bluuebunny/crossref_metadata_2025 huggingface dataset:
JTE‐013, an antagonist of sphingosine 1‐phosphate receptor 2, inhibits ovalbumin‐induced allergic asthma in Balb/c mice
Authors: Park Soojin , Kang Saeromi , Im Dongsoon
Published: November 2023

Background

Activation of sphingosine kinases 1 and 2 and secretion of sphingosine 1‐phosphate (S1P) have been implication in allergic responses, asthma and anaphylaxis. Type 1 and 2 receptors for S1P have been suggested as targets of secreted S1P functions in mast cells. However, functions of S1P receptors in vivo has not been investigated in an allergic asthma model.

METHODS

Antigen‐induced degranulation was measured in RBL‐2H3 mast cells by measuring b‐hexosamidase activity. An ovalbumin‐induced allergic asthma model was used for in vivo efficacy test of JTE‐013, a selective antagonist of type 2 S1P receptor.

RESULTS

JTE‐013 inhibited the antigen‐induced degranulation of beta‐hexosaminidase in RBL‐2H3 mast cells in a concentration‐dependent manner in vitro. Sensitization and challenge of ovalbumin induced allergic asthma responses, that is, the accumulations of eosinophils and increase of Th2 cytokine levels in bronchoalveolar lavage fluid and lung inflammation. Administration of JTE‐013 before ovalbumin sensitization or before ovalbumin challenge strongly inhibited the accumulation of eosinophils and lymphocytes to bronchoalveolar lavage fluid. JTE‐013 administration also inhibited expression and secretion of Th2 cytokines, that is, IL‐4 and IL‐13. Histologic studies showed that JTE‐013 reduced inflammatory signs in the lung.

CONCLUSION

JTE‐013 has anti‐allergic effects and may be utilized as a potent agent for the treatment of asthma.
Anti‐inflammatory effects of serotonin 5‐HT2A receptor activation in ovalbumin‐induced allergic asthma models
Authors: Flanagan Thomas Wayne , Sebastian Melaine Nicole , Nichols Charles David
Published: November 2023

Only recently has the full therapeutic value of serotonin [5‐hydroxytryptophan (5‐HT)] receptor activation begun to be explored. Currently there are two 5‐HT2A receptor agonists in human clinical trials for the treatment of depression and obesity. An exciting new therapeutic avenue in which 5‐HT2A agonists might be employed is the modulation of inflammation in allergic airways disease. Our lab has previously used an ovalbumin (OVA)‐induced asthma model to demonstrate that administration of (R)‐2,5‐dimethoxy‐4‐iodoamphetamine [(R)‐DOI] prior to allergen challenge prevents many of the symptoms of allergic asthma. Here we have utilized a modified protocol to test the effectiveness of (R)‐DOI in treating persistent allergic asthma. We demonstrate that administration of (R)‐DOI in a chronic model attenuates the elevated airway hyperresponsiveness (AHR) typically observed in an asthmatic response. We also have probed for the expression of inflammatory markers in the lung and BALF. We concurrently are testing for the impact psilocybin and other tryptamines have on AHR in rodents using our OVA model. Overall our strategy is to develop 5‐HT2A receptor agonism as a viable treatment modality against asthma and other inflammatory disorders.

Support or Funding Information

American Asthma Foundation Scholar Grant Award
The ‘Prophylactic’ and ‘Therapeutic’ Effects of β2‐Adrenoceptor Blockers in a House Dust Mite (HDM) Driven Murine Asthma Model
Authors: Joshi Radhika , Valdez Daniel , Kim Hosu , Knoll Brian J , Bond Richard A
Published: November 2023

Asthma is characterized by chronic airway inflammation, excess mucus production and airway hyper responsiveness (AHR). Our previous research suggested that some β2‐adrenoceptor (β2AR) blockers might be beneficial in chronic asthma therapy. Using an ovalbumin (Ova) allergen‐driven murine asthma model, we have previously shown that only certain β2AR blockers such as nadolol and ICI‐118, 551 attenuate the development of an asthma phenotype when administered prior to the antigen challenge (‘prophylactic’ effect); while others including carvedilol and propranolol are ineffective. Recently however, the Ova model has been criticized for its lack of clinical relevance. To address the limitations of this model, we tested the β2AR blockers, nadolol and carvedilol in a clinically more relevant model utilizing house dust mite (HDM) extract. Additionally, we investigated both the ‘prophylactic’ and ‘therapeutic’ (the drugs were administered after the development of asthma phenotype) potential of these β2AR blockers in the HDM model of asthma.

Male Balb/c mice were challenged with 25 μg HDM protein by intranasal delivery in two protocols designed to test the ‘prophylactic’ and ‘therapeutic’ effects of nadolol and carvedilol. In both protocols, chronic nadolol treatment significantly lowered the development of airway inflammation and mucus production in response to HDM, while chronic carvedilol treatment was ineffective. In addition, nadolol significantly attenuated the increased AHR response in the ‘prophylactic’ model but only moderately reduced the airway resistance in the ‘therapeutic’ model. Thus, the present studies support our previous findings in the ovalbumin model of asthma and provide further evidence to support nadolol's potential in asthma therapy.

