bluuebunny/crossref_metadata_2025 huggingface dataset:
Structure‐Based Design of IKKɛ and TBK1 Inhibitors for the Treatment of Obesity
Authors: Beyett Tyler S , Labudde Emily , Tesmer John J. G.
Published: November 2023

Obesity is a leading factor in the development of type 2 diabetes. It has been shown that inhibitor of NF‐κB kinase epsilon (IKKɛ) and TANK‐binding kinase 1 (TBK1) are upregulated in adipocytes of obese individuals. Knockdown of either kinase results in significant weight loss and increased insulin sensitivity. High‐throughput screening identified the FDA‐approved drug amlexanox as an ATP‐competitive inhibitor of both kinases which produces significant weight loss when administered to obese mice. Amlexanox has poor solubility and metabolic profile when administered orally, necessitating the design of improved analogs for clinical use. Docking simulations have not provided consistent binding modes for amlexanox due to the planar and somewhat symmetrical structure of the molecule, limiting rational design of improved analogs.

To better understand the molecular mechanism of inhibition and to guide further development, we sought to obtain co‐crystals structures of IKKɛ and TBK1 with amlexanox and related analogs. While crystallization of IKKɛ has been unsuccessful, we generated two crystal forms of TBK1 in complex with the potent inhibitor MRT67307 but were unable to obtain co‐crystals in the presence of amlexanox. Soaking these crystals in amlexanox resulted in a significant decrease in diffraction quality and partial density for the original inhibitor. TBK1·amlexanox crystals of a moderate resolution (3.7 Å) were obtained via microseeding and subsequent macroseeding. The resulting structure shows amlexanox binding along the hinge of the kinase and suggests that the carboxylate on amlexanox may form a salt bridge with Lys38 and/or Thr156. Differential scanning fluorometry with an K38A mutant shows a loss in thermal stabilization by amlexanox, and analogs with modifications to the carboxylate display decreased or abolished ability to inhibit TBK1. Knowledge of the potential importance of the carboxylate‐Lys38 interaction will guide the development of future amlexanox analogs. Presently, we have developed amlexanox analogs with improved efficacy in mouse models and are progressing them towards the clinic.

Support or Funding Information

National Institutes of Health, Pharmacological Sciences Training Program (T32‐GM007767), Rackham Graduate School at the University of Michigan
A Quantitative Morphometric Analysis of the Effects of Sub‐Chronic Cadmium Exposure on the Proximal Tubule of the Rat
Authors: Hublikar Ishaan , Edwards Joshua R , Lamar Peter C , Gasiorowski Joshua Z , Prozialeck Walter C
Published: November 2023

Cadmium (Cd) is a nephrotoxic environmental pollutant that causes insidious injury to the proximal tubule that results in polyuria and proteinuria. Previous studies from our laboratory have shown that the early stages of Cd‐induced kidney injury may involve disruption of the adhering junctions between epithelial cells of the proximal tubule. In the present study, we utilized the Image J computer program to quantify the effects of sub‐chronic Cd exposure on the morphology of the rat proximal tubule. Adult male Sprague‐Dawley rats (n=6) were treated with CdCl2 (0.6 mg/kg, in saline vehicle) by subcutaneous injection, 5 days per week for 12 weeks, while control animals (n=6) received saline vehicle alone. At the end of the treatment period, 24 hour urine samples were collected and assayed for levels of protein, cystatin C, beta‐2 microglobulin and kidney injury molecule 1 (Kim 1). In addition, the animals were euthanized and the kidneys were removed, fixed in formalin and processed for histopathologic analysis. Hematoxylin/eosin‐stained sections (4 microns thick) were viewed using a 40× objective lens, and digital images (n=15 control and 15 Cd‐treated) of the renal cortex were captured. The Image J computer program was used to identify, by hand drawing, cross‐sections of proximal tubules (n=5–8 per image). In addition, Image J was used to define individual components of the cross‐sectional images of proximal tubules. Specific components that were defined included: total cross sectional area, luminal cross sectional area, area of gaps between cells and total cellular area. The results showed that Cd caused an increase in urine volume and urinary levels of protein, Kim 1, beta‐2 microglobulin and cystatin C, with no change in creatinine excretion. The morphometric analyses showed that Cd had no significant effect on total cross sectional tubular area or cellular area. However, it caused a slight decrease (36%, p<0.01) in luminal cross sectional area and a marked increase (300%, p<0.001) in gap areas between cells. Additional studies indicated that at the time these changes were occurring, there was no evidence of necrosis and only a slight increase in the number of apoptotic cells in the proximal tubule. These results are consistent with previous findings suggesting that the early stages of Cd‐nephrotoxicity involve the disruption of epithelial cell‐cell junctions in the proximal tubule.

Support or Funding Information

Supported by restricted funds from the Department of Pharmacology, Midwestern University.
Neurotransmitter Systems in Mouse Airways
Authors: Kieffer Cameron M , Tu Yaping , Abel Peter W
Published: November 2023

