bluuebunny/crossref_metadata_2025 huggingface dataset:
Synthesis and biological evaluation of curcumin analogues without the beta‐diketone moiety
Authors: Li Xiaokun , Liang Guang , Xiao Jian , Huang Zhifeng , Zhou Huiping
Published: June 2021

Curcumin is an excellent lead compound with a variety of biological activities. However, its potential beneficial effects are limited due to its instability and low bioavailability in vivo. It is believed that presence of the beta‐diketone moiety attributes to the instability of curcumin under physiological condition, poor absorption and fast metabolism. In the present study, a series of curcumin analogues with more stable structures and better biological properties were synthesized. Methods: Curcumin analogues without the beta‐diketone moiety were synthesized by reacting relevant arylaldehydes and cyclopentanone in alkaline condition. The structures were determined by HNMR and MS. The crystal structures were analyzed by X‐ray diffraction. The antibacterial activities against seven Gram‐positive and Gram‐negative bacteria were tested in vitro and anti‐inflammatory activates were examined by measuring the inhibitory effect on LPS‐induced TNF‐alfa and IL‐6 expression in macrophages using ELSA. Results: Most of the analogues exhibited inhibitory activity against the ampicillin‐resistant Gram‐Enterobacter cloacae. Several analogues displayed better anti‐bacterial and anti‐inflammatory activities compared to curcumin. Conclusion: The properties and position of the substituent and the space of the linking strain determine the anti‐bacterial and anti‐inflammatory activities.
Inhibitory effects of propenone derivatives on angiogenesis in vitro and in vivo
Authors: Lee Jong Suk , Kim Jong Tae , Thapa Dinesh , Park Byung Chul , Choi Mi Kyoung , Lee Eung‐Seok , Kim Jung‐Ae
Published: June 2021

Previously, 1‐furan‐2‐yl‐3‐pyridin‐2‐yl‐propenone (FPP‐3) has shown an anti‐invasive activity through inhibitory effect on matrix metalloproteinase (MMP)‐2 and MMP‐9 that are important enzymes involved in angiogenesis. In this study, the propenone derivatives, 1,3‐diphenyl‐propenone (DPhP), 3‐phenyl‐1‐thiophen‐2‐yl‐propenone (PhT2P), 3‐phenyl‐1‐thiophen‐3‐yl‐propenone (PhT3P) and 1‐furan‐2‐yl‐3‐phenyl‐propenone (FPhP‐3) were investigated for an antiangiogenic activity. Among the propenone derivatives, 1,3‐diphenyl‐propenone (DPhP) and 1‐furan‐2‐yl‐3‐phenyl‐propenone (FPhP‐3) showed strong inhibitory effects on both VEGF‐ and interleukin (IL‐8)‐induced angiogenesis in chick chorioallantoic membrane (CAM) assay in vivo. We also found that DPhP and FPhP‐3 concentration‐dependently inhibited VEGF‐induced endothelial cell invasion, one of the important criteria for angiogenesis in vitro, by using matirgel‐coated transwell assay. In addition, DPhP and FPhP‐3 significantly inhibited VEGF‐induced proliferation and tube formation of endothelial cells. Taken together, our results suggest that DPhP and FPhP‐3, propenone derivatives, may be valuable drug candidates as an angiogenesis inhibitor. [This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MOST) (No. R11‐2007‐040‐02004‐0).]
Molecular targets of immunosuppressive pyrazole derivatives: From TRPC channels to mTOR
Authors: Ingraham Richard H. , Bananis Eustratios , Hansen Gale , Proudfoot John R. , Latli Bachir , Tibolla Annette , Akiba Isamu , Ueda Saisoku , Kameda Kinu , Barsoumian Edward L. , Jakes Scott , Papov Vladimir
Published: June 2021

Pyrazole‐containing immunosuppressive compounds exemplified in the literature by BTP2 have been identified in several labs. Published studies demonstrated that BTP2 can inhibit both receptor and store‐operated Ca2+‐influx in both native cells and cells transfected with members of the TRPC ion channel family. Here, we show the results of pull‐down studies performed with a biotinylated BTP2 analog attached to avidin beads. Lysates were prepared from Jurkat cells and HEK293 stably transfected with TRPC6, TRPC3, or TRPC5. Binding proteins were identified by mass spec, Western blots, or both. Identified binding proteins included TRPC3, TRPC5, TRPC6, STIM1, eIF3, PKCα, FKBP52, FKBP12, mTOR, calcineurin, and p70 S6 kinase. Many of the proteins in this group are known to complex with or regulate other members so it is not clear which proteins have actual binding interactions with the inhibitor probe. A potential link is the presence of FKBP proteins which are involved in regulation of both the TRPC and mTOR pathways. We observed direct probe binding to recombinant FKBP12 and an ability of PKC inhibitors to reverse Ca2+‐influx inhibition by BTP2 in HEK293‐TRPC6 cells. PKC phosphorylation of TPRC molecules inhibits their activity and activates TRPM4 activity. We propose that BTP2‐like compounds inhibit dephosphophorylation of TRPC to help impart their immunosuppressive effects.
Induction of Orphan Nuclear Receptor Nurr1 Expression by the Human EP1 Prostanoid Receptors
Authors: Ji Ruyue , Chen Xiaobo , Regan John W
Published: June 2021

The EP1 prostanoid receptor is a G‐protein coupled receptor that is activated by prostaglandin E2 (PGE2). Using gene microarray analysis, the expression of Nurr1 was found to be up‐regulated by PGE2 in HEK cells stably expressing human EP1 receptors. Nurr1 is an orphan nuclear receptor that has been found to play an important role in the development of midbrain dopaminergic neurons. The up‐regulation of Nurr1 expression was confirmed by immunoblot analysis and could be blocked using a dominant negative mutant of the cAMP response element binding protein (CREB) and by the NF‐kappa B inhibitor, BAY11‐7082. However, PGE2 did not stimulate intracellular cAMP formation suggesting that the PGE2 mediated induction of Nurr1 expression by the EP1 receptor involves the activation of NF‐kappa B and CREB signaling in a cAMP independent manner. Furthermore EP1 receptors have been reported to be expressed in the dopaminergic neurons of the rat substantial nigra. Our findings provide a basis for possible interactions between EP1 receptors and Nurr1 in dopaminergic signaling pathways.
Inhibition of proteolytic degradation and characterization of binding of natural angiotensins to AT1 receptors in brain and liver
Authors: Karamyan Vardan T. , Speth Robert C.
Published: June 2021

