bluuebunny/crossref_metadata_2025 huggingface dataset:
Naturally Occurring Mutations in Muscle‐Type Creatine Kinase Impact Tenofovir Monophosphate Phosphorylation
Authors: Mosher Eric P. , Wade Herschel , Bumpus Namandje N.
Published: June 2021

Tenofovir (TFV), administered as a tenofovir disoproxil fumarate prodrug, is a nucleotide analog reverse transcriptase inhibitor used in combination for the treatment and prevention of HIV. TFV requires two sequential phosphorylation events in order to become pharmacologically active. We previously found that in colon tissue, a putative site of HIV infection, the muscle‐type isoenzyme of creatine kinase (CKM) is responsible for the second TFV activating phosphorylation step, converting tenofovir monophosphate (TFV‐MP) to the active tenofovir diphosphate (TFV‐DP). Due to the similarity in structure between TFV‐MP and ADP, this is likely analogous to the endogenous activity of CKM, which regenerates ATP from ADP by phosphoryl transfer from phosphocreatine. In addition, we have previously discovered naturally occurring genetic variants of the CKM gene in humans. Some of these single nucleotide variants are predicted to cause single residue mutations in the translated protein, a fraction of which are predicted by bioinformatics programs to have deleterious effects on enzymatic function. In the present study, molecular docking was employed to investigate the potential effects of these mutations on TFV monophosphate (TFV‐MP) binding and phosphorylation by CKM. These results indicate CKM binds TFV‐MP similar to how it binds its natural substrate, ADP. Molecular docking simulation also predict a cluster of active site arginine residues at positions 130, 132, 236, 292, and 320 are likely important in the charge stabilization of the phosphates of TFV‐MP, which may impact substrate binding and phosphorylation. To directly investigate the impact of the aforementioned mutations on TFV‐MP phosphorylation, CKM wild type and mutant proteins were recombinantly expressed and purified. In vitro assays were then performed to compare the ability of the mutant proteins to convert TFV‐MP to TFV‐DP. Enzyme activity was measured by liquid chromatography‐tandem mass spectrometry using a method that separated and detected the phosphorylated tenofovir metabolites concomitantly. It was found that R130H and R132C mutations decreased enzymatic activity to less than 10% of that of the wild type protein. These results indicate that genetic variation in the CKM gene may impact tenofovir activation, potentially implicating a risk to patients possessing these genetic variants.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Bioengineering of single ncRNA molecule for multi‐targeting against NSCLC
Authors: Petrek Hannah , Ho Pui Yan , Tu Meijuan , Zhang Qianyu , Batra Neelu , Yu Ai‐Ming
Published: June 2021

Lung cancer is the second most common cancer among both men and women in the United States. The majority of lung cancer cases are classified as non‐small cell lung cancer (NSCLC). Current NSCLC treatments include resection surgery, radiation, and pharmacotherapy which all provide some benefits and have limitations. As a result, lung cancer caused deaths account for about 27% of all cancer deaths in the US. Therefore, new therapeutic agents are highly demanded for NSCLC. MicroRNAs (miRNA or miR) are a class of genome‐derived, small noncoding RNAs (ncRNA) in cells that govern target gene expression through mRNA degradation or translational inhibition. Some miRNAs such as miR‐34a, miR‐124 and let‐7c are commonly downregulated in NSCLC tissues/cells and target various oncogenes such as CDK6, STAT3, and Ras. Restoring the expression or function of such dysregulated, tumor suppressive miRNAs represents a new strategy for cancer therapy. Aiming at simultaneously introducing multiple miRNAs in NSCLC cells for multi‐targeting, we employed our recently developed novel ncRNA bioengineering technology to produce a single ncRNA molecule bearing multiple miRNAs, namely combination‐bioengineered RNA agent (CO‐BERA). Our data showed that a variety of target CO‐BERA molecules were successfully expressed in E. coli. We were able to purify recombinant CO‐BERAs to a high degree of homogeneity using anion exchange FPLC methods. We further demonstrated that treatment of human NSCLC cells with CO‐BERAs elevated cellular miR‐34a, miR‐124 and let‐7c levels, reduced protein levels of target genes including CDK6, STAT3 and Ras, and consequently inhibited NSCLC cell proliferation. CO‐BERAs were successful in inhibiting NSCLC patient derived xenograft (PDX) tumor growth in mice. Therefore, using single ncRNA agent for multi‐targeting may be more effective for the treatment of NSCLC.

Support or Funding Information

This study was supported by grants R01GM133888 and U01CA175315 from NIH.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Anticancer and Chemosensitizing Effects of WEE‐1 Kinase Inhibitor MK‐1775 in Triple Negative (MB‐231) Breast Cancer Cells
Authors: Aviles Nicole Marie Nieves , Mayol Noel , Gonzalez Maria T , Diaz Adriana , Salgado Iris K , Silva Walter , Maldonado Hector
Published: June 2021

