bluuebunny/crossref_metadata_2025 huggingface dataset:
A key role of AT1R in endothelial dysfunction during estrogen deficiency
Authors: Yung Lai Ming , Wong WT , Leung FP , Yao XQ , Chen ZY , Huang Y
Published: June 2021

We aim at examining mechanisms underlying endothelial dysfunction in estrogen deficiency. Changes in vascular reactivity in isolated aortas were studied in organ baths, while alterations in protein expression levels of biomarkers were examined by Western blot, including endothelial nitric oxide synthase (eNOS), phosphorylated eNOS, angiotensin converting enzyme (ACE), angiotensin II type 1 (AT1R) and type 2 receptors (AT2R), gp91phox and nitrotyrosine. Angiotensin II level was detected by immunohistochemical staining. Relaxations were impaired in the aorta from ovariectomized rats; which was prevented by acute treatment of losartan (AT1R blocker). Phenylephrine‐induced tension was higher in ovariectomized rats. Western blot showed that estrogen deficiency (i) decreased p‐eNOS level without affecting total eNOS; (ii) increased expressions of ACE, AT1R, gp91phox and nitrotyrosine; and (iii) increased angiotensin II level in aorta from ovariectomized rats. The results of the present study show that estrogen deficiency reduced bioavailability of NO, related to the elevated oxidative stress probably through up‐regulation of AT1R‐mediated vascular dysfunction.
Oxidative inactivation of the proteasome: a potential link between oxidative stress and upregulation of IL‐8
Authors: Shang Fu , Fernandes Alexandre F. , Zhou Jilin , Zhang Xinyu , Sparrow Janet , Taylor Allen , Pereira Paulo
Published: June 2021

Oxidative stress and inflammation have been implicated in the pathogenesis of a variety of age‐related diseases. Stress‐induced overproduction of inflammatory cytokines, such as interleukin‐8 (IL‐8), is one of the early events of inflammation. The objective of this study is to determine potential links between oxidative stress and overexpression of IL‐8 in retinal pigment epithelial (RPE) cells. Exposing RPE cells to H2O2 or A2E‐mediated photo‐oxidation resulted in overexpression of IL‐8 and inhibition of the proteasome. The secretion of IL‐8 was initially reduced following proteasome inhibition. However, prolonged treatment with proteasome inhibitors (>4 h) resulted in a dramatic increase in IL‐8 production, indicating that oxidative stress‐induced upregulation of the proinflammatory IL‐8 is related to proteasome inactivation. Prolonged proteasome inhibition (>2 h) resulted in the activation of p38 MAPK via activation of MKK3/6. Consistently, the proteasome inhibition‐induced upregulation of IL‐8 was blocked by a p38 MAPK specific inhibitor. Taken together, these data indicate that oxidative inactivation of the UPP and the related alteration in p38 MAPK signaling pathways provide a potential link between oxidative stress and overproduction of proinflammatory cytokines, such as IL‐8.
The Effect of Nicotine on the Epididymis and Testis of Prepubertal Sprague Dawley Rats
Authors: Ramadan Hana'a Mohammed , Afifi Nahla M. , Al‐Naemi Hamda
Published: June 2021

The majority of smokers begin tobacco use before they reach adulthood. Therefore, the effects of early administration of nicotine on the testes & epididymes were studied. Forty pre‐pubertal male Sprague Dawley albino rats 4 weeks age weighing between 40–65 grams were divided into 4 groups, each of 10 rats. G1 the control group injected with saline, G2 rats injected with nicotine with a dose of (6.25 ng/g) both groups injected for one week, G3 control group injected with saline and G4 injected with nicotine with a dose of (6.25 ng/g) both groups injected for two weeks. All groups were injected intraperitoneally. Results showed a significant decrease in the total weight gain (P<0.05) in G4. Histological studies of testes and epididymes showed different forms of degeneration in both G2 & G4. Plasma lipid profile showed a significant decrease in cholesterol (P≤0.01) and HDL‐C (P≤0.001) in both G2&G4, while triglyceride levels significantly increased (P<0.05) in G4. Plasma testosterone concentration showed a significant decrease in both G2 & G4 (P<0.05). However, FSH showed a significant decrease in concentration in G4 only. X‐ray microanalysis studies showed a significant increase in sodium in G2 (P<0.05) and G4 (P<0.001) in the testes while it significantly increased in the epididymis in G4 (P<0.01) only. These results indicates that nicotine exposure increase the risk of damage that occurs in the testes and epididymis with increasing the period of exposure.
Therapeutic Potential of Intramyocardial Transplanted CD34+ Cells in Myocardial Ischemia
Authors: McKee Jeff , Palmer Lora , Rice Debbie , Anderson Laura , Gass Jerome , Motlagh Delara , Pokropinski Sharon , Rausa Francisco , McCallister Brian , Jasani Bhumi , Kemshead John , Amrani David
Published: June 2021

Baxter's Isolex 300i Magnetic Cell Selection System isolates patient's CD34+ cells from peripheral blood. The mononuclear cell fraction is incubated with anti‐CD34 antibodies and captured using sheep anti‐mouse (SAM) IgG coated beads. The therapeutic potential of intramyocardial transplanted, autologous, SAM‐isolated CD34+ cells is under clinical investigation. Due to the theoretical potential for prion transmission and limited supply of the source sheep, a rat anti‐mouse (RAM) IgG coated bead has been developed. In vitro, RAM and SAM were comparable in their ability to isolate CD34+ cells. CD34+ cells demonstrated comparable yield, purity, viability, apoptosis, and cell subsets. CD34+ cells exhibited comparable clonogenic potential and migratory capacity in a dose‐dependant manner to VEGF and SDF‐1. In vivo, intramyocardial transplantation of RAM‐isolated human CD34+ cells improved cardiac performance in an athymic rat, left anterior descending coronary artery occlusion model. Echocardiographic evaluation demonstrated improvements in left ventricular fractional shortening (p<0.001) and left anterior wall thickening (p<0.01), and decreased ventricular fibrosis, similar to that reported by Kawamoto et al (2003) in support of the IND for SAM. In conclusion, these data suggest equivalent preclinical efficacy of RAM and SAM‐isolated human CD34+ cells. Baxter provided all research support.
Association between the polymorphisms in protein L‐isoaspartate (D‐aspartate) O‐methyltransferase gene and premature ovarian failure
Authors: Pyun Jung A , Kang HyunJun , Lee Seung Ku , Kim Min‐ho , Kim Hyoun Geun , Kim Kyoung‐Yeon , Kim Jeong‐Geun , Oh Ah Reum , Won Seongsik , Kang Eun Joo , Kwack KyuBum
Published: June 2021

