bluuebunny/crossref_metadata_2025 huggingface dataset:
atRA induces cholesterol accumulation through CYP7A1 repression in the liver
Authors: Won Kyoung‐Jae , Jeong Hyunyoung
Published: June 2021

All‐trans retinoic acid (atRA) is one of the most frequently used retinoids to treat certain cancers or dermatological diseases. Common side effects of atRA include hypercholesterolemia, leading to discontinuation of atRA uses. Previous studies have shown that atRA downregulates CYP7A1 (the rate‐liming enzyme converting cholesterol to a bile acid intermediate), potentially responsible for hypercholesterolemia. However, the detailed molecular mechanisms in atRA‐induced hypercholesterolemia is unclear. atRA increased cholesterol levels in a dose dependent manner in HepaRG cells, and this was accompanied by dramatic decrease in CYP7A1 mRNA and protein levels. CYP7A1 overexpression by lentiviral system reversed atRA‐induced cholesterol accumulation in HepaRG cells, suggesting that the cholesterol accumulation is mediated by CYP7A1 repression. CYP7A1 promoter reporter assays in HepG2 cells revealed that the HNF4α binding site in the promoter (−149/−118) is essential for atRA‐mediated CYP7A1 repression. HNF4α depletion by siRNA abrogated CYP7A1 repression by atRA in HepaRG cells. Western blot analysis revealed that atRA activates MAPKs signaling pathways including JNK, ERK, and p38 MAPKs in HepaRG cells. Pharmacological inhibition of JNK and ERK pathways but not p38 pathway by using a specific inhibitor (i.e., SP600125, PD98059, and SB203580 for JNK, ERK, and p38, respectively) attenuated atRA‐mediated CYP7A1 repression and cholesterol accumulation. Overexpression of AP‐1 (c‐Jun/c‐Fos), a downstream target of JNK and ERK, repressed CYP7A1 expression in HepG2 cells. In DNA pull‐down assay, AP‐1 exhibited sequence‐specific binding to the HNF4α binding site in CYP7A1 promoter following atRA treatment in HepaRG cells. Chromatin immunoprecipitation assay also showed that AP‐1 binding to the CYP7A1 promoter (−181/−34) was increased following atRA treatment, whereas HNF4α binding was decreased. Collectively, results from this study indicate that atRA‐activated JNK and ERK pathways and downstream target AP‐1 represses HNF4α transactivation of CYP7A1 promoter. These results suggest an important role of CYP7A1 in atRA‐induced cholesterol accumulation and provide a basis to establish future strategy in cholesterol disorder by atRA.

Support or Funding Information

This study was supported by R01 HD089455.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Suppression of Polycyclic Aromatic Hydrocarbon (PAH)‐Mediated Pulmonary Carcinogenesis in Mice by Omega‐3‐Fatty acids
Authors: Moorthy Bhagavatula , Jiang Weiwu , Wang Lihua , Maturu Paramahamsa , Zhou Guodong
Published: June 2021

3‐Methylcholanthrene (MC) and benzo[a]pyrene (BP) are potent polycyclic aromatic hydrocarbons (PAHs) carcinogens. PAHs are present in cigarette smoke, diesel exhausts, and charcoal broiled meats, etc. Cytochrome P450 (CYP) 1A/1B1 enzymes play key roles in the activation of PAHs to carcinogenic metabolites, which initiate carcinogenesis by binding covalently to DNA, and the adducts, if not repaired, could lead to tumorigenesis. In this study we tested the hypothesis that pre‐treatment of mice with omega‐3‐fatty acids, i.e. [eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) will lead to attenuation of PAH‐mediated pulmonary DNA adduct formation and pulmonary tumorigenesis. Twelve week old male and female A/J mice received EPA (60 mg/kg) and DHA (40 mg/kg) from day 1 to day 24. Control mice were treated with vehicle corn oil. On day 3, mice were treated with BP (40 μmol/kg) or 3‐methylcholanthrene (MC, 40 μmol/kg) by i.p. In the short‐term experiment (DNA adduct studies), 3–5 mice from each group were terminated at day 10 (7 days after BP or MC treatment). EPA/DHA significantly suppressed formation of BP‐DNA and MC‐DNA adducts in lung and liver of both male and female mice. In the long‐term experiment (tumor studies), 8 mice for each group continued to receive EPA/DHA twice per week till end of experiment (16 weeks). Pulmonary tumor incidence and multiplicity was significantly suppressed in mice given EPA+DHA, compared to those given the PAHs, BP or MC alone. PAH treatment led to enhancement of expression of epigenetic genes such as DNMT and EZH2 and repression of RUNX3 in lung. Mice given a combination of PAHs and EPA;/DHA attenuated DNMT and EZH2, but augmented RUNX3. These results suggest that EPA/DHA contribute to the suppression of PAH‐mediated mechanisms in part by modulating epigenetic genes. These studies also suggest that the omega 3‐fatty acids could be developed as novel candidates for the prevention of PAH‐mediated lung cancers in humans.