Current breast cancer classification included cells that express estrogen (ER) and progesterone (PR) receptors, those with ERBB2 (HER‐2/Neu) amplification, and those without expression of ER, PR, or amplification of ERBB2 (referred to as triple‐negative or basal‐like breast cancer). Triple negative breast cancers are generally more difficult to treat, are known to be more aggressive and with poor prognosis. Several molecular mechanisms have been identified as potential targets for development of drugs to treat breast cancer including cell cycle checkpoints kinases. The G2/M cell‐cycle protein tyrosine kinase, Wee‐1, plays a crucial role in the checkpoint arrest for DNA repair before mitotic entry. Under normal conditions, cells repair damaged DNA during G1 arrest; however, cancer cells often have a deficient G1/S (p53) checkpoint and depend on a functional G2/M checkpoint for DNA repair. MK‐1775 is a well‐characterized Wee‐1 kinase inhibitor with significant evidence supporting its potential use as anti‐cancer and/or chemosensitizing drug. Working hypothesis proposed for this project was that use of Wee‐1 kinase inhibitors in breast cancer cells can result in profound and significant anticancer and chemosensitizing effects. In order to test this hypothesis, we show previously in a series of dose response experiments using DNA‐Damaging agents or treatments (DNA‐dt: Ex. Cisplatin, 5‐Fluororacil and Doxorubicin) alone and/or in combination with MK‐1775. To continue testing this hypothesis an Immunoblotting bioassay is currently developed for continue monitoring the effects of MK‐1775 in several biochemical markers in triple negative breast cancer cell line. Preliminary results indicated that biochemical marker in triple negative cell line, are time dependent with the different combination of DNA‐dt alone and/or in combination with MK‐1775. Initial preliminary results indicated taken together, our preliminary results are consistent with a potential role of Wee‐1 kinase inhibitors like MK‐17175 as anticancer and/or chemosensitizing agents against triple‐negative or basal‐like breast cancer cells.

Support or Funding Information

The project described was supported by NIGMS/INBRE award P20 GM103475; U.S. Department of Education Title V grant number P031S130068 and Puerto Rico Science Technology and Research Trust. The content is solely the responsibility of the authors and does not necessarily represent the official view of any of the supporting agencies.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Antifungal Agents Induce Apoptotic and Non‐Apoptotic Cell Death in Human Melanoma Cells
Authors: Koh David W , Eskender Anna H , Daghistani Ayah T , Jira Lukas R , McKamey Shelby G
Published: June 2021

Patients with melanoma, the most aggressive form of skin cancer, have poor survival rates due to the resistance of advanced stage tumors to chemotherapy. This resistance appears to involve the dysregulation of apoptosis, such that inhibition of apoptotic progression allows melanoma cells to survive after treatment. We previously reported a potentially novel treatment for melanoma, where antifungal agents led to profound decreases in growth and proliferation of human melanoma cells. Our objective here was to analyze cell death pathways induced in human melanoma cell lines following treatment with antifungal agents. Cell death pathways were investigated via flow cytometry, cell proliferation assays, and immunoblotting after treatments.

Several lines of human melanoma and normal skin cells were analyzed. Apoptosis, via flow cytometry and analysis of activated caspases and PARP1 cleavage, was observed in all melanoma lines after treatment with clotrimazole and econazole. Caspase‐independent cell death was also observed in most melanoma lines after similar treatments. No extensive cell death was induced by these treatments in noncancerous human skin cells. These results indicated that antifungal agents selectively induced cytotoxicity in human melanoma lines. Because varying levels of cell death were observed among different melanoma lines after antifungal treatment, further studies will be required to ascertain the reason for these differential responses to treatment. Taken together, these results indicated that clotrimazole and econazole initiated apoptotic and non‐apoptotic cell death in human melanoma cells.

In conclusion, this study demonstrated the induction of apoptotic and non‐apoptotic cell death in human melanoma skin cell lines after treatment with antifungal agents. Differential levels of various cell death pathways were observed among all melanoma lines. This was corroborated by the various levels of efficacious treatment produced by these agents. Taken together, we conclude that clotrimazole and econazole induce cell death in human melanoma cells. These agents thus appear to have in vitro efficacy toward the treatment of human melanoma.

Support or Funding Information

This research was supported in part by the Bower, Bennet & Bennet Endowed Chair Award from the Ohio Northern University College of Pharmacy and the Ohio Northern University Summer Research stipend.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Constraining Multi‐Drug Resistance in Breast Cancer Cells by Energy Restriction
Authors: Omar Hany A. , Zaher Dana M. , Tolba Mai F. , Al‐Tel Taleb H.
Published: June 2021

The resistance of breast cancer to chemotherapy constitutes a major roadblock for a successful treatment. The development of multidrug resistance (MDR) is one of the common mechanisms for resistance to various chemotherapeutic agents including doxorubicin (DOX). In this context, we tested the hypothesis that energy restriction mimetic agents (ERMAs) such as OSU‐GC5 could synergize the antitumor effects of DOX through restraining the availability of energy that is essential for the activity of MDR efflux carriers. Subclones of MCF‐7 and MDA‐MB231 breast cancer cell lines resistant to DOX have been established by subjecting the cells to increasing concentrations of DOX over 6 months. The MDR phenotype was verified via the upregulated expression of ATP binding cassette (ABC) protein ABCC1, ABCB1 and ABCG2 together with a reduced cellular accumulation of DOX. Fluorescence‐activated cell sorting analysis indicated that the percentage of CD44+/CD24− stem cell‐like population was enriched in the resistant subclones. Combination analysis demonstrated a synergistic antitumor interaction between OSU‐CG5 and DOX accompanied by augmented activation of mitochondrial apoptosis and AMPK signaling as verified by Western blot analysis. Co‐treatment with OSU‐CG5 and DOX significantly reduced cellular ATP content and NADP+/NADPH ratio, while it significantly enhanced ROS production. Concomitant exposure to ROS scavenger, N‐acetylcysteine, eliminated the synergy between OSU‐CG5 and DOX. This study recommended OSU‐CG5 as a promising adjuvant to overcome MDR resistance. This combinatorial strategy can be extrapolated to breast cancer treatment regardless of the hormone receptor expression status, which encourages future research in syngeneic murine tumor xenograft models.

