bluuebunny/crossref_metadata_2025 huggingface dataset:
Enhanced Mitochondrial Biogenesis for the Treatment of Spinal Cord Injury
Authors: Scholpa Natalie E , Narang Aarti , Wang Wenxue , Corum Daniel , Tomlinson Stephen , Schnellmann Rick G
Published: November 2023

Spinal cord injury (SCI) is characterized by an initial trauma followed by a progressive cascade of damage referred to as secondary injury. It has been suggested that pharmacological interruption of this secondary injury could improve neuron survival, allowing for enhanced functional recovery. A hallmark of secondary injury is vascular disruption leading to vasoconstriction and decreased oxygen delivery, directly reducing the ability of mitochondria to maintain homeostasis, leading to loss of ATP‐dependent cellular functions, calcium overload, excitotoxicity and oxidative stress, further exacerbating injury. While therapeutics targeting restoration of single facets of mitochondrial function have proven ineffective post‐SCI, pharmacologically stimulating mitochondria biogenesis (MB) is a previously unexplored strategy. This study examined the effects of formoterol, an FDA‐approved β2‐adrenergic receptor agonist, and LY344864 (LY), a selective 5‐HT1F receptor agonist, on spinal cord MB and functional recovery following SCI in mice. Female C57BL/6 mice were subjected to moderate SCI using a force‐controlled pneumatic impactor‐induced contusion model (80 Kdyn), and administered daily formoterol or LY (i.p) beginning 1 h post‐SCI. Both treatments induced MB in the injury and peri‐injury sites post‐SCI, as evidenced by increased mitochondrial DNA content and nuclear‐encoded mitochondrial genes, as well as 2–3‐fold increases in various mitochondrial‐encoded genes. Drug‐treated mice also exhibited less histological damage than vehicle‐treated mice at 72 h post‐SCI, namely decreased loss of white matter and structural preservation of the central canal and anterior median fissure. The Basso‐Mouse Scale (BMS) was used to assess locomotor capability, where a BMS score of 0 indicates no movement of the hind limbs (observed 24 h following SCI) and a score of 9 indicates normal function (sham controls). Importantly, five days post‐SCI, LY‐treated mice demonstrated improved motor function compared to vehicle‐treated mice (BMS = 1 v 0.2). Following seven days of administration, the locomotor capability of both LY‐ and formoterol‐treated mice were greater than that of the vehicle group (BMS = 1.8 v 1.2 v 0.5). Fifteen days post‐injury, both formoterol‐ and LY‐treated mice exhibited the same overall improvement in motor function, with BMS scores twice that of vehicle‐treated mice (BMS = 4 v 2). We suggest that MB is a good drug target for the treatment of SCI and that LY‐ and formoterol‐induced MB as a therapeutic avenue for the treatment of SCI.

Support or Funding Information

South Carolina Spinal Cord Injury Research Fund
Brevetoxin Induced Dendritic Arborization and Functional Recovery in a Murine Post‐Stroke Model
Authors: Sequeira Erica , Pierce Marsha , Gomez Dina , Murray Thomas
Published: November 2023

RATIONALE

Stroke is a leading cause of long term disability rendering most survivors incapable of walking without assistance. This condition triggers structural and physiological changes in neuronal circuits adjacent to the infarct region, termed peri‐infarct area, that plays a key role in recovery and represents a potential therapeutic target. Pyramidal neurons are abundant in the cerebral cortex and form excitatory glutamatergic synaptic contacts. These glutamatergic neuron dendritic domains play a role in excitability and neuronal plasticity. Synaptic plasticity is largely mediated through N‐methyl D‐aspartate receptors (NMDARs) and calcium [[Ca2+]] dependent signaling pathways. Intracellular sodium [[Na+]i] acts as a signaling molecule in as much as it exerts and regulates NMDAR channel activity. Brevetoxin‐2 (PbTx‐2) is a voltage‐gated sodium channel (VGSC) gating modifier that increases [Na+]i, upregulates NMDAR function and increases downstream Ca2+ dependent signaling pathways.

OBJECTIVE

Purpose of this study was to develop a sensitive and a reproducible behavioral test to evaluate motor movements post‐stroke in mice and to test the effect of PbTx‐2 on recovery of movement. In addition, this study aimed to examine the effect of PbTx‐2 on dendritic arbor complexity of cerebral cortical layer V pyramidal cells.

METHODS

A stroke was produced by administration of an intraperitoneal injection of Rose Bengal dye followed by a 12.5 min light illumination of the caudal forelimb area of the motor cortex region. Hydrogel surgery was then performed on Day 5 post stroke which involves drilling the skull to inject Hydrogel vehicle or Hydrogel + 100 pmol PbTx‐2 using a Hamilton syringe. A Pasta Matrix Reach Task was used to determine the number of pieces of pasta retrieved post‐stroke and post‐Hydrogel treatment from a 5×5 matrix of pasta. Brain slices were imaged using Leica TCS SP8 MP confocal microscope that produces 3D images which were then analyzed with Imaris 8.3 software to measure dendritic arbor complexity by Sholl analysis.

RESULTS

The results demonstrated that the number of pastas retrieved was significantly impaired on Day 1 post‐stroke with a subsequent improvement following treatment with 100 pmol PbTx‐2 as compared to the control mice. In addition, PbTx‐2 treated layer V pyramidal neurons also had a greater dendritic arbor complexity in comparison to controls.

CONCLUSION

Preclinical research in a murine stroke model provided a behavioral assessment of neural insult‐induced functional deficits and PbTx‐2 induced motor recovery. The Pasta Matrix Reach Task proved to be a sensitive behavioral measurement for the study of neural impairment and recovery. Increased dendritic arbor complexity in layer V pyramidal cells indicates that PbTx‐2 can potentially aid neuroplasticity at the peri‐infarct region.

