bluuebunny/crossref_metadata_2025 huggingface dataset:
Integrin alpha 7 interacts with HtrA2 to induce prostate cancer cell death
Authors: Luo Jian‐Hua , Zhu Ze‐Hua , Yu Yan P
Published: June 2021

Integrins are a family of receptors for extracellular matrix proteins that have critical roles in human tissue development. Previous studies identified down‐regulation and/or mutations of integrin alpha 7 (ITGA7) in prostate cancer (PC), liver cancer, soft tissue leiomyosarcoma (STL) and glioblastoma multiforme. Here we report that expression of ITGA7 induced apoptosis in the human prostate cancer cell lines PC3 and DU145. Yeast two‐hybrid analysis revealed that the C‐terminus of ITGA7 interacts with HtrA2, a serine protease with a critical role in apoptosis. Expression of ITGA7 increases the protease activity of HtrA2 both in vitro and in vivo. Deletion of the HtrA2 interaction domain abrogates the cell death activity of ITGA7, while down regulation of HtrA2 dramatically reduced cell death mediated by ITGA7. In addition, site‐directed protease‐null mutant HtrA2S306A expression blocked apoptosis induced by ITGA7. Interestingly, interaction between ITGA7 and its ligand laminin 2 appears protecting against cell death, since depleting laminin beta 2 with a small interfering RNA (siRNA) significantly exacerbated apoptosis induced by ITGA7 expression. This report provides a novel insight into the mechanism by which ITGA7 acts as a tumor suppressor.
Role of Nrf2/antioxidant response element in mammary carcinogenesis
Authors: Becks Lisa , Prince Misty , Burson Hannah , Christophe Christopher , Itoh Ken , Yamamoto Masayuki , Orchard Elysse , McLarty Jerry , Zhang Songlin , Kleiner‐Hancock Heather E.
Published: June 2021

Nuclear factor erythroid 2‐related factor (Nrf2) is a positive regulator of the antioxidant response element (ARE). We hypothesized that Nrf2 knockout (KO) mice would be more susceptible to mammary carcinogenesis than their wild‐type littermates (WT). Female mice were dosed with medroxyprogesterone acetate and 7,12‐dimethylbenz[a]anthracene for a premalignant study (n=10) and a tumor study (34 WT, 30 KO mice). After 10 weeks (premalignant study), all mice treated with carcinogen exhibited marked intraductal proliferation, ductal carcinoma in situ and increased phospho‐p65‐ NF‐κB expression. However, no difference was found between the KO vs. WT mice. In striking contrast, the KO mice (tumor study) exhibited ~2‐fold increase in palpable mammary tumors and multiplicity, as well as a 10‐fold increase in tumor volume, particularly early on in the study. These results imply a possible link between Nrf2/ARE and mammary carcinogenesis. Also, since the premalignant lesions showed no apparent difference between KO and WT mice, NF‐κB activation was an early change, whereas other oncogenic factor(s) may be involved in the rapid malignant conversion in the KO mice. Supported by: ASPET; LSU Health Sciences Foundation; BRF; Intramural Award, and NCI 1K22CA102005‐01A2.
Capsaicin displays anti‐proliferative activity of human small cell lung cancer via recruitment of E2F4 to downstream target genes
Authors: Dasgupta Piyali , Brown Kathleen C. , Witte Ted R. , Luo Haitao , Hardman W. Elaine , Chen Yi C.
Published: June 2021

Capsaicin the pungent ingredient of chili peppers can inhibit the growth of several types of human cancers in both cell culture and animal models. Depending on the nature of the cells, capsaicin induces cell cycle arrest at the G1/S boundary or causes apoptosis. Although, the signaling pathways underlying the apoptotic effects of capsaicin are well studied, very little is known about the molecular mechanisms underlying capsaicin‐induced cell cycle arrest. Our study showed that capsaicin displays anti‐proliferative activity in human small cell lung cancer cells (SCLC). The treatment of human SCLC cells with capsaicin resulted in downregulation of E2F‐responsive S‐phase genes like cdc6, cdc25A and cyclin E. The dietary administration of capsaicin led to reduction in the size of established SCLC tumors in nude mice. Furthermore, capsaicin suppressed tumor growth in CAM assays. The anti‐proliferative effect of capsaicin was mediated via E2F4 and resulted in enhanced recruitment of E2F4 and p130 on E2F‐responsive proliferative promoters, leading to repression of their transcription. Our findings suggest that the anti‐proliferative effects of capsaicin could be useful in the therapy of human SCLCs. Funding for our study was supported by a COBRE Pilot Grant Research, the PhRMA Foundation, an MU‐ADVANCE Fellowship, and the Flight Attendant Medical Research Institute YCSA Grant.
Mucopolysaccharidosis Type IIIB and GalNAc Transferase Double Knockout Mice Manifest Early Onset Fatal Leukodystrophy
Authors: Whitley Elizabeth M. , Snella Elizabeth M. , Dierenfeld Ashley D. , Echevarria Franklin D. , Ellinwood N. Matthew
Published: June 2021