Metabolism of angiotensins in receptor binding assays is a continuing problem. The suggestion that brain AT1 receptors (AT1R) differ from peripheral AT1R necessitates accurate appraisal of brain AT1R binding. The goal of this study was to establish experimental conditions that preserve 125I‐Ang II and ‐III, to characterize their binding to rat hypothalamic and liver AT1R. Negligible specific binding of 125I‐Ang III and ‐II is observed in hypothalamic membranes (45 or 60min at 24oC in 5mM EDTA, 150mM NaCl, 0.1mM bacitracin, 50mM NaPO4, pH 7.2 buffer + 10 μM PD123319) due to severe proteolytic degradation, as < 5% of bound 125iodine corresponds to 125I‐Ang III or ‐II. In liver membranes, 125I‐Ang III is severely degraded, whereas 125I‐Ang II shows high affinity saturable binding to AT1R with > 50% of bound 125I corresponding to 125I‐Ang II. Addition of 1 mM o‐phenanthroline & phenylmethylsulfonyl fluoride, and 3 mM puromycin & glutamate phosphonate to the assay buffer increased bound 125I‐Ang II and ‐III to > 80%, both in liver and hypothalamic membranes revealing abundant, high‐affinity binding to AT1R. The affinity of both ligands for brain AT1R was less than for liver. 125I‐Ang III showed a smaller Bmax for AT1R in liver than 125I‐Ang II. These results indicate that both peptides bind with higher affinity for liver AT1R than brain AT1R, but that 125I‐Ang II binds to a larger population of AT1R in liver than does 125I‐Ang III.
Further characterization of the novel, non‐AT1, non‐AT2 brain‐specific angiotensin binding site in the mouse brain
Authors: Rabey Felicia M. , Karamyan Vardan T. , Speth Robert C.
Published: June 2021

To further characterize the non‐AT1, non‐AT2 binding site in mouse brain, differential centrifugation was used to assess its subcellular distribution. The fraction enriched in plasma membranes (P2) from both cerebral cortex (CTX) and hypothalamus (HT) showed a higher density of 125I‐Ang II binding relative to the nuclear/mitochondrial/debris (P1) fraction: CTX P1 Bmax= 21.9±3.6 fmol/mg protein, CTX P2 Bmax = 63.1±11.1 fmol/mg protein; HT P1 Bmax = 24.5±7.6 fmol/mg protein, HT P2 Bmax = 38.3±9.8 fmol/mg protein. The enriched binding in CTX and HT plasma membranes (71 and 66% of total binding, respectively) suggests that the non‐AT1, non‐AT2 angiotensin binding site is primarily localized to plasma membrane. Since p‐chloromercuribenzoate (PCMB), the agent that unmasks this binding site, may simulate pathophysiological conditions in the brain associated with an altered redox state, we examined the effect of reduced glutathione (GSH) on the ability of PCMB to unmask the binding site in P2 membranes. GSH at concentrations ranging from 0.3 to 10 mM inhibited 125I‐Ang II binding to the non‐AT1, non‐AT2 binding site from 78 to 87% in a concentration‐related manner. A small residual amount of 125I‐Ang II binding to the non‐AT1, non‐AT2 binding site in the absence of PCMB was also inhibited by 0.3 to 10 mM GSH by 51 to 70%. Functionality of the non‐AT1, non‐AT2 binding site may thus depend on redox state of the brain.
Relationship between Ser363 phosphorylation and internalization of the delta opioid receptor
Authors: Bradbury Faye , Zelnik Jennifer , Traynor John
Published: June 2021

Phosphorylation of Ser363 in the cytoplasmic tail of the delta opioid receptor (DOR) usually precedes receptor internalization, but is not necessarily a prerequisite for internalization. In these studies we measured phosphorylation of DOR Ser363 and DOR internalization in HEK 293 cells stably expressing FLAG‐tagged DOR in response to exposure to a variety of agonists. These agonist‐mediated responses were compared with their efficacy in the [35S]GTPγS assay. The agonists DPDPE and deltorphin were fully efficacious in the [35S]GTPγS assay and produced extensive phosphorylation and internalization. Morphine was a partial agonist in the [35S]GTPγS assay and failed to induce phosphorylation or internalization. In contrast, TAN‐67 a putative DOR1 agonist with a high degree of efficacy in the [35S]GTPγS assay, caused a high degree of Ser363 phosphorylation but very little internalization. A further disconnect between Ser363 phosphorylation and internalization was observed in FLAG‐tagged DOR cells where overexpression of GRK 2, 5, or 6 increased morphine‐induced phosphorylation of DOR Ser363, but did not increase morphine‐induced internalization. Conversely, overexpression of dominant negative GRK2‐K220R decreased DPDPE‐induced internalization. These results confirm a disconnection between phosphorylation of Ser363 and internalization. Supported by DA04087 and DA07261.
Molecular regulation of lysophosphatidic acid receptor endocytosis and desensitization
Authors: Aziziyeh Adel Isam , Pape Cynthia , Pampillo Macarena , Possmayer Fred , Di Guglielmo John , Babwah Andy , Bhattacharya Moshmi
Published: June 2021

Lysophosphatidic Acid (LPA) is a major constituent of serum and is involved in a variety of physiological and pathophysiological processes. LPA signals via the G‐protein coupled receptors (GPCRs), LPA1–3, but the mechanisms regulating LPA receptor desensitization and endocytosis are unknown. GPCRs can internalize via clathrin‐dependent mechanisms and/or lipid rafts and caveolae. We show that Flag‐LPA1 and ‐LPA2 receptors do not partition into lipid rafts of LPA treated human embryonic kidney (HEK) 293 cells suggesting that LPA1 and LPA2 do not internalize via caveolae. Surprisingly, the expression of dynamin‐1 and ‐2 mutant proteins does not affect receptor internalization. We also examined whether RalA and phospholipase Ds, shown to regulate the endocytosis of certain GPCRs, contribute to LPA receptor endocytosis. Although these proteins interact constitutively and co‐localize with both LPA1 and LPA2 in endocytic vesicles, expression of wild‐type or mutant forms of these proteins did not affect receptor internalization. Finally, we observed that the expression of GRK2 and βarrestins mediated the desensitization of LPA1 and LPA2 by attenuating LPA induced inositol phosphate production. In summary, RalA and PLDs may not mediate LPA1 or LPA2 endocytosis, but may act as scaffolding proteins linking the receptors to other signaling pathways.