Support or Funding Information

R01ES 009132 and R01HL129794

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Obesity Alters the Expression of Suppressive Factors in Intra‐Tumoral Dendritic Cells Following Combinatorial Immunotherapy
Authors: Turbitt William , Boi Shannon , Orlandella Rachael , Gibson Justin , Norian Lyse
Published: June 2021

A growing literature suggests that obesity induces dysregulated antitumor immunity. We found previously that diet‐induced obesity (DIO) increases the abundance of intra‐tumoral regulatory dendritic cells (DCs) in a murine model of renal cancer. Furthermore, DIO reduces the efficacy of an immunotherapy consisting of intra‐tumoral delivery of replication‐deficient adenovirus (Ad) encoding tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) (AdT) plus class B oligonucleotides (CpG1826). This therapy primes endogenous T cells by targeting DCs that process and present tumor‐derived antigens released by TRAIL‐mediated tumor cell death. Our current study evaluated the efficacy of AdT/CpG combined with either anti‐PD‐1 or anti‐CTLA‐4 immune checkpoint blockade, in the absence or presence of DIO. BALB/c mice (7–8‐weekold) were randomized to standard chow or high‐fat diet for 20 weeks prior to the orthotopic injection of Renca‐Luc renal cancer cells into the left kidney and were maintained on diets until day 28 post‐tumor injection. At day seven post‐tumor injection, mice were re‐injected in the left kidney with saline or AdT/CpG. Mice were further randomized to receive intra‐peritoneal isotype control, anti‐PD‐1, or anti‐CTLA‐4. Both AdT/CpG+anti‐PD‐1 (p<0.001) and AdT/CpG+anti‐CTLA‐4 (p<0.001) resulted in a significant reduction in renal tumor outgrowth and spontaneous lung metastasis in lean mice on standard chow. AdT/CpG increased the accumulation of intra‐tumoral DCs independent of dietary or immunotherapy intervention, with the conventional DC two (cDC2) subset favored over the conventional DC one (cDC1) subset. Expression of PD‐1 and PD‐L1 on DC subsets was unchanged in response to diet or immunotherapy intervention; however, untreated tumor‐bearing DIO mice had elevated PD‐1 expression on cDC1s (p=0.022) compared to untreated lean mice or lean mice receiving AdT/CpG+anti‐PD‐1. Lastly, Arginase‐1 expression in cDC1s was not significantly different between groups; however, Arginase‐1 expression was elevated in response to DIO on cDC2s (p=0.006) compared to both lean and DIO AdT/CpG+anti‐PD‐1 treated mice. Future studies are needed to characterize the suppressive function of intra‐tumoral DC populations, as well as the T cell priming capacity of DCs within tumor‐draining lymph nodes in response to immunotherapy in the absence or presence of DIO. In conclusion, the differential response to immunotherapy and alteration in suppressive factors in intra‐tumoral DCs in DIO mice warrants further investigation, as targeting these pathways may ultimately enhance treatment strategies in overweight and obese cancer patients.

Support or Funding Information

RO1CA181088 and T32DK062710

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Central Cholinergic Pathways Diminish the Hypotensive and Cardiac Autonomic Depressant Effects of Endotoxemia in Male Rats: Role of Medullary NFκB/α7/α4β2 nAChR Signaling
Authors: Sallam Marwa M. , El‐Gowilly Sahar M. , Fouda Mohamed A. , Abd‐Alhaseeb Mohamed M. , El‐Mas Mahmoud M.
Published: June 2021

We have recently shown that systemically administered nicotine improves endotoxic manifestations of hypotension and cardiac autonomic dysfunction in rats. In this report, integrative and molecular studies were employed to investigate whether medullary antiinflammatory pathways of α7 and α4β2 nicotinic acetylcholine receptors (nAChRs) modulate cardiovascular derangements induced by endotoxemia. The i.v. administration of nicotine (50 mg/kg) to conscious rats abolished the lipopolysaccharide (LPS, 10 mg/kg i.v.)‐evoked: (i) falls in blood pressure, (ii) decreases in spectral measure of cardiac sympathovagal balance (ratio of the low‐frequency to high‐frequency power, LF/HF) and (iii) increases in the immunohistochemical protein expression of NFκB in medullary neurons of the nucleus tractus solitarius (NTS). These favorable influences of nicotine were replicated in rats treated intracisternally (i.c.) with PHA‐543613 (selective α7 nAChR agonist; 2 μg/rat), or 5‐iodo‐A‐85380 (5IA, selective α4β2 nAChRs agonist; 0.1 μg/rat). Measurement of arterial baroreflex activity by the vasoactive method revealed that nicotine, PHA, or 5IA reversed the depressant effect of LPS on reflex bradycardic, but not tachycardic, activity. Pharmacologic antagonist studies showed that the reversing effect of nicotine on LPS hypotension was partly inhibited in rats treated concomitantly with i.c. methyllycaconitine citrate salt hydrate (α7 nAChR antagonist, 10 μg/rat) or dihydro‐β‐erythroidine hydrobromide (α4β2 nAChR antagonist, 10 μg/rat), whereas the associated increases in LF/HF ratio remained unaltered. The data signifies the importance of the NTS inflammatory pathways in cardiovascular endotoxic manifestations and the capacity of brainstem circuits of α7 and α4β2 nAChRs in ameliorating these responses.

Support or Funding Information

Supported by the Science and Technology Development Fund, Egypt (STDF Grant No. 14895)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Central Effects of Angiotensin‐(1‐7) Treatment on Medullary MAPK and PI3K Pathways of Antenatal Glucocorticoid Exposed Adult Sheep are Sex Dependent
Authors: Hendricks Alexa S. , Shaltout Hossam A. , Chappell Mark C. , Diz Debra I.
Published: June 2021

Introduction and Hypothesis

Fetal exposure to betamethasone (BMX) that results from the clinical administration of the glucocorticoid agonist to women threatening premature delivery may lead to deleterious long‐term effects on central autonomic regulation. In the adult offspring of pregnant sheep administered antenatal BMX, mean arterial pressure (MAP) is higher and baroreflex sensitivity (BRS) is impaired. These responses are associated with lower brain medullary expression of Angiotensin‐(1‐7) [Ang‐(1‐7)]. Moreover, we observed sex differences in two key signaling pathways implicated in cardiovascular dysfunction and stimulation of oxidative stress in the dorsal brain medulla (DMM) that include higher mitogen‐activated protein kinase (MAPK) in BMX adult male offspring and an attenuated phosphoinositide 3‐kinase (PI3K) pathway in BMX adult female offspring. To establish whether reduced central Ang‐(1‐7) tone contributes to the BMX‐induced cardiovascular effects, we assessed both MAPK and PI3K pathways in the DMM of BMX sheep following 2‐week intracerebroventricular infusion with Ang‐(1‐7) or artificial CSF (aCSF). We hypothesized that in both BMX male and female sheep, Ang‐(1‐7) therapy will correct the observed disturbances in these two pathways.