Support or Funding Information

Supported by R01AI110007
Determination of the P450‐ and UGT‐dependent Biotransformation of the Anti‐HIV Drugs Cabotegravir and Dolutegravir
Authors: Madeen Erin , Bumpus Namandjé
Published: November 2023

Cabotegravir (CAB) and Dolutegravir (DTG) are FDA approved viral integrase strand transfer inhibitors in oral formulation as antiretroviral therapeutics, while CAB is additionally in clinical trials as pre‐exposure prophylaxis (PrEP) for the prevention of HIV transmission. The molecular structure of CAB and DTG are remarkably similar, only differing in the terminal ring, a pentane ring on CAB and a hexane ring on DTG. The impact of structure on the metabolism of CAB and DTG were investigated. In order to investigate CAB and DTG metabolite formation human liver microsomes (HLM), c‐DNA expressed cytochrome P450 isozymes (P450s: 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 3A4, and 3A5), or c‐DNA expressed UDP‐glucuronosyltransferase (UGT) isozymes (UGTs 1A1, 1A3, 1A4, 1A6, 1A9, 2B4, 2B7, 2B15, 2B17) were employed. Separation, detection and identification were performed using UPLC‐MS/MS. HLM incubation resulted in the formation of two mono‐oxygenated metabolites of each CAB and DTG. The P450s that produced the greatest abundance of the CAB mono‐oxygenated metabolite M1 were P450s 2D6, 3A4, and 3A5, while DTG mono‐oxygenated metabolite M1 was produced by P450s 2B6, 2D6, 3A4, and 3A5. The P450 that produced the greatest abundance of mono‐oxygenated CAB metabolite M2 was CYP3A4 while DTG M2 was produced by P450s 1A2 and 3A4. Both CAB and DTG were directly glucuronidated in HLM incubations. The primary UGTs responsible for CAB direct conjugation were found to be UGT1A1, 1A3, and 1A9. In addition, a mono‐oxy‐CAB‐glucuronide conjugate was detected and formed by UGT2B7, while no glucuronide conjugate species of mono‐oxy‐DTG were detected. These data suggest that, despite the similarity of the molecular structure of these compounds, there are differing routes of P450 and UGT mediated metabolism.

Support or Funding Information

R01 GM103853
Development of novel substrate cocktail to applicate in vivo drug‐drug interaction study
Authors: Kim Sunjoo , Jo Jung Je , Na Ann‐Yae , Lee Sangkyu
Published: November 2023

Cocktail approach is very useful method for investigating drug‐drug interaction that can reduce a number of sacrificed animals and rapidly identify the interacted cytochrome P450 (CYP) isoforms. In this study, we aimed to assess whether drug interactions have influence on metabolism of 5 CYP enzymes using five probe drugs; caffeine for CYP1A, bupropion for CYP2B, omeprazole for CYP2C, dextromethorphan for CYP2D and midazolam for CYP3A, respectively. The cocktail, consist of caffeine (2 mg/kg), bupropion (20 mg/kg), omeprazole (2 mg/kg), dextromethorphan (10 mg/kg) and midazolam (2 mg/kg) is administered orally to ICR mice. Heparinized blood samples are collected and the plasma concentrations of the probe cocktail drugs and their metabolites are simultaneously quantified by LCMS/MS. In conclusion, the concentration of cocktail substrates and method of LC/MS/MS had been optimized in mice. Now we can use this study to investigate CYP interaction when herbs or other drugs are co‐administered with cocktails.

Support or Funding Information

BK21 Plus KNU Multi‐Omics based Creative Drug Research Team
A multidimensional FPLC approach for the purification of recombinant non‐coding RNAs
Authors: Duan Zhijian , Ho Pui Yan , Jilek Joeseph , Batra Neelu , White Ralph DeVere , Wun Theodore , Lara Primo , Yu Aiming
Published: November 2023

Translational and clinical research on RNA therapeutics for the treatment of cancer diseases requires the use of high‐purity RNA agents. Here we present a new, single‐run, multidimensional fast performance liquid chromatography (MD‐FPLC) approach for the purification of non‐coding RNAs that are bioengineered in bacteria using a novel tRNA/pre‐miRNA carrier we have invented recently. Total RNAs are separated on a combination of columns including size exclusion, weak anion exchange and strong anion exchange in a single run using a multidimensional NGC Chromatography System. The MD‐FPLC workflow is fast and highly reproducible. This MD‐FPLC platform is reliable, allowing a rapid and consistent production of multi‐milligrams of biological RNAs in a single run and offering an overall yield of 10–15% (target RNA/total RNAs). The purified ncRNAs are highly homogenous (> 97%, determined by a HPLC assay) and have minimal endotoxin activities (< 20 EU/mg RNA). In addition, the resultant biologic RNA products (e.g., let‐7c, miR‐34a and miR‐124a) are active in the regulation of target gene expression (e.g., BCL‐2/BCL‐xL, CDK6 and STAT3, respectively) and suppression of human cancer cell proliferation. Therefore, this MD‐FPLC approach offers a robust platform for the production of high‐purity biologic RNAs for research and development.