This study is funded by NSERC grant awarded to Dr. M Bhattacharya
Agonist‐promoted internalization of the P2Y1 receptor in Madin‐Darby Canine Kidney (MDCK) cells
Authors: Qi Ai‐Dong , Houston Dayle , Naruszewicz Izabela , Morris Dionne , Harden T. Kendall , Nicholas Robert A.
Published: June 2021

Nucleotide‐activated P2Y1 receptors (P2Y1‐R) regulate a variety of physiological processes, including ion flux in epithelial cells. We utilized radioimmunoassays (RIA) and radioligand‐binding assays with [32P]MRS2500, a P2Y1‐R‐selective antagonist, to quantify agonist‐promoted internalization of the P2Y1‐R. Treatment of MDCK epithelial cells, which endogenously express the P2Y1‐R, with the P2Y1‐R‐selective agonist 2MeSADP resulted in a 40–50% decrease in cell‐surface [32P]MRS2500 binding sites with a t½ of ~12 min. Importantly, HA‐tagged human P2Y1‐R stably expressed in MDCK cells exhibited identical kinetics of agonist‐induced internalization. Deletion of residues 340–373 in the C‐terminus of the P2Y1‐R resulted in loss of agonist‐promoted internalization, and mutational analysis revealed that S352 and S354 were largely responsible for agonist‐promoted internalization. Interestingly, mutation of Ser336 and Thr339, which are predicted to be PKC phosphorylation sites, in the sequence S336RAT339 to Ala (ARAA) resulted in receptors that internalized faster (t1/2 = 4 min) and to a greater extent (70–80%) in response to agonist than wild‐type P2Y1‐R. In contrast, agonist‐promoted internalization was abolished when the SRAT sequence was mutated to DRAD. These data indicate that the C‐terminal tail is critical for both agonist‐induced endocytosis and plasma‐membrane‐retention of the P2Y1‐R.
Differential Regulation of the M1 Muscarinic Acetylcholine Receptor by Orthosteric and Allosteric Ligands
Authors: Thomas Rachel , Langmead Christopher , Wood Martyn , Challiss John
Published: June 2021

AC‐42 (4‐n‐butyl‐1‐[4‐(2‐methylphenyl)‐4‐oxo‐1‐butyl] piperidine hydrogen chloride) is a selective M1 muscarinic receptor agonist with an allosteric mechanism of action. Here, we have investigated whether AC‐42 and two orthosteric muscarinic agonists (oxotremorine‐M (oxo‐M) and pilocarpine) cause internalization and down‐regulation of the M1 muscarinic receptor. Oxo‐M (EC50 0.8 μM) caused the greatest increase in [35S]‐GTPγS‐Gαq/11 binding in membranes from Chinese hamster ovary cells stably expressing the M1 muscarinic receptor, whereas AC‐42 (EC50 1.6 μM) and pilocarpine (EC50 2.4 μM) stimulated similar responses that were approx. 30% of the oxo‐M response. Using intact cell [3H]‐NMS and [3H]‐QNB binding assays, we next showed that after 24 h, oxo‐M (100 ìM) caused 68 ± 3% receptor internalization and 42 ± 3% receptor down‐regulation. Over a similar time‐course, pilocarpine (1 mM) caused 46 ± 7% internalization and 26 ± 8% down‐regulation. In contrast, AC‐42 did not cause significant receptor internalization or down‐regulation. These data suggest that while AC‐42 is able to activate selectively Gq/11‐dependent signaling through the M1 mACh receptor, it does not cause receptor internalization or down‐regulation, at least over the time‐course studied here.
Role of adenylyl cyclase type VII in signaling through CRFR1α receptors and HPA axis activation during ethanol intoxication: study using male and female animals
Authors: Pronko Sergey , Pronko Hanna , Saba Laura , Hoffman Paula L
Published: June 2021

Ethanol (Eth) can increase plasma ACTH and glucocorticoids even though Eth is thought to be anxiolytic. Studies using corticotrophs, have indicated that CRF 1α receptors (CRF‐R1α) initiate ACTH release through coupling to adenylyl cyclase type 9 and type 7 (AC7). Prior studies have shown that Eth enhances AC7 responsiveness to Gsα. We hypothesized that Eth through increasing the affinity of the AC7 for Gsα, can increase CRF‐R1α signaling, leading to increased ACTH levels.

We measured plasma ACTH and corticosterone (CS) levels after ip injection of Eth (2.25 g/kg) at time points of 0‐75 min in male and female AC7 transgenic (TG) and heterozygous knockdown (HET) mice. TG mice with transgene (human) AC7 mRNA had significantly higher (≈38%) AC7 protein in the pituitary, as measured with our antibody to AC7. HET mice had ≈50% lower levels of AC7 mRNA and significantly lower (≈26%) pituitary amounts of AC7 protein compared to WT mice.

After ip Eth TG male mice displayed significantly higher plasma ACTH levels compared to WT and HET group. The HET group displayed significantly lower ACTH levels in plasma. Over‐ or underexpression of AC7 in brains of mice may be a key factor in differential ACTH response of these animals to alcohol.

ACTH and especially CS levels were markedly higher in female mice compared to male mice despite the same blood alcohol levels.