Mucopolysaccharidosis type IIIB (MPS IIIB) causes progressive mental degeneration and early death due to lysosomal accumulation of heparan sulfate resulting from deficient activity of N‐acetyl‐α‐D‐glucosaminidase. Lysosomal accumulation of GM2 and GM3 gangliosides by unknown mechanism also occurs. We tested the benefit of eliminating GM2 storage in MPS III, by breeding MPS IIIB/GalNAc transferase double knockout (KO) mice. Rather than an improved outcome, double KO mice were short lived (p < 0.0001); 20 ± 3.4 wks, versus 45 ± 6.9 wks (MPS IIIB). Double KO mice had hind limb ataxia and hyper‐extension in contrast to single KO or normal mice, which appeared normal. Brain tissue of MPS IIIB‐affected, GalNAcT deficient, double KO, or wild‐type mice was examined histologically. In double KO mice, white matter showed prominent vacuolization and degeneration of nerve fibers. In contrast, MPS IIIB mice showed only intracytoplasmic vacuoles of many neurons and glia. Double KO mice likely succumbed to axonopathic and neuropathic effects in white matter tracts. This double KO model may prove useful as an improved model to investigate neuropathology and to assess therapy for MPS IIIB. Funded by the Sanfilippo Children's Research Foundation, the Lysosomal Storage Disease Research Consortium, the Center for Integrated Animal Genomics at ISU, and the State of Iowa Board of Regents.
AAV‐mediated sFLT‐1 gene therapy ameliorates retinal lesions in Ccl2/Cx3cr1 deficient mice
Authors: Tuo Jingsheng , Pang Ji‐Jing , Cao XiaoGuang , Shen Defen , Zhang Jun , Scaria Abraham , Wadsworth Samuel C , Pechan Peter , Hauswirth William W , Chan Chi‐Chao
Published: June 2021

To test the effect of subretinal delivery of adeno‐associated virus (AAV)‐sFLT‐1 (soluble VEGF receptor‐1) on retinal lesion in ccl2−/−/cx3cr1−/− mice, a murine model of age‐related macular degeneration (AMD), the AAV5‐sFLT‐1 was injected subretinally to the right eyes of ccl2−/−/cx3cr1−/− mice. The left eyes were untreated and served as controls. Three months after the injection, the treated eyes showed significant improvements in the retinal lesions as compared to the left eyes. The average lesion scores obtained by analyzing sebsequent fundus pictures were 0.23±0.14 in the right eyes versus 0.85±0.19 in the left eyes. The histological scores between the right and left eyes from 10 pairs of eyes revealed a decrease in score in 6 pairs, no change in score in 2 pairs, and an increase in score in 2 pairs. Retinal ultrastructure also showed fewer lipofuscin granules and better preserved photoreceptors in the right eyes than the left eyes. A2E was lower in the right eyes than the left eyes. We concluded that local AAV5‐CBA‐ sFLT‐1 can either reverse or stabilize the progression of retinal lesions in Ccl2/Cx3cr1 deficient mice. Lowering retinal expression of VEGF‐A by trapping excess VEGF‐A using sFLT‐1 may result in an amelioration of the diseased retina. The findings support the beneficial effects of sFLT‐1 gene therapy for AMD. Supported by the Intramural research program of NEI
Amelioration of STZ induced Type I diabetic nephropathy in rats by a phytomedicine: Gmelina arborea
Authors: Kulkarni Yogesh Anant , Veeranjaneyulu Addepalli
Published: June 2021

Diabetic nephropathy is one of the main causes of end stage renal disease (ESRD) leading to morbidity and mortality. Gmelina arborea is an important phytomedicine used in traditional system for treatment of diabetes. The objective of the present study was to investigate the effects of methanol extract of Gmelina arborea bark (ME) in streptozotocin (STZ) induced diabetic nephropathy in rats.

Diabetes was induced by streptozotocin (55 mg/kg, i.p.) in Wistar rats. After 4 weeks of STZ administration, the animals were divided into three groups‐ Group I diabetic control, Group II and III, ME treated (250 and 500 mg/kg/day, for 4 weeks). Age matched control rat received sodium citrate buffer.

Different biochemical parameters like glucose, albumin, blood urea nitrogen (BUN) and creatinine were assessed. The oxidative stress was measured by renal malonaldehyde, reduced glutathione and the anti‐oxidant enzymes superoxide dismutase and catalase. The histopathological study of kidney was carried at the end of the treatment. Results indicated significant improvement in BUN, creatinine, albumin and glucose after treatment with ME. Treatment for 4 weeks with ME significantly reduced the oxidative stress parameters. The kidneys of diabetic rats showed marked histological changes like glomerular thickening, tubular vacuolations and moderate interstitial fibrosis. The treatment with ME at selected doses of 250 and 500 mg/kg decreased the glomerular and interstitial alterations diabetic kidney. The results of the present study suggested, Gmelina arborea treatment can be effective in diabetic nephropathy.
Toll‐like receptor 4 deficiency attenuates insulin resistance and endothelial dysfunction associated with obesity and diabetes in mice
Authors: Liang CF , Xu Aimin , Vanhoutte Paul M
Published: June 2021

The present study analyzes, in mice with loss‐of‐function mutation of TLR4, the role of TLR4, a target for saturated fatty acids, in modulating metabolism and endothelial function.

A type‐2 diabetes model with double knockout (DKO) of leptin receptors (Lepr) and TLR4, was obtained by crossing Leprdb/+ and TLR4−/−− mice. Glucose and insulin tolerance tests were performed. Isometric tension was measured in carotid artery rings with or without endothelium.

DKO mice had lower fasting serum levels of glucose, triglycerides and cholesterol and better insulin sensitivity than Leprdb/db control mice. Acetylcholine‐induced endothelium‐dependent contractions (EDCF‐responses) of the carotid arteries were enhanced by genetic obesity, but were attenuated in DKO mice. They were inhibited by indomethacin and SC560, suggesting the involvement of COX‐1. Apocynin, MnTMPyP, catalase, DPI, DETCA, but not deferoxamine and tiron inhibited EDCF‐responses, suggesting a role for H2O2. Inhibitors including SP600128 [JNK], PD98058 [ERK] and SB203508 [MAPK] reduced the contractions in arteries of control but not of DKO mice indicating a mechanism downstream of TLR4. Rotenone and antimycin A inhibited the EDCF‐response implying that mitochondria are involved.