Results

Although presented before, two weeks of Ang‐(1‐7) administration lowered blood pressure and improved baroreflex sensitivity and heart rate variability. In BMX male sheep Ang‐(1‐7) administration was associated with a significant reduction in MAPK activation in the DMM as detected by phosphorylated ERK 1/2 compared to the aCSF‐treated BMX male controls (0.20 ± 0.07 vs 1.04 ± 0.31; p = 0.01), with no change in the PI3K pathway as determined by comparable phosphorylated Akt expression (0.25 ± 0.088 vs 0.36 ± 0.019; p = 0.34). In contrast, BMX female sheep administered Ang‐(1‐7) showed no change in either phosphorylated ERK 1/2 (0.46 ± 0.16 vs 0.70 ± 0.24; p = 0.31) or phosphorylated Akt expression (0.36 ± 0.06 vs 0.33 ± 0.05; p = 0.50) despite that BMX alone reduced PI3K expression.

Conclusion

Central administration of Ang‐(1‐7) constitutes a potential therapeutic approach to improve autonomic dysfunction in both BMX exposed sheep; however, distinctly different signaling mechanisms in the DMM of male and female BMX sheep appear to be stimulated by Ang‐(1‐7).

Support or Funding Information

HD 047584

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Chronic Angiotensin‐(1‐7) Treatment Prevents Obesity Hypertension in Mice
Authors: Miller Amanda Joy , White Melissa C. , Bingaman Sarah S. , Arnold Amy C.
Published: June 2021

Obesity is associated with overactivation of the renin‐angiotensin system hormone angiotensin (Ang) II, which increases blood pressure via multiple mechanisms. Conversely, Ang‐(1‐7), a vasodilatory hormone that opposes the actions of Ang II, is deficient in animal models of obesity. Our previous data show that in obese mice chronic systemic Ang‐(1‐7) administration can attenuate weight gain by increasing energy expenditure as well as improve insulin sensitivity. In this study, we hypothesized that Ang‐(1‐7) would prevent the hypertension and increased sympathetic tone also associated with this mouse model of obesity. To test this, adult male C57BL/6J mice received 12‐week Ang‐(1‐7) (400 ng/kg/min) or saline infusion via subcutaneous osmotic mini‐pumps. Immediately following mini‐pump implantation, mice were placed on a 60% high fat diet (HFD) or a standard chow diet. The three groups of mice in this study were chow diet saline (chow; n=6), HFD saline (HFD; n=3) and HFD Ang‐(1‐7) [n=8]. Blood pressure was measured during the last week of treatment via a carotid artery catheter connected to a strain‐gauge transducer and blood pressure analyzer. Cardiac sympathetic and parasympathetic tone and sympathovagal balance were calculated from the blood pressure signal using spectral analysis frequency domain methods. Consistent with our previous studies, Ang‐(1‐7) attenuated weight gain in HFD mice (36±2 chow vs. 44±2, HFD vs. 38±1 grams HFD Ang‐(1‐7); P=0.013). Ang‐(1‐7) also prevented the rise in systolic blood pressure with HFD (105±6 chow vs. 134±9 HFD vs. 111±5 mm Hg HFD Ang‐(1‐7); P=0.031), with a similar trend for diastolic blood pressure (P=0.084). There was no significant effect of Ang‐(1‐7) on heart rate (P=0.973). The Ang‐(1‐7)‐mediated blood pressure lowering in HFD mice was not associated with significant changes in cardiac sympathetic tone (P=0.303), parasympathetic tone (P=0.115), or sympathovagal balance (P=0.521). These data suggest that Ang‐(1‐7) prevents obesity hypertension in mice perhaps through autonomic‐independent mechanisms. Overall, these findings provide rationale for targeting Ang‐(1‐7) for treatment of obesity‐related hypertension.

Support or Funding Information

HL122507

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Delayed Therapy with A Hydrogen Sulfide Donor, JK1, Protects against Pressure Overload ‐ Induced Heart Failure
Authors: Li Zhen , Kang Jianming , Xian Ming , Lefer David J.
Published: June 2021

Background and Hypothesis

Hydrogen sulfide (H2S) protects against heart failure by ameliorating oxidative stress, improving mitochondrial function, increasing nitric oxide bioavailability, and attenuating apoptosis. However, at present there is insufficient evidence supporting the cardioprotective effects of H2S in delayed‐treatment settings which more closely mimics the heart failure in human. In current study, we tested the efficacy of delayed therapy with a novel H2S donor, JK1, in a murine model of transverse aortic constriction (TAC) induced heart failure (HF).

Methods

Male C57/BL6J mice at 10 weeks (wks) of age were subjected to TAC for 18 weeks. JK1 (200 μg/kg/d, n=20) or the control compound that is devoid of H2S (200 μg/kg/d, n=18) was administered via i.p. injection twice per day starting at 3 wks post TAC. Echocardiography was performed at baseline and tri‐weekly for 18 weeks post TAC to assess LV structure and function. LV hemodynamics, cardiac fibrosis, vascular reactivity (isolated thoracic aorta), and exercise capacity were measured at the endpoint.