Support or Funding Information

This study was supported by grants R01GM133888, U01CA175315, and P30CA093373 from NIH.
Mechanisms of Jzu4Me, a novel inhibitor of vascular endothelial growth factor induced angiogenesis and tumor angiogenesis
Authors: Huang Shiu‐Wen , Huang Tur‐Fu
Published: November 2023

Background

Angiogenesis is the formation of new blood vessels. This process involves the migration, growth, and differentiation of endothelial cells. It plays a critical role in the growth and spread of cancer, due to the new vessels supply of oxygen and nutrients for tumors to grow, allowing the cancer cells to invade nearby tissue, leading to tumor metastases. Therefore, novel effective therapeutic agents of anti‐angiogenesis are greatly needed. Further studies established that vascular endothelial growth factor (VEGF)‐A is an attractive anti‐angiogenesis target for therapeutic interventions in cancer.

Material and methods

In an effort to develop inhibitors to block VEGF signaling and angiogenesis, benzimidazole derivatives were designed and synthesized, and their angiogenesis inhibitory effects both in vitro and in vivo were evaluated.

Results

Among these benzimidazole derivatives, Jzu4Me significantly inhibited VEGF‐A‐induced endothelial cells proliferation, migration, invasion and tubular formation, and it also suppressed ex vivo microvessels formation of rat aorta ring induced by VEGF‐A. Moreover, in Matrigel plug implantation mice, Jzu4Me dose‐dependently suppressed the VEGF‐A or tumor‐induced neovascularization in vivo. We also found that Jzu4Me may suppress VEGF‐A signaling in modulating vascular endothelial cell remodeling.

Conclusions

Our findings reveal that Jzu4Me inhibits VEGF‐induced angiogenesis via downregulation of VEGFR2‐mediated signaling, suggesting that Jzu4Me has high potential to be developed into a novel therapeutic agent for treating angiogenesis.
A Dock‐Derived PEDF Mimic Targeting Laminin Receptor Recapitulates PEDF Signaling
Authors: Umbaugh Charles Samuel , Diaz‐Quinones Adriana , Figueiredo Marxa
Published: November 2023

Pigment epithelium derived factor (PEDF) is regarded as an exceptionally potent inhibitor of angiogenesis. Our lab and others have used PEDF as a novel prostate cancer therapy, as PEDF inhibits tumor establishment, vascularization, and alters malignant signaling. PEDF has been identified as a binding partner for the cancer associated protein Laminin Receptor (LAMR1). LAMR1 overexpression is common in a variety of cancers including breast, colon, and prostate. Recently, we attempted to mimic the protein‐protein interaction between PEDF and LAMR1 using a structure based drug design approach. From our 14,000 compound screen, we identified a hit compound with anti‐proliferative capacity against several prostate cancer cell lines.

We tested this hit compound in a medium‐throughput wound healing assay using the program tscratch and explored mechanisms of action at the molecular level. Cell pellets from treated and untreated prostate cancer cells were submitted for proteomic analysis using an Orbitrap Fusion and a label free quantitative approach coupled with MaxQuant and Perseus analysis. Significantly changed proteins were analyzed at the pathway level using Ingenuity Pathway Analysis.

High doses of our hit compound were able to markedly reduce wound‐healing in a scratch based assay. This compound also elicits proteome wide changes to metabolic, proliferation, and genome maintenance systems. Most notably, a 10 uM dose of hit compound was able to active P53 associated pathways while inactivating MYC associated pathways. We will orthogonally validate our results using western blotting and confirm biochemical binding of our lead compound to LAMR1 in a scalable binding assay.

Support or Funding Information

CTSI Core Grant

Purdue Cancer Research Center

Department of Basic Medical Sciences
Combating Colon Cancer through Modulation of the Tumor Microenvironment and Gut Microbiota
Authors: Ko Joshua K
Published: November 2023