Thus, TLR4 deficiency can prevent insulin resistance and endothelial dysfunction possibly by decreasing oxidative stress in mitochondria.
Ruboxistaurin attenuates hypertriglyceridemia in diabetic rats: Comparison with the antioxidant N‐acetylcysteine
Authors: Liu Yanan , Lei Shaoqing , Liu Hui‐min , Mao Xiaowen , Wong Gordon T , Vanhoutte Paul M , Irwin Michael G , Xia Zhengyuan
Published: June 2021

Hyperglycemia‐induced oxidative stress plays a critical role in the development of diabetic metabolic complications including hypertriglyceridemia. The beta isoform of Protein kinase C (PKCβ) is over‐expressed in various organs of diabetic rodents and can cause or exacerbate oxidative stress by activating NADPH oxidase, a major source of superoxide production in the cardiovascular system. We postulated that selective PKCβ inhibition with ruboxistaurin (RXT) may attenuate hypertriglyceridemia in diabetes by reducing oxidative stress. Control or streptozotozin‐induced diabetic rats were either untreated (C, D) or treated with RXT (1 mg/kg/day, D+RXT) or with the antioxidant N‐acetylcycsteine (NAC) (1.5g/kg/day, D+NAC) delivered by oral gavage for four weeks. Levels of 15‐F2t‐isoprostane (IsoP), a specific marker of oxidative stress, were significantly increased in D group in both the myocardium and plasma. This was accompanied by a marked increase in plasma triglycerides (P<0.05 vs. C). RXT and NAC, respectively, prevented the increase of IsoP and significantly attenuated the increases of triglycerides. However, triglycerides in the D+NAC group was lowered further than that in the D+RXT group (P<0.05). Despite similar antioxidant properties, ruboxistaurin seems to be inferior to NAC in reducing hypertriglyceridemia in diabetes.

Supported by RGC/GRF grants (781109 M to ZX, 766709 M to MGI).
Co‐regulation of Cardioprotective Gene Expression by NF‐kappaB, AP‐1, and STAT3 in Late Phase Ischemic Preconditioning
Authors: Tranter Michael , McGuinness Myc , Paulding Waltke , Haar Lauren , Ren Xiaoping , Jones W. Keith
Published: June 2021

Introduction

We have shown that NF‐κB regulates the expression of over 200 genes in late ischemic preconditioning (IPC) and its activation is necessary for the cardioprotection of late IPC. We have also demonstrated that Hsp70.3 is one of these NF‐κB regulated genes whose expression is necessary to elicit late IPC protection.

Results

Using a novel non‐viral DNA delivery agent to apply transcription factor decoys to the in vivo murine heart, we show that, in addition to NF‐κB, transcriptional blockade of AP‐1, or STAT3 prior to IPC abrogates the late phase of cardioprotection. We hypothesize that NF‐κB, AP‐1, and STAT3 co‐regulate a common subset of genes, including Hsp70.3, that are critical for late phase IPC cardioprotection. Results using in vitro reporter assays demonstrate coordinated regulation of the Hsp70.3 promoter by NF‐κB, AP‐1, and STAT‐3. We also used PCR based gene microarrays to examine in vivo expression changes of 238 previously identified NF‐κB‐dependent genes under conditions of AP‐1 and STAT3 inhibition after IPC to identity additional genes co‐regulated by this trio of IPC requisite transcription factors.

Conclusions

The set of genes whose expression is co‐dependent on multiple cardioprotective transcription factors after IPC are likely to underlie common critical pathways of cardioprotection and may represent novel therapeutic targets for the treatment of ischemic heart disease.

This work was supported by NIH grant R01 HL63034 (WKJ).
Myocardial TRPV activation associated with high fat diet and cardioprotection
Authors: Haar Lauren Lynn , Rubinstein Jack , Tranter Michael , Jiang Min , Jones W Keith
Published: June 2021

Clinical data suggests obese patients have improved outcomes after myocardial infarction as compared to non‐obese patients. It has been shown that cardioprotective effects of high fat diet are dependent upon transient receptor potential vanilloid 1 (TRPV1) channel activation. However, there are six TRPV channels and, to date, little is known about the expression or function of TRPV 2–6 in the heart. The goal of this study was to investigate the expression of the six TRPV channels in the myocardium and to evaluate their potential role in the link between obesity/high fat diet and cardioprotection. Our results show expression of all six TRPV genes in the myocardium. In addition, mice subjected to ischemic stimuli or maintained on a high fat diet display increases in TRPV channel expression. We hypothesize that TRPV channel activation by high fat diet plays a role in modulating myocardial function and cell survival. To understand the role of TRPV channels in the myocardium, we utilized echocardiography and examined the effect of TRPV channel activation, using relatively selective pharmacological agents, on in vivo cardiac function. We also investigated the expression and effect of TRPV channel activation on the function of isolated cardiomyocytes. TRPV channels play an important role in regulating cardiac function and may represent potential therapeutic targets for the treatment of cardiac pathologies.

This work was supported by NIH grant R01 HL63034 (WKJ).
SR Ca2+‐handling proteins and PKC: a role in low molecular weight FGF2‐mediated cardioprotection
Authors: Oloizia Brian , Bodmer Janet , Schultz Jo El
Published: June 2021

Fibroblast growth factor 2 (FGF2) protects against cardiac dysfunction caused by ischemia‐reperfusion (I/R). Our lab has shown that mouse models overexpressing all FGF2 isoforms or expressing only the low molecular weight (LMW) isoform (high molecular weight knockout, HMWKO) improved cardiac function following I/R, while ablation of FGF2 (FGFKO) decreased function. Protein kinase C (PKC), a modulator of Ca2+ homeostasis via phosphorylation of sarcoplasmic reticulum (SR) proteins, contributes to FGF2‐induced cardioprotection; yet the role of PKC and SR proteins in LMW FGF2‐mediated cardioprotection is unknown. Here, expression and phosphorylation of multiple SR proteins, including phospholamban (PLB), were measured in non‐ischemic WT, FGFKO, and HMWKO hearts to determine if these proteins may prime against cardiac I/R injury. Also, WT, FGFKO and HMWKO hearts underwent an ex vivo work‐performing heart model of I/R, and phospho‐Thr17 PLB was measured in the absence or presence of a PKCε inhibitor, to elucidate what role phospho‐PLB and PKCε may play in LMW FGF2‐induced protection against cardiac dysfunction. Significant changes in phosphorylation of SR proteins in HMWKO hearts prior to and during I/R suggest that Ca2+ handling proteins may play a role in LMW FGF2‐induced protection against myocardial dysfunction and that these changes may be mediated by PKC. Work supported by NIH T35 DK 60444 and HL075633.
Endothelin‐1 blockade with bosentan attenuates15‐F2t‐Isoprostane adverse effects on postischemic rat hearts
Authors: Xia Zhengyuan , Liu Hui‐min , Xia Zhong‐yuan , Liu Yanan , Lei Shaoqing , Mao Xiaowen , Irwin Michael G
Published: June 2021