Premature ovarian failure (POF) is defined as the occurrence of amenorrhea before the age of 40 years. It includes primary amenorrhea characterized by absent menarche and secondary amenorrhea represented cessation of ovarian function before the age of 40 years. POF is a polygenic disease, so we performed a whole genome association study to reveal some important genes associated with POF. In this study, we found several regions which were associated with POF. One of the regions strongly associated with POF was on chromosome 6 and encoded protein L‐isoaspartate (D‐aspartate) O‐methyltransferase (PCMT1). We identified that four polymorphisms in PCMT1 gene encoding a protein repair enzyme were associated with POF. Then three polymorphisms formed a haplotype block and were in perfect linkage disequilibrium (LD) through LD test. Haplotype1 had significantly higher frequency in the POF group, while haplotype 2 had significantly higher frequency in the matched control group. Therefore we suggest that ht1 was significantly associated with susceptibility to POF occurance. In contrast, ht2 was significantly associated with resistance to it.
Targeting FAAH and COX to Treat Visceral Pain
Authors: Lichtman Aron H , Naidu Pattipati S , Booker Lamont , Boger Dale L , Cravatt Benjamin F
Published: June 2021

Genetic deletion or pharmacological inhibition of fatty acid amide hydrolase (FAAH), the primary enzyme responsible for catabolism of the endogenous cannabinoid anandamide, leads to a CB1 receptor mediated decrease in nociceptive behavior in a variety of inflammatory and neuropathic pain models. However, there are presently no published reports that have evaluated whether FAAH blockade reduces nociception in visceral pain, though direct‐acting CB1 receptor agonists as well as cyclooxygenase (COX) inhibitors are highly effective in visceral pain assays. Therefore, in the present study we evaluated whether FAAH blockade will block nociceptive behavior in the mouse acetic acid writhing model. Additionally, we examined whether co‐administration of a FAAH inhibitor and COX inhibitor would synergize to block visceral pain. FAAH (‐/‐) mice as well as wild type mice treated with FAAH inhibitors (i.e., URB597 and OL‐135) or the non‐selective COX inhibitor diclofenac displayed a decrease in acetic acid‐induced writhing. URB597 (1–10 mg/kg; ED50 = 2.1 mg/kg) and diclofenac (3–30 mg/kg; ED50 = 9.8 mg/kg) produced dose‐dependent antinociception in the writhing test. The CB1 receptor antagonist rimonabant blocked the analgesic effects in the FAAH‐compromised mice, but failed to attenuate the actions of diclofenac. Interactions between URB597 and diclofenac were analyzed using an isobolographic approach. Combined administration of URB59597 and diclofenac in ratios of 1:1 [ED50s = 0.24 and 1.18 mg/kg], 1:3 [ED50s = 0.28 and 4.30 mg/kg], or 3:1 yielded [ED50s = 0.44 and 0.72 mg/kg] yielded synergistic interactions. Taken together, the results of the present study suggest that FAAH represents a promising target to treat visceral pain and combination of FAAH inhibitors and NSAIDs may have greater therapeutic utility than either class of drug given alone.
Dynamic In vivo Imaging of NADPH Oxidase Gene Expression to Monitor its Involvement in Morphine's Actions
Authors: XIA Min , Hull Lynn C , Thomas Cindy , Gabra Bichoy H , Li Ningjun , Li Pin‐Lan , Dewey William L
Published: June 2021

The present study examined the applicability of using in vivo molecular imaging to monitor the daily expression of locally transfected NADPH oxidase 4 (Nox4) gene in periaqueductal gray (PAG) of mice. A mixture of luciferase expression plasmids and transfection reagent with vectors containing Nox4 was injected into the lateral ventricle at 2 mm rostral and 2 mm lateral with at a 45° angle from the bregma. Luciferase expression was monitored daily using an IVIS 200 in vivo imaging system. The in vivo bioluminescent signals were detectable as early as 24 hours after gene transfection into mouse brain and were maintained for 7‐days. On days 2 to 7, the antinociceptive efficacy of morphine was significantly increased in Nox4 vector‐injected compared to empty vector‐injected and naïve mice. After in vivo monitoring, the mice were euthanized and brain slices were obtained for ex vivo imaging. Strong signals of transfection of luciferase genes were seen in the PAG area. By real‐time RT‐PCR, Nox4 mRNA abundance was also found doubled in the dissected PAG, but not in the brain cortex. These results show that the in vivo imaging system used in these experiments provides a powerful tool for daily non‐invasive dynamic visualization of gene expression and will be useful for future studies of the role of other genes in drug effects in the brain. (Supported by DK54927, DA01647, DA020836 and HL89563)
BICARBONATE INCOMPLETELY REVERSES THE INHIBITORY EFFECT OF CHLORPROCAINE ON BUPIVACAINE IN A SCIATIC BLOCK IN SPRAGUE‐DAWLEY RATS
Authors: Yung E. , Lahoti T. , Jafari S. , Weinberg J. , SchianodiCola J. , Yarmush J. , Ray S. D.
Published: June 2021