Autonomic and sensory nerves containing a variety of neurotransmitters innervate mouse lungs and control airway diameter. The objective of this study was to characterize the neurotransmitter systems that cause constriction of murine airways. Precision cut lung slices (PCLS) from CF‐1 mice were exposed to electric field stimulation (EFS) using parameters known to activate neurotransmitter release (1.0 ms, 50 V, 1–30 Hz). To measure constriction, changes in airway luminal area in response to EFS were captured as video images and quantified using ImageJ. EFS caused airway constriction in a frequency‐dependent manner that was best fit by a biphasic curve. PCLS taken from human lungs showed a similar biphasic fit. Neuron‐specific stimulation was verified by Mg2+ blockade. Maximum airway constriction to 30 Hz EFS in murine PCLS was 51.8 ± 3.0%. Interestingly, the muscarinic receptor antagonist atropine (10 μM) blocked 99.5 ± 7.2% of EFS induced constriction at 1 Hz, but only blocked 23.3 ± 3.8% of EFS induced constriction at 30 Hz and eliminated the first phase but not the second phase of the biphasic EFS response. These data are consistent with parasympathetic constriction of airways by acetylcholine at lower EFS frequencies. Treatment with capsaicin to deplete sensory neurotransmitters also altered the EFS response supporting the presence of sensory neurotransmitter systems. Taken together these data provide evidence for a robust, multi‐component innervation of mouse lungs. Elucidating the neurotransmitter systems causing nerve mediated constriction and dilatation of airways may identify novel therapeutic targets for treatment of asthma, COPD, and other lung diseases.

Support or Funding Information

State of Nebraska LB595
Phosphatidylinositol‐3,4,5‐trisphosphate‐dependent Rac exchange factor 1 (P‐Rex1) Exacerbates Asthmatic Airway Smooth Muscle Cell Proliferation via Rac1‐dependent Signal Pathways
Authors: Huang Yapei , Xie Yan , Jiang Haihong , Abel Peter W. , Panettieri Reynold A. , Casale Thomas B. , Tu Yaping
Published: November 2023

Background and Objective

Human airway smooth muscle (HASM) hyperplasia and remodeling are characteristic features of airways that contribute to their irreversible obstruction in patients with severe asthma. Accumulating evidence suggests that cell proliferation contributes to airway smooth muscle hyperplasia and remodeling. Although the role of HASM hyperplasia and remodeling in asthma is well established, the molecular mechanisms regulating the proliferation of HASM cells in asthma‐related conditions are poorly understood. P‐Rex1, a Rac‐selective guanine nucleotide exchange factor (GEF), plays important roles in both normal physiology and pathological conditions. The objective of our study was to define the pathological importance and mechanisms of upregulated P‐Rex1 in development of ASM hyperplasia and remodeling of severe asthma.

Methods

We used immunohistochemistry to compare P‐Rex1 protein expression in lung tissues between human asthmatic and control subjects. Western blot was used to determine changes in P‐Rex1 expression in primary HASM cells isolated from asthmatic and non‐asthmatic subjects. P‐Rex1 expression levels in HASM cells were manipulated by overexpression of recombinant P‐Rex1 or its specific siRNA. The proliferation rate of HASM cells was measured by 5‐Bromo‐2‐deoxyUridine (BrdU) assays.

Results

Immunohistochemistry staining revealed that P‐Rex1 was expressed in the epithelial layer of the bronchioles. Positive staining was also observed in HASM cells. However, samples from asthmatic subjects had a 4‐fold higher density of P‐Rex1 protein compared to those from control subjects (p < 0.01). Western blot assay also showed 3‐fold higher expression of P‐Rex1 protein in asthmatic HASM cells as compared to non‐asthmatic HASM cells (p<0.01). Overexpression of P‐Rex1 in non‐asthmatic HASM cells increased platelet‐derived growth factor (PDGF)‐stimulated cell proliferation by more than 2‐fold. Conversely, silencing P‐Rex1 in asthmatic HASM cells reduced PDGF‐stimulated cell proliferation by over 70%. Interestingly, treatment with Rac1 inhibitor NSC23766 not only significantly inhibited PDGF‐stimulated cell proliferation in 4 different asthmatic HASM cells but also largely abolished recombinant P‐Rex1 augment of proliferation in non‐asthmatic HASM cells.

Conclusion

Increase of P‐Rex1 protein promotes HASM cell proliferation via Rac1‐dependent pathways, which in turn causes HASM hyperplasia and remodeling, leading to irreversible airway obstruction.

Support or Funding Information

This work was supported by grants from the National Institutes of Health (5P20GM103489 and R01HL116849) and Nebraska State LB595 research program
Characterization of UDP‐glycosyltransferase 3A (UGT3A) Variants in Tobacco Carcinogen Metabolism
Authors: Vergara Ana G , Watson Christy J , Lazarus Philip
Published: November 2023

The UDP‐glycosyltransferase (UGT) family are phase II enzymes important in the metabolism of endogenous and exogenous substances including steroids and a variety of different carcinogens and drugs. The UGT3A subfamily consists of UGTs 3A1 and 3A2, which may play a role in the local detoxification of tobacco carcinogens. The objective of the present study is to identify UGT3A protein expression in human aerodigestive tract tissues, determine their glycosylation activity against tobacco carcinogens, and assess the potential functional role of UGT3A variants in tobacco‐related cancer susceptibility. As determined by Western blot analysis, UGT3A1 exhibited the highest expression in liver, followed by lung and several aerodigestive tract tissues. UGT3A2 exhibited the highest expression in floor of mouth, followed by trachea and other aerodigestive tract tissues. Of the tissues tested, liver exhibited the least UGT3A2 expression. Microsomes prepared from HEK293 cells overexpressing wild‐type UGT3A1 demonstrated glycosylation activity, as observed by reverse‐phase ultra‐pressure liquid chromatography, against polycyclic aromatic hydrocarbons (PAHs) including 1‐hydroxybenzo(a)pyrene (1‐OH‐B(a)P) and 8‐OH‐B(a)P. Microsomes prepared from HEK293 cells overexpressing wild‐type UGT3A2 exhibited significant glycosylation activity against a variety of PAHs including, 1‐OH‐B(a)P), 3‐OH‐B(a)P, 7‐OH‐B(a)P, 8‐OH‐B(a)P and 9‐OH‐B(a)P; additional PAHs and other tobacco carcinogens are currently being tested for both UGT3A enzymes. Preliminary kinetic analysis demonstrated a low Km, in the range of 130 – 330 μM, against all PAHs tested by UGT3A2, similar to that observed previously for UGT2A1 (Bushey RT, 2011). In addition, seven variants (>1% prevalence) for each UGT3A enzyme have been overexpressed in HEK293 cells and are currently being screened for altered activity against PAHs in vitro. These data suggest that UGT3A enzymes are important for the local detoxification of tobacco carcinogens, specifically PAHs. Inter‐individual differences in UGT3A expression and/or activity may result in variations in the detoxification of tobacco carcinogens and may play a role in tobacco‐related cancer risk.