15‐F2t‐Isoprostane (IsoP), a specific marker of oxidative stress, is increased after myocardial ischemia. It exerts deleterious effects on postischemic myocardium. We hypothesized that IsoP exacerbates post‐ischemic myocardial injury by stimulating endothelin‐1 (ET‐1) production. Adult rat hearts were perfused by the Langendorff technique. Global myocardial ischemia was induced by stopping perfusion for 40 min followed by 60 min of reperfusion. Hearts were randomized to one of five groups: untreated (C), treated with IsoP (100 nM) or the ET‐1 receptor A/B antagonist bosentan (1 μM) alone or in combination 10 min prior to ischemia and for 15 min during reperfusion or treated with IsoP as above plus delayed bosentan administration 15 min after reperfusion. Coronary effluent ET‐1 levels in the IsoP group were higher than those in the C group during reperfusion accompanied with increased release of cardiac‐specific creatine kinase, reduced cardiac contractility and increased myocardial infarct size (all p<0.05 vs. C). Bosentan administration during early reperfusion exacerbated the IsoP deleterious effects while delayed administration attenuated it. It is concluded that IsoP‐induced ET‐1 production during later reperfusion is detrimental to functional recovery of damaged myocardium while ET‐1 increase during early reperfusion seems to improve it.

Supported by NSFC grants (30872447 and 30672003)
Preconditioning by sub‐lethal ischemia/reperfusion or glibenclamide ameliorates lactate dehydrogenase activity to different extends in rat hippocampus and cerebral cortex (brain) subjected to ischemia/reperfusion injury
Authors: Nassar Noha N , Abdallah Dalaal M. , Abd‐El‐Salam Rania M , Abdel‐Rahman Abdel A
Published: June 2021

Sub‐lethal short term ischemic exert adaptive responses which may protect from lethal ischemia, a phenomenon referred to as ischemia preconditioning (IPC). In the CNS, IPC causes increased tolerance to lethal ischemia reperfusion (I/R) injury; however, the precise mechanism is not fully understood. Similarly, glibenclamide, a selective blocker of K+(ATP) channels, was reported to protect against I/R injury via a preconditioning mechanism. Hence, the present study aimed to investigate the role of free radicals as well as pro‐ and anti‐inflammatory cytokines in rats exposed to a I/R injury. To this end, 32 male Wister Rats were divided into 4 groups, group I served as sham operated control, group II was subjected to 15 min ischemia by occlusion of both carotid arteries, followed by 60 min reperfusion, group III injected glibenclamide (1 mg/kg, i.p) 10 min before ischemia (15 min) followed by reperfusion (60 min), and in group IV, preconditioned (IPC) rats were subjected to 3 episodes of ischemia (3 min) followed by reperfusion (10 min) afterwards animals underwent ischemia (15 min) followed by reperfusion (60 min). I/R increased LDH activity, as well as lipid peroxides level, and decreased total antioxidant capacity (TAC) and IL‐10 level in the hippocampus; however, just cortical LDH and TCA levels were deranged. IPC and glibenclamide before I/R improved only LDH activity in the cerebral cortex and partly in the hippocampus. Hence, the study shows that both IPC and glibenclamide impart neuroprotection independent of free radical scavenging nor cytokine production in rats subjected to I/R injury.
Interaction of tricyclic antidepressants with the human (h)α4β2 nicotinic receptor
Authors: Blaty Doug E , Arias Hugo R , Rosenberg Avraham , Targowska‐Duda Katarzyna M , Feuerbach Dominik , Jozwiak Krzysztof , Moaddel Ruin , Wainer Irving W
Published: June 2021

The interaction of tricyclic antidepressants (TCAs) with human (h)α4β2 nicotinic receptors (AChRs) was compared to that for mecamylamine. The results established that: (a) [3H]imipramine binds to hα4β2 AChRs with relatively high affinity, but imipramine does not differentiate between desensitized and resting AChRs, (b) although TCAs inhibit (±)‐epibatidine‐induced Ca2+ influx with potencies in the same concentration range as mecamylamine, TCAs inhibit [3H]imipramine binding to hα4β2 AChRs with affinities >100‐fold higher than mecamylamine. This can be explained by our docking results where neutral imipramine and mecamylamine interact with leucine (position 9′) and valine (position 13′) rings by van der Waals contacts, whereas protonated mecamylamine interacts electrostatically with the outer ring (position 20′), (c) van der Waals interactions are in agreement with the enthalpic and entropic components of imipramine binding. In conclusion, our data indicate TCAs and mecamylamine efficiently inhibit the ion channel by interacting at different luminal sites. Support: Science Foundation Arizona.
Effects of varenicline on the discriminative stimulus effects of nicotine in female cynomolgus monkeys
Authors: Blaylock Brandi L , Nader Susan H , Nader Michael A
Published: June 2021