Support or Funding Information

NIH R01NS053398
Identifying a Phospholipase C Epsilon Region that Interacts with Gbeta Gamma
Authors: Madukwe Jerry , Malik Sundeep , Smrcka Alan
Published: November 2023

Many hormones and neurotransmitters function by activating isozymes of Phospholipase C (PLC) family through G protein coupled receptors (GPCRs). PLCs hydrolyze membrane phospholipids to generate downstream signaling molecules essential for normal function of the cell. Chronic PLC activation has been implicated in diseases that affect the body. The PLCɛ isoform has been shown by our laboratory to be scaffolded at the nuclear envelope yet responds to plasma membrane (PM) endothelin 1 (ET‐1) receptor stimulus. ET‐1 stimulates phosphatidyl inositol 4‐phosphate (PI4P) hydrolysis in the perinuclear Golgi apparatus generating diacyglycerol and activating a downstream nuclear pool of PKD in neonatal cardiac ventricular myocytes (NRVM). This signaling process leads to reactivation of the fetal gene program and pathologic myocyte hypertrophy (Zhang et al. Cell, 2013).

Small GTPases and Heterotrimeric G protein activate PLCɛ. We have shown that the PM endothelin‐1 receptor stimulation of Golgi PI4P depletion is dependent on Gβγ dimer (Malik et al. Mol. Biol. Cell, 2015). Other groups have shown that PLCɛ co‐expressed with Gβγ and a select group of constitutively active small GTPases produce marked increases in inositol phosphate accumulation in COS7 cells. Domains of PLCɛ that interacts with small GTPases for its activation have previously been defined. However, it is not known whether PLCɛ interacts with Gβγ directly for its activation, or which region is required. Our laboratory has previously shown that Gβγ directly interacts with PLCβ2 in vitro in a region within its catalytic domain. We hypothesize that a region within the catalytic domain of PLCɛ interacts with Gβγ directly and propose to identify this region.

We reexamined PLCβ2 and PLCɛ activation by Gβγ, Gq and small GTPases (hRasG12V) in COS7 cells co‐transfection assay and observed marked increases in inositol phosphate accumulation. We then identified a region of PLCɛ that aligns with the catalytic domain of PLCβ2. We are currently making mutations within the catalytic domain and other regions of PLCɛ which will be tested for Gβγ activation using the COS7 cell co‐transfection assay. We will also determine if purified Gβγ can interact with and activate PLCɛ in vitro.
Quercitrin, a natural flavonoid glycoside, protects against cisplatin‐induced injury in rats without hindering cisplatin beneficial cytotoxic activity
Authors: Arafa Elshaimaa A. , Mohamed Wafaa , Shehata Basim , Hassan Hossam , Raslan Mai , Elsherbiny Gamal , Elgendy Abelnasser
Published: November 2023

Objectives

The present study aimed to evaluate the possible nephroprotective and hepatoprotective effects of quercitrin (a natural flavonoid glycoside) against cisplatin‐induced toxicity in rats. The study extended to evaluate the effect of quercitrin on the beneficial cytotoxic activity of cisplatin.

Background

Cisplatin is a widely used cytotoxic agent whose benefits are limited by its nephrotoxic and hepatotoxic potentials. Quercitrin has a wide variety of pharmacological actions including anti ‐ oxidant, anti‐inflammatory and immunomodulatory activities.

Methods

Cisplatin toxicity was induced by a single 7.5 mg/kg i.p injection. Quercitrin (10 and 20 mg/kg) was orally administrated for 1 week starting one day before cisplatin. Assessment of cisplatin injury was performed by measuring serum creatinine (Cr) and blood urea nitrogen (BUN) as renal injury markers, while serum alanine transaminase (ALT) and aspartate transaminase (AST) were assessed as hepatic injury markers. In addition, serum total cholesterol (TC) and triglycerides (TG) were determined as dyslipidemic markers. Renal and hepatic tissue malondialdehyde (MDA), reduced glutathione (GSH) and total nitrate/nitrite (NOx) levels were measured as oxidative and inflammatory markers., Western blot analysis of renal and hepatic phospho‐Akt (p‐Akt) and nuclear factor‐kappa B (NF‐κB) expression were determined as tissue inflammatory and immunological markers, coupled with histopathological examination of renal and hepatic tissues.

Animal handling protocol was approved by Ethics Committee of the Faculty of Pharmacy, Beni‐Suef University, following guidelines of the National Institute of Health (NIH) Guide for Care and Use of Laboratory Animals.

Results

Quercitrin significantly ameliorated cisplatin‐induced renal and hepatic injury evidenced by corrections of serum Cr, BUN, ALT, AST, TC and TG levels as well as renal and hepatic MDA, GSH, NOx contents and p‐Akt and NF‐κB expression levels, in addition to correction of histopathological alterations. Cytotoxicity assay on breast adenocarcinoma (MCF‐7) cells using the thiazolyl blue tetrazolium bromide (MTT) revealed that quercitrin did not inhibit the cytotoxic activity of cisplatin in vitro.

Conclusions

Our data showed the ability of quercitrin to protect against cisplatin‐induced nephrotoxicity, hepatotoxicity and dyslipidemia without interfering with Cisplatin's beneficial anticancer activity, suggesting the effective use of quercitrin during cisplatin course treatment permitting dose scaling required in some resistant cancer types.
In Vitro Inhibition of Human UGT Isoforms by Anti‐HIV Boosting Drugs, Ritonavir and Cobicistat
Authors: Alam Novera , Algeelani Sara , Hossain Amin , Greenblatt David
Published: November 2023