Support or Funding Information

This study is supported by the National Institute of Environmental Health Sciences (NIEHS) of the National Institutes of Health to P Lazarus (Grant R01‐ES025460) and a NIEHS Research Supplement to Promote Diversity in Health‐Related Research to AG Vergara (Grant R01‐ES025460‐02S1).
Human Fetal Sulfotransferase 1C4: Unique Properties and Novel Substrates
Authors: Guidry Amber L. , Runge‐Morris Melissa , Falany Charles N.
Published: November 2023

Sulfoconjugation of xenobiotics and endogenous compounds by the human cytosolic sulfotransferases (hSULTs) modifies the substrates' functions and increases the compounds' excretion from the body. The hSULT1C subfamily, in contrast with most of the other human SULTs, is primarily expressed during human fetal development; however, the biological role of these fetal isoforms remains uncharacterized. The purpose of this study was to generate pure hSULT1C4 and to characterize the enzyme's structure, activity, and physiological substrates. Based on hSULT1C4's native biochemical properties, a novel purification protocol utilizing affinity chromatography and ion exchange chromatography was established. The small phenolic compound 1‐naphthol was analyzed in combination with the obligate sulfonate donor PAPS (3′‐phosphoadenosine 5′‐phosphosulfate) to optimize hSULT1C4 activity assays. To begin characterizing hSULT1C4, the affinity of the enzyme for PAPS and the reaction product PAP (3′, 5′‐diphosphoadenosine) was determined by measuring the Michaelis constant of PAPS (7.0 ± 1.0 μM) and the dissociation constant of PAP (2.8 ± 0.2 μM) and PAPS (4.8 ± 0.8 μM). Human SULT1C4 has a reduced affinity for PAPS compared to PAP. Previous studies have shown hSULT1C4 sulfonates catechol estrogens; therefore, endogenous and environmental estrogenic compounds were screened as substrates for sulfonation by hSULT1C4. The hSULT1C4 activity assays revealed that all of the screened estrogenic compounds (genistein, daidzein, apigenin, chrysin, 6,4′‐dihydroxyflavone, estradiol, and bisphenol A) are substrates for hSULT1C4. Based on the enzyme's expression in the human fetus and its affinity for sulfonating estrogenic compounds, hSULT1C4's physiological function may be to metabolize various dietary compounds and hormonal signaling molecules during fetal development.

Support or Funding Information

This work was supported by the National Institutes of Health Grant GM038953 and National Institutes of Health Grant ES022606.
Stereochemistry of the Carbonyl Reduction of MSDC‐0602, An mTOT‐modulating Insulin‐sensitizer for the Treatment of Nonalcoholic Steatohepatitis (NASH)
Authors: Adams Wade J , Yousey Thad , Bourdon Kelly , Talay Susan , Colca Jerry R
Published: November 2023

The objective of this study was to determine the stereochemistry of the carbonyl reduction of MSDC‐0602 in human, monkey and rat plasma using a validated LC‐MS/MS analytical method developed for the analysis of the four diastereomers of the major MSDC‐0602 reduction product, MSDC‐0597. The R‐ and S‐diastereomers of MSDC‐0597 have different in vitro binding affinities for mTOT and significantly different abilities to activate PPARγ, which has been implicated as the cause of the side effects of PPARγ insulin‐sensitizers. It is thus important to determine the stereochemistry of the major pharmacologically active metabolite of MSDC‐0602, MSDC‐0597, particularly with respect to its potential to have off target activity to activate PPARγ. A supported liquid extraction (SLE) procedure using either 100 μL or 25 μL (rat) of plasma was used for the analysis with the analytes eluted with methyl tert‐butyl ether (MTBE). The eluate was evaporated and the residue taken up in acetonitrile: water for injection on a Diacel CHIRALPAK® IA column. Analytes were eluted by isocratic chromatography using acetonitrile:methanol:2‐propanol:water, 67:12:18:3, v:v:v:v) at 50°C and the column effluent was monitored by negative ESI MS on SCIEX API 4000 or API 5500 instruments with an Analyst data system. Selected plasma samples were assayed from a human repeated dose study (Day 1 at 0, 4 and 24 hr, and Day 7 at 4 and 24 hr), a monkey repeated dose study (Day 1 at 0, 4 and 24 hr, and Day 182 at 4 and 24 hr), and a rat repeated dose study (Day 1 at 0, 4 and 24 hr, and Day 181 at 4 and 24 hr). No clinically significant gender differences in diastereomer percentages were apparent in any species. Overall mean R‐MSDC‐0597 concentrations in human, monkey and rat were 87.0±9.1%, 93.9±1.8% and 67.3±10.0%, respectively, of total MSDC‐0597. Overall mean R‐MSDC‐0597 percentages at 24 hr post‐dose were modestly lower than at 4 hr post‐dose in rat and human, but not in monkey. Owing to the more than ten‐fold lower in vitro ability of R‐MSDC‐0597 to bind to activate PPARγ than pioglitazone (ACTOS™), and its higher in vitro binding affinity for mTOT than pioglitazone, it is expected that MSDC‐0602 will have a better therapeutic ratio than pioglitazone in the treatment of NASH.
Ontogenic expression of key ADME genes and their roles in imatinib‐induced toxicity in juvenile rats
Authors: Wang Yedong
Published: November 2023