Orthodox anticancer strategies have been focused on targeting the tumor itself, which could result in unsatisfactory treatment outcome and drug resistance. Some cytotoxic tumoricidal agents may even cause major systemic toxicities. Our earlier research had revealed the synergistic anticancer effect of novel adjuvant agent with first‐line chemotherapeutic drug in colon cancer therapy (Carcinogenesis 28:1347–55, 2007). We determined that such action is due to alleviation of the cytotoxic drug's hematologic toxicity through immunomodulation (Am J Chin Med 44:579–93, 2016). In order to effectively control cancer growth and progression, we need to consider carcinogenesis as a process involving complex heterotypic multi‐cellular interactions within a tumor microenvironment, comprising tumor‐associated macrophages (TAM), cancer‐associated fibroblasts (CAF), leukocytes, bone marrow‐derived cells, vascular endothelial cells and mesenchymal stem cells. Investigation of drug actions in such tumor microenvironment could enable us to identify the way to cut off the connection between cancer cells and its microenvironment, providing a target‐specific treatment that is free from major side effects in the host. Proliferation and migration of human colon cancer cells were markedly increased when they were cultured in activated macrophage‐conditioned medium (CM). We demonstrate that novel chemotherapeutic agents attenuate the polarization and activation of TAM and CAF and inhibit colon cancer progression to be promoted by TAM and CAF. These include inhibition of growth inhibition and apoptosis, prevention of cell invasion and migration of colon cancer cells that used to be provoked when co‐cultured with TAM or CAF. Besides, the overexpressed proangiogenic and metastatic factors (such as MMPs and VEGF) and altered signaling pathways in activated TAM and CAF as well as in colon cancer cells previously interacting with CM would be downregulated and rectified after drug modulation. Other than the tumor microenvironment, increasing evidences have shown that the gut microbiota is also important in the development of intestinal tumorigenesis. Their genotoxic metabolites could bind to specific intestinal cell surface receptors and consequently disturb intracellular signal transduction. Understanding the association between gut microbiota and the development of colon cancer will be crucial to rectify the altered molecular pathways during the carcinogenic process. We have explored the the restorative potential of novel chemotherapeutic agent on gut bacteria homeostasis in nude mice implanted with colon tumor, with maintenance of the commensal bacterial colonies such as lactobacillus. Such activity could bring along with antitumor effect of the drug, which may involve modulation of the tumor microenvironment during the course. These findings suggest that mediation of the tumor microenvironment and gut microbiota could serve as a potential therapeutic approach in the treatment of colon cancer.
The role of microtubule‐associated protein 3 in angiogenesis
Authors: Yang Xiaoyan , Sun Ying , Saqib Uzma , Banerjee Avik , Geyer Melissa , Gaponenko Vadim , Rosenblatt Mark , Malik Asrar , Komarova Yulia
Published: November 2023

Pathological angiogenesis contributes to the progression of various diseases including cancer, benign and malignant angiogenic tumors, and retinopathies. We previously demonstrated that microtubule‐associated end‐binding protein 3 (EB3), a core component of the microtubule plus end protein network, binds to inositol 1,4,5‐trisphosphate receptors (IP3Rs), promotes assembly of IP3‐induced IP3R clusters, and organizes calcium signaling in endothelial cells. Based on analysis of the EB3 ‐ IP3R binding interface and computational alanine scanning mutagens, we have developed an allosteric inhibitory peptide (EBIN) that interacts with EB3 and inhibits its function. Furthermore, our in vitro experiments have demonstrated that EBIN attenuates agonist‐induced calcium and nitric oxide signaling in endothelial cells. Consistent with the known role of calcium signaling in VEGF‐induced cell migration and proliferation, treatment of animals with EBIN prevented capillary leakage induced by intradermal injection of hVEGF‐A as well as inhibited the ingrowth of new vessels into matrigel plugs implanted under the skin. Since choroidal neovascularization (CNV) is a major cause of vision loss in the elderly population, we next tested the pharmacological effect of EBIN using a laser‐induced CNV model in mice. We found that EBIN treatment (via intravitreal injection or eye drops) significantly reduced choroidal leakage and neovascularization. Our studies suggest that EBIN is a promising anti‐angiogenesis agent that may represent a novel strategy for treating pathological angiogenesis.
Haptoglobin prevents atherosclerosis by inflammation regulation: A prevention role of haptoglobin Hp for atherosclerosis
Authors: Cheng Tsai‐Mu , Chu Hsueh‐Liang , Pan Ju‐Pin
Published: November 2023

Coronary artery disease is the leading cause death all over the world. Dysfunction of the endothelial cells in blood vessels is one of the major events in the development of atherosclerosis. Inflammation involve in the initiative of atherosclerotic lesion process. In the progression, monocytes recruitment contributes to the early stage of atherosclerosis. Oxidation stress is another remarkable proinflammatory factor will induce endothelial cell inflammation and stimulate monocytes recruitment and differentiated to resident macrophages in vascular wall.

In this study, we estimate that haptoglobin have strong anti‐oxidant activity and decrease the differentiated adhesive monocytes amount in vitro. In addition, we find haptoglobin inhibit monocyte adhesion molecular vascular cell adhesion molecule 1 (VCAM‐1) and Intercellular Adhesion Molecule 1 (ICAM‐1) expression in PMA‐induced inflammation endothelial cells. According, haptoglobin has the abilities to prevent LDL oxidation to mm‐LDL and ox‐LDL and decrease inflammation driven monocyte recruitment and differentiate into resident macrophages in vascular wall intimal space. Furthermore, we find haptoglobin enhance human umbilical vein endothelial cells (HUVECs) proliferation, migration, and angiogenesis in vitro. The abilities can accelerate wound healing process in the oxidative and inflammation initiated atherosclerotic lesion and improve cardiomyocyte function in ischemic damaged lesion.