Chloroprocaine (CP) and bupivacaine (BP) bind to the intracellular portion of Na‐channels and block Na influx into neurons, preventing depolarization. CP is a fast acting local anesthetic and BP is long acting. They have been coadministered to yield a block with rapid onset and long duration, with limited success. The lack of success was hypothesized to be due to the low pH of CP, or an inhibitory effect of CP or its metabolite on the action of BP at the Na‐channel. A recent unpublished study demonstrated the efficacy of a block with bicarbonated CP followed by BP administration. In order to understand the role bicarbonate (BC) plays in the success of the block, the sciatic nerve in 40 Sprague‐Dawley rats (250gm to 350gm) was blocked with CP and epinephrine (EPI), with or without BC, followed by BP and EPI. The time to onset and duration of analgesia were measured. The data show that addition of BC to CP and EPI, followed by BP and EPI, hastened the onset (1.38 ± 0.13 min vs. 2.73 ± 0.32 min; p < 0.001) and prolonged the duration (129.63 ± 5.24 min vs. 110.28 ± 5.56 min; p = 0.013) of the block. Controls show that adding BC to CP alone decreased the onset time and duration of block. BP alone had a longer duration than CP with BC followed by BP. These observations suggest that the apparent inhibitory effect of CP on BP‐induced block is a function of both the low pH of clinically available CP solutions and a direct effect of the CP molecule or its metabolite.
PKC and PKA inhibitors reverse acute tolerance to low‐ and moderate‐ but not high‐efficacy mu‐opioid agonists
Authors: Hull Lynn C , Gabra Bichoy H. , Smith Forrest L. , Dewey William L.
Published: June 2021

The present study was designed to investigate possible differing mechanisms of tolerance to opioids of various efficacies. We, as well as others, have shown that the administration of highly selective inhibitors of protein kinase C (PKC) and A (PKA) completely reversed morphine antinociceptive tolerance in mice. We now have investigated whether these inhibitors reverse tolerance to other opioids of low and high efficacy. An 8‐hr model of acute tolerance was used in which the test opioid was administered repeatedly at a dose which yielded maximum antinociception in naïve mice. Animals were then challenged with increasing doses of the test opioid with and without the PKC and PKA inhibitors to construct a dose response curve. The 8‐hr tolerance to the low‐efficacy μ‐agonist meperidine and the moderate‐efficacy μ‐agonists, morphine and fentanyl was fully reversed by the PKC inhibitors Bisindolymaleimide I, and Gö6976 as well as the PKA inhibitor PKI 14‐22. However tolerance to DAMGO, a highly efficacious μ‐opioid agonist, was not reversed by injection of either the PKC inhibitor Gö6976 or the PKA inhibitor PKI 14‐22 suggesting that μ‐opioid receptor desensitization induced by high‐efficacy μ‐agonists is independent of PKC or PKA activation. It can be concluded that the mechanisms underlying μ‐opioid receptor desensitization may be determined by the agonist activating the receptor. Supported by DA01647 and DA020836.
Regulation of retinal amino acid neurotransmitters by neuroprostanes and isoprostanes, in vitro and ex vivo
Authors: Opere Catherine Atieno , Zhao Min , Ohia Sunny E
Published: June 2021

Isoprostanes (isoPs) can regulate the release of exogenously applied glutamate (measured as [3H]D‐aspartate) in isolated bovine retinae (Opere et al., Neurochem Res 30:2005). It is unknown whether the isoP‐like compounds, neuroprostanes (NPs) regulate neurotransmitter release. We now report an effect of A4‐NP and 8‐isoPGE2 on K+‐evoked release of [3H]D‐aspartate release and endogenous amino acid levels in bovine retinae in vitro and ex vivo. Isolated bovine retina were incubated in Krebs solution containing 200 nM of [3H]D‐aspartate and then prepared for neurotransmitter release studies using the Superfusion Method. For ex vivo studies, freshly isolated bovine eyeballs were injected intravitreally with A4‐NP and 8‐isoPGE2. After 30 mins, retina was isolated for measurement of amino acids using HPLC. A4‐NP (0.1μM) enhanced K+‐evoked [3H]D‐aspartate release by 41%. Likewise, intravitreally injected A4‐NP (10μM) enhanced retinal glutamate and glycine levels by 20 % and 18% (p<0.01), respectively. 8‐isoPGE2 reduced K+‐evoked [3H]D‐aspartate release and endogenous levels of glutamate and glycine. For instance, 8‐isoPGE2 (1 and 100μM) reduced glutamate by 34% and 48 %(P<0.001) and glycine by 20% and 36%, respectively. 8‐isoPGE2 (0.1μM) reduced K+‐evoked [3H]D‐aspartate release by 10%. Thus NPs and isoPs have a differential action on release/availability of retinal amino acid neurotransmitters. Support: NIH EY 013967
Peroxynitrite Decomposition Catalysts as Adjuncts to Morphine for the Management of Chronic Pain States
Authors: Salvemini Daniela , Batini‐Haberle Ines , Cuzzocrea Salvatore , Reboucas Julio , Masini Emanuela , Matuschak George , Spasojevic Ivan
Published: June 2021

Severe pain reduces life quality in patients with inflammatory and neoplastic diseases (i.e lung cancer), partly because reduced analgesic effectiveness of chronic opiate therapy leads to escalating doses and distressing side effects. The mechanisms involved are poorly understood. Peroxynitrite (ONOO−), a potent pro‐inflammatory and pro‐apoptotic reactive species, is now implicated in hyperalgesia of several etiologies. In mice we showed that repeated morphine administration leads to nitrotyrosine (NT) formation in the dorsal horn of the spinal cord, with NT as a reliable marker for ONOO−. The role of ONOO− was proven by showing that co‐administering morphine with novel and potent ONOO− decomposition catalysts such as Mn(III) ortho alkylpyridylporphyrins, attenuated NT formation and prevented antinociceptive tolerance at doses devoid of behavioural side effects and up to 300 fold‐lower than previously used Fe or Mn porphyrins. These effects coincided with: 1) nitration of MnSOD, a glutamate transporter, and glutamine synthase; 2) increased formation of pro‐inflammatory cytokines; and 3) oxidative DNA damage and activation of the nuclear factor poly(ADP‐ribose) polymerase. These findings support our central hypothesis that ONOO− is critical to the development of morphine‐induced antinociceptive tolerance providing the rationale towards the development of ONOO− decomposition catalysts as adjuncts to opiates for the management of chronic pain.
Ethanol lowers blood pressure in conscious female rats via enhancing the phosphorylation of myocardial neuronal nitric oxide synthase
Authors: El‐Mas Mahmoud M , Fan Ming , Abdel‐Rahman Abdel A
Published: June 2021