Imatinib is an oral tyrosine kinase inhibitor that is approved for the treatment of pediatric chronic myeloid leukemia. Prepubertal growth impairment has been reported to be a major neurologic adverse effect of long‐term imatinib treatment in children. Numerous studies have demonstrated that cytochrome P450 3A4 and ABCB1 are major ADME genes that play critical roles in biotransformation and brain disposition of imatnib. In the present study, we first evaluated the relationships between ontogentic expression of ADME genes and exposure of imatinib in a neonatal rat model. The expression of Cyp3a1 and Abcb1 of rats from different postpartum days (PPD) were determined at mRNA and protein levels, and results revealed that their expression increased with age. Pharmacokinetic studies after single oral dose of imatinib (30 mg/kg) given to 7‐, 14‐, 21‐, 28‐ or 35‐day‐old rats demonstrated that brain distribution of imatinib decreased with age, with > 20 fold decrease in PPD35 rats compared with PPD7 rats, consistent with mRNA and protein expression levels of key ADME genes. Juvenile rat toxicity studies with oral imatinib were conducted to examine the relationship between Cyp3a1 and Abcb1 gene expression and growth (in particular bone formation) retardation. Preliminary data suggest a distinctive inhibitory effect of imatinib on bone growth in neonatal rats. Mechanistic investigations using osteoblasts and in vivo neonatal rat model are on‐going to delineate regulatory network between imatinib disposition and bone formation.

Support or Funding Information

The current work was supported in part by a grant from Soochow University, Suzhou, China [Grant No. Q413200711], and a grant from the National Science Foundation of China [Grant No. 81473278].
Leading the charge in sprouting angiogenesis: The roles of PKA and PDE4D in regulating tip cell invasion
Authors: MacKeil Jodi
Published: November 2023

During angiogenic sprouting, vascular endothelial cells (VECs) either act as “tip cells” that guide blood vessel formation, or as the follower “stalk cells”, which proliferate to lengthen the newly developing vessel. While cAMP‐signaling regulates numerous VEC functions required for angiogenic sprouting, few studies have attempted to deconstruct the influences of this ubiquitous second messenger on individual functions of tip and/or stalk cells. Thus, this study was undertaken to address whether cAMP‐signaling selectively acted on VEC expressing tip or stalk phenotypes, and to begin to explore how individual components of the cAMP‐signaling system coordinated these events. Specifically, a spheroid model of angiogenic sprouting was used to assess whether knocking down the cAMP effectors expressed in human arterial VECs (HAVECs) (i.e. PKA, Epac1), or the dominant cAMP PDEs expressed by these cells (i.e. PDE4D, PDE3B), impacted tip cell matrix invasion. Using this approach, we show that while knocking down PKA‐Cα expression in HAVECs markedly enhanced tip cell invasion, knocking down EPAC1 had no effect. With respect to the dominance of individual PDE‐family variants in these processes, knocking down PDE4D expression promoted angiogenic sprouting as measured by tip cell invasion, while knocking down PDE3B markedly inhibited these processes. Indeed, tip cell matrix invasion was nearly abolished in PDE3B knockdown spheroids. Our data are consistent with the notion that individual PDE isoenzymes are involved in distinct regulatory pathways, and suggest that PDE3B and PDE4D differentially regulate angiogenic sprouting in response to pro‐angiogenic stimuli, potentially through PKA.

Support or Funding Information

Canadian Institutes of Health Research (CIHR)
Memantine, the dual α7‐nAChR/NMDAR antagonist displays potent anti‐angiogenic activity in lung cancer
Authors: Robateau Zachary R , Brown Kathleen C , Chen Yi C , Egleton Richard D , Dasgupta Piyali
Published: November 2023

Non‐small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) bear a strong etiological association with smoking habits. Nicotine, the addictive component of cigarettes, promotes angiogenesis in lung cancer via the α7‐nicotinic acetylcholine receptor (α7‐nAChR) subunit in human lung endothelial cells. Therefore, we conjectured that α7‐nAChR‐antagonists should display potent anti‐angiogenic and antitumor activity in lung cancers. Memantine is a dual α7‐nAChR/NMDAR antagonist, which is used in the clinic for the treatment of patients suffering from mild‐to‐moderate Alzheimer's disease. Receptor binding assays have shown that the affinity of Memantine for α7‐nAChR is greater than NMDAR. Here we show that memantine attenuates nicotine‐induced angiogenesis in human microvascular endothelial cells of the lung (HMEC‐Ls). Most interestingly, the levels of α7‐nAChR in tumor‐associated endothelial cells was greater than normal lung endothelial cells. The anti‐angiogenetic activity of memantine was mediated by the α7‐nAChR (and not by NMDARs) on lung endothelial cells. Furthermore, the α7‐nAChR antagonist memantine displayed potent anti‐angiogenic activity in the chicken chorioallantoic membrane (CAM) model. Our studies show that α7‐nAChR antagonists may be useful anti‐tumor agents relevant for the treatment of human lung cancer.