Supported by NIAAA and Banbury Foundation.
The role of p53 pro‐apoptotic target, PERP, induces mitochondrial permeability and apoptosis in hypoxic renal cells
Authors: Singaravelu Kurinji , Padanilam Babu
Published: June 2021

PERP, a direct target of p53, has lately been implicated in apoptosis, and we reported earlier a p53‐dependent upregulation of PERP in proximal tubular epithelia subsequent to ischemia/reperfusion injury (IRI); nonetheless, the role of PERP in IRI is unknown. To test our hypothesis that PERP alters mitochondrial membrane permeability (MMP) to induce apoptosis following renal IRI, we developed renal tubular epithelial cell lines (LLCPK1) stably overexpressing PERP (P‐Epi) or control vector (C‐Epi) and subjected to 4 hour (hr) hypoxia in a glucose‐free buffer followed by 1hr normoxia with normal medium replacement. By 1hr post‐injury, the mitochondrial membrane potential is significantly altered in P‐Epi cells compared to C‐Epi or uninjured cells (p< 0.001; n=3) as analyzed by flow cytometry with JC‐1 probing. In addition, AIF and cytochrome c were released from mitochondria earlier by 3hrs post‐injury in P‐Epi cells compared to C‐Epi cells. Further, the number of P‐Epi cells undergoing apoptosis by 3hrs post‐injury was higher compared to control cells (p< 0.05; n=3). Thus we conclude, upregluation of PERP accelerates the release pro‐apoptotic molecules by altering MMP after IRI in renal tubular epithelia. Targeting a p53 downstream molecule rather than p53 per se may offer a specific therapeutic strategy to inhibit p53‐mediated apoptosis in acute kidney injury. Support by Univ Nebraska Med Cntr and NKF Nebraska.
α‐CGRP diminishes infarct size in a murine model of myocardial ischemia‐reperfusion injury
Authors: Katki Khurshed A , Bhardwaj Ashwani K , DiPette Donald J , Supowit Scott C
Published: June 2021

The efferent function of the peripheral sensory nervous system is mediated by the release of neuropeptides, such as α‐calcitonin gene‐related (CGRP). CGRP, a potent vasodilator, has been suggested to play a beneficial role in myocardial ischemia/reperfusion (I/R) injury. Therefore, we examined the role of α‐CGRP in I/R injury, using a mouse model of acute myocardial I/R. CGRP knockout (KO; n=6/group) and wild type (WT; n=6/group) mice received 30 minutes of occlusion of the left anterior descending artery, followed by 24 hours of reperfusion. Hearts were evaluated for infarct size by Evan's Blue and TTC staining. Plasma troponin and cardiac isoprostanes were also assessed. After I/R, the area at risk (AAR) was increased 55% in both the KO and WT mice. The α‐CGRP KO mice exhibited a significantly increased area of infarct (IA; 35%) compared to their WT counterparts (28%). Plasma troponin, a marker of ischemic injury, was elevated in both KO (0.8±0.4 ng/ml) and WT (0.9±0.6 ng/ml) mice after I/R, compared to the respective sham animals (KO, 0.5±0.08 ng/ml; WT, 0.5±0.2 ng/ml). The α‐CGRP KO mice exhibited a 30% rise in cardiac isoprostanes (a marker of oxidative stress) compared to sham animals, while the WT mice showed an increase of 20% compared to sham animals. Therefore, these data indicate that deletion of α‐CGRP gene makes the heart vulnerable to I/R injury possibly due, at least in part, to increased oxidative stress.
Cardiac Overexpression of Constitutive Active Form of Zea maize Rac‐D in Transgenic Mice Deteriorates Postischemic Contractile Recovery
Authors: Moustafa‐Bayoumi Moustafa , Talukder Hassan , Alhaj Mazin , El‐Sayed Osama , Abouelnaga Zenib , Hassona Mohamed , Zweier Jay , Goldschmidt‐Clermont Pascal , Hassanain Hamdy
Published: June 2021

The present study investigated the effect of increased reactive oxygen species (ROS) in the heart on myocardial injury. Our hypothesis is that transgenic mice overexpressing Rac 1 in the heart will have increased ROS generation and poor contractile recovery during ischemia reperfusion (I/R). We have generated a transgenic mouse model that expresses the cDNA of a constitutively active mutant of Zea maize Rac‐D gene in the heart of FVB/N mice using a mouse α‐myosin heavy chain promoter. IHC analysis confirmed the expression of Rac‐D protein in the heart of transgenic (TG) mice. Echocardiographic analysis showed significantly low ejection fraction in TG mice compared to control mice [20±4 (TG) vs. 62±5 (control), P<0.05]. I/R studies were performed in isolated hearts from age matched control and TG mice. Rac‐D TG mice demonstrated markedly poor post.ischemic contractile recovery [59±4% (TG) vs. 99±5% (control), P<0.001] with frequent abnormal ventricular rhythms compared to age matched control. These results suggest an important role of Rac 1 in NADPH‐ROS mediated myocardial injury during I/R. In conclusion, the Rac‐D transgenic animal model provides an advantage to investigate the signaling mechanisms regulating ischemic heart diseases. In addition, it is a unique model for in vivo studies utilizing novel therapeutic approaches targeting Rac 1 to reduce ischemic heart diseases.
The combination of protein kinase C epsilon activator (PKC ε +) and tetrahydrobiopterin (BH4) exerts cardioprotective effects in ischemia/reperfusion injury (I/R) when given during reperfusion
Authors: Pershad Sailesh Ram , Chen Qian , McGraw Sloane , Adams Jovan , Burkhart Samantha , Young Lindon Howard
Published: June 2021

In the presence of polymorphonuclear leukocytes (PMNs) during reperfusion, following myocardial ischemia, cardiac contractile dysfunction is observed. Isolated ischemic (20 min) and reperfused (45 min) rat hearts in the presence of activated PMNs were studied. A cell permeable peptide (PKC ε+) that enhances interaction of PKC ε with endothelial nitric oxide synthase (eNOS) to increase nitric oxide (NO) release, and BH4, an essential cofactor of eNOS, were given during the first five min of reperfusion. In hearts given PKC ε+ (10 μM) and BH4 (5 μM) (n=7) with PMNs, left ventricular developed pressure (LVDP) significantly recovered to 92% ± 11% of baseline values compared to I/R + PMN hearts (n=5) (48% ± 7%) (p<0.05) and the maximal rate of LVDP (+dP/dtmax) significantly recovered to 83% ± 7% compared to I/R + PMN hearts (34% ± 4%) (p<0.01) at 45 min reperfusion. The cardioprotective effects of PKC ε+ and BH4 were associated with an approximate 50% decrease in PMN infiltration in post‐reperfused hearts as confirmed by myeloperoxidase assay. These results suggest that the combination of PKC ε+ and BH4 attenuates PMN‐induced cardiac contractile dysfunction after I/R, in part by promotion of eNOS to increase NO release and inhibition of PMN infiltration in post‐reperfused hearts. This study was supported by NHLBI Grant 1R15HL‐76235‐01 and the Center for the Chronic Disorders of Aging at PCOM.
Blocking mitochondrial Ca2+ uniport activity during activated Na+/H+ exchange reduces mCa2+ loading but does little to better protect function on reperfusion
Authors: Aldakkak Mohammed , Stowe David F , Heisner James S , Haumann Johan , Camara Amadou KS
Published: June 2021