Results

Delayed treatment with JK1 preserved left ventricular (LV) ejection fraction (LVEF 54.6 ± 1.4% vs. 29.7 ± 2.9%, p < 0.01), reduced LV dilation (p < 0.01), and attenuated eccentric hypertrophy (p < 0.01). JK1 treated mice displayed lower LV end‐diastolic pressure (LVEDP 11.4 ± 0.8 mmHg vs. 22.9 ± 1.2 mmHg, p < 0.01) and improved relaxation time constant (Tau 7.2 ± 0.4 ms vs. 10.05 ± 0.6 ms, p < 0.01). Global cardiac fibrosis was markedly reduced in mice received JK1 treatment (5.8 ± 1.5% vs. 17.1 ± 2.4%, p < 0.01). In addition, acetylcholine‐mediated vasorelaxation was significantly improved in mice received JK1 treatment (Max. Relaxation, 66.1 ± 4.6% vs. 31.5 ± 3.6%, p < 0.01; EC50, 605.3 ± 115.9 nM vs. 161.8 ± 31.5 nM, p < 0.01). More importantly, mice received JK1 treatment had greater exercise capacity when compared to those received control (Distance, 0.520 ± 0.083 km vs. 0.089 ± 0.006 km, p < 0.05).

Conclusion

These results demonstrate that delayed administration of a novel H2S donor, JK1, significantly preserves systolic LV function, attenuates fibrotic remodeling, and improves LV hemodynamics. JK1 treatment provides benefits beyond the heart as it significantly ameliorates the damage to endothelium caused by HF. In addition, JK1 treatment markedly enhances the exercise capacity in HF. Studies are currently underway to define the molecular mechanisms involved in JK1‐induced cardiovascular protection.

Support or Funding Information
Kolaviron abrogates ischaemic heart reperfusion injury through enhancement of Akt/PKB and down‐regulation of p38MAPK/PARP/Caspase 3 signalling pathways
Authors: Oyagbemi Ademola Adetokunbo , Bester Dirk , Esterhuyse Johan , Farombi Ebenezer Olatunde
Published: June 2021

The study was designed to investigate the ameliorative effect of Kolaviron (KV) on ischaemic‐reperfusion injury in experimental rat models. Twenty male Wistar rats were randomly divided into 2 groups: Group one received corn oil as a vehicle and rats in group two were orally administered KV at 200 mg/kg for four weeks. After four weeks of KV pre‐treatment, hearts were excised and mounted on the Working heart perfusion system. Western blot analysis was carried out on frozen heart samples. From this study, there was a significant (p<0.05) reduction in the activity of catalase, superoxide dismutase and glutathione peroxidase with concomitant reduction in oxygen radical absorbance capacity (ORAC) in ischaemic rat heart of animals that received corn oil without KV pre‐treatment. Similarly, pre‐treatment of rats with KV led to a significant (p<0.05) decline in intracellular reactive oxygen species (ROS) and malondialdehyde. Western blots analyses revealed that KV significantly increased the expressions of total Akt/PKB, phosphorylated Akt/PKB at serine 473 and also caused a significant reduction in p38 MAPK, Caspase 3, and cleaved PARP. Combining all, we propose that KV offered notable cardioprotection via quenching of ROS and up‐regulation of pro‐survival sinalling pathways. KV may therefore offer a novel therepeutic window for cardiovascular and other heart related conditions

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
GPER Attenuates Angiotensin II‐Induced Oxidative Stress via cAMP‐Mediated Regulation of NOX4
Authors: OGOLA BENARD O. , Zimmerman Margaret A. , Sure Venkata N. , Katakam Prasad V.G. , Lindsey Sarah H.
Published: June 2021

Our previous work shows that the G protein‐coupled estrogen receptor (GPER) is protective in the vasculature and kidneys during angiotensin (Ang) II‐dependent hypertension, in part by inhibiting oxidative stress. In addition, we find that acute estrogenic signaling via GPER involves activation of the cAMP signaling pathway in vascular smooth muscle cells. Ang II stimulates the production of reactive oxygen species (ROS) via NADPH oxidase (NOX) complex activation, but whether GPER interferes with NOX activation and whether this occurs through cAMP signaling is not known. Therefore, the goal of the current study was to assess the molecular mechanisms by which GPER attenuates Ang II‐induced oxidative stress in vitro. A7r5 rat aortic smooth muscle cells were treated overnight with 100 nM Ang II in the presence of vehicle or 100 nM of the GPER agonist G‐1. ROS was measured via electron spin resonance spectroscopy (e‐scan) and CellRox™ Green fluorescence. NOX activity and expression were measured by NADP/NADPH ratio, real time RT‐PCR, and immunoblotting. Both e‐scan and CellRox experiments showed that Ang II increased oxidative stress, while co‐administration of the GPER agonist G‐1 prevented this increase (4.3 × 105 vs 1.9 × 105 AU/mg protein, P<0.001, n=7–8). Similarly, NOX activity was elevated in response to Ang II but normalized by G‐1 (189% vs. 83% RLU, P<0.0001, n=5). When the production of cAMP was inhibited with the adenylyl cyclase inhibitor SQ22536, G‐1 was unable to block Ang II‐induced NOX activity (153% vs. 142% RLU, P=0.39, n=6). Ang II increased NOX4 mRNA over time, with a maximum increase at 4h (3.5‐fold change, P<0.05, n=3). Ang II also increased NOX4 protein (1.2 vs 2.1 ADU/β‐actin, P<0.01, n=2). In contrast, administration of G‐1 for 4h decreased NOX4 mRNA by 60% (P<0.05, n=3) with no significant impact on protein expression (P=0.91, n=2). Preliminary studies using male wildtype and knockout mice show higher expression of NOX4 in response to genetic deletion of GPER. These experiments indicate that GPER inhibits NOX4 transcription and suppresses NOX activity, thereby limiting the production of ROS by Ang II. The impact of GPER on NOX activity was dependent on the production of cAMP. Taken together with our previous work, this study provides insight into how acute estrogen signaling provides cardiovascular protection during Ang II‐induced hypertension and potentially other diseases that are characterized by increased oxidative stress.