Support or Funding Information

Funding for our study was supported by the an NIH R15‐AREA Grant (2R15CA161491‐02), WVU‐MU Health Partnership Grant and by ACTSI Grant from Marshall University
Novel role of Septin2 in Endothelial Cell Podosome Formation and Matrix Degradation
Authors: Collins Kerrie B. , Kang Hojin , Klomp Jennifer , Malik Asrar B , Karginov Andrei V.
Published: November 2023

In the initial phases of angiogenesis endothelial cells must degrade the extracellular matrix before migrating to alternative sites to form nascent vessels. The degradation of the matrix is performed by podosomes, actin rich structures that are regulated by Src family kinases. Here we report that the filamentous GTPase Septin2 is phosphorylated upon activation of Src and plays a critical role in Src‐mediated podosome formation and matrix degradation. We have identified that activation of Src induces phosphorylation of Septin2 and its translocation to the podosome structures in endothelial cells. Super resolution microscopy analysis further revealed that Septin2 localizes to the borders of podosome rosettes on the basal side of the cell. We have found that downregulation of Septin2 expression in endothelial cells inhibits Src‐mediated podosome formation as well as matrix degradation. Additionally, we have shown that Septin2 expression is necessary for endothelial cell invasion into a 3D collagen matrix, indicating a possible role for Septin2 in angiogenesis via regulation of podosome function. We are currently investigating the role of Septin2 in angiogenesis as well as the functional significance of Septin2 phosphorylation secondary to Src activation.
RNA Receptor RIG‐I Stimulation Impairs Endothelial Barrier Function through Inflammasome Activation Independent of NLRP3
Authors: Pitzer Ashley L , Zhang Yang , Bhat Owais , Li Pin‐Lan
Published: November 2023

Retinoic acid‐inducible gene‐I (RIG‐I) as a putative cytosolic RNA sensor plays a critical role in mediating innate immune and inflammatory responses. The role of RIG‐I in vascular regulation under physiological and pathological conditions remains poorly understood. The present study tested a hypothesis that cellular RIG‐I activity regulates vascular endothelial barrier dysfunction by activation of an NLRP3‐indepdendent inflammasome. Using confocal microscopy, stimulation of RIG‐I in mouse endothelial cells (ECs) with specific agonist, 5′‐triphosphate double‐stranded RNA (3pRNA, 0.5 mg/L) was found to induce co‐localization of RIG‐I, but not NLRP3, with inflammasome components ASC (apoptosis speck‐like protein) and pro‐caspase‐1. Inflammasome activation detected via ELISA analyses showed that 3pRNA significantly increased IL‐1β production by 10 folds. Interestingly, this activation of RIG‐I inflammasome decreased expression of tight junction proteins ZO1 and ZO2 as well as adheren protein VE‐cadherin, as shown by immunofluorescence detection and Western blot analysis. To test functional changes, a permeability assay imploring an FITC conjugated dextran showed significantly increased leakage of dextran across of the EC layer during 3pRNA treatment, which was abolished by inhibition of caspase‐1 and HMGB‐1 and by ASC gene silencing. Together, these data implicate involvement of RIG‐I inflammasome activation in the disruption of vascular endothelial barrier function that triggers an early molecular event leading to vascular dysfunction.

Support or Funding Information

Supported by NIH grants HL057244, HL075316 and HL122937
Inhibitory effect and mechanisms of paroxetine, a selective serotonin reuptake blocker, on 5‐HT‐induced cell proliferation and migration of vascular smooth muscle cells
Authors: Lin Yi‐Chen
Published: November 2023

Restenosis (or neointimal hyperplasia) was characterized as abnormal proliferation and migration of vascular smooth muscle cells (VSMCs). Serotonin (5‐hydroxytryptamine; 5‐HT), a mitogen for VSMCs, may contribute to the progression of balloon angioplasty‐induced neointima formation. Paroxetine, a selective serotonin reuptake inhibitor (SSRI), has been used to treat depression, obsessive‐compulsive disorder, and anxiety disorders. The purpose of this study was to understand the potential pathological roles of serotonin and paroxetine in the development of neointimal hyperplasia. Cell viability and migration were examined by MTT and wound healing assays, respectively. The protein and mRNA expressions were monitored by Western blot and RT‐PCR, respectively. Experimental data showed that paroxetine can significantly reduce 5‐HT stimulated VSMC growth and migration. Our study suggested that selective serotonin reuptake inhibitors, such as paroxetine, might be a potential remedy for restenosis prevention.
Evidence that NaHS inhibits the tachycardic responses induced by stimulation of the preganglionic sympathetic outflow in pithed rats
Authors: Centurion David , Castillo‐Santiago Shirley V , Becerril‐Chacón Maria E , Torres‐Perez Jose A , de la Cruz Saul Huerta , Sanchez‐Lopez Araceli
Published: November 2023

In anesthetized rats, i.v. administration of NaHS, a donor of H2S, elicited dose‐dependent bradycardia, although the mechanisms are unknown. In this regard, we hypothesized that the inhibition of the cardioaccelerator sympathetic outflow may be partially involved. Thus, this study was designed to determine the potential capability of NaHS to mediate inhibition of the tachycardic responses induced by preganglionic sympathetic stimulation (C7‐T1). For this purpose, Wistar rats were anesthetized, pithed and cannulated for drug administration. In animals pre‐treated with gallamine, the effect of i.v. infusion of NaHS (310 and 560 μg/kg min) or its vehicle (phosphate buffer saline; 0.02 ml/min) was determined on the tachycardic responses induced by: (1) sympathetic stimulation (0.1–3.2 Hz); or (2) i.v. bolus injections of exogenous noradrenaline (0.03–3 μg/kg). The tachycardic responses induced by preganglionic sympathetic stimulation were dose‐dependently inhibited by i.v. infusion of NaHS (310 and 560 μg/kg min), but not by vehicle, particularly at high frequencies. In marked contrast, the tachycardic responses to exogenous noradrenaline were not inhibited by the above doses of NaHS or its vehicle. These results, taken together, demonstrate that NaHS inhibited the tachycardic responses induced by preganglionic sympathetic outflow by a prejunctional mechanism. This is the first evidence demonstrating this effect by NaHS that may contribute, at least in part, to the bradycardia induced by NaHS.