For more than a decade ritonavir has been the only available pharmacokinetic boosting agent for HIV protease inhibitors. Another boosting agent, cobicistat, is now available, and reportedly has efficacy comparable to ritonavir. Cytochromes P450 (CYPs) and uridine 5′‐diphospho‐glucuronosyltransferase (UGTs) are major metabolizing enzymes that catalyze the majority of drug biotransformation reactions. Cobicistat and ritonavir both are strong inhibitors of CYP3A, which is the isoenzyme involved in the metabolism of most HIV protease inhibitors. Consequently, they increase the concentrations of anti HIV protease inhibitors in the blood. However, they can also increase blood levels of many other drugs. Since data from the manufacturer suggested that cobicistat was a more selective CYP3A4 inhibitor than ritonavir, differences are expected with co‐medication drug‐drug interaction profiles. The inhibitory effect of ritonavir and cobicistat on human UDP glucuronosyltransferase (UGT) enzymes is not established. Here, we have studied the inhibition of different human UGTs Isoforms by ritonavir and cobicistat, along with their IC50 values for 5 different human liver microsomes (HLMs) including pooled HLMs. Acetaminophen and ibuprofen have been used as nonspecific substrates to evaluate metabolic activity of the principal UGT isoenzymes (UGT1A1, UGT1A6, UGT1A9, UGT1A3, UGT2B7, and UGT2B4). The formation of the UGT isoform‐specific metabolites was monitored by HPLC following incubation of index substrates with HLMs at different inhibitor concentrations. A decrease in the formation of the metabolites compared to vehicle control is used to calculate an IC50 value. Probenecid and estradiol served as positive control inhibitors of the formation of 4‐acetamidophenol glucuronide and ibuprofen acyl‐β‐D‐glucuronide. Probenecid inhibited the glucuronidation of acetaminophen (IC50 0.7 ± 0.5 mM) but not of glucuronidation of ibuprofen. This indicated that probenecid is a relatively selective inhibitor of some UGT isoforms. However, estradiol was a potent inhibitor of ibuprofen glucuronidation (IC50 17 ± 2 μM). Neither ritonavir nor cobicistat meaningfully inhibited in vitro formation of glucuronide conjugates of acetaminophen or ibuprofen. The minimum inhibitory concentrations of cobicistat and ritonavir were at least 50 μM, which is much higher than typical clinical plasma levels. The potential for time‐dependent (mechanism‐based) inhibition was evaluated through studies in which inhibition was determined without and with pre‐exposure of enzymes to inhibitor prior to addition of substrate. Inhibition was not enhanced by preincubtion, thereby providing no evidence of mechanism‐based or irreversible inhibition. Taken together, the findings indicate that ritonavir and cobicistat are unlikely to produce clinically important drug interactions involving drugs metabolized to glucuronide conjugates by UGT isoforms 1A1, 1A3, 1A6, 1A9, 2B7 and 2B7.
Fructose diet decreases APAP‐induced hepatotoxicity in mice
Authors: Tripathi Ashutosh , Cho Sung‐Joon , Chlipala George E. , Green Stefan , Jeong Hyunyoung
Published: November 2023

Acetaminophen (APAP) is a commonly used over‐the‐counter analgesic and antipyretic. APAP overdose can cause hepatotoxicity, and is responsible for ~50% of acute liver failure in US. However, risk factors for severe liver injury after APAP dose remain unclear. Previous studies have shown conflicting effects of fructose diet on the susceptibility to APAP‐induced hepatotoxicity (1, 2). The aims of the present study were to evaluate the role of fructose diet in modulating the extent of APAP‐induced liver injury and explore potential roles of differential gut microbiota in any effects. Male C57BL/6J mice (8 weeks old) were given 30% (w/v) fructose in water (or regular water) for 8 weeks, followed by oral administration of APAP (600 mg/kg) or vehicle control. The animals were sacrificed 24 h after APAP dosing, and tissues were collected. APAP‐induced liver injury (determined by serum levels of liver enzymes) was decreased by two‐fold in mice pretreated with fructose. Fructose‐treated group exhibited (~1.5 fold) higher basal glutathione levels and (~2 fold) lower basal (mRNA and activity) levels of CYP1A2 and CYP2E1, suggesting decreased bioactivation of APAP and increased detoxification of toxic metabolite in fructose‐treated mice. Also, mRNA expression levels of GCLC (glutamate‐cysteine ligase; a rate‐limiting enzyme in synthesis of glutathione) exhibited a trend of increase in fructose group. mRNA expression of HSP70 (heat shock protein 70; a chaperone protein previously known to have a protective effect against APAP hepatotoxicity) was also found increased in fructose group. High‐throughput, next‐generation sequence (NGS) analysis of bacterial 16S rRNA gene amplicons from the cecal samples of vehicle group showed that fructose diet altered the composition of gut microbiota, leading to increased α‐diversity (Mann‐Whitney, p=0.043) and a significant difference in overall bacterial community (Bray‐Curtis, ANOSIM: R = 0.581, p = 0.001). However, no bacteria taxa were found to be significantly correlated with hepatic mRNA level of CYP2E1 or CYP1A2, or glutathione levels. These findings suggest that fructose diet decreases APAP‐induced liver injury in mice, in part by reducing metabolic activation of APAP and inducing detoxification to toxic metabolites. Fructose diet also alters gut microbiota composition in mice although its role in modulation of APAP toxicity remains unclear.