Support or Funding Information

This work was supported by a research grant from Al Jalila Foundation, United Arab Emirates (Grant number AJF201610)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A PF‐3845‐induced novel lipid fraction in mouse medullary interstitial cells causes diuresis and natriuresis via a CB1‐mediated pathway
Authors: Daneva Zdravka P , Dempsey Sara , Ahmad Ashfaq , Ritter Joseph
Published: June 2021

The relationship between the endocannabinoid system in the renal medulla and the long‐term regulation of blood pressure is not understood. To investigate the possible role of the endocannabinoid system in renomedullary interstitial cells, mouse medullary interstitial cells (MMICs) were obtained, cultured and characterized for their responses to treatment with a selective inhibitor of fatty acid amide hydrolase (FAAH), PF‐3845. Treatment of MMICs with PF‐3845 increased cytoplasmic lipid granules detected by Sudan Black B staining and multilamellar bodies identified by transmission electron microscopy. HPLC analyses of lipid extracts of MMIC culture medium revealed a 205nm‐absorbing peak that showed responsiveness to PF‐3845 treatment. The biologic activities of the PF‐3845‐induced product (PIP) isolated by HPLC were investigated in anesthetized, normotensive surgically‐instrumented mice. Intravenous and intramedullary infusions of PIP at low doses resulted in a vasodepressor, diuretic and natriuretic effects. Pharmacological and genetic ablation of cannabinoid 1 receptors resulted in the loss of the vasodepressor, diuretic and natriuretic effect of PIP. Radioactive binding assays showed that PIP competes for binding to CB1 receptors in the presence of a full agonist (CP 55,940). PIP did not elevate urine and sodium excretion in a genetic knockout mouse model of FAAH. A PIP‐induced decrease in blood pressure at high doses of PIP was similar between FAAH WT and FAAH KO mice. These data support a model whereby PF‐3845 treatment of MMICs results in increased secretion of a neutral lipid which acts directly to promote diuresis and natriuresis via a CB1‐mediated mechanism. The diuretic and natriuretic responses to PIP are eliminated by inhibition of FAAH, suggesting an undergoing sensitization to PIP in FAAH KO mice. Efforts to identify the structure of the PF‐3845‐induced lipid and its relationship to the previously proposed renomedullary antihypertensive lipids are ongoing.

Support or Funding Information

This work was supported by the National Institute of Diabetes and Digestive and Kidney Disorders grant [grant R01DK102539] and the National Institute on Drug Abuse [grant P30DA033934]. S.D. was supported by the National Institute on Drug Abuse [training grant T32DA007027].

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Luteolin attenuates glycerol‐induced acute renal failure through modulation of Kim‐1/NF‐κB /Nrf2 signaling pathways
Authors: Oyagbemi Ademola Adetokunbo , Omobowale Temidayo Olutayo , Ola‐Davies Olufunke Eunice , Asenuga Ebunoluwa Racheal , Ajibade Temitayo Olabisi , Adejumobi Olumuyiwa Abiola , Afolabi Jeremiah Moyinoluwa , Ogunpolu Blessing Seun , Falayi Olufunke Olubunmi , Hassan Fasilat Oluwakemi , Nabofa Williams Enivwenaye , Saba Adebowale Benard , Adedapo Adeolu Alex , Yakubu Momoh Audu
Published: June 2021

Acute renal failure (ARF) has been documented as a life‐threating disease with high morbidity and mortality. We investigated the protective effect of Luteolin against ARF. In the study, forty‐male Wistar albino rats were randomly divided into four groups (n = 10). Group A received normal saline. Group B received glycerol (10 ml/kg BW, 50% v/v in sterile saline, i.m.). Groups C and D were pre‐treated with Luteolin 100 and 200 mg/k for 7 days, and thereafter administered Glycerol (10 ml/kg BW, 50% v/v in sterile saline, i.m.). Administration of glycerol significantly increased blood pressure, renal oxidative stress, serum creatinine and reduced nitric oxide (NO) bioavailability. Glycerol administration enhanced expressions of kidney injury molecule 1 (Kim‐1), nuclear factor kappa beta (NF‐κB) and nuclear factor‐erythroid‐2‐related factor 2 (Nrf2). Luteolin pre‐treatment normalized blood pressure, reduced markers of oxidative stress, renal damage, and improved NO bioavailability. Together, Luteolin attenuates glycerol‐induced acute renal failure through modulation of Kim‐1/NF‐κB /Nrf2 signaling pathways. Hence, Luteolin might be a novel therapeutic target for the treatment of acute renal failure.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Lysosome Dysfunction and Medial Calcification in the Arterial Wall of Smooth Muscle Cell‐Specific Smpd1 Transgenic Mice: A Ceramide‐Mediated Vasculopathy
Authors: Bhat Owais M , Yuan Xinxu , Lohner Hannah , Li Pin‐Lan
Published: June 2021