Support or Funding Information

The authors acknowledge to Conacyt (Grant No. 252702) for their financial support.
Sirt1 plays a pivotal role in TLR2‐mediated Mac‐1 expression in monocytes
Authors: Lee Seung‐Jin
Published: November 2023

Sirtuin (Sirt) 1 has been demonstrated to exerts protective effects in the progression of atherosclerosis, however, the underlying mechanisms are unclear. Thus, this study investigated the the role of Sirt1 in TLR2‐mediated monocyte adhesion to vascular endothelium, which is a key early event in macrophage foam cell formation. The atherosclerotic lesion formation in aortic sinus was significantly increased in Pam3CSK4‐treated wild type (WT) mice, which was significantly inhibited in Sirt1 Tg mice group. An en face immunohistochemistry of endothelial surfaces revealed a marked increase in monocyte adhesion to aortic endothelium in WT mice treated with Pam3CSK4, which was significantly attenuated in Sirt1 Tg mice. Likewise, the adhesion capacity of primary monocytes isolated from Pam3CSK4‐treated WT mice was higher than those of monocytes from Sirt1 Tg mice. In in vitro study, Pam3CSK4 increased monocyte adhesion to endothelial cells with an enhanced Mac‐1 expression. These were attenuated by a recombinant Sirt1, as well as by molecular overexpression of Sirt1 in monocytes. In addition, we found that Pam3CSK4‐reduced Sirt1 expression in monocytes was regulated by the transcription factors, NF‐kB and CREB. The role of NF‐kB and CREB in Pam3CSK4‐reduced Sirt expression was confirmed by siRNA knockdown of Sp1 and NF‐kB. Sirt1 supresses atherosclerosis progression by TLR2‐mediated monocyte adhesion to endothelium through Mac‐1 expression, which is mediated by NF‐kB and CREB pathways in monocytes, consequently leading to the development of therapeutic interventions for regulating Sirt1 expression in atherosclerosis.
Amelioration by GABAB receptors of the cyclosporine‐evoked counteraction of endotoxic cardiovascular manifestations in conscious rats
Authors: Sallam Marwa Y , El‐Gowilly Sahar M , Abdel‐Galil Abdel‐Galil A , El‐Mas Mahmoud M
Published: November 2023

We have previously shown that cyclosporine (CSA) counteracts the GABA receptor‐sensitive endotoxic cardiovascular manifestations such as hypotension and cardiac autonomic dysfunction. In this study we investigated whether the facilitation of central GABAergic neurotransmission blunts these favorable influences of CSA. The effects of CSA on LPS hemodynamics were determined in the absence and presence of pharmacologic maneuvers that activate GABA receptors or elevate synaptic GABA levels in conscious rats. Intravenous administration of lipopolysaccharide (LPS, 10 mg/kg) decreased blood pressure (BP), increased heart rate, and impaired cardiac autonomic activity as suggested by the reductions in time‐ and frequency domain indices of HRV (measures of cardiac autonomic control). The consequent i.v. administration of CSA (10 mg/kg) blunted the LPS‐evoked hypotension and tachycardia and associated reductions in HRV indices. The ability of CSA to reverse the LPS effects disappeared in rats treated intracisternally (i.c.) with baclofen (selective GABAB agonist, 2 mg/rat) but not muscimol (selective GABAA agonist, 1 mg/rat), indicating a preferential compromising action for central GABAB receptors on the CSA/LPS interaction. Moreover, the improvement by CSA of LPS‐evoked cardiovascular derangements was eliminated after concurrent i.c. administration of vigabatrin (GABA transaminase inhibitor, 200 mg/rat) or tiagabine (GABA reuptake inhibitor, 100 mg/rat). Overall, these results demonstrate that the activation of central GABAB receptors either directly via baclofen or indirectly following interventions that boost GABA concentrations in central synapses counterbalances the advantageous effects of CSA on endotoxemia.

Support or Funding Information

Supported by the Science and Technology Development Fund, Egypt (STDF Grant No. 14895).
Peak Total‐[Hemoglobin + Myoglobin] During Incremental Dynamic Handgrip Exercise and Post Occlusive Hyperemia
Authors: Didier Kaylin D , Ade Carl J , Hammer Shane M , Barstow Thomas J
Published: November 2023

Introduction

Near infrared spectroscopy (NIRS) has been used previously to measure changes in the total hemoglobin + myoglobin (Total‐[Hb+Mb]) signal, thought to reflect changes in microvascular hematocrit (HCT). During rest the hematocrit of the microvasculature is lower compared to the systemic hematocrit. However, during exercise microvascular hematocrit increases towards that seen in the systemic circulation. This increase in hematocrit provides an increase in the surface area available for O2 exchange. Post‐occlusive reactive hyperemia (PORH) tests have also utilized changes in NIRS signals, including Total‐[Hb+Mb], to evaluate function/dysfunction of the circulatory system. However, it is currently unclear how the peak Total‐[Hb+Mb] observed transiently following cuff release during PORH might relate to the peak Total‐[Hb+Mb] seen at task failure during incremental exercise (active hyperemia). These results could provide insight into the underlying mechanisms which control microvascular HCT.

Purpose

The purpose of this investigation was to determine how the peak Total‐[Hb+Mb] achieved during incremental dynamic handgrip exercise compared to that seen during PORH.