Support or Funding Information

This study was supported by Chicago Biomedical Consortium.
Capsaicin suppresses the metastasis of human NSCLC in vivo
Authors: Nolan Nicholas A , Akers Austin T , Colclough Kate W , Hurley John D , Williams Cathryn D , Rollyson William D , Brown Kathleen C , Denning Krista L , Brown Linda G , Egleton Richard D , Dasgupta Piyali
Published: November 2023

Non‐small cell lung cancer (NSCLC) is characterized by aggressive clinical course, rapid doubling time and a propensity for extrapulmonary metastasis. A substantial proportion of NSCLC presents with metastatic disease at the time of their diagnosis. Metastasis is a complex multistep process. One of the earliest events of the metastatic process is the invasion of malignant cells through the surrounding stroma into the blood and lymph. The long‐term goal of our laboratory is to identify nutritional compounds (as single agents or in combination with standard chemotherapy), which will suppress metastasis of NSCLCs. Capsaicin is the pungent ingredient of chili peppers. Several convergent studies have shown that capsaicin inhibits the invasion and metastasis of several types of human cancers including melanoma, prostate cancer, and cholangiosarcoma. The present study examines the anti‐invasive properties of capsaicin and two capsaicin‐like compounds, capsiate and capsiconiate, found in a select variety of chili peppers on NSCLC. We measured the anti‐invasive activity of these compounds by the Boyden chamber assay and spherical invasion assays. We found that capsaicin and capsiate displayed equivalent anti‐invasive activity in NSCLC cells. In contrast, capsiconiate did not suppress the invasion of NSCLC cells. Furthermore, we tested the anti‐metastatic activity of capsaicin in a syngenic mouse model of metastasis. We observed that the dietary administration of capsaicin potently decreased the area of lung metastatic foci relative to control untreated mice. Our data may form the basis of novel nutrition‐based therapeutic regimens in NSCLC metastasis.

Support or Funding Information

Funding for our study was supported by the an NIH R15‐AREA Grant (2R15CA161491‐02), WVU‐MU Partnership Grant, and a NASA Undergraduate Fellowship to NAN
IGF‐1 and Ang II regulate expression of the G protein‐coupled estrogen receptor in vascular smooth muscle cells.
Authors: Duong Jennifer Lee , Lindsey Sarah H
Published: November 2023

Young premenopausal females have a lower incidence of many cardiovascular diseases compared to males, but this protection significantly declines with aging and menopause. We have previously shown that the G protein‐coupled estrogen receptor (GPER) mediates many beneficial effects of estrogen in the cardiovascular system. In addition, GPER expression and function is decreased in the vasculature of aging rats and mice. The goal of the current study was to identify a mechanism by which aging decreases vascular GPER expression. Since circulating levels of angiotensin II (Ang II) and IGF‐1 are altered with aging, we assessed the impact of these two factors on GPER expression in vitro. We hypothesized that IGF‐1 signaling would positively influence GPER expression, while Ang II would negatively regulate GPER. A7r5 cells, an embryonic rat aortic smooth muscle cell line, were exposed to Ang II (100 nM) or IGF‐1 (100 ng/ml) for 24h before cells were harvested and GPER protein was assessed by immunoblot. In comparison to our previous work with primary cultured vascular smooth muscle cells, GPER expression in A7r5 cells was stable throughout passaging. Treatment with IGF‐1 significantly increased GPER expression (0.34 ± 0.05 vs. 1.37 ± 0.14 P<0.05 n=2–3) Conversely, Ang II treatment significantly repressed the expression of GPER (1.04 ± 0.06 vs. 0.83 ± 0.01 P<0.05 n=3). Thus, we conclude that aging‐induced decreases in IGF‐1 signalling and/or increases in Ang II that occur with aging or cardiovascular disease may influence GPER expression. We propose that loss of GPER signalling will diminish the cardiovascular protective effects of estrogen in aging postmenopausal women.
PDE1C regulates the dynamics of actin‐based structures in migrating human arterial smooth muscle cells
Authors: Brzezinska Paulina , Payne Darrin , Rampersad Sarah , Mackeil Jodi , Burke‐Kleinman Jonah , Maurice Donald
Published: November 2023

Phenotypic remodeling and an associated increased migration by human arterial smooth muscle cells (HASMCs) are features of neo‐intimal hyperplasia that promote occlusive vascular diseases, including post‐angioplasty restenosis. Although it is established that cyclic adenosine 3′, 5′‐monophosphate (cAMP)‐mediated signaling inhibits overall HASMC migration, how this ubiquitous signaling system impacts the individual “steps” required for directed cell migration, including the dynamics of lamellipodial projections at their leading edges, is currently poorly defined. Using a modification of the trans‐well assay, our study highlights a critical role for a specific cyclic nucleotide phosphodiesterase, Phosphodiesterase 1C (PDE1C), in modulating lamellipodial dynamics in migrating HASMCs. Formation of HASMC lamellipodial projections in response to exposure of these cells to a chemotactic gradient (0.5% FBS) was assessed by quantifying the accumulation of actin‐rich structures on the underside of filters with 3 μm pores in 4hrs. When compared to vehicle‐treated controls, selective pharmacological inhibition of PDE1C (compound 33 (C‐33), Dart Neuroscience LLC) increased accumulation of lamellipodial structures significantly. Similarly, RNAi‐mediated knockdown of PDE1C using several distinct siRNA markedly increased accumulation of lamellipodial structures when compared to those formed in control siRNA transfected cells. In contrast, agents that increase intracellular cAMP by activating HASMC adenylyl cyclases (ACs) (i.e. forskolin) reduced accumulation of these actin‐rich structures in HASMCs exposed to a gradient of FBS. Most interestingly, lamellipodial projection accumulation in response to PDE1C inhibition, or knockdown, was also markedly antagonized by addition of forskolin. These results are consistent with the novel idea that PDE1C is acting locally at the leading edge of HASMCs to promote accumulation of lamellipodial structures and that this hyper‐localized effect is lost when HASMC cAMP levels are increased globally, using forskolin. Consistent with this, we show that PDE1C can associate with an A‐kinase anchoring protein AKAP79 and the cAMP effector Protein Kinase A (PKA). Overall, since each PDE1C and AKAP79 is found at the leading edge of migrating HASMCs, we suggest that PDE1C acts locally, via a PKA/AKAP79‐based signalosome, to regulate lamellipodial projection formation in migrating HASMCs.

Support or Funding Information

Canadian Institutes for Health Research (CIHR)
Differential βarrestin1‐Mediated Signaling in Spontaneously Hypertensive versus Normotensive Rat‐Derived Astrocytes
Authors: Negussie Shmuel , Lymperopoulos Anastasios , Clark Michelle A.
Published: November 2023

Objective

To investigate the role of βarrestin1 in angiotensin II (AngII) type 1 receptor (AT1R)‐mediated signaling in astrocytes isolated from the brainstem of spontaneously hypertensive rats (SHRs) or normotensive rats.