It has been reported that multivesicular body (MVB) trafficking and release of exosomes are a hallmark of vascular calcification. Since ceramide is a crucial regulator of lysosome function, the present study was performed to test whether lysosomal acid sphingomyelinase (its mouse gene code is Smpd1)‐derived ceramide contributes to the exosome secretion from smooth muscle cells (SMCs) and consequently lead to arterial calcification. In Smpd1trg/SMcre mice with SMC‐specific overexpression of Smpd1 gene, a high dose of Vit D (500,000 IU/kg/day) induced much more aortic and coronary arterial medial calcification compared to their littermates (Smpd1fl/fl/WT and WT/WT mice), as detected by Alizarin Red S and Von Kossa staining. It was also found that there was more significantly increased expression of RUNX2 and osteopontin in the coronary and aortic media of Smpd1trg/SMcre mice than their littermates, indicating a SMC phenotypic transition to osteogenic in these SMC‐specific Smpd1 transgenic mice. When these mice received treatment with amitriptyline, an Asm inhibitor, the calcification and phenotypic switch in both coronary and aortic medial SMCs were reversed. Furthermore, Smpd1trg/SMcre mice were found to have increased annexin 2 (AnX2) and alkaline phosphatase (ALP) levels (exosome markers) in the arterial wall, which was accompanied by reduced co‐localization of lysosome marker (LAMP‐1) with MVB marker (VPS16), a parameter for lysosome‐MVB interaction. All these changes related to lysosome function and exosome release from SMCs in Smpd1trg/SMcre mice were substantially attenuated by amitriptyline treatment. These data indicate that lysosomal ceramide plays a critical role in phenotype change and exosome release in SMCs, which may contribute to the control of mineral metabolism or deposition in the arterial wall.

Support or Funding Information

Supported by NIH grants HL057244, HL075316 and DK120491

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inhibition of α5β1integrin with the clinically validated small peptide, ATN‐161 stabilizes cerebral vasculature, reduces inflammation, and decreases blood‐brain barrier permeability after experimental ischemic stroke
Authors: Edwards Danielle , Salmeron Katie , Lukins Douglas , Trout Amanda , Fraser Justin , Bix Gregory
Published: June 2021

Stroke is the second leading cause of death and the leading cause of long‐term disability worldwide. Blood‐brain barrier (BBB) dysfunction exacerbates reperfusion‐induced injury after recanalization in ischemic stroke. Endothelial cell integrin receptors, specifically the β1 subtype, play a direct role in this BBB dysfunction through regulation of barrier‐forming tight junction (TJ) proteins. We hypothesize that inhibition of a specific β1 integrin subtype, α5β1, will stabilize the BBB, and thereby reduce infarct volumes and improve functional recovery after experimental stroke.

Method

Male C57BLJ6 mice underwent transient middle cerebral artery occlusion for 1 hour. Upon reperfusion, vehicle or the clinically validated α5β1 inhibitor, ATN‐161 (1mg/kg), was injected intraperitoneally and repeated 24 and 48 hours later. Infarct volume (TTC and MRI – DTI sequence) and edema volume (T2‐weighted MRI) was determined on PSD3. Functional recovery was determined by an 11‐point Neuroscore on PSD1‐PSD7. Physiological measurements, including pulse distention, heart rate and body temperature, were obtained before, during and after the initial dose of ATN‐161. Immunohistochemical analysis of α5β1, claudin‐5, CD45 (pan‐leukocyte), and IgG, expression was performed on PSD3. qPCR analysis on claudin‐5, collagen IV, MMP‐9, IL‐1β, and CXCL12 were performed on PSD3.

Results

ATN‐161 administration reduced infarct volume, edema volume, and functional deficit. ATN‐161 also resulted in cerebral vascular stabilization by a decrease in MMP‐9 and increases in claudin‐5, and collagen IV. Furthermore, IL‐1β and CD45 was reducedupon ATN‐161 administration, suggesting a decrease in the post‐stroke inflammatory response. Together, the cerebrovascular stabilization and decrease in inflammation resulted in less IgG extraversion, suggesting a more intact BBB. Collectively, administration of ATN‐161 post‐stroke produced significant benefits, and thus could represent a novel stroke therapy worthy of further investigation.

Support or Funding Information

This work was supported by NIH R01NS065842.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inhibitory Effect of Total Ginsenoside on Pulmonary Fibrosis Induced by Bleomycin is involved in Matrix Metalloproteinase
Authors: Deng Jiang , Chen Pan‐pan , Luo Min , Yang Lu , Shi Wan‐lan
Published: June 2021

Pulmonary fibrosis is a refractory disease of the respiratory system. As the active components of Panax ginseng, total ginsenoside (TG) have been reported to have antioxidant, anti‐inflammatory and immune modulatory activity. We previously showed that TG has protective effect on pulmonary fibrosis. The purpose of the study was to investigate whether the mechanism of TG against pulmonary fibrosis is involved in the regulation of matrix metalloproteinase (MMPs). The pulmonary fibrosis model of BALB/c mice was replicated by injecting BLM into the trachea. TG (40, 80, 160 mg/kg/day) were given for 28 consecutive days after surgery. Pulmonary fibrosis was determined by measuring lung coefficient, Haematoxylin and Eosin (HE) staining, Masson's trichrome staining, and expression of the alpha smooth muscle actin (α‐SMA, a gene related to pulmonary fibrosis) in lung tissues. Real‐time quantitative PCR and western blot were used to detect matrix metalloproteinase‐2 (MMP‐2), matrixmetalloproteinase‐9 (MMP‐9) and tissue inhibitor of metalloproteinase‐1 (TIMP‐1) mRNA and protein levels respectively. In the present study, TG (40, 80, and 160 mg/kg/d) treatment could evidently ameliorate pulmonary fibrosis and down‐regulate the expression of MMP‐2, MMP‐9 and TIMP‐1 mRNA and protein levels in BLM‐treated mice. The protective effect of TG on bleomycin induced pulmonary fibrosis in mice is related to the regulation of matrix metalloproteinases.