Support or Funding Information

This study was funded by National Institutes of Health grant HL133619 to S.H.L.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Enhanced expression of Egr‐1 in vascular smooth muscle cells from spontaneously hypertensive rats: role of Giα proteins
Authors: Polchtchikov Stephanie , Li Yuan , Anand‐Srivastava Madhu B.
Published: June 2021

Early growth response, Egr‐1, is known to activate the expression of several genes implicated in the development of vascular dysfunction. Earlier studies have shown that angiotensin II (AngII) enhances the expression of Egr‐1 and Giα proteins in vascular smooth muscle cells (VSMC). In addition, the role of enhanced levels of endogenous Ang II in enhanced expression of Giα proteins in spontaneously hypertensive rats (SHR) has also been shown. The present study was therefore undertaken to examine if VSMC from SHR also exhibit enhanced expression of Egr‐1 proteins and explore the underlying molecular mechanisms mediating this effect. The levels of Egr‐1 protein in VSMC from SHR were not different in 2 weeks old SHR as compared to WKY (Wistar‐Kyoto rats), however, it started increasing at 4 weeks and at 12 weeks, it was significantly increased by 80%. Knockdown of Giα‐2 proteins, using antisense and siRNA treatment, attenuated the enhanced expression of Egr‐1 proteins to control levels. In addition, the knockdown of Egr‐1 by siRNA also attenuated the expression of Giα‐2 and Giα‐3 proteins in VSMC from 12 week old SHR. The enhanced levels of Egr‐1 protein were attenuated by losartan (an AT1 receptor antagonist), BQ123 (an ETA receptor antagonist), BQ788 (an ETB receptor antagonist) but not by PD123319 (an AT2 receptor antagonist) in VSMC from 12 week SHR. These results suggest that VSMC from SHR exhibit an overexpression of Egr‐1 which is due to the enhanced levels of endogenous Ang II, ET‐1 and Giα proteins. In addition, Egr‐1 also regulates the expression of Giα proteins in VSMC from SHR and suggests a cross‐talk between Giα and Egr‐1.

Support or Funding Information

Supported by CIHR

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inhibitory effect and mechanisms of Crossostephium chinensis (L.) Makino on cell proliferation and migration of vascular smooth muscle cells
Authors: Wang Jieyu , Wu Chieh‐Hsi
Published: June 2021

Percutaneous transluminal coronary angioplasty (PTCA) is a common angioplasty used to relieve the narrowing and obstruction of coronary arteries. Restenosis occurs 30% to 50% of patients after PTCA. The mechanism of restenosis is principally neointinal hyperplasia and inflammatory, as stents resist arterial remodeling. Crossostephium chinensis, a traditional herbal medicine, is indicated on arthralgia, dehumidify and cure rheumatism. Previous studies showed that the extracts of C. chinensis have antioxidant, anti‐proliferative and anti‐inflammatory activities. Thereby, the purpose of this study was to explore the potential effect and mechanisms of C. chinensis on restenosis. Cell viability and migration were examined by MTT and trans‐well assays, respectively. The protein level was monitored by Western blot. Experimental data showed that extracts of C. chinensis can suppress the vascular smooth muscle cells (VSMCs) viability and migration. Protein levels of phospho‐AKT, mTOR, FAK, ERK, PDGFR‐β had been reduced by extract of C. chinensis. Our study suggested that C. chinensis can be a potential inhibitory remedy for restenosis.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Focal Inhibition of Thrombin Signaling With Anti‐Thrombin Nanoparticles Reduces Ischemia‐Reperfusion Injury in Acute Myocardial Infarction
Authors: Fazal Jawad , Chen Junjie , Weinheimer Carla , Kovacs Attila , Pan Hua , Wickline Samuel A.
Published: June 2021

Introduction

Although systemic anti‐coagulants are typically administered in acute coronary syndromes (either in anticipation of angioplasty or for secondary prevention of subsequent ischemic events), their role in mitigating potential damage directly associated with reperfusion is not clear. Here we test the hypothesis that inhibition of thrombin signaling selectively at the site of myocardial injury with anti‐thrombotic nanoparticles (NPs; which exert no systemic anticoagulant or platelet effects) could protect myocardium at risk and improve contractile function without bleeding.

Methods

About 13,000 copies of PPACK (phenylalanine‐proline‐arginine‐chloromethylketone) were conjugated per perfluorocarbon NP (~200 nm) and prepared by microfluidization. 32 mice underwent closed‐chest ischemia (LAD occlusion) for 90 min followed by reperfusion. 15 min before occlusion, either PPACK or control nanoparticles were injected intravenously. Echocardiography was performed (VEVO 2200) 24 hrs pre‐ and post‐ischemic reperfusion injury followed by harvesting heart tissue which was embedded in OCT for histological staining of H&E, Prussian blue, CD144/VE‐cadherin, p65, and thrombin.

Results

PPACK NP treated mice exhibited marked preservation of cardiac functional parameters as early as 24 hours after ischemia as compared to control mice according to changes in contractile metrics: 1) ΔEDV%: 40% ± 21% vs 77% ± 38% for treated vs control (p = 0.02) and 2) ΔESV%: 118% ± 43% vs 216% ± 108%, for treated vs control (p = 0.02) (Fig).