Background

The βarrestins (βarrestin1 and‐2) terminate G protein signaling by G protein‐coupled receptors (GPCRs) and, at the same time, they initiate their own “second wave” of signaling from GPCRs, independently of G proteins. The AT1R is one such GPCR that signals via both G proteins and βarrestins in various cell types. It is unknown whether the AT1R signals via βarrestins in the brain. We sought to examine the involvement of βarrestins in the AT1R‐mediated Extracellular signal‐regulated kinase (ERK1/2) mitogen‐activated protein kinase (MAPK) activation and angiotensinogen (AGT) expression in Spontaneously Hypertensive Rat (SHR)‐versus normotensive rat‐derived primary astrocytes.

Methods

Primary cultures of brainstem astrocytes were isolated from the brains of 2–3 days old pups. 100 nM AngII and 10 μM SII (an AngII peptide analog which is a βarrestin‐biased agonist) were used to study AT1R‐mediated ERK1/2 activation and AGT expression. Protein analysis was done using Western blotting and mRNA measurement was done using real‐time PCR. The results in SHR astrocytes were compared to normotensive Wistar rat astrocytes. Protein analysis was done using Western blotting and mRNA measurement using real time PCR.

Results

Our results indicate that βarrestin1 is the major βarrestin isoform in astrocytes from both SHR and normotensive rat brainstem astrocytes. AngII activates ERK1/2 in SHR astrocytes more robustly than in normotensive rat astrocytes. On the other hand, SII strongly activates ERK1/2 in normotensive rat astrocytes and only minimally in SHR astrocytes. In addition, SII, similarly to AngII, upregulates cytoplasmic AGT protein synthesis in both SHR and normotensive rat astrocytes. However, SII has no significant effects on AGT mRNA levels in brainstem astrocytes from either rat model.

Conclusion

In this study, we show that AngII‐AT1R‐induced ERK1/2 activation in SHR astrocytes appears to be almost exclusively G protein‐dependent, contrary to normotensive rat astrocytes. Moreover, the AT1R‐mediated βarrestin1‐dependent signaling pathway plays a role in the regulation of AGT protein synthesis in rat astrocytes derived from the brain stem region in both models.

Support or Funding Information

* This research was funded by Nova Southeastern University President's Faculty Research & Development Grant and Health Profession Division Grant.
Exploration of chemical components correlated with pharmacology of EHYT based on a LC‐HRMS metabolomics approach
Authors: Ha In Jin , Kim Eunok , Kim Kyuseok
Published: November 2023

HYT (Hyeonggaeyeongyo‐tang) is a thirteen‐herb Traditional Chinese Medicine (TCM) and Korean medicine formula. Its modified prescription (Eunhwayeongyo‐tang; EHYT) is an herb medicine formula composed of the HYT and Lonicerae Flos (LF). Those have been traditionally used for acne, sinusitis, and otitis media otitis, tonsillitis, and a variety of otolaryngology symptoms. Nowadays, those prescriptions are used to treat diseases alone or combined with western medicine. However, it lacks in‐depth research of the constituents of EHYT and HYT. Establishing botanical medicine formula as globally‐accepted polychemical medicines required comprehensive chemical and biological profiling correlated with pharmacology. In this study, the production of pro‐inflammatory cytokines (IL‐1a, IL‐1b, IL‐6 and IL‐8) via Toll‐like receptor 2 (TLR2) and TLR4 in HaCat cells was demonstrated by RT‐PCR analysis. RT‐PCR analysis reveals EHYT inhibit LPS induced expression of chemokine IL‐8 in HaCaT cells. HYT and LF were shown the lower inhibition effects although those were treated on the induced cells in higher concentration than EHYT. Also, ultra high performance liquid chromatography coupled with quadrupole‐time‐of‐flight mass spectrometry and metabolomics were established to characterize the chemical profiling and explore significantly potent metabolites correlated with bioactivity. In total, potent 243 candidates were identified based on MS and MS/MS, and 36 compounds were characterized among three formula. A dicaffeoylquinic acid group was characterized as one significantly differential component among them. It demonstrated that UHPLC‐QTOF and chemometrics processing approach could be successfully applied for screening and comprehensive analysis of chemical components in complex constituents of herbal medicine. It reveals that EHYT consisting of HYT and LF has shown synergistic effects on anti‐inflammatory. LF may potentiate the anti‐inflammatory activity of HYT.

Support or Funding Information

This study was supported by a grant from the Traditional Korean Medicine R&D Project, Ministry of Health & Welfare, Republic of Korea. (HI16C0801)
Study of Systemic and Microvascular Responses to Progressive Hemolytic Anemia in Rats
Authors: Nugent William H , Carr Danuel A , Ormond Aimee , Selwood Simon P , Song Bjorn K
Published: November 2023