Nicotine (NIC) is a widely abused drug, primarily consumed through tobacco products. Varenicline (VAR), a nicotinic acetylcholine receptor partial agonist, is an FDA‐approved pharmacotherapy for smoking cessation. Despite its clinical use, little preclinical research exists to understand how VAR reduces the abuse liability of NIC. Though VAR purportedly reduces the reinforcing effects of NIC, the effects of VAR on the subjective effects associated with NIC are not conclusive. To examine this relationship, we investigated the effects of VAR alone and in combination with NIC in a two‐choice, food‐reinforced, drug discrimination procedure in which ovariectomized female cynomolgus monkeys (n=4) were trained to discriminate 0.3 mg/kg (base) NIC from saline (i.m., 10‐min presession). Preliminary data suggest that VAR (0.03–0.3 mg/kg, base, i.m., 10‐min presession) does not engender NIC‐like discriminative stimulus effects when substituted for NIC, nor affect response rates. When administered 10 min prior to NIC, VAR (0.1–0.3 mg/kg, i.m.) potentiated the rate‐decreasing effects of NIC without affecting discrimination; however, VAR blocked the NIC‐like discriminative stimulus effects when administered 2 hours prior to the training dose of NIC. These results suggest VAR has biphasic effects – initially interacting in a synergistic manner with NIC, but antagonizing NIC at later time points. DA12460
The ability of repeated nicotine exposure to alter locomotor activity and prefrontal cortex CREB phosphorylation in adolescent rats depends on sensation‐seeking phenotype
Authors: Philpot Rex Montgomery , Engberg Melanie Elizabeth , Wecker Lynn
Published: June 2021

Individuals with a high sensation‐seeking (HSS) phenotype are more vulnerable to addiction than those with a low SS (LSS) phenotype. Differences in SS may involve alterations in cAMP response element binding protein (CREB) in the prefrontal cortex (PFC), which regulates both impulsivity and reinforcement. We determined whether repeated nicotine had phenotype‐specific effects on CREB phosphorylation in the PFC. Adolescent rats (PND 31), characterized as HSS or LSS based on initial activity in a novel open field, received 8 daily injections of saline or 0.4, 0.6, 1.2 or 1.6 mg/kg nicotine bitartrate (sc) from PND 35–42; activity in a familiarized open field was measured, and CREB and pCREB determined 18 hours after the final injection. Maximal locomotor activity of HSS rats was apparent following 4 days of 0.4 mg/kg nicotine, where LSS rat activity peaked following the 8th injection of 0.6 mg/kg nicotine. CREB in PFC from HSS or LSS controls did not differ and was unaffected by any dose of nicotine. pCREB was greater in PFC from control HSS than LSS rats, and nicotine increased pCREB, with a greater effect in PFC from LSS animals and a maximal effect equal to that in PFC from HSS animals. Results indicate that repeated nicotine exposure during adolescence has differential effects on both locomotor activity and CREB that mask initial phenotypic differences (Supported by NIAAA AA016449)
Acute donepezil treatment does not attenuate the deficit in learning observed in Ts65Dn mice, a model of Down syndrome
Authors: Wenger Galen R.
Published: June 2021

The Ts65Dn mouse, a model of Down syndrome, displays a deficit in learning when responding under an incremental repeated acquisition (IRA) schedule of milk presentation. This study examined whether acute administration of donepezil prior to testing would attenuate the learning deficit. Adult male, Ts65Dn and littermate control mice were trained to respond under an IRA schedule. When responding stabilized, an acute dose‐response curve was obtained for donepezil (1–5.6 mg/kg, ip). Following saline administration, Ts65Dn mice earned fewer milk presentations, responded at a lower percent accuracy and had a slower rate of responding than littermate controls. Low doses of donepezil ( 1 and 1.8 mg/kg, ip) given one hour prior to testing did not alter performance in any way. A dose of 3 mg/kg decreased the number of reinforcers earned, and the rate of responding in the Ts65Dn mice, but not in the littermate controls. Following 5.6 mg/kg, rate of responding and the number of milk presentations were decreased in Ts65Dn and littermate control mice. The 5.6 mg/kg dose also decreased the percent correct responding in Ts65Dn mice but not that of littermate controls. These results show that acute administration of donepezil does not attenuate the learning deficit in Ts65Dn mice or increase the learning performance of control mice responding under the IRA task. Supported by NIH grant HD047656.
Phenotypical characterization of NPS precursor knockout mice
Authors: Liu Xiaobin , Reinscheid Rainer K
Published: June 2021

Neuropeptide S (NPS) and its cognate receptor, NPSR, have been found to modulate arousal, anxiety, stress response, feeding behavior, and learning and memory in rodent animals. To investigate phenotypical consequences of NPS deficiency in vivo, we generated a knockout mouse model by homologous recombination in ES cells, deleting the third exon of the NPS precursor gene. No NPS peptide was found in brains of homozygous knockout mice using immunofluorescence. Initial characterization of behavioral phenotypes of NPSprecursor knockout mice showed that NPS−/− mice are more anxious, show reduced arousal in a novel environment, and display deficits in inhibitory avoidance learning. These observations support the hypothesis that activity of the endogenous NPS system is required for behavioral arousal, modulation of anxiety responses and fear memory. This research was supported in part by a grant from the National Institute of Mental Health (NIMH, MH‐71313).
Ligand‐specific roles for transmembrane 5 serine residues in D1 dopamine receptor binding and activation
Authors: Chemel Benjamin Reed , Bonner Lisa A. , Watts Val J. , Nichols David E.
Published: June 2021

In this study, we employed site‐directed mutagenesis to explore the complex hydrogen bonding interactions that are critical for D1 dopamine receptor activation. We stably expressed wild‐type and three transmembrane 5 serine to alanine mutants (S198(5.42)A, S199(5.43)A, and S202(5.46)A) in HEK 293 cells, and evaluated binding affinity and potency of cAMP production for many structurally diverse ligands. Each of these cell lines expressed similar levels of receptor (approximately 2,000 fmol/mg), and bound [3H]SCH‐23390. The radioligand exhibited significant losses of affinity, however, at S198A and S199A. Dopamine, tetracyclics (e.g. dihydrexidine) and cyclohexyl‐substituted bicyclics similarly suffered losses of affinity and potency at the mutant receptors, where S202A produced the largest disruption of binding and activity. Interestingly, the phenylbenzazepines (e.g. SKF‐38393) demonstrated reduced affinity and potency for each mutant, but were least affected by S202A. Uniquely, the non‐catechol ergolines (e.g. CY 208–243) were more potent at S198A, whereas S199A and S202A produced relatively minor losses of affinity and potency. These results demonstrate that these structurally diverse ligands uniquely interact with the D1 binding pocket, and present a new understanding of the complexities of D1‐ligand recognition and activation. This work was supported by NIH grants MH42705 and MH60397.
AT1AR‐β‐arrestin signaling confers PPARγ agonist‐mediated myocyte contractility
Authors: Tilley Douglas G , Nguyen Anny , Rockman Howard A
Published: June 2021

