bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of memantine and donepezil on object recognition memory and hippocampal acetylcholine levels in rats with cholinergic lesions
Authors: Ihalainen Jouni , Sarajarvi Timo , Kemppainen Susanna , Keski‐Rahkonen Pekka , Lehtonen Marko , Banerjee Pradeep K. , Tanila Heikki
Published: June 2021

The combination of donepezil (DON) and memantine (MEM) produces greater amelioration of Alzheimer's disease (AD) symptoms than DON treatment alone. We sought to elucidate mechanisms underlying that phenomenon.

Rats with cholinergic denervation of the hippocampus (fornix lesion) were treated with DON (2.5 mg/kg/day), MEM (30 mg/kg/day), or placebo in drinking water for 3 weeks, then challenged with a single i.p. dose of MEM (5 mg/kg) or DON (2.5 mg/kg). In both treatment phases, animals were subjected to an object recognition task (ORT) and microdialysis measurements of hippocampal ACh levels. Cholinergic denervation was assessed by immunohistochemistry.

Fornix lesions resulted in a loss of >50% cholinergic fibers and significantly impaired ORT performance. Three weeks of MEM treatment restored ORT performance and tended to increase ACh levels, whereas DON treatment produced no significant ORT improvement. The MEM challenge following DON or vehicle treatment significantly elevated ACh release, whereas the DON challenge did not affect ACh release. Both challenges improved ORT performance.

Our data suggest that MEM restores recognition memory and increases hippocampal ACh release in ACh‐deficient rats. This latter effect, likely resulting from NMDA receptor antagonism, in addition to the normalization of glutamatergic transmission, may underlie the benefits of MEM in DON‐treated patients with AD.
Activation of TRPA1 channels by Fenamate NSAIDs
Authors: Herz Jeffrey Mark , Hu Hongzhen , Tian Jinbin , Wang Chunbo , Xiao Rui , Wood Jackie , Zhu Michael X.
Published: June 2021

TRPA1 forms non‐selective cation channels in polymodal nociceptors and can be activated by electrophilic prostaglandins and pungent chemicals through covalent modification. In the present study, we tested the activity of a group of non‐steroidal anti‐inflammatory drugs (NSAIDs) using electrophysiological techniques and intracellular Ca2+ measurements. We found that specific NSAIDs can act as full agonists of TRPA1, while others are partial agonists or inactive. Extracellularly applied flufenamic, niflumic, and mefenamic acid, as well as flurbiprofen, ketoprofen, diclofenac, and indomethacin rapidly activated rat TRPA1 expressed in Xenopus oocytes and human TRPA1 in WI‐38 fibroblasts. These same NSAIDs activated human TRPA1 inducibly expressed in HEK293 cells, but the responses were absent in uninduced and parental HEK293 cells. Responses to fenamate agonists were blocked by TRPA1 antagonists, AP‐18, HC‐030031, and ruthenium red. Washout of agonist led to a rapid transition to a resting state. At subsaturating concentrations, the fenamate NSAIDs potentiated the activation of TRPA1 by AITC, cinnamaldehyde, and cold, demonstrating positive synergistic interactions. The stimulatory effect was specific to TRPA1 because flufenamic acid inhibited TRPV1, TRPV3 and TRPM8. We conclude that fenamate NSAIDs are a novel class of potent and reversible direct agonists of TRPA1.
Desensitization of the Dopamine D1‐D2 Receptor Heteromeric Complex in Primary Striatal Neurons and Bi‐functional Regulation by GRK2
Authors: Verma Vaneeta , Hasbi Ahmed , O'Dowd Brian F. , George Susan R.
Published: June 2021

Co‐activation of a Gq‐linked heteromeric D1‐D2 dopamine receptor signaling complex results in phospholipase C‐dependent intracellular calcium release. We have specifically identified that the D1 agonist, SKF83959, selectively activates the D1‐D2 receptor heteromer, while SKF83822 only activates the D1 homooligomer. These agonists were used to evaluate agonist induced desensitization of the D1‐D2 receptor heteromer mediated calcium signal. Pre‐treatment with each agonist for 5 min led to rapid desensitization of the calcium signal both in cells and in primary striatal neurons. Desensitization was abolished by the D1 antagonist, but not by the D2 antagonist. Expression of G protein‐coupled receptor kinase 2 (GRK2) led to a decrease in the calcium signal. This was mediated by both the catalytic and RGS domains of GRK2. Our results indicate that desensitization of the D1‐D2 receptor heteromer mediated calcium signal occurs by D1 receptor occupancy with or without signal activation and is regulated by two distinct functions of GRK2. Intriguingly, occupancy of the D1 receptor binding pocket by SKF83822 that was unable to activate the D1‐D2 receptor complex still permitted desensitization of the calcium response, suggesting that activation of the receptor was not a prerequisite and occupancy of the receptor binding pocket with its associated conformational changes could lead to decreased signaling.