Conclusion

Anti‐thrombin NP therapy preserves myocardial function in the setting of acute myocardial injury without bleeding risk and implicates thrombin as a promising target for the prevention of ischemia‐reperfusion injury.

Support or Funding Information

This work was supported by NIH grant HL073646‐13.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Heparins Derived from Ovine (Sheep) Mucosa are Interchangeable to their Porcine (Pig) Counterparts
Authors: Kouta Ahmed , Abro Schuharazad , Clarizio Destini , Farooqui Ambar , Siddiqui Fakiha , Siddiqui Zafar , Bouchard Olivia , Fareed Jawed
Published: June 2021

Background and Purpose

Heparin is the most widely used anticoagulant drug for parenteral indications including surgical, interventional, and medical usage. Currently available heparin is obtained from porcine (pig) intestine. For Muslim countries, an alternate source of heparin, such as ovine (sheep) heparin, from non‐porcine origin may be more acceptable for religious reasons. The purpose of this study is to compare ovine heparin with currently available porcine heparin to demonstrate their biosimilarity.

Materials and Methods

Several batches of powdered porcine heparin were commercially obtained. Powdered ovine heparins were provided by Ronnsi Pharmaceutical Co., China. These heparins were compared for the anticoagulant (ACT, aPTT, TT), antiprotease (Anti‐Xa, Anti‐IIa), and USP potency cross‐referenced against USP reference heparin. Molecular profile studies were carried out as specified by U.S. FDA. Protamine titration studies were carried out for both preparations using various tests. The relative interactions of these heparins with HIT antibodies were also investigated using HIT antibodies obtained from patients employing platelet aggregation studies. Thrombin generation inhibition studies were also carried out.

Results

The molecular profile of porcine heparins were comparable and ranged from 15–18 kDA(p<0.05). However, the ovine heparins showed a slightly lower molecular weight in comparison to porcine heparins (14–16 kDA). The anticoagulant activities in all the assays were also comparable for the ovine and porcine heparins. The USP potency of several batches of porcine heparin ranged from 170–200 U/mg (mean=182+/−12) whereas the ovine heparin exhibited 160–210 U/mg (mean=184+/−16) potency (p<0.05). In the ACT studies both the bovine and porcine heparin provided similar results. In the protamine titration curves, both heparin preparations provided superimposable results. Similarly, in the HIT antibody assays, the behavior of ovine and porcine heparin was identical.

Conclusions

These studies clearly demonstrated that the ovine heparins exhibit comparable molecular profiles and anticoagulant activities. Furthermore, in the USP assay, the potency of these preparations were similar. Thus, ovine heparin should be considered biosimilar to its porcine counterpart and is expected to provide similar results in various clinical indications.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Heat shock protein 90 promotes morphine anti‐nociception in the spinal cord, but not in the brain, in a murine cancer induced bone pain model
Authors: Stine Carolyn , Lei Wei , Streicher John M.
Published: June 2021

Heat shock protein 90 (Hsp90) is a ubiquitous and highly expressed regulator for various signaling pathways. Hsp90 has been well studied as a potential cancer therapeutic target, and Hsp90 inhibitors such as 17‐AAG have been tested as anti‐cancer therapeutics in preclinical studies and clinical trials. However, there have been no reported studies as to whether or not treatment with an Hsp90 inhibitor could affect pain or opioid therapy in cancer patients. Our laboratory previously found that Hsp90 promotes opioid‐induced anti‐nociception in the brain and represses it in the spinal cord in tail flick, paw incision, and HIV neuropathic pain models, suggesting potential effects in cancer pain treatment. This study was designed to investigate the regulatory activity of Hsp90 on opioid induced anti‐nociception in cancer pain using a model of cancer induced bone pain (CIBP). To achieve this goal, female Balb/c mice were injected with 66.1 murine breast cancer cells in the intramedullary space of the right femur. Pain behaviors, including mechanical allodynia, spontaneous flinching, and guarding, were assessed prior to, 7 days, and 13 days after the surgery. Thirteen days after the injection with cancer cells, mice were given intracerebroventricular (icv) or intrathecal (it) injections with either 0.5 nmol 17‐AAG or 1% DMSO vehicle, followed by a 24 hour recovery. After 24 hours, all of the mice were subcutaneously injected with 10 mg/kg morphine and pain behaviors were assessed for two hours. We found that it administration of 17‐AAG greatly reduced morphine anti‐nociception in mechanical allodynia (~64% reduction) without changing spontaneous pain. Meanwhile, treatment with 17‐AAG via icv showed no effect on von Frey, flinching, or guarding. These results indicate that Hsp90 in the spinal cord, but not in the brain, modulates opioid‐induced anti‐nociception in cancer pain. These results also suggest that Hsp90 inhibitors given for cancer therapy could make opioid treatment less effective in cancer patients.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Epiligulyl oxide from Saussurea lappa inhibited allergic asthma using in vivo and in vitro experiments
Authors: Huang Jin , Lee Bo‐Kyung , Park Soo‐Jin , Kang Saeromi , Im Dong‐Soon
Published: June 2021

Background

The root of Saussurea lappa is used in Asian traditional medicine to treat asthma, rheumatism, and other conditions. Extracts of Saussurea lappa were reported to alleviate house dust mite‐induced atopic‐like dermatitis in Nc/Nga mice, and sesquiterpene lactones were isolated. To elucidate whether epiligulyl oxide, a sesquiterpene lactone has beneficial effects on allergic asthma, anti‐asthma effects were studied using female Balb/c mice and rat RBL‐2H3 mast cells

METHODS

Antigen‐induced degranulation was measured in vitro by measuring β‐hexosaminidase activity. A murine ovalbumin‐induced allergic asthma model was used to test the in vivo efficacy of epiligulyl oxide.