Peroxisome proliferator‐activated receptor γ (PPARγ) agonists are commonly used to treat cardiovascular diseases, and are reported to have several effects on cardiovascular function that may be due to PPARγ‐independent signaling events. Select angiotensin receptor blockers (ARBs) interact with and modulate PPARγ activity, thus we hypothesized that a PPARγ agonist may exert physiologic effects via the angiotensin II type 1A receptor (AT1AR). In AT1AR‐overexpressing HEK 293 cells, both angiotensin II (Ang II) and the PPARγ agonist troglitazone (Trog) induced AT1AR internalization. A fluorescence assay to measure diacylglycerol accumulation showed that only Ang II induced Gq protein‐mediated signaling, however, immunoprecipitation studies showed both Ang II and Trog caused and recruitment of β‐arrestin (β‐arr) to AT1AR. In isolated mouse cardiomyocytes, Trog increased both % and rate of cell shortening, effects which could be blocked with an ARB. Additionally, these effects were found to be β‐arr‐dependent, as cardiomyocytes isolated from βarr2‐KO mice showed blunted contractile responses to Trog. These findings suggest that PPARγ agonists may act as biased ligands at the AT1AR to exert β‐arr‐dependent physiologic effects distinct from PPARγ activation. Funding was provided by a JCHP New Investigator Program Grant (DGT) and the National Institutes of Health (HAR).
Identification of the Nuclear Localization Sequence in Beta Arrestin1: functional inplications in NF‐(kappa)B activation
Authors: Hoeppner Crystal Zoe , Cheng Ni , Ye Richard D
Published: June 2021

A mounting body of evidence has shown the importance of βArrestin1 in the nucleus, however the mechanism of how βArrestin1 enters the nucleus to mediate its functions is not known. Despite a high homology between the two members of the βarrestin family, they do not localize to the same subcellular compartments. βarrestin1 can be seen ubiquitously throughout the cell while βarrestin2 is excluded from the nucleus by a functional NES in its C terminus. While no nuclear import signal has been identified in either βarrestin, it has been noted that an intact N‐terminal domain is required for nuclear localization of both members. To explore this further, we visualized the localization of full‐length βarrestin1 as well as N‐terminal domain truncation and deletion mutants. Based on the results, a seven‐residue candidate nuclear localization sequence was found in βarrestin1. Mutation of these residues led to a loss of βarrestin1 nuclear localization by inhibiting its binding to the nuclear import machinery. Functionally, expression of wild type βArrestin1 is able to enhance the transcriptional activity of NF‐κB following bradykinin stimulation and the nuclear localization of βArrestin1 is critical for this effect. Loss of nuclear localization of βArrestin1 led to an inhibition of NF‐κB mediated gene transcription by affecting the post‐translational modification profile of p65/RelA following bradykinin stimulation, which led to a decrease in effective promoter binding. In conclusion, this study identifies structural determinants for the nuclear localization of βArrestin1, and demonstrates its role in regulating NF‐κB activation.
Estrogen‐induced resistance artery relaxation is nNOS‐ and age‐dependent in postpartum rats
Authors: Royal Crista , White Richard
Published: June 2021

Our hypothesis is that pregnancy and parturition alter how resistance vessels respond to the vasoactive hormone 17β‐estradiol (E2). We performed isometric contractile force recordings on mesenteric resistance arteries from female Sprague Dawley rats. Normally, E2 is a vasodilator. We found that relaxation to E2 was impaired in younger (≤19 weeks old) postpartum rats as compared to age‐matched nulliparous, or virgin, controls. Arteries from animals older than 20 weeks exhibited no significant impairment. We had previously demonstrated that E2 relaxes arterial smooth muscle by stimulating nitric oxide production from neuronal nitric oxide synthase (nNOS) expressed in vascular smooth muscle. Pretreating vessels with 10−4M L‐NPA, a nNOS selective inhibitor, inhibited E2‐induced relaxation arteries from control rats by 102.17±6.12%, and by 93.65±3.11% in arteries from older postpartum animals. In contrast, L‐NPA had no effect on arteries from younger postpartum animals that exhibit impaired relaxation. These findings suggest that dysfunction of normal nNOS‐mediated relaxation may contribute to the diminished responsiveness of mesenteric microvessels from young postpartum rats to E2. HL073890
Ginkgo Biloba Extract Ameliorates the Formation of Foam Cells by Regulating the Expression of SR‐A and ABCA1 in Macrophage: Role of Heme Oxygenase‐1
Authors: Su Kuo‐Hui , Tsai Jin‐Yi , Kou Yu Ru , Chiang An‐Na , Lee Tzong‐Shyuan
Published: June 2021

Objective

Accumulation of foam cells in intima is the hallmark of early stage atherosclerotic lesion. Ginkgo Biloba extract (EGb761) has been reported to exert excellent anti‐oxidative and anti‐inflammatory properties in the development of atherosclerosis, yet the effect and the molecular mechanisms of EGb on the formation of macrophage foam cells are not fully understood.