Hemolytic Anemia (HA) is characterized by an increase in the ratio of red blood cell destruction to production, which results in reduced oxygen carrying capacity, tissue hypoxia, inflammation, and corresponding clinical manifestations. The microvasculature is the primary site of vascular resistance and gas exchange, and is well suited to studying the subclinical progression of HA. HA was induced in male Sprague Dawley rats by intraperitoneal administration of phenylhydrazine (PHZ) per the following dosing schedule: Group 1 (100 mg/kg on Day 1) and Group 2 (100 mg/kg on Day 1, and 50 mg/kg on Day 2). Group 3 (baseline; BL) was an untreated time control. Rats were surgically prepared for the following systemic and microvascular measurements: total hemoglobin (tHb), mean arterial pressure (MAP), heart rate, pulse pressure (PP), arteriolar contractile response to three doses of phenylephrine (PE; 0.01, 0.1, 1 mg/kg; I.V), and tissue oxygenation (PISFO2). PHZ reduced tHb by 47 and 75% for Groups 1 and 2 respectively. The 47% decrease in tHb for Group 1 was associated with the following changes from BL (Group 3): MAP decreased by 12%, PP increased by 121%, and PISFO2 decreased 23%. A 75% decrease in tHb (Group 2) resulted in the following changes from BL: MAP decreased by 34%, PP was no different, and PISFO2 decreased 40%. Heart rate remained unchanged between the three groups. The arteriolar contractile response profile to increasing concentrations of PE was right shifted, or less responsive, as tHb dropped between groups. The data indicate a robust cardiovascular compensatory response to severe hemolytic anemia, which serves to prevent peripheral tissue hypoxia. At a 75% reduction in tHb, though, this system begins to show signs of compromise as arteriolar diameters lose responsiveness to contractile stimuli and compensatory elevations in PP begin to decay. Yet, PISFO2 remains normoxic suggesting that as tHb is reduced, tissue hypoxia occurs precipitously not progressively.

Support or Funding Information

Support: Song Biotechnologies
Impaired Reactivity of Basilar and Circle of Willis Arteries in Osteoarthritis and Ischemic Heart Disease Patients
Authors: Al‐Khazraji Baraa K. , Badrov Mark B. , Vording Jennifer L. , Birmingham Trevor B. , Shoemaker J. Kevin
Published: November 2023

Regional brain perfusion is supplied by flow from subcortical conduit vessels, namely the anterior, middle and cerebral arteries, and the functionality of these vessels is largely characterized by vasoreactivity to physiological stimuli. Patients with cardiovascular disease, such as ischemic heart disease (IHD), are at greater risk for stroke, a risk determined in part by cerebrovasoreactivity tests (i.e., dilation or constriction). Patients with osteoarthritis (OA) express greater incidence of cardiovascular disease. However, whether these patients express impaired cerebrovascular reactivity, and how such changes are manifest in each subcortical conduit vessel, remain unanswered. This study tested the hypothesis that, compared to healthy individuals (CTL), those either IHD or OA exhibit heterogeneous yet impaired reactivity (change in cross‐sectional area, ΔCSA) in the basilar and all Circle of Willis conduit in response to elevated levels of end‐tidal CO2 (ETCO2). In 22 participants (CTL: 6, OA: 7, IHD: 9; age range: 40–75 years), we collected simultaneous subcortical vascular responses (i.e., vasoreactivity) during normocapnia and hypercapnia (gas mixture of 5% CO2, 95% oxygen; ADInstruments Powerlab) conditions using a 3 Tesla Siemens MAGNETOM Fit scanner and T1‐weighted pulse sequence (0.7 mm isotropic). The relative change in cross‐sectional area (%ΔCSA) in response to CO2, measured at a site perpendicular to the direction of flow, of the basilar, left and right internal carotid, anterior, middle, and posterior cerebral arteries (9 vessels total) were measured using OsiriX software. The cross‐sectional vasoreactivity to changes in ETCO2 (ΔETCO2) was calculated as %ΔCSA/ΔETCO2. In all 9 vessels, OA and IHD patients exhibited similar vasoreactivity; yet, relative to CTL, OA and IHD patients had lower reactivity (%/mm Hg; mean ± S.D.) in the right anterior cerebral (CTL: 16.3 ± 6.5, OA: 3.8 ± 4.1, IHD: 4.4 ± 9.4; p<0.05), right internal carotid (CTL: 8.9 ± 4.9, OA: 3.9 ± 2.9, IHD: 1.5 ± 1.5; p<0.05), and basilar arteries (CTL: 7.8 ± 4.6, OA: 0.7 ± 0.8, IHD: 2.9 ± 3.0; p<0.05). Additionally, relative to CTL, OA patients expressed less left anterior cerebral artery reactivity (CTL: 11.6 ± 6.5, OA: 2.3 ± 1.2, IHD: 3.5 ± 8.3; p<0.05), and IHD patients had diminished right middle cerebral artery reactivity (CTL: 22.4 ± 17.4, OA: 5.6 ± 6.4, IHD: 3.7 ± 4.8; p<0.05). Therefore, heterogeneous patterns of dilation across subcortical vessels was observed in all groups, but the patient groups exhibited significantly impaired vasoreactivity in many of these vessels compared to healthy individuals.

Support or Funding Information

Supported by The University of Western Ontario Bone and Joint Institute Catalyst grant and the Canadian Institute of Health Research grant (201503MOP‐342412‐MOV‐CEEA).
ATP‐Sensitive Potassium Channels Contribute to Progressive Coronary Vasodilation in Response to Acute Isovolemic Hemodilution
Authors: Kiel Alexander M , Goodwill Adam G , Barnard April L , Sturek Joshua T , Noblet Jillian N , Sassoon Daniel J , Tune Johnathan D
Published: November 2023