Intragastric (i.g.) ethanol lowers blood pressure (BP) in female rats via a reduction in cardiac output (CO) and stroke volume (SV). We tested the hypothesis that enhancement of myocardial nNOS and/or eNOS activity constitutes a cellular mechanism for ethanol‐evoked reductions in CO and BP. We investigated the effect of inhibition of nNOS (Nω‐propyl‐L‐arginine, NPLA) or eNOS [N5‐(1‐iminoethyl)‐L‐ornithine, L‐NIO] on hemodynamic, biochemical (nitrate/nitrite levels, NOx), and myocardial nNOS/eNOS phosphorylation elicited by ethanol (1 g/kg i.g.) in proestrus female rats. In saline‐pretreated rats, the hemodynamic effects of ethanol (reductions in BP, CO, and SV) were coupled with increases in plasma NOx and myocardial p‐nNOS, but not p‐eNOS, expression. nNOS inhibition by NPLA attenuated the hemodynamic effects of ethanol and associated increases in plasma NOx and cardiac p‐nNOS. eNOS inhibition by L‐NIO attenuated ethanol hypotension, but not concomitant CO/SV reductions. L‐NIO uncovered a dramatic increase in total peripheral resistance (TPR) in response to ethanol, which appeared to have offset the reduction in CO. These findings highlight: (i) myocardial nNOS as the cellular mechanism that underlies, in part, reductions in BP and CO caused by ethanol in female rats, and (ii) vascular eNOS‐NO signaling counterbalances ethanol‐induced increases in TPR.

Supported by Grant R01 AA014441 from NIAAA.
Brown Norway chromosome 16 protects the SS‐16BN/Mcwi consomic model of left ventricular hypertrophy from progressing to heart failure
Authors: Kriegel Alison Jessica , Greene Andrew S.
Published: June 2021

Left ventricular hypertrophy (LVH) is a well established risk factor for cardiovascular disease however the determinants of progression to heart failure (HF) have been incompletely characterized. Regulatory pathways are difficult to study in humans due to the interaction of genetic and environmental factors. Selective breeding of animal models allows for tight control of these factors. We find that introgression of chromosome 16 from the normotensive Brown Norway (BN) rat onto the genetic background of the hypertensive SS/Mcwi (Dahl Salt‐Sensitive (SS)) both reduces blood pressure and prevents HF in the consomic SS‐16BN/Mcwi (SSBN16) rat, a model of idiopathic LVH. LV fibrosis and ejection fraction (EF) were both measured at 18 and 36 weeks of age in the SS, SSBN16 and BN rats as indicators of progressive HF. At 18 weeks the percent of the LV showing fibrosis did not differ between strains, but by 36 weeks both the SS and BN rats exhibited a significant increase (122% and 78% respectively; p < 0.05, ANOVA). There was no change in fibrosis in the SSBN16. EF was preserved near 85% in the SSBN16 and BN rats between 18 and 36 weeks, while it dropped in the SS rats from 84.81% ± 2.45 to 60.74% ± 4.11. This data suggests that the interaction of one or more genes on BN chromosome 16 with the SS genetic background protects the LV from developing phenotypes associated with HF and may be part of a HF regulatory pathway. Support HL 29587; HL 66579
In vitro Cytochrome P450 Reaction Phenotyping for Metabolism‐linked hemotoxicity of NPC1161B, a novel 8‐aminoquinoline antimalarial agent
Authors: GANESAN SHOBANA , TEKWANI BABU L , TRIPATHI LALIT M , NANAYAKKARA DHAMMIKA , WALKER LARRY A
Published: June 2021

8‐Aminoquinolines are important class of drugs, holding promise in many infectious diseases. Primaquine is a prototype of this class, while its 5‐dichlorophenoxy derivative, NPC1161B has been identified as a promising candidate with enantioselective toxicity and efficacy profiles. Use of 8‐AQs is restricted due to their hematological toxicities, for which biotransformation seems to be a prerequisite. Earlier metabolic studies have been hindered due to the unstable nature of hematotoxic intermediates. An in vitro metabolism‐linked hematoxicity assay has been employed to evaluate generation of methemoglobin and other hematotoxic responses by 8‐AQs. Direct incubation of the drug with microsomes or recombinant human CYP isoforms afforded in situ generation of toxic metabolites that mediate a robust hemoglobin oxidation. Three main approaches of reaction phenotyping i.e., correlation analysis, chemical inhibition and recombinant CYPs were employed to investigate hematoxicity of NPC1161B. CYP3A4 selectively metabolized NPC1161B leading to methemoglobin toxicity. It also correlated with CYP3A4 content of different batches of human liver microsomes. Inhibitors of CYP3A4 dramatically reduced methemoglobin formation in in vitro and in vivo studies. Data provide the basis for a mechanism based approach to abrogate hematotoxicity of NPC1161B, currently under evaluation for clinical advancement.
Evaluation of P‐glycorptotein mediated interaction of the atypical antipsychotic agents risperidone and paliperidone in vitro
Authors: Zhu Hao‐Jie , DeVane C. Lindsay , Markowitz John S.
Published: June 2021

The atypical antipsychotic risperidone (RSP) and its major active metabolite paliperidone (PALI) are both substrates and inhibitors of the drug transporter P‐glycoprotein (P‐gp). This study was undertaken to investigate potential P‐gp mediated drug‐drug interactions of RSP and PALI in vitro. Incubation of RSP in concentrations ranging from 2‐100 μM significantly increased the uptake of co‐incubated PALI in LLC‐PK1/MDR1 cells when PALI was adopted as a substrate at the concentrations of 10 and 50 μM. Additionally, the influence of PALI on RSP transport was determined by incubating RSP (2 μM and 10 μM) with various concentrations of PALI. The increases of RSP uptake in LLC‐PK1/MDR1 cells were observed only when 2 μM RSP was incubated with PALI at or above 10 μM. When the concentration of RSP was increased to 10 μM, PALI did not influence RSP accumulation even at the highest concentration tested (100 μM). Furthermore, the interaction of P‐gp mediated transport of RSP and PALI was investigated by transwell assay. The P‐gp function in LLC‐PK1/MDR1 monolayers was inhibited by 10 M RSP resulting in decreased transport of PALI in the basolateral to apical direction. However, 100 μM of PALI had no influence on the transport of RSP across cell monolayers. Therefore, it is unlikely that PALI affects the transport of RSP via P‐gp inhibition in vivo. Additional studies are needed to determine whether the inhibition of P‐gp mediated PALI transport by RSP could result in a clinically significant influence on PALI disposition in the various P‐gp expressing tissues. This work was supported by NIH grant MH071811‐02A1.
Anti‐metastasis effect of PA‐22 in oral squamous cell carcinoma HSC‐3 cells
Authors: Hsieh Wen‐Tsong , Chen Yi‐Tin , Lin Hei‐Yi , Chung Jing‐Gung
Published: June 2021