Support or Funding Information

This research was supported by the Social Program for Tackling Key Problems of National Natural Science Foundation of China (NO.81360660 and 81860732.)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Type II Diabetes Mellitus in the Goto‐Kakizaki Rat Impairs Microvascular Function and Contributes to Premature Skeletal Muscle Fatigue
Authors: Frisbee Jefferson , Lewis Matthew , Kasper Jonathan , Chantler Paul , Wiseman Robert
Published: June 2021

Despite extensive investigation into the impact of metabolic disease on vascular function and, by extension, tissue perfusion and organ function, interpreting results for specific risk factors can be complicated by the additional risks present in most models. To specifically determine the impact of type II diabetes without obesity on skeletal muscle microvascular structure/function, and on active hyperemia with elevated metabolic demand, we used 17‐week old Goto‐Kakizaki rats (GK) to study microvascular function at multiple levels of resolution. Gracilis muscle arterioles demonstrated blunted dilation to acetylcholine (both ex vivo proximal and in situ distal arterioles) and elevated shear (distal arterioles only). All other alterations to reactivity appeared to reflect compromised endothelial function associated with increased TxA2 production and oxidant stress/inflammation rather than alterations to vascular smooth muscle function. Structural changes to the microcirculation of GK were confined to reduced microvessel density of ~12%, with no evidence for altered vascular wall mechanics. Active hyperemia with either field stimulation of in situ cremaster muscle or electrical stimulation via the sciatic nerve for in situ gastrocnemius muscle was blunted in GK; primarily due to blunted functional dilation of skeletal muscle arterioles. The blunted active hyperemia was associated with impaired oxygen uptake (VO2) across the muscle and an accelerated muscle fatigue. Acute interventions to reduce oxidant stress (TEMPOL) and TxA2 action (SQ‐29548) or production (dazmegrel) improved muscle perfusion, VO2, and muscle performance. These results suggest that T2DM in GK impairs skeletal muscle arteriolar function apparently early in the progression of the disease and potentially via an increased ROS/inflammation‐induced TxA2 production/action on network function as a major contributing mechanism.

Support or Funding Information

American Heart Association and National Institutes of Health (USA); Canadian Institutes for Health Research and Natural Sciences and Engineering Research Council (Canada)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Impact of analgesics on macro‐ and microcirculatory responses to hemorrhage
Authors: Xiang Lusha , Calderon Alfredo S , Connor Brian S , Hinojosa‐Laborde Carmen , Ryan Kathy L
Published: June 2021

BACKGROUND

Peripheral vasoconstriction in responseto hemorrhage is critical for blood pressure compensation and vital organperfusion. Opiates and ketamine have been demonstrated to have direct vasoactive effects, but effects on peripheral vasoconstriction response to hemorrhage are unknown for these analgesics. We hypothesize that opioid(fentanyl) impairs blood pressure compensation following hemorrhage due toblunted peripheral vasoconstriction, while ketamine preserves pressure compensation and vasoconstriction.

METHOD

Sprague Dawley rats(11–13wk) were randomly assigned to saline (control), fentanyl, or ketamine‐treated groups (n = 9 for each group). Rats were anesthetized within actin and the spinotrapezius muscles were prepared for microcirculatory observation. The arcade arterioles were topically challenged with saline, fentanyl, or ketamine at concentrations relevant to analgesic doses and vascular responses were recorded. After procedurally‐challenged arteriolar diameters returned to baseline, 30% of total blood volumes were removed during a 25 min period. Ten minutes after hemorrhage, rats were treated with a single intravenous(IV) injection of an analgesic dose of fentanyl or ketamine or a comparable volume of saline. Blood pressure, respiratory rate, and arteriolar responses were recorded throughout the experiment.

RESULTS

Baseline parameters were not different among groups (Table 1). Topical fentanyl induced vasodilation (17 ± 2%, p < 0.01)but ketamine caused vasoconstriction (−15 ± 4%; p < 0.01). IV ketamine and fentanyl failed to affect blood pressure compensation compared with the control group (Figure A). Neither saline nor ketamine altered respiratory rate. However, IV fentanyl induced a prolonged suppression in respiratory rate (>10 minutes, with a peak inhibition at 57 ± 8% of baseline, Figure B). A persistent and similar arteriolar constriction to hemorrhage was observed in the control and ketamine treated groups. However, this compensatory vasoconstriction was absent in the fentanyl‐treated group, which instead exhibited continuous vasodilation (peak change 42 ± 10% of baseline immediately after injection, p <0.01, Figure C). While no mortality occurred with IV saline or ketamine, fentanyl caused an 18% mortality.