Currently, anti‐oxidant, anti‐inflammation, and angiogenesis enhance agents are the major studies trend for coronary artery diseases therapeutic application. In this study, we find haptoglobin has the activities to prevent atherosclerotic process and to improve functional activities in damaged atherosclerotic lesion. In conclusion, haptoglobin has strong potential as a protein drug for atherosclerosis prevention and therapeutic application.

Support or Funding Information

This work was supported by the grants MOST 105‐2314‐B‐038‐016 and MOST 105‐2119‐M‐038‐001 from Ministry of Science and Technology, Taiwan.
The Effect of MCPIP1 Deficiency on Age‐related Lipid Profile Changes
Authors: Yang Chalen , Deaton Evan , Chang Yingzi
Published: November 2023

Background

Chronic inflammation is associated with metabolic disorders, such as impaired lipid homeostasis, which can lead to atherosclerotic plaque formation and ultimately myocardial infarction or stroke. Monocyte chemotactic protein‐induced protein‐1 (MCPIP1) is a negative regulator in inflammatory processes. Previous studies have shown that MCPIP1 gene deficient mice display phenotypic changes, such as splenomegaly, lymphadenopathy, growth retardation, decrease adipose deposit, and increased pro‐inflammatory cytokines. Our current project is to investigate the age‐related lipid profile changes in MCPIP1 deficient mice.

Methods

Plasma and liver tissues are collected from wild‐type (WT) and MCPIP1 knockout (KO) C57/BL6 mice at ages 2, 4, and 6 weeks. Plasma HDL, LDL‐VLDL, and liver total cholesterol are measured by using HDL and LDL/VLDL quantitation kit from Sigma‐Aldrich. Plasma and liver total triglycerides are quantified by using Cayman Chemical Triglyceride Assay Kit.

Results

We found that MCPIP1 deficiency significantly: 1) lowered plasma HDL levels in mice at the age of 6 weeks; 2) caused age‐related reduction of HDL‐cholesterol; 3) increased plasma LDL‐VLDL in mice at ages of 2 and 4 weeks; and 4) increased total plasma triglyceride in the mice at the age of 6 weeks. No differences of total triglyceride and cholesterol were observed between WT and KO livers.

Conclusion

The data collected thus far suggest that MCPIP1 is an important regulator in maintaining plasma lipid homeostasis. MCPIP1 deficiency may contribute to the age‐related changes of lipid profile changes.

Support or Funding Information

ATSU Masters of Biomedical Sciences Program
Distinct roles of extracellular vesicles from macrophages and endothelial cells in regulation of endothelial inflammation
Authors: Lin Xingguang , Zuo Ping , Khanal Grishma , Zhang Youzhi , Li Xiang , Zhang Yang
Published: November 2023

Background and aims

Extracellular vesicles (EVs) play an important role in maintaining endothelial cell (EC) homeostasis. Here, we examined the effects of exosomes and microvesicles, two main subtypes of EVs, derived from either ECs or macrophages on endothelial inflammation.

Methods

Exosomes and microvesicles were isolated from the cell culture medium from mouse microvascular ECs and J774 macrophages by sequential ultracentrifugation. Exosomes were detected by Western blotting analysis of marker protein CD63 and AIP‐1. Microvesicles were stained with FITC‐annexin V and Hoechst 33342 and analyzed by flow cytometry. Endothelial inflammation was examined by analyzing mRNA and protein expression of VCAM‐1 and monocyte adhesion using real time RT‐PCR and fluorescence microscopy.

Result and conclusion

Both macrophages and ECs spontaneously released CD63 and AIP‐1 positive exosomes under resting condition, whereas the expressions of these exosome markers were not affected by TNFα stimulation. In contrast, TNFα markedly increased the release of annexin V+ microvesicles from either ECs or macrophages. Interestingly, among these microvesicles, we identified a novel subpopulation of microvesicles that are positively stained with both annexin V and Hoechst 33342. This result suggests that TNFα increased the release of a unique subset of microvesicles containing DNA fragments. Functionally, EVs (microvesicles and exosomes) derived from TNFα‐treated macrophages, but not from TNFα‐treated ECs, increased VCAM‐1 expression and monocyte adhesion in naïve ECs. Together, these results suggest that EVs may exert distinct effects (pro‐inflammatory v.s. anti‐inflammatory) on endothelial homeostasis depending on their subtypes and/or cellular sources.

Support or Funding Information

Supported by NIH grants HL122769 and HL122937
Arachidonic acid metabolites induce TRPV1‐mediated inflammation
Authors: Hamers Alexander Jozua Pedro , Ahluwalia Amrita
Published: November 2023

Introduction

Transient receptor potential cation channel subfamily V member 1 (TRPV1) is a non‐specific cation channel localised to sensory C‐fibres. Opening of the TRPV1 channel leads to cell membrane depolarization, resulting in the release of neuropeptides including calcitonin gene‐related peptide and substance P (SP). These neuropeptides play an important role in vasodilatation, vascular leakage and cell migration during acute inflammatory responses [1, 2]. Endogenous activators of TRPV1 are thought to include the arachidonic acid (AA) metabolites 12‐hydroperoxyeicosatetraenoyl acid (12‐HpETE) and 20‐hydroxyeicosatetraenoic acid (20‐HETE) [3]. However, whether these lipids underlie TRPV1 activation in inflammatory responses is unknown.