This study was designed to identify mechanisms responsible for coronary vasodilation in response to progressive decreases in arterial oxygen content. Isovolemic hemodilution was produced in open‐chest, anesthetized swine via concurrent removal of 500 ml of arterial blood and the addition of 500 ml of 37°C synthetic plasma expander (Hespan, 6% hetastarch in 0.9% sodium chloride) up to a total sequential volume exchange of 4.02 ± 0.19 L. Arterial blood pressure, heart rate, left anterior descending coronary blood flow, and coronary arterial and venous blood gases were measured following stabilization after each fluid exchange. To establish content dependent effects, data were analyzed based on binned arterial oxygen contents of 18.0–13.0, 12.9–10.0, 9.9–8.0, 7.9–6.0, 5.9–4.0 ml O2/dl. Despite eliciting no change in mean arterial pressure, heart rate, or myocardial oxygen consumption, progressive hemodilution demonstrated clear reductions in arterial oxygen content (14.7 ± 0.4 to 4.7 ± 0.1 ml O2/dl; P < 0.001) and hematocrit (32 ± 1 to 10 ± 1%; P < 0.001). An accompanying increase in coronary flow was observed (0.39 ± 0.05 to 1.63 ± 0.16 ml/min/g; P < 0.001) which was sufficient to maintain oxygen delivery (58 ± 7 to 76 ± 8 μl O2/min/g; P = 0.46). Inhibition of nitric oxide synthase (L‐NAME: 25 mg/kg, iv) resulted in significant increases in mean arterial pressure (87 ± 2 to 122 ± 3 mmHg; P < 0.001) and myocardial oxygen consumption (47 ± 2 to 57 ± 3 μl O2/min/g; P < 0.001), however, this treatment did not significantly alter the coronary response to progressive anemia (P = 0.41). Inhibition of KV channels (4‐aminopyridine: 0.3 mg/kg, iv) also did not significantly alter coronary responses to decreasing arterial oxygen content. Alternatively, blockade of KATP channels with glibenclamide (3 mg/kg, iv) significantly reduced the coronary blood flow response from 1.87 ± 0.11 ml/min/g to 1.12 ± 0.08 ml/min/g (P < 0.001) when arterial oxygen content was lowered from 15.1 ± 0.3 to 4.7 ± 0.1 ml O2/dl. This glibenclamide‐induced decrease in coronary flow diminished myocardial oxygen delivery from 69.4 ± 4.3 to 52.0 ± 4.3 μl O2/min/g (P < 0.001). Taken together, these data support that KATP channels, but not KV channels or nitric oxide, contribute to coronary vasodilation in response to progressive, acute isovolemic hemodilution.

Support or Funding Information

This study was supported by U01HL118738 and the Purdue University Ross Fellowship. Ms. Noblet was supported by TL1 TR000162 (A. Shekhar, PI) from the National Institutes of Health, National Center for Advancing Translational Sciences, Clinical and Translational Sciences Award.
Traumatic brain injury impairs myogenic constriction of cerebral arteries: role of mitochondria‐derived H2O2, TRPV4 and calcium‐activated potassium channels
Authors: Szarka Nikolett , Pabbidi Mallikarjuna R. , Amrein Krisztina , Czeiter Endre , Pohoczky Krisztina , Helyes Zsuzsanna , Ungvari Zoltan , Buki Andras , Koller Akos , Toth Peter J
Published: November 2023

Traumatic brain injury (TBI) impairs pressure‐induced myogenic constriction of cerebral arteries, which contributes to TBI‐related autoregulatory dysfunction and the development of secondary brain injury. TBI has been shown to lead to mitochondrial oxidative stress in cerebral vessels, which likely contributes to secondary brain injury, as well. We tested the hypothesis that TBI‐induced mitochondrial oxidative stress contributes to the impaired myogenic response of cerebral arteries after TBI and aimed to establish the underlying mechanisms. We induced traumatic brain injury in WKY rats using the weight‐drop technique, and 24 hours after severe TBI we found that myogenic responses of isolated middle cerebral arteries (MCAs) of TBI rats were blunted compared to control. The impaired myogenic responses of MCAs were restored after inhibiting mitochondrial oxidative stress (mitoTEMPO), eliminating H2O2 (PEG catalase) and blocking large‐conductance calcium‐activated potassium (BKCa) channels and transient receptor activated vanilloid channel 4 (TRPV4). We also found that H2O2‐induced dilation of MCAs involves the activation of BKCa and TRPV4 channels, and that H2O2‐induced BK currents on vascular smooth muscle cells depend on the activation of TRPV4 channels. Our results suggest that after TBI excessive mitochondria‐derived H2O2 activates BKCa channels via TRPV4 channel activation in the cerebrovascular wall, which diminishes pressure‐induced constriction of cerebral vessels. Future studies should elucidate the potential of inhibiting these vascular pathways to restore autoregulatory function after TBI in order to prevent TBI‐related secondary brain damage in patients suffered traumatic brain injury.

Support or Funding Information

This work was supported by grants from the Marie Curie Actions SMARTER 7th Framework Program of the European Union 606998 (to NSz and AK), the Hungarian Academy of Sciences (the Bolyai Research Scholarship BO/00634/15 to PT), the Hungarian Brain Research Program (Grant No. KTIA_13_NAP‐A‐II/8 to PT, ECz and AB), National Center for Complementary and Alternative Medicine (R01‐AT006526 to ZU) and the Hungarian Scientific Research Fund (K108444 to AK).
An Automated, Label‐free Method for Quantifying Intercellular Gap Formation in Images of Cell Monolayers
Authors: Francis Christopher Michael , Morrow Kyle Adam , Renema Phoibe , Teague Douglas Alex , Agwaramgbo Ezinne , Langham Geri , Stevens Trevor C , Williams Chris Caesar , Alvarez Diego F , Audia Jonathon P , Balczon Ronald D , Stevens Troy
Published: November 2023

Within cell monolayers, specifically the vascular endothelium, intercellular gap formation is an important phenomenon involved in response to infection, wound healing, force transduction, and barrier regulation. Because intercellular gap formation is studied extensively in the context of biological research, there exists a need for analysis tools that enable the quantification of intercellular gaps. However, existing tools to assess cell gaps are limited by the requirement of exogenous cell markers and fluorescent probes to label cellular regions, or by cell damage‐associated markers that incompletely reflect the dynamic process of physiological gap formation and repair. Currently, there is no method of quantifying intercellular gaps in unlabeled light micrographs. Therefore, we developed an automated, algorithmic solution to the quantification of cell gaps, which works by iterative contrasting of cellular and non‐cellular regions within images. We vetted this algorithm by analyzing dose‐dependent cytotoxicity responses in pulmonary microvascular endothelial cell (PMVEC) monolayers after Pseudomonas aeruginosa infection, and compared our data to the conventional lactate dehydrogenase (LDH) cell cytotoxicity assay. We found that our algorithm was comparably sensitive to the LDH assay. However, it was faster and required no additional reagents. Additionally, our approach eliminated the user bias inherent to manual analysis techniques, and was robust to deviations in image background intensity, color, blur, and cell morphology. We conclude that this novel technique represents an accurate and sensitive approach to the measurement of intercellular gap formation. We next plan to develop and implement a user interface in order to disseminate this technology to the scientific community at large.