CONCLUSION

These results indicate that ketamine preserves, but fentanyl impairs, respiration and peripheral vasoconstrictor response to hemorrhage, although minor effects on initial compensation of systemic hemodynamics appear to be similar. The study suggests that microcirculatory responses may be more sensitive than systemichemo dynamics to predict outcomes during pain management following hemorrhage.

Support or Funding Information

This study was funded by the US Army Medical and Materiel Command, Clinical and Rehabilitative Medicine Research Program.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Age‐related Peripheral Vascular Dysfunction Predicts Cognitive Decline in Healthy Individuals
Authors: Csipo Tamas , Fulop Gabor Aron , Lipecz Agnes , Tarantini Stefano , Hand Rachel , Ngo Bich‐Thy , Dzialendzik Mikita , Balasubramanian Priya , Kiss Tamas , Yabluchanska Valeriya , Csiszar Anna , Ungvari Zoltan , Yabluchanskiy Andriy
Published: June 2021

BACKGROUND

Age‐related vascular cognitive impairment and dementia (VCID), defined as cognitive decline caused by cerebromicrovascular pathologies, is a major public health issue that affects >10 million individuals aged 65 and older in the US with annual healthcare costs totaling >$100 billion. Aging is a major risk factor for the development of VCID and current strategies for VCID treatment are limited to targeting cardiovascular risk factors such as hypertension, diabetes mellitus, etc. Here, we hypothesized that aging, similarly to other risk factors, affects vasculature uniformly and that age‐related changes in the peripheral circulation can be used as an important tool to predict cognitive decline.

METHODS

For our study, we have enrolled 55 healthy 22–92 yo individuals and divided them into two groups of young (<45 yo) and aged (>65 yo). Peripheral macrovascular and microvascular endothelial function was measured using a standard flow‐mediated dilation (FMD) and Laser Speckle Contrast Imaging tests (LSCI) in response to increased flow‐mediated shear stress after a 5‐min of blood flow restriction with arterial cuff. Pulse Wave Analysis was used to evaluate the augmentation index (AIx) as a measure of arterial stiffness. A principal component analysis (PCA) approach was used to create a vascular health index (VHI) that included all vascular measurements for each participant. Cognitive function including domain of learning, memory and executive function was measured via a selection of tests from the Cambridge Cognition CANTAB cognitive panel. A PCA approach was used to generate a cognitive impairment index for each participant.

RESULTS

Subjects from the aged group showed significantly impaired macrovascular endothelial function (FMD, 5.4±0.7% vs. 8.5±0.6% in young, p<0.01) and increased arterial stiffness compared to young (AIx 28.9±1.7% vs 4.5±2.6% in young, p<0.01). Microvascular endothelial function showed significantly lower skin perfusion in aged subjects (2.8±0.1% vs 3.4±0.1% in young, p=0.03) that correlated with FMD measurements (r=0.34, p=0.014), and significantly lower reperfusion rate in aged individuals (25.8±2.2PU/s vs 35.1±2.7 PU/s in young, p=0.01). PCA‐generated Vascular Health Index (VHI) showed a significant negative correlation with age (r=−0.55, p<0.01). VHI correlated with the Cognitive Impairment Index (r=−0.44, p<0.01).

CONCLUSION

Our study demonstrates that deterioration of peripheral macro‐ and microvascular endothelial function is associated with and can predict VCID in healthy aged individuals.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Kidney hyperfusion upregulates major histocompatibility complex expression in glomerular endothelial cells through c‐Jun N‐terminal kinase
Authors: Shi Yi , Zhu Dong , Tang Qunye , Meng Mei , Liu Wenjie , Zhu Tongyu , Zhang Yi
Published: June 2021

Objectives

The endothelium is the first line of defense while contacting with the immune system. The present study was investigated roles of antigen representing in glomerular endothelial cells.

Methods

C57/BL6 mice were randomly grouped into sham‐operated (2K) and 5/6 nephrectomy groups (5/6Nx). After whole body irradiation, mice were received splenic lymphocytes from either C57/BL6 or Balb/c mice. Human glomerular endothelial cells were cultured in vitro. Blood and renal cortex, and glomeruli were collected for measurement of kidney function, Western blotting, fluorescence‐activated cell sorting and enzyme‐linked immunosorbent assays.

Results

Four weeks after nephrectomy 5/6Nx mice had a normal renal function, but a significantly higher level of renal blood flow, compared with 2K groups, indicating that mice in 5/6Nx have hyper‐perfusion in the kidney. Both mRNA and protein expressions of major histocompatibility complex (MHC) II, CD80, CD86, and interferon γ were upregulated in glomerular endothelial cells of the 5/6Nx mice. Mice injected with lymphocytes from Balb/c mice, but not C57/BL6, had significantly higher levels of serum creatinine, confirming that kidney hyper‐perfusion initiate the immune reaction and the allograft lymphocytes transplant accelerates renal dysfunction. In cultured glomerular endothelial cells, interferon γ significantly upregulated protein expression of MHC II and MHC class I chain‐related genes A (MICA), but not MHC I. Interferon γ also increased transcription factor CIITA protein expression, as well as phosphorylation JNK. JNK inhibitor, SP600125 or JNK peptide, reduced protein expressions of CIITA, MHC II and MICA in cultured glomerular endothelial cells. In the 5/6Nx mice SP600125 or JNK peptide, reduced MHC II expression, but not CD80 or CD86 in the 5/6Nx mice, suggesting that JNK signalling plays a role in the regulation of MHC II protein.