Aim

To investigate the role of TRPV1 in the acute inflammatory response and to determine whether arachidonic acid metabolites might play a role in its endogenous activation.

Methods

Lipopolysaccharide (LPS, 100 μg Ecoli 055:B5), zymosan activated serum (ZAS) (50%), SP (1 nmol) or AA (30, 100 and 300 nmol)‐induced skin oedema formation were determined in TRPV1 knockout (KO) and wild type (WT) littermate mice using a modified Miles assay method. The roles of 12‐HpETE and 20‐HETE were investigated by treating animals with the 12‐HpETE synthesis inhibitor cinnamyl‐3, 4‐dihydroxy‐a‐cyanocinnamate (CDC) (8 mg/kg, i.p.) or the 20‐HETE synthesis inhibitor 17‐octadecynoic acid (17‐ODYA, 35 mg/kg,i.p.). The effects of pharmacological blockade of TRPV1 and the SP receptor neurokinin 1 (NK1) were tested using AMG‐9810 (30 mg/kg, i.p.) and SR‐140333 (0.1 μmol/kg i.v) respectively.

Results

The oedema response to LPS (P<0.01) and ZAS (P<0.05) were reduced by ~ 53 and 43% respectively in TRPV1 KO animals compared to WT mice (LPS: 24.6 ± 3.8 μl, ZAS: 48.4 ± 9.0 μl n=13). In WT mice treatment with AMG‐9810 (n=13) reduced the LPS (P<0.01), ZAS (P<0.01) and AA (P<0.001) response, by ~ 43, 22 and 36% respectively compared with vehicle treated mice (LPS: 19.4 ± 2.2, ZAS: 42.2 ± 3.2, AA: 47.6 ± 5.9 μl n=13). 17‐ODYA (n=15) significantly reduced LPS (P<0.05), ZAS (P<0.01) and AA (P<0.05,) induced oedema by ~ 47, 24 and 29% compared to vehicle treated mice (LPS: 19.6 ± 2.7, ZAS: 48.1 ± 4.7, arachidonic acid: 47.0 ± 6.6 μl n=13). NK1 receptor blockade reduced AA‐induced (300 nmol) oedema from 43.4 ± 3.9 μl to 29.0 ± 1.8 μl (n=13, p<0.001).

Conclusion

Our findings suggest that TRPV1 plays an important role in vascular permeability during an acute inflammatory response induced by diverse stimuli. In addition our findings suggest that the endogenous activator of TRPV1 in these scenarios is 20‐HETE. Furthermore, the NK1 receptor plays a key role in mediating the AA oedema response induced by endogenous TRPV1 activation, indicating that 20‐HETE is the endogenous activator and SP is the downstream mediator of TRPV1.

Support or Funding Information

A.H. is supported by The Trustees of the Professor Derek Willoughby PhD Fund for Inflammatory Research.
Nicotine induced endothelial dysfunction through cathepsin B‐dependent activation of endothelial Nlrp3 Inflammasome
Authors: Chen Yang , Li Xiang , Li Pinlan , Zhang Yang
Published: November 2023

Recent studies indicate that endothelial dysfunction occurs in association with several cardiovascular risk factors and endothelial Nlrp3 inflammasomes play a crucial role in the pathogenesis of cardiovascular diseases. But the precise innate immunity mechanisms that endothelium cell how to been trigger or promote the inflammatory response during the development of early stage of smoking‐associated vascular complications remains poorly understood. The present study explored the role of endothelial Nlrp3 inflammasome as a major injurious factor in mediating gap junction disruption, an important event of endothelial barrier dysfunction leading to endothelial hyperpermeability during initiate stage of smoking addiction. In vivo study, confocal microscopy analysis demonstrated that nicotine stimulation had significantly increased endothelium gap junction disruption and Nlrp3 inflammasome induced HMGB1 release compared with animals fed normal diet (ND). And the Nlrp3−/− mice significantly attenuated the endothelium gap junction disruption and HMGB1 release. In vitro, these Nlrp3 inflammasome molecules could be aggregated to form an inflammasome complex on stimulation of nicotine, as shown by fluorescence confocal microscopy. Correspondingly, nicotine significantly increased caspase‐1 activity and IL‐1β and HMGB1 release in microvascular endothelial cells, indicating an activation of Nlrp3 inflammasomes. Further pharmacological inhibition and genetic intervention experiments showed that the nicotine‐induced endothelium cell permeability was abolished in MVECs when cells were treated with either a HMGB1 inhibitor glycyrrhizin (GLY), caspase‐1 inhibitor or Nlrp3 gene silencing. In order to further study the mechanism, we used fluorescence staining experiments. It's showed that nicotine‐induced endothelium cell cathepsin B activation was abolished in MVECs when pretreatment the lysosome stabilizer (Dexamethasone). The present study revealed that nicotine‐induced endothelium Nlrp3 inflammasome formation and activation is mediated through cathepsin B pathway and the injurious effect of nicotine on the endothelium is associated with the HMGB1 via Nlrp3 inflammasome activation. Endothelial Nlrp3 inflammasome system may serve as an important pathological mechanism in the development of early stage of smoking‐associated vascular complications.