Methods

Eleven men completed two testing protocols; 1) Incremental dynamic handgrip exercise to failure and 2) PORH. Exercise and PORH were performed in a supine position, with the right arm abducted to ~80° at heart level. During incremental exercise the work rate progressively increased 0.5 Watts every 30 sec until the subject could not maintain the correct contraction rate for at least 3 consecutive contractions despite verbal encouragement. The PORH test included: 1 min of baseline, 5 min occlusion of the brachial artery, and 3 min recovery after cuff release. Near‐infrared spectroscopy (NIRS) measurements were made continuously during both tests, from which the Total‐[Hb+Mb] signal was derived.

Results

There was no difference in the mean values for peak Total‐[Hb+Mb] during the incremental test (93.0 ± 20.3 μM) and that observed during the PORH test (93.1 ± 17.7 μM) (p = 0.992). Further, peak Total‐[Hb+Mb] during PORH was significantly correlated to peak Total‐[Hb+Mb] during incremental handgrip exercise (R2 = 0.657, p = 0.002). The slope of the regression line for Peak PORH vs. Incremental was 0.930.

Conclusion

These data suggest that in a given subject, similar peak values of microvascular Total‐[Hb+Mb], and thus microvascular HCT, are achieved during active and reactive hyperemia.
Microvascular Perfusion Heterogeneity Impairs Oxygenation and Contributes to Peripheral Vascular Disease in Metabolic Syndrome
Authors: McClatchey P. Mason , Woo Fan , Olfert I. Mark , Eliis Christopher , Goldman Daniel , Reusch Jane , Frisbee Jefferson
Published: November 2023

Development of the metabolic syndrome in obese Zucker rats (OZR) is associated with impaired fatigue‐resistance of in situ skeletal muscle paralleling a blunted functional hyperemia. However, recent studies suggest that reduced bulk oxygen delivery to skeletal muscle with elevated metabolic demand may not explain the compromised muscle performance. Using novel experimental data and recent insight into altered microvascular perfusion in OZR, we developed a simulation for tissue oxygenation with increasing metabolic demand in lean (LZR) and OZR skeletal muscle using physiologically‐realistic data and relationships. With elevated metabolic demand, blood flow to, and oxygen uptake by, in situ skeletal muscle increased in both strains, although the response was blunted in OZR. However, venous blood oxygen tension (PvO2) draining the muscle of OZR was elevated versus LZR across metabolic demands; a paradoxical response given assumptions of increased microvascular residency time in OZR. Using a microvascular network model of multiple Krogh cylinders supplied by a network with homogeneous flow distribution at bifurcations (γ=0.5), we were unable to simulate tissue oxygenation and PvO2 differences between LZR and OZR. However, with introduction of increasing perfusion asymmetry (γ>0.5), changes to microvascular hematocrit and increased plasma skimming throughout the network, our ability to simulate the experimental results was much improved. As a result, our data suggest that increased perfusion asymmetry (γ) within the muscle microcirculation is not only a defining characteristic of metabolic syndrome, it is required to effectively model and understand alterations in blood‐tissue oxygen exchange in this highly translationally relevant model of human disease risk.

Support or Funding Information

National Institutes of Health and American Heart Association
Beneficial Pleiotropic Anti‐Depressive Effects of Cardiovascular Disease Risk Factor Interventions in Metabolic Syndrome
Authors: Frisbee Jefferson , Stranges Saverio , Duncan Audra , Frisbee Stephanie
Published: November 2023

While the increased prevalence and severity of clinical depression and elevated cardiovascular disease risk represent two vexing public health issues, the growing awareness of their combined presentation presents a special challenge for not only integrated health outcomes, but also for effective treatment. The obese Zucker rat (OZR), a model of the metabolic syndrome with significant elevations in CVD risk spontaneously develops significant depressive symptoms in parallel with the progression of the metabolic syndrome; thus representing a particularly compelling model for study. To begin to interrogate relationships between CVD risk factors and depressive symptom severity, we chronically treated OZR with traditional clinically relevant interventions against CVD risk to determine impacts on not only vascular function but also on behavioral outcomes and depressive symptom severity. While most of the interventions (chronic exercise, anti‐hypertensive, anti‐dyslipidemia, anti‐diabetic) were differentially effective at improving vascular function, only those interventions that also resulted in a significant improvement to oxidant stress, inflammation and arachidonic acid metabolism (PGI2 vs. TxA2) and their associated sequelae were effective at also blunting depressive symptom severity. Using multivariable analyses, discrimination between the effectiveness of treatment groups to maintain behavioral outcomes was dependent upon maintaining arachidonic acid metabolism and vascular oxidant stress together, with preventing changes in vascular stiffness, mean arterial pressure and plasma cortisol concentration (together) also being important. This initial study provides for a very compelling framework from which to further interrogate the links between CVD risk and cognitive/behavioral status, and begins to provide insight into mechanistic links and potentially effective avenues for intervention.