Conclusion

In the present study, nephrectomy induces hemodynamic changes in the kidney, leading to increases in major histocompatibility complex protein expression in glomerular endothelial cells. Inhibition of JNK signalling reduces MHC expression in glomerular endothelial cells in vitro and in vivo.

Support or Funding Information

This work was supported by the National Nature Science Foundation of China (No 81573418) and ZSYXRC‐014

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Resting Coronary Blood Flow Velocity by Transthoracic Coronary Echocardiography is Higher in Patients with Cardiovascular Diseases Compared with the Healthy Adults
Authors: Gao Zhaohui , Blaha Cheryl , Cauffman Aimee , Sinoway Lawrence I.
Published: June 2021

Transthoracic Coronary Echocardiography (TCE), defined as blood velocity detection mainly in the left anterior descending artery (LAD) by noninvasive Doppler echocardiography, has been employed to measure coronary blood flow velocity and coronary flow reserve in research labs and in clinical settings. Most prior studies focused on the coronary flow reserve data, and normal values for resting coronary artery flow velocity have rarely been reported. The purpose of this study was to examine resting coronary blood flow velocity in healthy subjects and patients with cardiovascular disease. We retrospectively collected resting coronary blood flow velocity data by TCE from 69 healthy subjects (42 M, 27 F, 60 ± 8 yrs) and 37 patients (27 M, 10 F, 63 ± 10 yrs) with various cardiovascular diseases (14 peripheral arterial disease, 5 Pre‐TAVR, 8 hypertrophy cardiomyopathy, 10 CABG). The patient group had higher systolic blood pressure (135 ± 18 vs. 120±14 mmHg, P value <0.001), mean blood pressure (94 ± 11 vs. 89 ±10 mmHg, P value <0.05), and Rate‐Pressure Product (RPP, an index of myocardial oxygen demand) (8617 ± 1702 vs. 7249 ± 1453. P value < 0.001). Resting coronary blood flow velocities early (CBV1) and late (CBV2) in diastole were significantly higher in the patient group (CBV1: 27.6±8.6 vs. 19.0 ± 4.9 cm/s, P value < 0.001; CBV2: 17.6 ± 5.5 vs. 13.4 ± 3.2 cm/s, P value <0.001). After controlling for oxygen consumption (CBV/RPP), resting coronary blood flow velocity during the early diastolic period was still significantly higher in patients than in the healthy group (Adjusted CBV1: 3.3 ± 1.4 vs. 2.7 ± 0.8 au, P value < 0.001). There was no significant difference in adjusted coronary blood flow velocity during the late diastolic period (Patient group vs. healthy group, 2.1 ± 0.8 vs.1.9 ± 0.5 au, P value = 0.105). Further studies are needed to explore the clinical implication and importance of higher resting coronary blood flow velocity data by TCE in patients.

Support or Funding Information

Supported by National Institutes of Health Grants P01 HL134609 (Sinoway), and UL1 TR002014 (Sinoway).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Role of P62 Protein in Acute Oxidative Stress‐induced Ventricular Arrhythmias
Authors: Hwang Hyun Seok , Koh Yeojung , Rodriguez Jose , Salazar Gloria
Published: June 2021

Introduction

Chronic ischemia depletes myocardial cells of intracellular defenses from excessive oxidative stress often leads to ventricular arrhythmias. Recently, we reported that autophagy adapter protein, p62 modulate the SR Ca handling in myocytes under chronic myocardial injury. Interestingly, oxidant‐induced beta‐adrenergic signaling involved, it is little known how p62 protein contributes to oxidative stress‐related ventricular arrhythmias in the hearts.

Purpose

To investigate the role of p62 protein on acute ischemia‐induced arrhythmogenesis in the intact heart.

Methods

Isolated heart from wild‐type (WT) and p62 knockout (KO) mice (n=8 per group) were used at 10–12 weeks of age. Global ischemia protocol applied in isolated heart preparation. Electrocardiography (ECG) recorded during 15 min steady‐state, 30 min ischemia, and 60 min reperfusion to detect heart rate and arrhythmias (premature ventricular contractions; PVCs). At the end of the experiment, ventricles harvested to measure the expression level of proteins, p62, LC3‐I/LC3‐II, and oxi‐CaMII.

Results

The heart rate (sinus rate) at baseline was not different between the groups. After 30 min ischemia, sinus rate tends to decrease in only p62 KO hearts during the reperfusion. The incidence of arrhythmias significantly attenuated in the p62 KO heart compared to WT heart during the first 15 min of reperfusion (Premature ventricular contraction, PVC/min; WT, 2.71±1.16* vs. 0.26±0.10,* <0.05 vs. WT, Fig.). The deletion of the p62 gene has a beneficial effect on acute oxidative stress‐induced ventricular arrhythmias in the intact hearts.

Discussion

Our results suggest that p62 protein involve the oxidative stress‐induced arrhythmogenesis in the hearts, and it may be associated with the intrinsic beta‐adrenergic mechanism.