We reported that alkaline buffer activated Na+/H+ exchanger (NHE) in isolated hearts and increased mitochondrial Ca2+ (mCa2+) and ROS production, which were abated by inhibiting NHE. We tested if blocking mCa2+ uniporter with ruthenium red (RuR, 2.5 μM) before and after ischemia, would reduce mCa2+ overload and ROS, and restore NADH and cardiac function during activated NHE. Guinea pig isolated hearts were perfused (HEPES buffer) with or without RuR at pH 7.4 (control), pH 8 to activate, or pH 6.5 to inhibit, NHE for 10 min before, after, and during 35 min ischemia. NADH, mCa2+, and ROS were assessed at the LV wall by fluorescence. We found that mCa2+ increased less during ischemia in all groups in the presence of RuR. NADH was higher with pH 8+RuR vs pH 8 alone, but RuR had no added effect in other pH groups. ROS was higher after pH 8 alone than in other groups, but adding RuR did not decrease ROS. pH 8+RuR did not protect LVP function or restore coronary flow better than pH 8, but pH 7.4+RuR was better than pH 7.4, pH 7.4 was better than pH 8±RuR, and pH 6.5 was most protective. Results indicate that RuR reduced mCa2+ loading (all pHs) and improved redox state (pH 8), did not reduce ROS (all pHs), and improved function (pH 7.4). These data show that blocking the mCa2+ uniporter reduces mCa2+ loading at all NHE activities but suggest that reducing mCa2+ may be less important than reducing ROS production for improving cardiac function after ischemia.
Metallothionein (MT) expression increases in skeletal muscle following tourniquet‐induced ischemia/reperfusion (I/R) injury
Authors: Urso Maria Laina , Szelenyi Eric R , Walters Thomas J
Published: June 2021

Increased MT and extracellular matrix (ECM) metalloprotease (MMP) activity has been documented in skeletal muscle following trauma. MTs bind zinc and MMPs, possibly affecting activation of the zinc‐dependent mTOR pathway and MMP‐dependent tissue repair post‐injury. Tourniquet (TK) application is used to control bleeding post‐injury, although I/R episodes associated with TK use result in skeletal muscle proteolysis. This study investigated TK‐induced I/R injury on MT, MMP, and mTOR pathway activation in rat extensor digitorum longus (EDL). A TK was applied to the hind limb of 14 male rats for 3 h, and then deflated, resulting in I/R injury. EDLs were harvested 2 h post‐reperfusion for analysis. I/R resulted in a significant (p<0.05) upregulation of MT (4.7‐ fold) and MMP‐9 (2.6‐ fold) mRNA, with no change in MMP‐2. Immunoblot analysis revealed that protein levels of MT were increased 24 ± 11.1 % (p<0.05). The mTOR protein level, its phosphorylation on Ser2448, and the activity of S6K1 were unaffected 2 h post‐I/R injury. Immunohistochemistry confirmed results. MT production is likely related to I/R‐induced oxidative‐stress, which potentially affects ECM integrity, explaining our observed increase in MMP‐9. These data suggest that oxidative‐mediated ECM disruption is a potential mechanism for increased skeletal muscle proteolysis in response to TK‐induced I/R‐injury.
Anoxia/reoxygenation‐induced cardiac myocyte apoptosis: role of HMGB1
Authors: Yang Min , Wu Jun , Martin Claudio , Rui Tao
Published: June 2021

It has been proposed that ischemia/reperfusion (I/R)‐induced myocardial dysfunction is due to myocytes apoptosis. However, the mechanism(s) involved in myocyte apoptosis after I/R is not completely clear, but appears to involve cytokines. High mobility group box 1 (HMGB1) is a nuclear non‐histone DNA binding protein that has been implicated in cell apoptosis. The aim of the present study was to assess the role of HMGB1 in myocyte apoptosis‐induced by anoxia/reoxygenation (A/R, in vitro counter part of I/R). Cultured murine cardiac myocytes were challenged with a 30 min of anoxia followed by 48 hrs of reoxygenation. The A/R challenge increased myocyte apoptosis (Caspase 3 activity and Cell Death ELISA). Concomitantly, there was an increase in HMGB1 expression in cardiac myocytes. Further, the A/R‐induced increase in myocyte apoptosis was prevented when the myocytes were pretreated with a HMGB1 inhibitor (glycyrrhizic acid). Finally, exposure of the cardiac myocytes to medium containing HMGB1 (1 μg/ml) for 24 hrs resulted in myocyte apoptosis. Taken together, our findings indicate that HMGB1 production by cardiac myocytes plays an important role in A/R‐induced myocyte apoptosis. (CIHR MOP 81303).
The Loss of MCP‐1 Attenuates Cutaneous Ischemic‐reperfusion Injury
Authors: Hasegawa Minoru , Saito Yuki , Fujimoto Manabu , Takehara Kazuhiko , Steeber Douglas A , Sato Shinichi
Published: June 2021