Oral squamous cell carcinoma (OSCC) is one of the most common head and neck cancers. In the present study, human oral squamous cell carcinoma (OSCC) cell lines HSC‐4 were used to evaluate the effects of PA‐22 on the cell cycle and the invasion activity. PA‐22, is isolated from Chinese medicine herb physalis anagata with respect to tumor growth, invasion in OSCC model. PA‐22 treated HSC‐4 cells dose and time‐dependently by MTT assay, Flow cytometry, DAPI assay, DNA Ladder assay, Comet assay, and Western blot. PA‐22 treatment triggered the mitochondrial apoptotic pathway indicated by a change in Bax/Bcl‐2 ratios, resulting in mitochondrial membrane potential loss, cytochrome c release, and active caspases. We also found Matrigel invasion assay revealed that the invasiveness of HSC‐3 was suppressed by treatment with PA‐22. It down‐regulate the secretion of matrix metalloproteinase (MMP) to culture supernatant as well as the expression of MMP proteins. These results indicate that PA‐22 is a novel candidate agent for the treatment of oral cancer.
Induction of vascular endothelial growth factor by peptidyl‐prolyl isomerase Pin1 in breast cancer cells
Authors: Kim Mi Ra , Kang Keon Wook
Published: June 2021

Pin1, a peptidyl prolyl isomerase, is overexpressed in most types of cancer tissues and plays an important role in oncogenesis. We found that Pin1 increases the transcriptional activity and protein level of vascular endothelial growth factor (VEGF) in the human breast cancer cell line, MCF‐7. Reporter gene analyses showed that Pin1 overexpression increased the reporter activities in cells transfected with reporters containing the VEGF gene promoter or with minimal reporters of activator protein‐1 (AP‐1) and hypoxia response element (HRE). VEGF reporter gene activity was significantly inhibited by either hypoxia inducible factor‐α(HIF‐1α) siRNA or AP‐1 decoy ODN. Moreover, the reporter activities of the VEGF promoter, AP‐1 and HRE in Pin1−/− mouse embryonic fibroblast (MEF) cells were decreased compared with those in wild‐type Pin1 MEF cells. These results imply that Pin1 stimulates VEGF expression by activating HIF‐1ƒÑ and AP‐1, and suggest that Pin1ƒnis a potential therapeutic target of angiogenesis during cancer development.
The utility of monitoring skeletal muscle troponin I to detect drug‐ induced skeletal muscle injury
Authors: Knapton Alan D , Espandiari Parvaneh , Morales Azorides , Moussazadeh Mitra , Robins Terry , Herman Eugene
Published: June 2021

Drug‐induced myotoxicity is a continuing problem. Available myotoxicity biomarkers are of limited value. Recently, novel assays were developed to detect the slow and fast isoforms of skeletal muscle troponin (sTnI) released from type I (fast sTnI) and type II (slow sTnI) muscle fibers. The present study, using this assay, sought to determine whether monitoring serum levels of sTnI provided a better means to identify myotoxicity. Adult male or female Sprague‐Dawley rats were dosed with 25, 50 or 75 μ mole/kg 2, 3, 5, 6 tetramethyl p‐phenylenediamine (TMP) (sc) daily for 2 days or a single 200 mg/kg dose of cyclophosphamide (CP) (ip). Female rats were more sensitive to the toxic effects of TMP (mortality and body weight). Degenerative skeletal muscle changes were observed in both male and female rats. Increases in fast sTnI were noted in both male and female rats treated with 25 (2/5 male (avg 6 ng/ml) vs 3/5 female (avg 20 ng/ml) and in all animals given 50 μ moles/kg or higher. The levels of fast sTnI were significantly higher in female than male rats at the 50 μ mole/kg dose (avg 503 vs 64 ng/ml). Low levels of slow sTnI were detected in both female (25 and 50 μmole/kg) and male (75 μmole/kg). CK levels were increased over baseline only at the highest tolerated doses. No changes were noted in CP‐treated rats. Thus, sTnI appears to be a sensitive biomarker that could be used to detect drug‐induced myotoxicity.
Prevention of free fatty acids/high fat diet‐induced hepatic lipotoxicity by 18β glycyrrhetinic acid
Authors: Wu Xudong , Zhang Luyong , Shang Jiang , Gurley Emily , Studer Elaine , Hylemon Phillip B , Pandak William M , Sanyal Arun J , Zhou Huiping
Published: June 2021

Free fatty acids (FFAs) are key players in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). Inhibition of FFAs‐induced hepatic toxicity represents a potential therapeutic strategy. Glycyrrhizin (GL), the major bioactive component of licorice root extract, has been used to treat hepatitis to reduce liver inflammation and hepatic injury; however, the mechanism underlying the protective effect of GL remains unknown. GL exhibits its pharmacological functions through its biologically active metabolite, 18β glycyrrhetinic acid (GA), which is formed by presystemic hydrolysis. The aim of the present study was to examine if GA is able to prevent FFAs‐induced lipotoxicity and identify the potential cellular mechanisms. Both in vitro FFAs‐induced HepG2 NAFLD model and in vivo high fat diet (HFD)‐induced rat NAFLD model were used in the current studies.

Results: GA not only prevented FFAs‐induced lipoapoptosis in HepG2 cells, but also prevented HFD‐induced liver injury in in vivo rat models. Furthermore, stabilization of lysosomal membrane, inhibition of cathepsin B expression and enzyme activity, inhibition of mitochondrial cytochrome c release, and reduction of oxidative stress represent the major cellular mechanisms underlying its protective effects on FFAs/HFD‐induced hepatic lipotoxicity.