Methods and results

Treatment with EGb761 resulted in a dose‐dependent decrease in oxLDL‐mediated cholesterol accumulation in macrophages, a result that was due to a decrease in cholesterol uptake and an increase in cholesterol efflux. Additionally, EGb treatment significantly decreased the mRNA and protein expression of class A scavenger receptor (SR‐A), while EGb had no effect on the expression of CD36, SR‐BI or ATP‐binding cassette (ABC) transporter G1. Furthermore, EGb increased the protein stability of ABCA1 by reducing calpain activity without affecting ABCA1 mRNA expression. Moreover, small interfering RNA (siRNA) targeting heme oxygenase‐1 (HO‐1) abolished the EGb‐induced protective effects on the expression of SR‐A and ABCA1 and calpain activity. Consistently, EGb‐mediated suppression on lipid accumulation in foam cells was also abrogated by HO‐1 siRNA.

Conclusion

EGb confers the protection from the formation of foam cells by an HO‐1‐dependent regulation on cholesterol homeostasis in macrophages.
Turning on inflammatory response to homocysteine through activation of infammasomes in podocytes
Authors: Zhang Chun , Xia Min , Wang Zheng‐Chao , Hu Jun‐Jun , Boini Krishna M , Li Ningjun , Li Pin‐Lan
Published: June 2021

The inflammasome is a multiprotein complex responsible for the activation of caspases 1 or 5, which has been found to be switched on by some endogenous danger signals. The present study was designed to test whether activation of this inflammasome contributes to homocysteine (Hcys)‐induced inflammatory injury in podocytes. By RT‐PCR and immunocytochemistry, we detected the expression of 3 major inflammasome components in mouse podocytes including NALP3, apoptosis‐associated speck‐like protein (ASC), and caspase‐1. All these 3 molecules were abundant in cytoplasma, and ASC was located around nuclei. Under fluorescent confocal microscope, no colocalization of three proteins could be observed under control condition. However, incubation of podocytes with L‐Hcys (80 μM) for 12 hours significantly increased colocalization of NALP3 with ASC or caspase‐1, which was associated with ASC trafficking towards the cell periphery, indicating the formation of a NALP3 inflammasome. To determine the activity of this inflammasome, colorimetric measurement of caspase‐1 activity was conducted. It was found that L‐Hcys increased caspase‐1 activity by 6 folds. These results indicate that activation of inflammasomes to produce pro‐inflammatory factors may turn on the inflammatory response to Hcys in podocytes, producing podocytes dysfunction and glomerular injury (supported by NIH grant HL057244 and DK54927).
Effects of a 4 Hour Exposure to Conventional Peritoneal Dialysis Fluids on the Murine Hepatic Microcirculation
Authors: Patrick Amanda , Fox‐Robichaud Alison E.
Published: June 2021

Peritoneal dialysis (PD) is associated with structural and functional changes in the peritoneal and liver vasculature limiting its efficacy. Our objective was to examine the impact of high osmolar PD solutions and a 4hr dwell time on the liver microcirculation. Mice were given TNF 500ng or normal saline 50 μl (NS) IP, then 30 min later 2 ml of PD solution was injected. The hepatic microcirculation was examined by intravital microscopy. The PD fluids were a glucose‐based, bicarbonate buffered Physioneal, icodextrin‐based lactate and bicarbonate buffered Extraneal and dextrose‐based, lactate buffered Dianeal, compared to lactated Ringer's.

In TNF‐treated mice, Dianeal and Extraneal caused an increase in leukocyte rolling, compared to NS (P>0.004). Adhesion was higher with TNF compared to NS in all groups (P>0.05); however, there was no significant difference between PD solutions. Comparing the 4 hr to a 1 hr dwell, Physioneal only was associated with a 6‐fold increase in adhesion (0.8±0.3 vs 5.2±0.7, P<0.001) and a decrease in leukocyte rolling (8.5±2.1 vs 3.2±0.8, P<0.04) with peritoneal TNF exposure.

With a clinically relevant exposure time, TNF, combined with a glucose based, bicarbonate buffered PD solution, induced an increase in leukocyte adhesion in the hepatic microcirculation. Replacement with icodextrin may alleviate the effects of high glucose and cytokine stimulation.
Interaction of (α4 β1) integrin/VCAM‐1 induces Myosin Light Chain phosphorylation in monocytes and endothelial cells
Authors: Haidari Mehran , Zhang Wei , Chen Zhenping , Gangehei Leila , Dixon Richard
Published: June 2021

We undertook this study to investigate the role of actin cytoskeleton and Myosin Light Chain (MLC) phosphorylation in transendothelial migration (TEM) of monocytes. Our studies demonstrated that exposure of THP‐1 to integrin‐activating antibody induced both Extracellular Signal‐Regulated Kinase (ERK) and MLC phosphorylation. These inductions were more pronounced in the presence of α4β1 ligand, CS‐1fibronectin. Exposure of THP‐1 cells to chemokines (MCP‐1, SDF‐1) induced MLC‐phosphorylation by activation of MLC kinase and inactivation of MLC phosphatase. The chemokine‐induced MLC phosphorylation was accompanied by ERK‐phosphorylation and was inhibited by pre‐exposure of the cells to the inhibitors of MLC kinase, protein kinase C and MEK/ERK. The integrin‐mediated clustering of VCAM‐1 on the endothelial cells (ECs) is mimicked by the crosslinking of antibodies to VCAM‐1. Our studies demonstrated that crosslinking of VCAM‐1 on ECs induces ERK and MLC phosphorylation. The induction of phosphorylation of MLC was attenuated when the cells were pre‐exposed to the inhibitors of MLC kinase, protein kinase C and MEK/ERK. Experiments in the transwell system showed that TEM of monocytes were reduced and enhanced after inhibition and induction of MLC phosphorylation in THP‐1 cells. Our studies demonstrated that interaction of α4β1/VCAM‐1 leads to a critical reaction in TEM of monocytes, phosphorylation of MLC.
Effect of Cuscuta chinensis on renal function in ischemia/reperfusion‐induced acute renal failure rats
Authors: Shin Sun , Lee An Sook , Kim Eun Ju , Kang Dae Gill , Lee Ho Sub
Published: June 2021