The formation of pressure ulcers is dependent on multiple factors including ischemic‐reperfusion (IR). This study assessed the mechanism of a previously reported murine model of cutaneous IR injury. Three cycles of IR (day 1–3) by external application of two magnetic plates were performed to induce pressure ulcer formation. Increased infiltration of neutrophils and macrophages, and augmented expression of proinflammatory cytokines and inducible nitric oxide synthase (iNOS), were observed during IR cycles. In this model, monocyte chemoattractant protein‐1 (MCP‐1) was remarkably increased at day 1 in the skin followed by inflammatory cell infiltration. Therefore, IR cycles were performed in MCP‐1 deficient (MCP‐1−/−) mice to evaluate the role of this chemokine in pressure ulcer development. MCP‐1−/− mice showed reduced macrophage infiltration and expression of tumor necrosis factor‐alpha and iNOS during IR cycles leading to attenuated apoptosis and skin injury. Importantly, MCP‐1 played a role in apoptosis and injury via inducing iNOS during the reperfusion rather than the ischemic period. These findings indicate that MCP‐1 may be a critical factor for macrophage recruitment and subsequent skin inflammation and injury during IR cycles. We propose that this is a useful model for investigating the mechanism of pressure ulcer formation using various transgenic mice.
Normal saline resuscitation induces increased sinusoidal MIP‐2 expression in early sepsis
Authors: Patrick Amanda L , Fox‐Robichaud Alison E
Published: June 2021

Fluid resuscitation is an integral part of the treatment of patients with severe sepsis. Common fluids administered within the first 6 hours of diagnosis include normal saline and Ringer's lactate, however, currently there is no clear evidence that support the use of one resuscitation fluid over another. Previous work in our lab demonstrated that normal saline increases leukocyte‐endothelial cell interactions. The objective in this study was to determine the mechanisms for saline‐induced hepatic leukocyte recruitment.

Sepsis was induced by cecal ligation and perforation in C57Bl/6 mice. Septic, sham‐operated or naïve mice received an IV bolus (20mL/kg) of Ringer's lactate or normal saline (0.9% NaCl). Six hours later the hepatic microcirculation was examined by confocal intravital microscopy to visualize the neutrophil chemokine macrophage inflammatory protein‐2 (MIP‐2) expression using an Alexafluor conjugated monoclonal antibody.

MIP‐2 expression, as measured by mean fluorescent intensity, was significantly higher in the hepatic sinusoids of septic mice treated with normal saline compare to septic mice given Ringer's lactate. The use of normal saline as an intravenous resuscitation fluid was associated with increased chemokine expression in the both sinusoids and post‐sinusoidal venules.

These data suggest that saline treatment in early sepsis augments neutrophil recruitment by increasing endothelial cell chemokine expression.
Free radical generation in germinal matrix hemorrhage
Authors: zia muhammad tahir , ungvari zoltan , csiszar anna , Ballabh Praveen
Published: June 2021

Germinal matrix hemorrhage‐Intraventricular hemorrhage (GMH‐IVH) is a major complication of prematurity. In adult animal model, intracerebral hemorrhage is attended by free radical generation resulting from activation of NADPH oxidase, which contributes to brain injuries. Therefore, we hypothesized that there might be greater generation of reactive oxygen species (ROS) in brain with IVH than controls and that suppression of NADPH oxidase might attenuate free radical formation. We tested our hypotheses in a rabbit pup model. We delivered pups prematurely and induced IVH by producing intracranial hypotension with IP glycerol at 2h age. We measured production of O2. ‐ and H2O2 in the periventricular and the cerebral cortical area at 6h postnatal age. To determine the source of free radical generation, we used inhibitors for NADPH oxidase (apocynin), xanthine oxidase, COX‐2 or NO synthase. We found that O2. ‐ and H2O2 generation were significantly greater in both the periventricular area and cortex of pups with IVH than controls (P<0.05). Only apocynin inhibited ROS generation in both brain areas (P<0.05). Our data suggest that free radicals are generated more in the periventricular area than in the cortical mantle in IVH and that the activation of NAPDH oxidase is the predominant mechanism of oxidative stress. Suppression of ROS production by NADPH oxidase inhibitor might reduce brain injuries in premature infants.
Contribution of Leukocytes to Capillary Hemostasis in the Spontaneously Hypertensive Rat
Authors: Tran Edward D , Schmid‐Schonbein Geert W
Published: June 2021

Our recent evidence suggests that leukocytes in hypertensives are involved in capillary stasis, which may lead to capillary rarefaction. Because the spontaneously hypertensive rats (SHR), a glucocorticoid‐dependent form of hypertension, have more activated circulating leukocytes with pseudopods, we hypothesize in SHR glucocorticoids impair normal leukocyte passage through capillaries and causes stasis. The objective of this study was to examine the effect of glucocorticoids on circulating leukocytes of the SHR and the normotensive Wistar‐Kyoto (WKY) rat and capillary blood flow. Small aliquots of blood (about 8% of blood volume) from SHR or WKY were incubated with or without supplement of dexamethasone (DEX 5 μM in 2 hours). The cells were fluorescently labeled, and transfused into SHRs, WKYs, or Wistar rats. Selected mesenteric networks were continuously observed over 2 hours. The labeled cells were traced with digital fluorescent microscopy. Without central pressure reduction, capillary stasis was initiated by immobilized leukocytes. Untreated SHR and WKY leukocytes did not increase stasis, but DEX‐treated leukocytes increased stasis significantly. DEX‐treated SHR leukocytes induced higher levels of stasis than DEX‐treated WKY leukocytes. These results suggest that the abnormal leukocyte response to glucocorticoids may serve as mechanisms for capillary stasis in SHR. Supported by HL10881.
Homocysteine decreases bone blood flow in rats
Authors: Fleming John , Vacek Thomas , Tyagi Suresh
Published: June 2021

Elevated levels of homocysteine (Hcy) known as hyperhomocysteinemia (HHcy) are associated with osteoporosis. A decrease in bone blood flow is a potential cause of compromised bone mechanical properties. We hypothesize that HHcy decreases bone blood flow. To test this hypothesis sexually mature male Sprague Dawley rats were treated with Hcy (0.067 mg/ml) in drinking water for 16 weeks. Age matched rats served as controls. At the end of the treatment period the rats were anesthetized. The systolic blood pressure was measured from the right carotid artery. A small incision was made to expose the antero‐medial surface of the tibia. A laser Doppler flow probe was placed against the bone to provide an index of flow. The results suggest that though there was no significant differences in the blood pressure in controls and Hcy treated rats, the flow index of the tibia of control rats was 0.78±0.09 (flow unit) compared to 0.51±0.09 in Hcy‐treated rats. The tibia mass was 1.1±0.1grams in controls and 0.9±0.1 was in Hcy‐treated rats. These results suggest that Hcy causes reduction in bone blood flow and may decrease bone density.