RESULTS

Antigen‐induced degranulation was inhibited by epiligulyl oxide. Potency Sensitization and challenge of ovalbumin induced allergic asthma responses. Administration of epiligulyl oxide decreased numbers of inflammatory cells, especially eosinophils, and reduced the expression and secretion of Th2 cytokines, including IL‐4 and IL‐13, in lung tissues and bronchoalveolar lavage fluid. Histologic studies showed administration of epiligulyl oxide reduced inflammatory signs and mucin production in lungs.

CONCLUSION

These findings provide evidence that the epiligulyl oxide has potential use as anti‐asthma therapeutics.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The comparative study of anti‐oxidative and anti‐melanogenic effects by Angelica tenuissima extract and its biotransformed
Authors: Sohn Eunsoo , Park Yuna , Lee Jin Woo , Lim Hyosun , Kim Youn Kyu , Koo Hyun Jung , Namkoong Seung , Jang Seon‐A , Sohn Eun‐Hwa
Published: June 2021

Angelica tenuissimae Nakai (Umbelliferae) has been used as a traditional medicine in Asia because of its various pharmaceutical efficacies. Fermentation produces different compounds from the original one and is secondary trial to augment the contents or compositions of beneficial components. Here, we retrieved 56 articles regarding to A. tenuissima from the SCOPUS database and analyzed recent R&D status and the biological activities of principal ingredients by bibliometric methods, and then investigated the anti‐oxidative and anti‐melanogenic activities of A. tenuissimae ethanol extract (ATE) and fermented ATE (FATE). HPLC was used to evaluate changes in the levels of biogenic amines in ATE and FATE. Depigmenting effects of ATE and FATE were also examined with B16F10 melanocytes. As a result, FATE had no cytotoxicity until 2000 ug/ml, and was superior to ATE in regulating melanin production and tyrosinase activity in α‐MSH‐stimulated B16F10 cells in dose‐dependent manners. Both of ATE and FATE showed a similar degree of anti‐oxidant activities. Therefore, our results suggest that FATE could be used as an ingredient in cosmeceutical agent with whitening and anti‐oxidative effects.

Support or Funding Information

This research was supported by the Ministry of Trade, Industry and Energy (MOTIE), Korea Institute for Advancement of Technology (KIAT) through the Encouragement Program for The Industries of Economic Cooperation Region, and Korea Institute of Science and Technology Information (KISTI).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Troponin Subunits are Expressed in the Rat Afferent Arteriole
Authors: Walsh Michael Patrick , Kathol Iris , Sutherland Cindy , Wang Xuemei , Loutzenhiser Rodger , Takeya Kosuke
Published: June 2021

Vascular smooth muscle cells of the renal afferent arteriole are unusual: they must be able to contract very rapidly in response to a sudden increase in systemic blood pressure, thereby protecting the downstream glomerular capillaries from catastrophic damage. We showed that this could be accounted for in part by exclusive expression, at the protein level, of the “fast” (B) isoforms of smooth muscle myosin II heavy chains in the afferent arteriole, in contrast to other vascular smooth muscle cells such as the rat aorta and efferent arteriole, which express exclusively the “slow” (A) isoforms (Shiraishi M et al (2003) FASEB J 17: 2284–2286). Since contraction of the more rapidly‐contracting striated (skeletal and cardiac) muscles is regulated by the thin filament‐associated troponin (Tn) system, we hypothesized that Tn or a Tn‐like system may exist in afferent arteriolar cells and contribute to the unusually rapid contraction of this tissue in response to increased intraluminal pressure. We examined the expression of TnC (Ca2+‐binding subunit), TnI (inhibitory subunit) and TnT (tropomyosin‐binding subunit) in vascular smooth muscle cells of the rat renal afferent arteriole at the mRNA level. Fast skeletal muscle and slow skeletal muscle/cardiac TnC isoforms and slow skeletal muscle and cardiac TnI isoforms were detected by RT‐PCR and confirmed by cDNA sequencing. Furthermore, cardiac and slow skeletal muscle TnI isoforms, but not fast skeletal muscle TnI were detected in isolated afferent arterioles at the protein level by proximity ligation assay. We conclude that, in addition to Ca2+‐mediated phosphorylation of myosin regulatory light chains, contraction of the afferent arteriole may be regulated by a mechanism normally associated with the much more rapidly contracting cardiac and skeletal muscles, which involves Ca2+ binding to TnC, leading to alleviation of inhibition of the actomyosin MgATPase by TnI and tropomyosin and rapid contraction of the vessel.

Support or Funding Information

Supported by the Canadian Institutes of Health Research

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Novel non‐canonical regulation of soluble VEGF/VEGFR2 signaling by mechanosensitive ion channel TRPV4 in endothelial cells
Authors: Kanugula Anantha Koteswararao , Adapala Ravi K. , Midha Priya , Cappelli Holly C. , Meszaros Gary j. , Paruchuri Sailaja , Chilian William M. , Thodeti Charles K.
Published: June 2021