Support or Funding Information

National Institutes of Health, American Heart Association
A tri‐phasic role for hydrogen peroxide in blood‐brain barrier endothelial cells
Authors: Shaji Chinchusha Anasooya , Robinson Bobby , Yeager Antonia , Beeram Madhava , Isbell Claire L. , Tharakan Binu
Published: November 2023

In microvascular endothelial cells, hydrogen peroxide (H2O2) plays an important role as a signaling second messenger as well as an inducer of oxidative stress in various pathologies such as ischemic and traumatic injuries. However, it is unclear how H2O2 influences various cellular functions differentially, particularly in microvascular endothelial cells of the blood‐brain barrier (BBB). We hypothesized that the change in cellular concentrations of H2O2 is a major contributor in regulation of angiogenesis, endothelial cell permeability and cell death/apoptosis. Further, we postulate that these differential effects are dependent upon the changes in the integrity of BBB tight junctions and alterations in cytoskeletal organization. Rat brain microvascular endothelial cell (RBMEC) grown on Transwell inserts in a monolayer were exposed to various concentrations of H2O2 (0.001μM to 100mM). Monolayer permeability was studied based on the passage of FITC‐dextran added in the upper chamber of the monolayer plate followed by its fluoromteric measurement form the lower chamber. Angiogenesis was studied using cells grown in Matrigel in 96 well plates that were exposed to H2O2 followed by microscopy. The changes in cell viability and apoptosis were studied using Calcein AM fluorometeric assay and caspase‐3/7 Green visualization assay, respectively. The changes in tight junction integrity and actin cytoskeletal assembly were studied using zonula occludens‐1 (ZO‐1) immunofluorescence and rhodamine phalloidin staining for f‐actin, respectively. The cellular levels of H2O2 were visualized using a fluorescence visualization assay. Exposure of RBMEC with increasing concentrations of H2O2 followed by its visualization confirmed the concentration‐dependent rise in intracellular levels of H2O2. The angiogenesis assay demonstrated that H2O2 at low concentrations (0.01μM and 1μM) increased tube formation, an indicator of angiogenesis, whereas the high concentrations showed no obvious effects. Monolayer permeability assay demonstrated that H2O2 at 100μm and above induced hyperpermeability compared to all other lower concentrations tested (p<0.05). H2O2 at 10mM and above induced loss of cell viability (p<0.05) and induced apoptosis. The lower concentrations where ineffective in inducing cell death/apoptosis. Immunofluorescence localization of ZO‐1 demonstrated its localization at the tight junctions but showed discontinuous distribution starting at 100μm. Rhodamine phalloidin labelling of f‐actin demonstrated cytoskeletal alterations starting at 100μm. These results demonstrate that H2O2 influences angiogenesis, permeability and cell death/apoptosis differentially and in a concentration‐dependent manner. This suggests a triphasic role for H2O2 in regulating multiple cellular functions in microvascular endothelial cells of the blood‐brain barrier.
Inflammation‐Induced Syndecan Shedding Contributes to Endothelial Barrier Dysfunction
Authors: Jannaway Melanie , Yang Xiaoyuan , Meegan Jamie E , Yuan Sarah Y
Published: November 2023

The endothelial glycocalyx (GCX) is a complex matrix of glycosaminoglycans and proteoglycans such as syndecans (SDCs), which form a protective layer on the vascular luminal surface contributing to the endothelial barrier function. Ectodomain shedding of transmembrane proteoglycans or degradation of GCX components can damage barrier integrity. The objective of this study was to investigate the involvement of a disintegrin and metalloproteinase 15 (ADAM15) in GCX shedding, and the effects of SDC fragments on endothelial barrier function. A confocal microscopic imaging analysis of mouse lungs demonstrated reduced SDC expression on microvascular endothelial surface following lipopolysaccharide (LPS) treatment, an effect blunted in ADAM15−/− mice, implicating ADAM15 in GCX injury. ELISA analysis of human lung perfusate collected following LPS perfusion showed increased soluble SDC concentration, indicating LPS‐induced SDC shedding. Furthermore, when analyzed via transmission electron microscopy, microvessels within these LPS‐perfused samples displayed evidence of GCX injury. Together, these results suggest that an inflammatory state can lead to the shedding of endothelial SDCs, likely through the activities of proteases and sheddases including ADAM15.
Ojeoksan suppressed TNF‐α‐induced vascular inflammation in human umbilical vein endothelial cells
Authors: Han Byung Hyuk , Kim Hye Yoom , Ahn You Mee , Jin Xian Jun , Jeong Da Hye , Lee Yun Jung , Lee Ho Sub , Kang Dae Gill
Published: November 2023

Endothelial dysfunction is regarded as an important role in vascular inflammation. Ojeoksan (OJS), originally recorded in an ancient Korean medicinal book named “Donguibogam”(), is a well‐known blended traditional herbal formula. OJS is consist of seventeen natural herbal plants. This study was carried out to investigate the effect of OJS on tumor necrosis factor‐alpha (TNF‐α)–induced vascular inflammation in human umbilical vein endothelial cells (HUVECs).

Pretreatment of OJS suppressed TNF‐α‐induced expression of cell adhesion molecules, such as intracellular adhesion molecule‐1, vascular cell adhesion molecule‐1, and endothelial selectin in TNF‐α‐stimulated HUVECs. OJS also decreased the adhesion of HL‐60 cells to HUVECs. Moreover, OJS inhibited TNF‐α‐induced matrix metalloproteinase‐2/‐9 (MMP‐2/‐9) activity; significantly decreased TNF‐α‐induced production of intracellular reactive oxygen species (ROS); and inhibited the phosphorylation of IκB‐α in the cytoplasm. Pretreatment with OJS also inhibited the translocation of NF‐κB p65 to the nucleus. OJS increased the phosphorylation of endothelial nitric oxide synthase (eNOS) and Akt (protein kinase B) which are related with NO production. In addition, OJS increased the protein expression of Nrf2 and HO‐1 in TNF‐α‐stimulated HUVECs, suggesting its protective effect against vascular inflammation.

In conclusion, these results of this study suggested that OJS, the traditional herbal formula, has a protective effect on vascular inflammation. Therefore, OJS might be a potential therapeutic agent for early atherosclerosis.

Support or Funding Information

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (2008‐0062484).