Support was provided by NIH #HL‐71010.
Estrogen replacement treatment (ERT) potentiates post‐ischemic brain inflammation in diabetic rats via the aldose reductase pathway
Authors: Xu Haoliang , Vetri Francesco , Pelligrino Dale
Published: June 2021

We examined whether ERT in ovariectomized (OVX) rats increased the expression and activity of aldose reductase (AR). The hypothesis was that ERT potentiates transient forebrain ischemia (TFI)‐induced inflammation represented by post‐ischemic pial venular leukocyte adhesion (PVLA) not only via the receptor for advanced glycation end products (RAGE), but also via AR activation in diabetics. The rats were divided into three groups: diabetic OVX females (OVX+D), diabetic OVX females with estrogen replacement for 1 week (OVE2+D), and non‐diabetics (ND). Each group was further divided into two subgroups. One subgroup was received AR antisense oligodeoxynucleotide (AS) through a cranial window that was prepared 24 h before TFI. The other subgroup was treated with AR missense ODN (MS) as a control. The percentage of venular area occupied by adherent rhodamine 6G‐labeled leukocytes was measured up to 10 hrs of reperfusion using intravital microscopy. In AR MS‐treated rats, all diabetics showed higher levels of PVLA compared to their ND controls. Moreover, starting at 4 hours afterischemia, PVLA in the OVE2 females roseto significantly higher levels compared with the OVX group, and leukocyte extravasation(at ≥ 6 h of reperfusion) was observed in a majorityof the OVE2 animals, with limited or no extravasationseen in the OVX group. With AR AS treatment, TFI‐induced PVLA was significantly suppressed in all diabetic groups. No significant effect of AR AS was observed in ND groups. Furthermore, RT‐PCR analysis revealed a more than 2‐fold higher expression of AR mRNA in OVX+D vs ND rats. With ERT, AR mRNA was further increased (4‐fold greater expression vs ND females). These findings suggest that AR contributes to the pro‐inflammatory effects of ERT in diabetics subjected to TFI.
Microvascular Sphingosine Kinase Isoforms in Cerebral Hypoxic Preconditioning
Authors: Wacker Bradley K , Gidday Jeffrey M
Published: June 2021

Hypoxic preconditioning (HPC) provides robust neuroprotection against ischemia‐induced neurovascular dysfunction and cell death following stroke and may depend on augmentation of Akt and eNOS signaling pathways. Studies of preconditioning in myocardium reveal sphingosine kinase (SphK) activity could be involved. To test the hypothesis that cerebral HPC may also require SphK, we preconditioned Swiss‐Webster ND4 mice for 4 h at 8% oxygen and measured SphK1 and SphK2 expression in cortical microvessel lysates by immunoblotting. Although no change in SphK1 level was detected, SphK2 increased 2‐fold versus sham (P<0.05) 2 h post‐HPC. No change was seen prior to 2h and SphK2 returned toward baseline after 2h. The SphK2 product S1P induces Akt and eNOS phosphorylation, suggesting that SphK2 upregulation may contribute to ischemic protection promoted by HPC. SphK activity and knockdown experiments to identify the role of SphK2 in HPC signaling by Akt and eNOS are underway in a cerebral endothelial cell culture model of HPC. These studies indicate SphK2 may be important in establishing a vasculo‐protective phenotype in the cerebral microcirculation in response to HPC. Supported by NIH grant HL79278.
Blood‐brain barrier retains its functional integrity to glutamate in diet‐induced obesity
Authors: Hawkins Richard Albert , Mokashi Ashwini , DeJoseph Mary Regina , Fernstrom John D.
Published: June 2021

The consequence of type II diabetes on the permeability of the blood‐brain barrier (BBB) was studied in rats with diet induced obesity. Male Sprague‐Dawley rats were fed a high‐energy diet for a period of 2 weeks whereupon they were separated into three groups on the basis of weight‐gain. The heaviest rats were selected as diet induced obese (DIO), the lightest as diet resistant (DR). The DIO and DR rats continued to be fed the high‐energy diet. The rats of intermediate weight gain were given normal rat chow (Chow). After 6 weeks the rats were infused with a tracer quantity of [14C]glutamate or with [14C]glutamate as well as sufficient glutamate to raise the concentration to more than 2 mM. Autoradiographs of the brains showed that glutamate did not pass the BBB except in circumventricular organs that have fenestrated capillaries. Insulin, leptin and the weight of fat pads (epidymal, mesenteric and retroperitoneal) were also measured. The weight of fat was closely correlated with plasma leptin concentration (r2=0.98) and correlated with plasma insulin concentrations (r2=0.34) The ratio of insulin‐to‐glucose in DIO rats was significantly higher than the Chow or DR rats indicating they were insulin resistant. Thus, in this model of diet‐induced insulin resistance and diabetes, the normal integrity of the BBB toward glutamate was maintained. Sponsored by IGTC.
Effects of Alpinia officinarum on LPS‐induced brain injury
Authors: Seok Sun Mi , Park Tae Yeoup , Kang Chan Sun , Lee Tae Gu , Moon Hyo‐Eun , Jung Yi‐Sook , Baik Eun Joo , Moon Chang‐Hyun , Lee Soo Hwan
Published: June 2021

The galangal (the rhizome of Alpinia officinarum, AO) is well known as a traditional herbal medicine in Asia. The present study reported that the chloroform extracts (AOC) of galangal could potentially protect against the blood brain barrier (BBB) collapse by lipopolysaccharide (LPS). Particularly, we confirmed that the increase of BBB permeability caused by LPS was effectively reduced by AOC in vitro BBB model. Moreover, in the increase of leukocyte adhesion caused by LPS, we found that AOC reduced LPS‐induced ICAM‐1, VCAM‐1 and E‐selectin expression at the levels of mRNA and protein in mouse brain microvascular endothelial cells (bEnd.3) and consequent LPS‐induced leukocyte‐adhesion. In addition, we found that the inhibition of these adhesion molecules resulted from the down‐regulation of NF‐κB activity by AOC. These data suggest that AOC may potentially protect from LPS‐induced brain inflammation in bEnd.3.