Support or Funding Information

This study was supported by grants from the National Institutes of Health (HL057244, HL122769 and HL122937) and the National Natural Science Foundation of China (NO.81603587).
Inhibitory Effects of Growth Differentiation Factor 11 on Phenotypic Transition Associated with Autophagy Deficiency in Mouse Coronary Arterial Myocytes
Authors: Yuan Xinxu , Bhat Owais , Lee RaMi , Lohner Hannah , Zhang Yang , Li Pin‐Lan
Published: November 2023

Growth‐differentiation factor 11 (GDF11) as an anti‐aging factor may reverse age‐related cardiac hypertrophy in mice and cause youthful regeneration of cardiomyocytes. The role of GDF11 in the growth and phenotypic transition of vascular smooth muscle cells remains poorly understood. The present study first examined the effects of GDF11 to induce differentiation of cultured mouse coronary arterial myocytes (CAMs). By real time RT‐PCR and Western blot analysis, GDF11 was found to increase CAMs differentiation, as shown by increased expression of various muscle cell differentiation markers such as α‐SMA, myogenin, MyoD, and MyHC, but decreased expression of dedifferentiation markers such as vimentin and proliferating cell nuclear antigen (PCNA). Upregulation of GDF11 gene by trichostatin A (TSA) or specific CRISPR‐cas9 activating plasmids also stimulated the differentiation of CAMs with a remarkable decrease in the ratio of vimentin vs. SM22α. In CAMs treated with 7‐ketocholesterol (7‐keto), a phonotypic transition into dedifferentiation status was observed with significant increases in the ratio of vimentin vs SM22α, which was companied by autophagosome (AP) accumulation with large increase in LC3‐II. This 7‐keto‐induced CAMs dedifferentiation and AP accumulation were almost completely blocked by GDF11 or TSA treatment. Similarly, in CAMs pretreated with lysosome function inhibitor, bafilomycin or chloroquine, both GDF11 and TSA markedly attenuated the phenotypic transition and AP accumulation. Moreover, in CAMs from mice lacking CD38 gene that produces lysosome trafficking regulator, NAADP, GDF11 was also shown to inhibit phenotype transition to dedifferentiation status with or without 7‐keto pretreatment. It is concluded that both exogenous and endogenous GDF11 promote CAMs differentiation and prevent a phenotypic transition of these cells by reduction of lysosome accumulation induced by different pathological stimuli.

Support or Funding Information

Supported by NIH grants HL057244, HL075316 and HL122937
Droplet Digital PCR Reveals Substantial Tissue‐Specific Differences in Estrogen Receptor Expression Profiles
Authors: Hutson Dillion D , Duong Jennifer L. , Sato Ryousuke , Lindsey Sarah H.
Published: November 2023

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

Support or Funding Information

National Institute of General Medical Sciences IDeA Program (COBRE, P30GM103337)
Characterization of Acid Ceramidase and Its Regulation by HMGB1 in Mouse Coronary Arterial Myocytes
Authors: Lee RaMi , Meng Nan , Yuan Xinxu , Li Pin‐Lan
Published: November 2023

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

Support or Funding Information

Supported by NIH grants HL057244, HL075316 and HL122937
Oroxylin‐A suppresses adhesion molecules expression and endothelial barrier disruption in endotoxemic arteries
Authors: Tseng Tzu‐Ling , Chen Mei‐Fang , Liu Chin‐Hung , Hsu Yung‐Hsaiang , Lee Tony J. F.
Published: November 2023

Vascular inflammation plays a critical role in the pathogenesis of sepsis, which is characterized by disruption of endothelial junctions, increasing vascular permeability and leakage, and promoting leukocytes and inflammatory exudates to enter the injured area. Here we assess the beneficial effects of post‐treatment of Oroxylin‐A (OroA), a flavonoid, in ameliorating lipopolysaccharide (LPS)‐induced adhesion molecules expression, endothelial barrier dysfunction, and vascular inflammation. Rats were injected with LPS (10 mg/kg, ip) to induce systemic inflammation, and OroA (15 mg/kg, iv) was given 6 hours after LPS treatment. Biochemical changes in the circulation, and morphological/histological alternations associated with inflammation in the aorta were investigated. LPS significantly enhanced proinflammatory cytokines release, increased vascular cell adhesion molecule (VCAM)‐1 and intercellular adhesion molecule (ICAM)‐1 expressions, disrupted endothelial tight junction zona occludens (ZO)‐1 surface expression, reduced stabilization of adherens junction vascular endothelial cadherin (VE‐cadherin) of endothelial barrier, and increased macrophage infiltration and accumulation in aortic arteries. All these changes were reversed by OroA (15 mg/kg, iv) post‐treatment. This post‐treatment also blocked the LPS‐activated aortic NF‐κB. Moreover, this post‐treatment suppressed interleukin‐1‐receptor‐associated‐kinase (IRAK)‐4‐targeted IKKα/β phosphorylation without directly affecting interaction between LPS and toll‐like receptor (TLR)‐4 receptor. These results obtained from studying OroA suggest that beside anti‐inflammation via inhibiting IRAK‐4‐mediated NF‐κB activation, reversal of adhesion molecules expression and endothelial barrier dysfunction may be another useful strategies in managing endotoxemic shock.