Support or Funding Information

Supported by NIH HL66299 and NIH HL60024.
Racial Differences in Cell Adhesion Molecule Expression in HUVECs
Authors: Adeyemo Adelola , Ling Chenyi , Cook Marc D. , Grimm Heather , Brown Michael D.
Published: November 2023

Background

Endothelial dysfunction is both a root cause and consequence of hypertension (HTN) and cardiovascular diseases (CVD). It is characterized by nitric oxide (NO) deficiency and altered release of vasoactive substances including inflammatory cytokines and adhesion molecules. African Americans (AAs) have higher rates of these vascular disruptions and develop hypertension at higher rates than any other racial/ethnic group in the United States. C‐Reactive Protein (CRP) levels are greater in AAs and CRP has been shown, in vitro, to promote endothelial dysfunction.

Purpose

Therefore, we examined the impact of CRP stimulation on AA and Caucasian (CA) endothelial cell (EC) activation.

METHODS

CRP with and without receptor blockade (IgG) was applied to confluent ECs. Gene and protein expression of FcγRIIB receptor, Intracellular Adhesion Molecule‐1 (ICAM‐1), and Vascular Cell Adhesion Molecule‐1 (VCAM‐1) was assessed. HUVECs were pretreated with CRP receptor FcγRIIB antibodies for additional inhibitory experiments.

RESULTS

CRP significantly upregulated VCAM‐1 and ICAM‐1 gene and protein (>0.5 fold increase, p< .01) expression. Significant racial differences in VCAM‐1 protein were only seen under CRP incubation at 10 μg/mL (0.25 fold difference, p< .005; Fig 3). Strong racial differences in ICAM‐1 gene expression existed within all conditions (p< .05; Fig 2), but CRP significantly increased ICAM‐1 expression in AA HUVECs but not CA. Blockade of the CRP FcγRIIB receptor decreased the relative CRP‐induced differences in VCAM‐1 and ICAM‐1 between AA and CA HUVECs (p< .05; Fig 1). However, ICAM‐1 remained significantly elevated in AA. FcγRIIB receptor gene expression was significantly greater in AA compared to CA HUVECs under basal and CRP conditions (p< .01; Fig 4).

CONCLUSION

CA and AA HUVECs respond differently to CRP, with AA HUVECs showing greater inflammatory adhesion molecule upregulation under basal and stimulated conditions. Elevated levels of adhesion molecules likely contribute to increased CVD risk in AAs considering the higher circulating CRP levels and the greater endothelial responses to CRP.

Support or Funding Information

ACSM Doctoral Student Research Grant to Chenyi Ling
Repeated Sprint Training in Hypoxia Improves Exercise Tolerance in Recreationally‐ Trained Men: A Randomized Single‐Blind Study
Authors: Wang Ran , Fukuda David H. , La Monica Michael B. , Starling Tristan M. , Stout Jeffrey R. , Hoffman Jay R.
Published: November 2023

Purpose

As a novel altitude training approach, repeated sprint training in hypoxia (RSH) is based on repetition of “all‐out” efforts of short duration separated by incomplete recovery. Although several investigations have reported that RSH provides additional benefits when compared with the same training under normoxia (RSN), the data regarding the effect of RSH on exercise tolerance is limited. Therefore, the primary purpose of this study was to investigate the effect of RSH on exercise tolerance in recreationally‐trained men.

Methods

Twenty‐six recreationally‐trained men (22.9 ± 3.1 y, 1.75 ± 0.76 m, 76.0 ± 9.3 kg) performed 8 sessions of repeated sprint training (each consisting of 3 sets of 5 × 10‐s sprints at a resistance of 7.5% of body mass, with 20‐s intervals of recovery) over 4 weeks (2 sessions per week) on a cycle ergometer either in normoxia (RSN, 300 m; FiO2 = 20.9%; n = 13) or normobaric hypoxia (RSH, 3000 m; FiO2 = 14.2%; n = 13). Anthropometry, graded exercise testing (GXT), repeated sprint testing (RST), and 3‐min all‐out critical power testing (3MT) was performed in normoxic conditions prior to and after completion of training. Changes in body composition, GXT peak power, maximal oxygen consumption (VO2max), respiratory compensation point (RCP), power output at RCP, mean power of all 5 sprints and the 5th sprint in RST, 3MT critical power and total work between RSH and RSN were compared using one‐way analysis of covariance (ANCOVA) on post‐test values with pre‐test values serving as the covariate.

Results

No group differences were observed in body composition (p = 0.502), GXT peak power (p = 0.170), VO2max (p = 0.124), and mean power of all 5 sprints in RST (p = 0.151), while significantly greater mean power of the 5th sprint in RST (p = 0.036, 8.655 vs 8.209 W· kg−1), 3MT critical power (p = 0.027, 2.829 vs 2.625 W· kg−1) and total work (p = 0.011, 0.741 vs 0.706 kJ· kg−1) were detected in RSH as compared to RSN. Additionally, a significantly greater improvement was found only in RCP (p = 0.019, 38.965 vs 36.173 ml· kg−1· min−1) following RSH. Though not significant, a trend towards a greater improvement was noted in power output at RCP (p = 0.076, 2.929 vs 2.806 W· kg−1).

Conclusion

The present findings demonstrate that greater improvement in exercise tolerance, together with enhanced working capacity, can be attained with RSH compared with RSN.

Support or Funding Information

This study was partially funded by a National Strength and Conditioning Association (NSCA) International Collaboration Grant.