This study was supported, in part, by Gyunggi‐do through CCRB‐GRRC.
Transient changes in blood‐brain barrier integrity, thermotolerance, and heat shock protein expression following brief hyperthermia in an in vitro model
Authors: Klein Courtney , Bobilya Dennis
Published: June 2021

Hyperthermia has potential as a means of opening the blood‐brain barrier (BBB) to enhance delivery of pharmaceuticals into the brain. An in vitro BBB model of porcine brain capillary endothelial cells was exposed to brief hyperthermia and transendothelial electrical resistance (TEER) was measured to assess BBB integrity. Hyperthermia for 10 s at 45, 48, and 51°C, and for 5 s at 54°C caused significant (P<0.0001) loss in BBB integrity compared to controls (10 s at 37°C). The relative loss of BBB integrity increased as the treatment temperature increased (r = 0.88, P<0.0001). All BBB models recovered their integrity (TEER) within 30 min. Thermotolerance was examined by applying a second hyperthermia (10 s at 51°C) 24 h afterward. All BBB models demonstrated thermotolerance by a significantly (P<0.001) less loss of BBB integrity. The degree of thermotolerance increased as the pre‐conditioning temperature increased (r = 0.42, P<0.0001). Heat shock protein (Hsp) 27 and Hsp70 were measured by Western blot analysis to test their role in this thermotolerance. The relative quantity of Hsp27 was not changed (P>0.10) by any of the heat treatments. Hsp70 increased (P<0.05) only after pre‐conditioning for 5 s at 54°C. In summary, heat treatment opened the BBB and induced thermotolerance to a subsequent heat treatment. However this thermotolerance cannot be completely explained by relative changes in Hsp27 and Hsp70 abundance.
Effect of hydrogen sulfide on methionine‐induced oxidative stress in brain endothelial cells
Authors: Tyagi Neetu , Vacek Thomas Paul , Kumar Munish , Sen Utpal , Moshal Karni S , Tyagi Suresh C
Published: June 2021

Hydrogen sulfide (H2S) is a gasotransmitter produced endogenously from methionine metabolism through homocysteine. Although a neuromodulatory role of H2S has been demonstrated, little is known about its biological functions. In present study, we investigated whether H2S has an affect on Met‐induced cytotoxicity and oxidative stress in mouse brain endothelial cells (MBEC). MBEC were exposed to Met in the presence or absence of NaHS (donor of H2S). Cell viability was determined by MTT assay. Reactive oxygen species (ROS) was detected using DCFH‐DA assay. O2‐ production was measured using chemiluminescence. The results showed that Met‐induced cytotoxity and increased levels of H2O2, ONOO‐, O2‐ in a concentration and time‐dependent manner. Low levels of NaHS decreased the production of H2O2, ONOO‐ and O2‐ in the presence of Met and protected MBEC from cytotoxicity. Our data suggested that H2S is an important modulator of endothelial cytotoxicity via redox pathway in hyperhomocysteimia. These observations indicate that H2S may have therapeutic potential in Met‐mediated oxidative stress and cellular damage.
ProRenin/Renin Receptor is Expressed in the Collecting Duct of Normal Rat Kidney
Authors: Prieto‐Carrasquero Minolfa C , Martin Victoria L , Botros Fady , Navar L. Gabriel
Published: June 2021

Tubular renin is present in principal cells of collecting ducts (CD) of human, mouse and rat kidneys. The recent cloned(pro)renin renin receptor (PRR), is localized in brain, lung, heart and kidney, and binds renin or (pro)renin, thus leading to increased renin catalytic activity, or allowing (pro)renin to become fully active. Within the kidney, PRR is expressed by glomerular mesangial cells, podocytes, and renal arteries. The presence of PRR in CD might be important for potential generation of tissue angiotensin peptides in the terminal nephron segment. We investigated the immunolocalization of PRR in normal rat kidneys. Immunoperoxidase technique was performed by using a goat anti‐PRR antibody on 4% paraformaldehyde fixed normal rat perfused kidney sections. As described, positive immunostaining of PRR was visualized in mesangial cells, podocytes, and smooth muscle cells of cortical and medullary renal arteries. Interestingly, positive PRR immunoreactivity was also observed in the apical aspects of the cortical and medullary CD cells. Our results demonstrate the presence of PRR immunoexpression in cortical and medullary CD cells. Co‐expression of renin and PRR in the distal nephron suggests that renin or (pro)renin secreted by principal cells of CD into the tubular lumen anchors to the PRR allowing increased efficiency of Ang I formation contributing to angiotensin peptide generation in the CD.
Pressor and renal haemodynamic responses to angiotensin II (ANG II) in conscious lambs: Role of nitric oxide (NO) and prostaglandins (PGs)
Authors: Ebenezar Kumar Kesavarao , Wong Andy Kin On , Smith Francine Gabriel
Published: June 2021

Previously, we showed in conscious lambs that pressor and renal haemodynamic responses to ANG II were greater at ∼six weeks as compared to newborns aged ∼one week. The present study investigated the roles of NO and PGs in modulating these responses to ANG II during post natal maturation. Two experiments were carried out at an interval of 24 h in two age groups of conscious, chronically instrumented lambs: ∼one week (N=8) and ∼six weeks (N=10). Acute arterial pressure (AP) and renal blood flow (RBF) responses to intra‐venous (I.V.) injection of EC50 ANG II were tested before and after administration of the L‐arginine analogue, NG‐nitro‐L‐arginine methyl ester (L‐NAME), in the presence of either vehicle (veh, experiment one) or indomethacin (indo, experiment two). In both age groups, L‐NAME had no effects on the AP and RBF responses to ANG II after veh. In contrast, after indo, AP and RBF responses to L‐NAME were increased at one week but not at six weeks. Therefore, there is an age‐dependent interaction between NO and PGs in buffering the pressor and renal haemodynamic effects of ANG II soon after birth.