Support or Funding Information

supported by the MOST, Tzu Chi Foundation, Tzu Chi University and Tzu Chi Medical Center
O‐GlcNAc‐eNOS is decreased in blood vessels from pregnant Wistar rats, but not in vessels from pregnant SHR
Authors: Troiano Jessica Antonini , Potje Simone Regina , Graton Murilo Eduardo , Silva Daniela Souza , Antoniali Cristina
Published: November 2023

Introduction

Late pregnancy in spontaneously hypertensive rats (SHR) is related to low blood pressure levels and decreased vasoconstrictors responses to alpha 1‐adrenergic drugs. In blood vessels, increased endothelial nitric oxide synthase (eNOS) phosphorylation and nitric oxide (NO) production are associated to pregnancy effects. Some post‐translational modifications, such as O‐glycosylation by N‐acetyl‐glucosamine (O‐GlcNAc) decreases eNOS activity leading to a lower NO bioavailability. O‐GlcNAc is involved on the OGT (O‐GlcNAc transferase) activity, which binds O‐GlcNAc on serine and threonine residues of target proteins and OGA (O‐GlcNAcase), which removes O‐GlcNAc from these proteins. We hypothesized that O‐GlcNAc‐eNOS is decreased in blood vessels of pregnant SHR. We evaluate the effect of PugNAc, OGA inhibitor, on vascular reactivity to phenylephrine (PE) in aortas of pregnant Wistar rats and in mesenteric artery of pregnant SHR.

Methods

Aortic rings (2 mm) of non‐pregnant (NP) and pregnant (P) Wistar rats (n= 5–7) and rings of the 2nd and 3rd branches of the mesenteric artery of NP and P SHR (n = 4–6) were incubated in DMEM in the presence of PugNAc (100 μM) or methanol (vehicle) for 24 hours. These rings, with and without endothelium, or with endothelium in the presence of L‐NAME (100 μM, 30 min incubation), were stimulated with increasing and cumulative concentrations of phenylephrine (PE, 1 nM to 10 μM). The results were compared between the groups (ANOVA, p <0.05).

Results

Pregnancy modulates the vascular reactivity of the blood vessels, reducing the maximum effect of PE curves in aortas from P Wistar rats (22.96 ± 1.36 mN) when compared to the NP Wistar (27.01 ± 0.68), and in mesenteric arteries from P SHR (12.04 ± 0.53 mN) when compared to NP SHR (24.12 ± 0.99 mN). Incubation with PugNAc did not alter the reactivity of NP Wistar aortas to PE (Emax: 26.16 ± 0.43 mN). However, aortas from P Wistar rats incubated with PugNAc showed greater maximum contractile effect to PE (32.75 ± 1.17 mN) when compared to non‐incubated aorta or to NP Wistar aortas. The increased endothelium modulation on PE vasoconstriction observed in aortas from P Wistar rats was reduced by PugNAc when compared to non‐incubated aortas. PugNAc did not alter mesenteric artery rings reactivity to PE in NP SHR (24.66 ± 0.69 mN) or P SHR (13.49 ± 0.71). PugNAc reversed the low reactivity to PE, which is dependent of eNOS activity, in aortas from pregnant Wistar rats, but not in mesenteric arteries from pregnant SHR.

Conclusion

These results suggest that O‐GlcNAc‐eNOS is decreased in blood vessels from pregnant Wistar rats, but not in vessels from pregnant SHR.

Support or Funding Information

Financial support: Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) [Grant 2015/08188‐4].
Mechanisms Linking Chronic Obstructive Pulmonary Disease and Stroke
Authors: Bernard Ashton Juan , Ku Jacqueline M , Bozinovski Steven , Vlahos Ross , Miller Alyson A
Published: November 2023

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

Support or Funding Information

Supported by NHMRC funding
Do Non‐C1 Cells In The Rostral Ventrolateral Medulla Increase Blood Pressure?
Authors: Dash Kiana Elizabeth , Stornetta Ruth L. , Holloway Benjamin B. , Guyenet Patrice G.
Published: November 2023

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

Support or Funding Information

University of Virginia Summer Research Internship Program