Support or Funding Information

Florida State University

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mapping neutrophil polarization over the myocardial infarction time continuum
Authors: Daseke M John , Valerio Fritz , Lindsey Merry
Published: June 2021

In response to myocardial infarction (MI), neutrophils are early responders that initiate the inflammatory reaction. However, whether they have other roles in cardiac wound healing has not been explored. We hypothesized that neutrophils may undergo polarization state changes over the MI time course. C57BL/6J male mice (3–6 months old) were subjected to permanent coronary artery ligation to induce MI, and neutrophils were isolated from the infarct region at days, 1, 3, 5, and 7 after MI. Day 0 served as negative no MI controls. Aptamer proteomics was performed on n=10–12 biological replicates for each time, and a total of 123 proteins passed quality control and were assessed. Day 1 MI neutrophils exhibited upregulation of cytokine signaling pathways (IL‐16), extracellular matrix (ECM) degradation (MMP‐8 and MMP‐9), and leukocyte recruitment (calgranulin B). Day 3 MI neutrophils exhibited a similar phenotype, with an increase in ECM reorganization and fibrinolysis (activin A, cathepsin D, and fibrinogen). At MI days 5 and 7, neutrophils displayed a more pronounced shift to ECM reorganization indicated by the continued increase in proteins directly involved in fibrotic pathways (fibrinogen, fibronectin, galectin‐3, and vitronectin). Our results indicate neutrophils show distinct protein expression profiles over the first week of MI. This is the first report to detail the changes in the neutrophil proteome over the first week of MI.

Support or Funding Information

NIH GM104357, GM114833, GM115428, HL051971, HL075360, HL129823, and VA 5I01BX000505

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Rosa laevigata Michx‐induced improvement of vascular dysfunction through PI3K/AKT‐mediated NO‐cGMP pathway in endothelial cells and 2K1C hypertensive rat
Authors: Kim Hye Yoom , Ahn You Mee , Hong Mi Hyeon Hong , Yoon Jung Joo , Lee Yun Jung , Kang Dae Gill , Lee Ho Sub
Published: June 2021

Rosa laevigata Michx has been used to treat chronic cough, arterial sclerosis, menstrual irregularities, and urinary incontinence. The purpose of the present study was to investigate the underlying cellular mechanisms of the nitric oxide (NO)‐mediated property of the aqueous extract of Rosa Laevigata Michaux (ARL). The roles of the NO signaling in the ARL‐induced effects were tested in HUVECs and aortic ring. HUVEC treated with ARL produced higher amount of NO compared to control. ARL increased in the expression and phosphorylation levels of eNOS and Akt in HUVECs, which were attenuated by L‐NAME, a NOS inhibitor, and wortmannin, a PI3K inhibitor. In addition, ARL‐induced dose‐dependent relaxation of phenylephrine‐precontracted aorta was abolished by removal of functional endothelium. Pretreatment with NO‐cGMP inhibited the ARL‐induced vasorelaxation. In addition, an upstream signaling molecule of eNOS, attenuated the ARL‐induced vasorelaxation. Incubation of endothelium‐intact aortic rings with ARL increased the production of cGMP, however, which were attenuated by L‐NAME and ODQ. Two‐kidney‐one‐clip (2K1C) hypertensive rats were established to investigate the hypotensive effect of ARL in renovascular hypertension. 2K1C rats were treated with ARL at dose of 100 mg/kg/day orally for 3 weeks. Consequently, ARL significantly lowered blood pressure, and ameliorates vascular relaxation in thoracic aorta. Taken together, the present study suggests that ARL ameliorates vascular dysfunction via the regulation of endothelium‐dependent activation of through the PI3K/Akt‐mediated NO‐cGMP signaling.

Support or Funding Information

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of Oryeongsan in 2K1C rats through regulation of renin‐angiotensin‐aldosterone system and ANP system
Authors: Ahn You Mee , Kim Hye Yoom , Kim Hyun Ju , Yoon Jung Joo , Lee Yun Jung , Lee Ho Sub , Cho Kyoung Woo , Kang Dae Gill
Published: June 2021

Oryeongsan (ORS) which was first recorded in SangHanLoon, the classic of traditional Chinese medicine describing treatments of disordered body fluid balance. Renal hypertension is caused by renovascular dysfunction through an activation of renin‐angiotensin‐aldosterone system (RAS). The RAS is one of the most important regulators in maintenance of body fluid homeostasis and in long‐term regulation of blood pressure. The purpose of this study is to identify effects of ORS on regulation of renal function, change of blood pressure and RAS in the two‐kidney one‐clip (2K1C) renal hypertensive rats. Experiments were performed in four experimental groups: sham rats, 100 mg/kg/day ORS in sham rats, 2K1C rats and ORS in 2K1C rats, respectively. After 4 weeks of operation, sham and 2K1C rats were administered with vehicle or ORS for 3 weeks. Urinary volume, urinary excretion of Na+, K+, Cl−, osmolality and creatinine were measured. The 2K1C rats showed significantly higher systolic blood pressure (SBP) compared with sham rats. ORS treatment for 3 weeks markedly decreased the SBP in 2K1C rats. ORS significantly increased urinary volume, urinary excretion of electrolytes, and clearance of creatinine (CCr) in 2K1C rats. ORS significantly suppressed plasma level of PRA, Angiotensin II and aldosterone in 2K1C rats. Also, ORS suppressed the upregulated RAS components in the kidney from 2K1C rats. However, ORS increased plasma level of ANP in 2K1C rats. These findings suggest that ORS may elicit renal and vascular effects via two ways: directly and indirectly through activation of the ANP system and inhibition of the RAS.

Support or Funding Information

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.