The kidneys play the central role in regulating the water, ion composition and excrete metabolic waste products into the urine. Cuscuta chinensis has been known as an important traditional Chinese medicine for controlling urination. Thus, we designed to examine whether aqueous extract of Cuscuta chinensis (ACC) has an effect on renal function parameters in ischemia/reperfusion (I/R)‐induced acute renal failure (ARF) rats. Administration of ACC (250 mg/kg/day) for 4 days showed that renal functional parameters including urinary excretion rate, osmolality, Na+, K+, Cl−, creatinine clearance, solute‐free water reabsorption and blood urea nitrogen were significantly recovered in I/R‐induced ARF. Periodic acid Schiff (PAS) staining showed that administration of ACC improved tubular damage I/R‐induced ARF. In immunoblot and immunohistological examination, I/R‐induced ARF decreased the expressions of water channel (AQP 2, 3) and sodium potassium pump (Na,K‐ATPase) in renal medulla. However, administration of ACC markedly incremented AQP 2, 3 and Na,K‐ATPase expression. Therefore, these data indicate that administration of ACC ameliorates regulating the urine concentration and renal functions in rats with I/R‐induced ARF.
Enhancing complement control on endothelial barrier reduces renal dysfunction following ischemia/reperfusion injury
Authors: Pushpakumar Sathnur Basappa , Perez‐Abadia Gustavo , Todnem Nathan , Soni Chirag , Fensterer Tathyana , Patibandla Phani K , Barker John , Maldonado Claudio
Published: June 2021

Excessive complement activation following ischemia and reperfusion (IR) of organs disrupts endothelial barrier, mediates inflammation and subsequent tissue injury. Previous strategies to reduce complement induced IR injury required systemic administration of agents, which can lead to increased susceptibility to infections/immune diseases. The objective of this project was to determine whether an increase in complement control defenses of rat kidney endothelium reduces IR injury. We hypothesized that increased complement control on the endothelial barrier reduces IR‐mediated complement activation and reduces kidney dysfunction. Fisher rats underwent left kidney ischemia for 45 min. and treatment with a novel fusogenic lipid vesicle (FLVs) delivery system to decorate endothelial cells with an anti‐complement protein, His6‐VCP followed by reperfusion for 24h. Assessment included renal function by serum creatinine and urea, myeloperoxidase assay for neutrophil infiltration, histopathology and immunohistochemistry. Animals in which the kidney endothelium was bolstered by His6‐VCP had better renal function with a significant reduction in serum creatinine as compared to controls (lactated ringer's only). Neutrophil infiltration showed a downward trend. We conclude that FLVs/His6‐VCP therapy was effective during the first 24h in reducing vascular and renal dysfunction following IR injury.
Simultaneous measurement of RBC velocity, flux, hematocrit and shear rate in vascular networks in vivo
Authors: Kamoun Walid Samy , Chae Sung‐Suk , Lacorre Delphine A. , Tyrrell James A. , Mitre Mariela , Gillissen Marijn A. , Fukumura Dai , Jain Rakesh K.
Published: June 2021

Blood flow directly affects nutrient and oxygen as well drug delivery in tumors. The distribution of blood within tumor is disturbed by structural and functional abnormalities, and may be further altered by therapy. In turn, this results in heterogeneous distribution of drugs and/or oxygen, with direct effects on the efficacy of cytotoxic therapies. These heterogeneities are poorly understood because of the inability of current techniques to characterize the function of individual vessels and their local network. To address this problem, we have developed a novel methodology for simultaneously quantifying blood flow (velocity, flux, hematocrit and shear rate) in extended networks at the single capillary level. Our approach relies on deconvolution of signals produced by labeled red blood cells as they move relative to the scanning laser of a confocal or multiphoton microscope, and provides fully‐resolved three‐dimensional flow profiles within vessel networks. Using this methodology, we show that blood velocity profiles are perturbed and asymmetrical near intussusceptive tissue structures in tumors. Furthermore, we show that subpopulations of vessels, classified by functional parameters, exist in different tissue regions, and that Angiopeitin‐1 can improve tumor perfusion by inducing arteriogenesis.
Insulin supplementation elevates systolic blood pressure and arteriolar tone in mice with Type 2 diabetes
Authors: Feher Attila , Yang Yang‐Ming , Sun Dong , Kaley Gabor , Bagi Zsolt
Published: June 2021

Hyperinsulinemia is frequently associated with hypertension but their interrelationship remains unclear. During the progression of Type 2 diabetes mellitus (T2DM) insulin secretion may decline due to loss of function of pancreatic β cells. We investigated the effect of insulin administration (daily i.p. injection of insulin, 1.67 mU/body weight (g) for 4‐wk) on blood pressure and also insulin‐induced changes in diameter of isolated resistance arteries in 36‐wk old db/db mice with T2DM. Compared to 12‐wk old db/db mice we found significantly reduced insulin levels (16.5±2.6 vs. 7.7±3.1 ng/ml) and severe hyperglycemia in 36‐wk old db/db mice. Also, compared to 12‐wk old db/db mice blood pressure (as measured by the tail cuff method) declined in 36‐wk old db/db mice and was found to be similar in aged‐matched control mice (118±13 vs. 119±5 mmHg). Interestingly, 4‐wk insulin treatment significantly elevated the blood pressure of 36‐wk old db/db mice (151±14 mmHg), to a level similar to what was found in 12‐wk old db/db mice (146±8 mmHg). In isolated skeletal muscle arterioles, incubation with insulin (30 min, 10 μg/ml) elicited significantly enhanced myogenic tone in 36‐wk old db/db mice (from 25±14% to 51±11% at 80 mmHg), but not in age‐matched controls. Thus we suggest that insulin, due to its vasoconstrictor effect on arterioles may elevate blood pressure in mice with Type 2 diabetes. (AHA 0735540T and NIH NHLBI 43023)