This work is supported by the NIH National Institute on Drug Abuse.
Rgs16 is an early marker for islet formation and beta cell expansion in diabetics
Authors: Ocal Ozhan , Villasenor Alethia , Wang Zhao , Rivera Lee , Asterholm Ingrid Wernstedt , Artyukhin Alexander , Scherer Philipp , Brekken Rolf , Cleaver Ondine , Wilkie Thomas
Published: June 2021

We seek to identify molecular markers of hormone signaling during early physiologic responses to metabolic stress. Regulators of G protein Signaling (RGS) proteins are feedback inhibitors of ligand‐activated G protein‐coupled receptors (GPCR). We characterized Rgs16 gene expression in BAC transgenic GFP reporter mice during pancreatic development and in diabetic mice. Rg16::GFP is first expressed in embryonic pancreas progenitor cells at E9 and then becomes restricted to endocrine cells during later differentiation. Islet expression of Rgs16::GFP is extinguished by two weeks of age (P14) but persists in Vessel‐ and Ductal‐Associated Cells (VDAC) until P28. Pancreatic expression in adults is reactivated in VDAC and beta cells in regenerating islets of hyperglycemic PANIC‐ATTAC mice (type 1 diabetes model) and in obese ob/ob mice (type 2 diabetes model). Elevated glucose and insulin levels correlate with increased Rgs16::GFP expression in ob/ob mice. Exendin‐4, a Glucagon‐like Peptide‐1 (GLP‐1) homolog that stimulates beta‐cell regeneration and normalization of blood glucose levels in type 1 diabetics, promotes pancreatic Rgs16::GFP re‐expression when co‐injected with glucose. Our results suggest that Rgs16 is likely to control aspects of islet progenitor cell activation, differentiation, and beta cell expansion in embryos and metabolically stressed adults.

Funding: NIH (GM061395) to TM Wilkie
Regulation of the RGS14/Gia1‐GDP signaling complex by phosphorylation
Authors: Cowan Donald Patrick , Shu Fengjue , Hepler John R
Published: June 2021

RGS14 is a poorly understood brain protein that we show acts as a scaffold to bind inactive Gia1/3‐GDP, active H‐Ras and Rap2, and Raf kinases to modulate MAPkinase signaling. Our previous work showed that RGS14 is phosphorylated by PKA to regulate its interactions with Gia1. Here we report that RGS14 is phosphorylated in cells by a different kinase when in complex with Gia1. Recombinant RGS14 localizes to the cytosol in cells, but is recruited to the plasma membrane when co‐expressed with Gia1 to form a stable RGS14/Gia1‐GDP complex. RGS14 post complex with Gia1‐GDP appears as a prominent second higher molecular weight (HMW) immunoreactive band by SGS‐PAGE. We hypothesize this ‘upper’ band is due to addition of a novel phosphorylated residue on RGS14. Consistent with RGS14 being phosphorylated, we find that treatment of samples with alkyline phosphatase eliminates the HMW form of RGS14. Mutation of known PKA and Erk phosphorylation sites on RGS14 fail to eliminate the phosphorylated form of RGS14, suggesting the involvement of different kinase(s). Ongoing studies are designed to identify the specific phosphorylation site(s), involved kinase(s), and the functional consequences of phosphorylation. A deeper understanding of the regulation of RGS14 by phosphorylation will be important to understand its role(s) as a novel integrator of G protein and MAPkinase signaling in brain.
BPP‐10c isolated from Bothrops jararaca venom has antithrombotic effect in rats
Authors: Ianzer Danielle Alves , Xavier Carlos H , Fraga‐Silva Rodrigo A , Faria Fernanda , Camargo Antonio C M , Santos Robson A S , Teixeira Mauro M
Published: June 2021

