcal toxic shock syndrome. Pathogenic bacteria like GAS express a multitude of virulence factors with diverse properties and modes of action that act in concert to overcome the host's innate immune defense mechanisms. Pore forming toxins (PFTs) are the most common bacterial cytotoxic proteins (25–30% of all cytotoxic proteins), which make them the single largest category of virulence factors. The PFT streptolysin O (SLO) is a well characterized virulence factor produced by nearly all GAS clinical isolates, and is necessary and sufficient to promote dermal necrosis, blood vessel contraction, increased capillary permeability, intravascular thrombosis, cardiotoxicity, and death during severe invasive infection. In the present work, we describe how the pharmacological presentation of red blood cell (RBC) derived biomimetic nanoparticles (“nanosponges”) can sequester SLO and block the ability of GAS to damage host cells, thereby preserving innate immune function and increasing bacterial clearance in vitro and in vivo. Nanosponge administration protected human neutrophils, macrophages and keratinocytes against SLO‐mediated cytoxicity. This therapeutic intervention prevented SLO‐induced macrophage apoptosis and increased neutrophil extracellular trap formation, allowing increased GAS killing by the respective phagocytic cell types. In a murine model of GAS necrotizing skin infection, local administration of the biomimetic nanosponges was associated with decreased lesion size and reduced bacterial colony‐forming unit recovery. Utilization of a toxin decoy and capture platform that inactivates the secreted SLO before it comes into contact with the host cell membrane presents a novel virulence factor targeted strategy that could be a powerful adjunctive therapy in severe GAS infections where morbidity and mortality are high despite antibiotic treatment.Support or Funding InformationUCSD Graduate Training Program in Cellular and Molecular Pharmacology: T32 GM007752
Computational approaches to understanding ARB resistance: a route to personalized ARB therapy
Authors: Anderson Shane D. , Nies Isaac , Santos Priscilla , Soong Tik Hang , Thu Yin Win , Botello‐Smith Wesley M. , Luo Yun Lyna , Andresen Bradley T. 
Published: November 2023

The binding of Angiotensin II to the Angiotensin II type 1 receptor (AT1Rs) plays a pivotal role in the regulation of blood pressure and other physiological processes. AT1R blockers (ARBs) are commonly prescribed anti‐hypertensive drugs; however, not all patients respond equally to ARBs and some patients are ARB resistant. One documented reason behind these clinical observations is non‐synonymous polymorphisms within the AT1R that alter ARB affinity. There are 103 known polymorphisms within the crystallized AT1R structure that we utilized for computational approaches to predict which polymorphisms contribute to ARB resistance. To accomplish this, a 150 ns molecular dynamic (MD) simulation was performed to create a stable‐empty AT1R in an 87% POPC and 13% cholesterol membrane. Each polymorphism was made in the MD generated AT1R via MOE and minimized prior to docking performed with autodock4. Each ARB, including the active metabolite of Losartan, was docked using an experimentally derived grid that produced affinities in the wild‐type AT1R comparable to published reports. In total, 93,600 drug‐receptor complexes were analyzed and the data indicates that polymorphisms fall into two general categories: ARB specific resistance and total ARB resistance. Polymorphic AT1Rs displaying ARB specific resistance display wild‐type affinities for a subset of ARBs and in some cases, such as C101Y, G196V, and F313C, not the common ARBs valsartan and losartan; whereas, total ARB resistance, such as W84C, V108I, H132Y, and C289W, indicates that all ARBs are likely ineffective. In conclusion, patients that appear ARB resistant should have their AT1R sequenced and the sequence could be used to determine which ARB may be effective or suggest discontinuing ARB therapy.Support or Funding InformationThis project is funded by internal WesternU funds.
IGF‐1 deficiency alters hypertension induced cerebrovascular remodeling in mice
Authors: Fulop Gabor Aron , Ramirez‐Perez Francisco I , Tarantini Stefano , Sjoelund Virginie , Martinez‐Lemus Luis A. , Sonntag William E. , Csiszar Anna , Ungvari Zoltan 
Published: November 2023

Hypertension induced cerebral microhemorrhages (CMHs) may contribute to cognitive decline and predict larger strokes. Previous studies of our group found evidence that IGF‐1 deficiency exacerbates hypertension‐induced cerebral microhemorrhages (CMHs) mimicing an age‐related phenotype, but the underlying mechanisms remain elusive. We hypothesized that increased cerebrovascular stiffness and altered hypertension induced remodeling of the vessels may contribute to higher number of CMHS in IGF‐1 deficiency. Liver‐specific knockdown of IGF‐1 (Igf1f/f + TBG‐iCre‐AAV8) and control mice were chronically treated with angiotensin‐IIleading to similar hypertension in both groups (CTRL syst.: 151.375±2.08, diast.:109.65±3.27; IGF syst.: 150.10±2.00, diast.: 106.48±2.53). Middle cerebral (MCA) arteries of both groups were examined, by fixing them to a canulated isobaric vascular system and loaded with calcein. Smooth muscle cell related characteristics likepressure induced alteration in wall thickness and vessel diameter were measured using confocal microscopy. We found impaired cellular hypertrophic response in the IGF‐1 deficient hypertensive (HT) group, leading to smaller increase in wall thickness (1.43±0.04 μm in CTRL HT vs. 1.25±0.04 μm in IGF‐1 deficient HT). Based on the wall thickness and vessel diameter data circumferential stress and circumferential strain were calculated at different pressure levels (5–160 mmHg). Circumferential stress was higher in the IGF‐1 deficient group at any given pressure comparing to wild type group, while there was no change in the circumferential strain. Young's elastic modulus (Emod) was calculated by linear fitting of the stress‐strain curve. The Emod at lower pressures (describes elastin based elasticity) was higher in IGF‐1 HT group (468.20±73.56 vs. 306.77±36.22) while there was no change in the Emod at higher pressures (describes collagen based elasticity). Based on these data we can conclude that age‐related IGF‐1 deficiency alters hypertension‐induced cellular and extracellular matrix remodeling, which may lead to increased stiffnening of microvessels, possibly leading to incrased risk of pressure‐induced rupture.Support or Funding InformationThis work was supported by grants from the American Heart Association, the National Center for Complementary and Alternative Medicine, and the National Institute on Aging