Conclusion: GA may be beneficial for preventing FFAs/HFD‐induced lipotoxicity or treating NAFLD.
Acute toxicity evaluation of a tepary bean (Phaseolus acutifolius) lectin
Authors: Lopez‐Sanchez Claudia , Picones‐Puebla Christopher , Gonzalez‐Arrollo Berenice , Rodriguez‐Mendez Adriana , Gallegos‐Corona Marco Alonso , Mendiola‐Olaya Elizabeth , Blanco‐Labra Alejandro , Garcia‐Gasca Teresa
Published: June 2021

As plant lectins have shown anticancer potential, we had previously tested the cytotoxic effect of a lectin from tepary bean on human cancer cells proliferation and the most sensible were colon cancer cells. Before testing the lectin against colon cancer in vivo, it is necessary to determine its toxicity, therefore the goal of the present work was to evaluate the acute toxicity of a concentrated tepary bean lectin fraction (TBLF). Female Sprague Dawley rats of 7 weeks old were administrated with the TBLF at doses of 5, 50, 300 and 2000 mg/kg body weight (423 OECD) by intragastric canula. Food intake was determined daily and body weight each 5 days, until 15 days. Highest doses caused diarrhea, lethargy and piloerection in the first 24 h, but a full recovery was observed after 48 h. After necropsy, no alterations were observed in liver, stomach, gut, colon, lungs, heart, thymus and spleen; no alteration was observed in the colon histology. Lectin adminstration increased leucocytes in 54% with 50 mg/kg (Tukey, p<0.05) and 2000 mg/kg decreased the number in 25%. TBLF by oral administration showed typical signs of lectins toxicity but it was not lethal and animals had a full recovery. Current studies are focusing on the evaluation of chronic toxicity.
Uncoupling protein 2 regulates mast cell activation
Authors: Tagen Michael , Shirihai Orian , Vasiadi Magdalini , Kalogeromitros Dimtrios , Makris Michalis , Theoharides Theoharis C.
Published: June 2021

Mast cells are critical for allergic reactions, but are increasingly implicated in many other inflammatory diseases. Yet, little is known about the regulation of mast cell activation. Uncoupling protein 2 (UCP2) is a mitochondrial protein associated with regulation of reactive oxygen species. Bone marrow mast cells (BMMC) from Ucp2 −/− mice showed significantly greater release of histamine in response to antigen (DNP‐BSA) and compound 48/80 and significantly greater production of IL‐6 and PGD2 after stimulation with DNP‐BSA or lipopolysaccharide. UCP2 was induced in human LAD2 mast cells by the peroxisome proliferator receptor gamma (PPARγ) agonist, 15‐deoxy‐Δ12,14‐prostaglandin J2 (15d‐PGJ2). Treatment with 15d‐PGJ2 inhibited histamine, TNF‐α and PGD2 release induced by antigen (anti‐IgE) and substance P (SP) stimulation. 15d‐PGJ2 also inhibited histamine release from +/+ BMMCs by 42%, but had no effect on Ucp2 −/− BMMCs. Lentiviral‐mediated overexpression of UCP2 produced similar results to 15d‐PGJ2 treatment. Moreover, lesional skin from psoriasis, a chronic inflammatory skin condition involving mast cell activation, had decreased gene expression of Ucp2 compared to unaffected or normal skin. These results indicate that UCP2 is a novel regulator of mast cell activation, its dysregulation may be involved in inflammatory disease and UCP2 may serve as a therapeutic target.
The Role of the Tumor Supressor‐p53 in Alpha1D‐Adrenergic Receptor (AR) Induce Apoptosis in Vascular Smooth Muscle Cells
Authors: Smith Jennifer Leigh , García‐Cazarín Mary Lolis , Simmerman Linda A , Hadley Robert W , Piascik Michael T
Published: June 2021

Stimulation of the alpha1‐adrenergic receptor (AR) associated human aortic smooth muscle cells (HASMC) with phenylephrine (PE) resulted in a dose‐dependent increase in the levels of reactive oxygen species (ROS). Prolonged exposure to PE resulted in HASMC apoptosis. Both the increase in ROS and apoptotic cell death were blocked by the non selective alpha1‐AR antagonist prazosin as well as the selective alpha1D‐AR antagonist BMY 7378 but not the alpha1A‐AR antagonist WB 4104. Increases in ROS and apoptosis were also blocked in a dose‐dependent fashion by the p38 MAP kinase inhibitor SB 202190 and the NAPDH oxidase inhibitor apocynin but not the ERK1/2 inhibitor PD 98059 nor the JNK inhibitor SP 600125. Pifithrin alpha, an inhibitor of the tumor suppressor protein p53, had no effect on ROS generation but did block alpha1‐AR‐induced apoptosis. Activation of the alpha1‐AR resulted in translocation of p53 to the mitochondria. SB 202190, apocynin and pifithrin all blocked p53 translocation. These data show that p38 and NADPH oxidase are involved in alpha1‐AR mediated increases in ROS. Alpha1‐AR apoptosis engages a p53 dependent pathway that includes the mitochondrial translocation of p53. The data also provide evidence of a linkage between the alpha1‐AR and p53.(HL‐38120)
Inhibition of arachidonic acid‐induced ER stress and mitochondrial dysfunction by novel dithiolethiones
Authors: Shin Sang Mi , Kim Sang Geon
Published: June 2021

The mitochondrial respiratory chain is a major source of reactive oxygen species (ROS) under pathological conditions. Generation of ROS can induce ER stress, and may lead to apoptosis. This study investigated whether arachidonic acid (AA), a representative proinflammatory ω‐6 fatty acid, causes ROS‐mediated mitochondrial impairment and ER stress, and if so, whether a new dithiolethione compounds (DTTs) protect cells from AA‐induced apoptosis. In HepG2 cells, DTTs inhibited ER expansion induced by AA. Moreover, DTT treatment abrogated Grp78 and CHOP induction in AA‐treated cells. Inhibition of AA‐induced ER stress by the agents was responsible for the decrease in apoptosis. DTTs effectively inhibited mitochondrial permeability transition promoted by AA in combination with Fe++, thereby protecting cells from ROS‐induced apoptosis. It appeared that AMPK activation by DTTs contributed to cell survival effects, which was partly supported by the reversal of DTTs's restoration of mitochondrial membrane potential by concomitant compound C treatment. As a same token, AICAR, an AMPK activator, inhibited AA + Fe++‐induced mitochondrial permeability transition with an increase in cell viability. Our data support the conclusion that new DTT compounds were capable of protecting cells from AA‐induced ER stress, ROS production, and mitochondrial dysfunction, which might be associated with AMPK activation.
Ethanol Withdrawal Provokes Mitochondrial Membrane Aging
Authors: Jung Marianna E , Wilson Andrew M , Metzger Daniel , Brown Nastassia , Mallet Robert T , Simpkins James W
Published: June 2021