VEGF and VEGFR2 signaling is the major regulator of endothelial functions such as proliferation, migration, and angiogenesis. While many studies on the modulation of angiogenesis focus on targeting VEGF signaling, mechanical forces also play critical role. We have recently shown that the mechanosensitive ion channel transient receptor potential vanilloid 4 (TRPV4) negatively regulates angiogenesis via modulation of Rho/Rho kinase‐dependent endothelial mechanosensing. Nevertheless, it is not known if a cross‐talk exists between TRPV4 and VEGF signaling. To explore this nexus, we silenced TRPV4 in endothelial cells (EC) and measured VEGFR2 expression, localization, and phosphorylation. Immunofluorescence analysis revealed significant localization of total‐VEGFR2 around the perinuclear (Golgi) compartment in control EC. Small interfering RNA (siRNA) knockdown of TRPV4 significantly decreased perinuclear localization of VEGFR2. Interestingly, we found increased phosphorylation of VEGFR2 at Y1175 and its localization to the plasma membrane in TRPV4 knocked‐down cells. We also found increased plasma membrane localization of phospho‐VEGFR2 Y1175 in TRPV4 null EC. Western blot analysis further revealed a significant increase in phospho‐VEGFR2 at Y1175 in whole cell lysates and membrane fractions of TRPV4 knocked‐down cells compared to control siRNA‐treated cells. Finally, TRPV4 deletion/knockdown also increased VEGF‐mediated migration with localization of phospho‐VEGFR2 at the leading edge of migrating EC. Taken together, our results suggest that TRPV4 channels regulate VEGFR2 localization and activation to identify novel cross‐talk between mechanical (TRPV4) and soluble (VEGF) signaling in EC.

Support or Funding Information

This work was supported by National Institutes of Health (NIH) R01HL119705 and National Cancer Institute (NCI) R15CA202847

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Microvascular Lattice: An Updated Paradigm of Flow Distribution Through Capillary Networks
Authors: Mendelson Asher , Ho Edward , Milkovich Stephanie , Goldman Daniel , Ellis Christopher
Published: June 2021

Background

Capillaries, as the smallest vessels in the microcirculation, represent the most essential site for oxygen delivery; yet, given their overwhelming density and complicated geometry, understanding how they integrate within the broader microvascular network has been challenging.

Classical microvascular flow modelling has used linear branching networks such that each capillary bundle (CB) corresponds to a unique arteriolar‐venular pair with one‐directional pathway of flow. However, it has long been observed in vivo that terminal arterioles supply multiple CBs and similarly, post‐capillary venules collect from multiple CBs. Such a network forms a lattice‐like structure with an alternating pattern of arterioles, CBs, and venules.

Objective

We aim to develop a mathematical model for the microvascular lattice that can be applied to study blood flow properties through capillary networks in skeletal muscle.

Methods/Results

Microvascular lattice networks are generated from our own rodent intravital videomicroscopy data and compared to data reported in the literature. Distinct CBs are selected and their anatomical features are delineated with 3‐D reconstruction software. Capillary hemodynamics (RBC velocity, RBC supply rate, hematocrit) are extracted from video data and used to characterize the flow within and between multiple CBs. The generalized architecture of a microvascular lattice is then extrapolated from our experimental data and applied to a dual‐phase steady‐state blood flow model. Flow properties of the lattice are compared to those of a classical linear branching network ‐ both containing the same number of CBs.

Conclusions/Impact

The microvascular lattice represents an updated paradigm for describing blood flow through capillary networks. This lattice structure raises important questions regarding how flow can be regulated to support local oxygen demand and how larger microvessels interact with capillaries within the microcirculation. Furthermore, CB functional data for a variety of microvascular conditions can be inputted into this lattice model to determine how alterations to capillary architecture affect overall flow through the microvascular network.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Role of Myocardial Endothelin‐Converting Enzyme in Promoting Coronary Arteriolar Constriction during Diabetes
Authors: Tsai Shu‐Huai , Hein Travis W. , Kuo Lih
Published: June 2021

Coronary blood flow is regulated, by adjusting the diameters of coronary arterioles, to meet the oxygen demand of the myocardium. The release of local metabolites by the myocardium upon metabolic activation can trigger nitric oxide (NO)‐dependent coronary arteriolar dilation. In diabetes, coronary blood flow regulation is compromised by impairing endothelium‐dependent NO‐mediated vasodilation. However, it is unclear whether the myocardium plays a role in altering coronary arteriolar constriction to endothelin‐1 (ET‐1), which has been shown to contribute to the development of diabetic cardiomyopathy. To address this issue, pig coronary arterioles were isolated with the surrounding myocardial tissue, cannulated and pressurized for functional study. The vasomotor function was analyzed using the videomicroscopic technique and compared with isolated arterioles without myocardium. Streptozocin‐injected pigs (2 wk of hyperglycemia; 524 ± 48 mg/dl; n = 7) or age‐matched control pigs (euglycemia; 91 ± 7 mg/dl; n = 7) were used to study the influence of myocardium on coronary vasomotor function under diabetes. Coronary arterioles with and without myocardial tissue from control pigs constricted to ET‐1 (0.1 nM) in a comparable manner. Without myocardium, incubating the arterioles with ET‐1 precursor big ET‐1 (0.1 μM) for 40 minutes caused a 32% constriction of the vessels. The vasoconstriction reached 53% in the presence of myocardial tissue. The enhanced big ET‐1‐induced vasoconstriction was inhibited 74% by ETA receptor antagonist BQ123, 24% by ETB receptor antagonist BQ788, and 87% by endothelin‐converting enzyme (ECE) inhibitor CGS35066. In diabetic pigs, coronary arteriolar constriction in response to big ET‐1 was enhanced by 26% compared with that from the euglycemic pigs, and the vasoconstriction was further increased in the presence of myocardial tissue. However, ET‐1‐induced coronary arteriolar constriction without surrounding myocardium was not altered by diabetes. The ECE1 protein expression was increased in the myocardium in diabetic pigs. In conclusion, myocardial ECE is capable of converting big ET‐1 to ET‐1 for coronary arteriolar constriction. Under diabetic condition, upregulated myocardial ECE1 expression appears to promote ET‐1 synthesis for coronary arteriolar constriction, and may contribute to coronary blood flow dysregulation and lead to insufficient blood supply to the heart.

Support or Funding Information

Supported by Baylor Scott & White Kruse Chair Endowment Fund, and NIH Grants R01EY023335 and R01EY024624

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.