We investigated whether abrupt withdrawal from chronic ethanol (EW) provokes mitochondrial aging. Five, 12, and 16 month‐old female rats consumed 6.5% ethanol for 3 mo, and then were abruptly withdrawn from the diet to impose EW stress. Mitochondria were isolated from cerebellum harvested at 24 h EW. Control rats consumed an isocaloric dextrin diet. Mitochondrial membrane swelling (MMS), an indicator of mitochondrial membrane fragility was assessed by measuring light absorbance of mitochondrial suspensions at 540 nm, and reported (Table: means ± SEM, n = 5–6) as time (min) to 50% decline in absorbance. While the control diet did not affect MMS at any age, EW accelerated MMS (*P < 0.05 vs. control diet), especially in the oldest rats. These results show that EW sensitizes mitochondrial membranes to aging in a manner that is more prominent in old than young rats. Mitochondrial membranes appear to be the target of a deleterious interaction between EW and aging brains of female rats. (Support: NIAAA/AA013864, NIAAA/AA015982).
Reactive oxygen species are required for normal endothelial function in coronary microvasculature
Authors: Kang Lori Sunhae , Muller‐Delp Judy Marie
Published: June 2021

Oxidant stress imparts deleterious effects on the endothelium, which is exemplified by the redundant production of superoxide (O2−). The beneficial effects of NO‐mediated vasodilation are quickly abolished in the presence of O2−, and this effect may be augmented with aging. Estrogen, conversely, may bestow endothelial protection by enhancing endogenous antioxidant function. We previously demonstrated an age‐induced impairment of flow‐induced dilation (FID) in rat coronary arterioles. Therefore, the purpose of this study was to determine the effects of O2− scavenging, as well as removal of H2O2, the byproduct of O2− scavenging, on FID in coronary resistance arterioles of young (4 mo) and aged (24 mo) male and female (YM, OM, YF, OF) Fisher 344 rats. Endothelium‐dependent vasodilation was evaluated by increasing intraluminal flow (5–60 nL/s) before and after treatment with the superoxide dismutase mimetic Tempol (100 μM), the H2O2 scavenger catalase (100U), or both Tempol + catalase. Removal of H2O2 with catalase reduced FID in YM, OM, and YF (p<.05), and Tempol diminished vasodilation in YF (69%, p<.05). Tempol + catalase blunted FID in young animals, but had no effect in aged groups. These data indicate that removal of oxidant radicals impairs FID, suggesting that basal levels of reactive oxygen species are necessary for normal endothelial function. Supported by NIH R01HL077224
Low Oxygen Affinity Hemoglobin HBOC‐201 enables increased Oxygen Uptake (VO2) In Bovine Pulmonary Artery Endothelial Cells (BPAEC)
Authors: Karaian John Edward , Mongan Paul D. , MacDonald Victor W. , Bunger Rolf
Published: June 2021

Hemoglobin‐based oxygen carriers (HBOCs) with various oxygen affinities are currently being evaluated for clinical use. We tested whether HBOCs with high vs. low oxygen affinities can affect cellular oxygen uptake (VO2), using a BPAEC model.

Methods:

Confluent BPAECs, passages 3–6, were maintained in RPMI media, supplemented with 10% FBS, L‐glutamine, and antibiotics. 5 ×107 cells, were placed in a respirometer, containing 100μl of room air saturated buffer in the absence or presence of 1.35 g/dl HBOC‐201® (p50=26 torr @ 21.5ºC) or 1.35 g/dl HbA0 (p50= 6.9 @ 21.5ºC). Uncoupling 0.3μM carbonyl cyanide 4(tri‐fluoromethoxy) phenylhyrdrazone (FCCP) was used to trigger maximal mitochondrial VO2. The HBOC‘s oxygen dissociation curves were corrected for pH and temperature (J Appl Physiol. 94(2):561–6.2003). VO2 was defined as use of (free + bound) O2 divided by the time to full O2 depletion.

Results:

Untreated BPAECs had a VO2 of 3.09 ± 3.9 nmoles O2/min, which increased to 7.34 ± 5.1 with FCCP. The VO2 of BPAECs treated with HBOC‐201 was 6.69 ± 2.5 nmoles O2/min, which increased to 10.43 ± 9.1 with uncoupler. When 2,3‐DPG‐free human HbA0 was combined with FCCP the VO2 was 4.41 ± 2.3 nmoles O2/min, higher than basal control VO2 but lower than that in presence of HBOC‐201 + FCCP.

Conclusion:

Low oxygen affinity HBOC‐201 facilitates VO2 by BPAECS relative to high oxygen affinity HBOC preparations such as human HbA0.
Nitric Oxide Production By Contracting Rat Mesenteric Lymphatic Vessels Is Primarily Within Valvular Regions
Authors: Bohlen Harold Glenn , Gasheva Olga , Gashev Anatoliy , Zawieja David
Published: June 2021

Nitric oxide produced by lymphatic endothelial cells is required for diastolic relaxation during lymphatic pumping. We propose increased lymph shear during contraction increases NO for relaxation. Additionally, valvular regions with endothelial‐coated valves might be a primary site for NO generation. Mesenteric lymph vessels of anesthetized rats were studied with NO microelectrodes during contractions. On both lymphatic bulb and downstream tubular regions, [NO] began to increase during each contraction and peaked after the contraction subsided. The valvular region [NO] was 1054+154 nM compared to 620+126 nM for tubular sites ∼ 500 um away (7 vessels, 4 rats). These [NO] are as high as that of nearby large arterioles. Outside the valvular wall by one adipocyte (∼40–60 um), the [NO] was 542+192 nanomolar, demonstrating the diffusion of NO away from the lymphatic to the surrounding tissue. As the frequency of contractions increased, the average [NO] increased and if spontaneous contractions declined, so did the average perilymphatic [NO]. The overall data indicate valvular areas of lymph vessels are capable of rapid NO responses to individual contractions and the average [NO] is contraction frequency dependent. Valvular regions may be the primary site of NO generation for the lymphatic vessels given the much higher [NO] than in downstream tubular regions. (Supported by HL70308)
Acute ischemic stress causes augmentation of delayed rectifier K+ and Na‐K‐pump currents and inhibition of inward rectifier K+ current in arteriolar smooth muscle cells
Authors: Ma Ke‐Tao , Yang Yu‐Qin , Jiang Zhi‐Gen
Published: June 2021

Ischemia generally induces a protective vasodilation in the most vascular beds but the underlying cellular mechanism remains controversial. Using conventional intracellular and whole‐cell recording from smooth muscle cells in situ arteriolar segments of the brain, mesenteric and the cochlear spiral modiolar arteries of guinea pigs, we analyzed the membrane actions of acute ischemic treatment (AIT, hypercapinic hypoxia, pO2 =0 mmHg, pH=6.0). The AIT for 2 to 30 min caused a reversible vasodilation and a hyperpolarization in the majority of cells recorded from all the three arterioles. ACh‐ and 10 mM K+‐induced dilation was significantly reduced, lost or reversed to a constriction during and after the AIT. At −40 mV, AIT induced a 10–50 pA outward current within 1–2 min followed by a slowly increasing outward current until ending of the AIT. During the AIT, the whole‐cell I/V curve shifted up‐ and left‐ward with a 15–35 mV hyperpolarization in the resting potential. Ouabain reduced the I/V shift. A computational modeling analysis indicated that at least three current mechanisms underlied the I/V shift. 1) The maximum conductance of delayed rectifier K+‐channels (Kdr) increased by 1–4 fold and its half‐activation voltage shifted leftward by 5–20 mV. TEA significantly attenuated this augmentation. 2) The Na‐K‐pump current was enhanced from a negligible to a substantial level. 3) The Kir current was reduced by 75 to 100%. Upon wash with normal bathing solution, the Kdr recovered in a few min but the changes of pump current and Kir recovered only partially after 1 h. We conclude that the acute ischemia induces hyperpolarization, dilation and loss of EDHF function by implicating activation of Kdr and Na‐K‐pump and inhibition of Kir currents. Supported by NIH NIDCD DC 004716.
Neointimal formation is exacerbated following vascular injury in p22phox overexpressing transgenic mice
Authors: Weber David S , Manogue Michael , Drake Douglas
Published: June 2021

These studies tested the hypothesis that transgenic mice overexpressing the p22phox subunit of the NAD(P)H oxidase selectively in smooth muscle (Tgp22smc) would exhibit exacerbated neointimal formation following mechanically induced transluminal vascular injury compared to wild‐type control mice. Tgp22smc mice have a two‐fold increase in p22phox expression and H2O2 production; thus providing a model to examine the effects of elevated vascular ROS production on VSMC function in vivo. To induce vascular injury, a 0.36 mm steel catheter was guided 4–5 mm into the left common carotid via the external carotid. Following one minute the catheter was removed and reperfusion verified. The degree of neointimal formation was assessed by morphological analysis of wall thickness (WT) and cross‐sectional wall area (CSWA) in male Tgp22smc mice and littermate controls 14 days following injury. WT following injury indicated significantly greater neoinitimal formation in Tgp22smc mice (54.3 ± 7.3 vs. 43.7 ± 4.1 μm), while non‐injured contralateral carotids were similar between groups (28.5 ± 3.3 vs. 33.8 ± 2.8 μm). Moreover, the increase in carotid artery CSWA following injury was approximately four‐fold greater in Tgp22smc mice compared to control animals. Findings from these in vivo studies strongly support in vitro data which has implicated ROS as a crucial mediator of both VSMC proliferation and migration. AHA 03300119N
Direct evidence for the role of PECAM‐1 in mechanotransduction and vascular remodeling
Authors: Chen Zhongming , Tzima Eleni
Published: June 2021

Flow‐induced shear stress plays an important role in the development of the vasculature, the maintenance of vascular integrity and vascular diseases, such as atherosclerosis. Disturbed flows near bifurcations and curvatures are associated with increased inflammation, leukocyte adhesion and atheroma formation. We have previously shown that platelet endothelial cell adhesion molecule‐1(PECAM‐1) plays an important role in mechanotransduction. Here, we investigate the role of PECAM‐1 in flow‐induced inflammation and flow‐mediated remodeling. PECAM‐1 knockout (KO) and reconstituted (RC) endothelial cells were subjected to oscillatory flow and their inflammatory response was assayed. Oscillatory flow induced sustained NF‐kB activation in endothelial cells expressing PECAM‐1; however this was not seen in KO endothelial cells. Prolonged oscillatory flow in RC cells promoted expression of the cell adhesion molecules ICAM‐1 and VCAM‐1, which resulted in increased leukocyte adhesion to the endothelium. These effects were significantly dampened in cells that lacked PECAM‐1. To test the role of PECAM‐1 in flow‐mediated remodeling in vivo, we performed partial ligation of the left carotid artery in PECAM‐1‐null and wild‐type mice. Importantly, we observed NF‐kB activation, increased ICAM‐1 and VCAM‐1 expression, intima‐media thickening of the left carotid artery and leukocyte transmigration in wild‐type mice. In contrast, the PECAM‐1 null mice showed no activation of NF‐kB, reduced expression of ICAM‐1 and VCAM‐1 and substantially decreased remodeling. These observations indicate that disturbed shear stress activates NF‐kB and inflammatory responses through PECAM‐1 in endothelial cells. These effects further induce arterial remodeling in vivo. Therefore, we conclude that disturbed shear stress initiates atherogenesis and vascular remodeling in vivo through PECAM‐1.