Bradykinin potentiating peptides (BPPs) were the first ACE inhibitors described. Recently, we identified new BPPs from B.jararaca venom and showed that some of them, including BPP‐10c, were able to reduce arterial pressure of SHR without affecting ACE activity. Male wistar (WT) and spontaneously hypertensive rats (SHR) were used to evaluate tail bleeding time after acute or chronic administration of vehicle (NaCl 0.9%) or BPP‐10c (71nmol/Kg) (n=7 each). We also assessed venous thrombus weight from vena cava after acute iv injections of vehicle or BPP‐10c (n=4). Both treatments with BPP‐10c increased bleeding time in WT (28.0±1.8 vs. 11.7±1.6; 27.3±2.3 vs. 16.8±3.2min; P<0.05, respectively) and SHR (15.3±1.6 vs. 5.8±0.9; 20.4±3.0 vs. 9.6±2.0min; P<0.05, respectively). BPP‐10c also reduced the thrombus weight of WT (−29%) and SHR (−48%) compared to control groups (7.6±0.9 vs. 10.7±0.8; 6.2±1.0 vs. 11.8±0.6mg; P<0.05, respectively). Prothrombin time, activated partial thromboplastin time and fibrinogen levels were not changed. In conclusion, BPP‐10c has antithrombotic effect and represents a potential pharmacological tool to recover homeostasis and treat both hypertension and coagulation diseases. Mechanisms involved require further investigation. Support: FAPESP and CNPq.
Gut Wall Compromise in the Presence of Pancreatic Enzymes Causes Circulatory Shock
Authors: Kistler Erik B. , Schmid‐Schonbein Geert W.
Published: June 2021

Pancreatic proteolytic enzymes (PEs) in the ischemic intestine play a central role in multisystem organ failure, which can be prevented by protease inhibition in the small intestine lumen. PEs, if allowed to circulate systemically may also result in shock. However, it is unclear whether enzyme liberation in an intestine with increased permeability alone leads to shock. To test this idea the non‐ischemic rat small intestinal lumen was perfused for two hours with either (A) PEs, (B) interventions designed to increase lumenal permeability (N‐acetylcysteine (NAC) + atropine + increased flow) or (C) both, and animals were observed for shock and organ failure. PEs perfused (Groups A and C) included trypsin, chymotrypsin, elastase, amylase and lipase at concentrations 2 log greater than baseline values. Group A (n=6) maintained baseline blood pressures as did other groups perfused with single enzymes alone. However all animals in Group C (n=6) developed hypotension, significant increases in gut permeability (p<0.001) and died (p<0.001). Group B (n=6) developed mild hypotension (NS) and increased gut permeability (p<0.05) compared to controls but there were no deaths. These experiments demonstrate for the first time that increased gut permeability in the presence of lumenal PEs is sufficient to induce shock. PEs, if allowed to penetrate the gut wall result in multiorgan failure and death. Supported by NIH Grant GM085072.
PI3K/Akt signaling pathway controls PGE2‐induced ICAM‐1 expression through activation of NF‐kB
Authors: Park Tae yeop , Jung Yi‐Sook , Baik Eun Joo , Moon Chang‐Hyun , Lee Soo Hwan
Published: June 2021

Prostaglandin E2(PGE2) is known to be the principal pro‐inflammatory prostanoid and play important roles in brain disease through binding to four G protein‐coupled receptor(EP1‐4). However, there have been contradictory reports on its actions in inflammation processes. For instance, PGE2 inhibits or stimulates ICAM‐1 expression depending on the cell type and experimental conditions. In the present study, we investigated whether EP receptor‐mediated elevation of cAMP is necessary for the effect of PGE2 on ICAM‐1 expression in bEnd.3 cerebrovascular cells. PGE2 as well as dbcAMP and forskolin stimulated ICAM‐1 expression, and induced PI3K‐dependent Akt phosphorylation. LY294002, a PI3K inhibitor, attenuated PGE2‐induced ICAM‐1 expression indicating possible involvement of PI3K in PGE2‐induced ICAM‐1 expression. This inference was further supported by the separate experiment using constitutively active and dominant negative Akt constructs. NF‐κB inhibitors such as BAY‐11 and MG‐132 diminished the PGE2‐induced ICAM‐1 expression implicating the role of NF‐κB. This result was further supported by increases in the IκB phosphorylation, translocation of NF‐κB into nucleus and the activity of NF‐κB‐responsive promoter construct. Interestingly, ectopic expression of dominant negative Akt resulted in the reduction of PGE2‐induced IκB phosphorylation. Taken together, these results suggest that PGE2‐induced ICAM‐1 expression is mediated by PI3K‐dependent NF‐κB activation.

This study was supported by grants from Ajou University School of Medicine and Gyunggi‐do through CCRB‐GRRC
Characterization of a novel liposome based therapy to reduce complement deposition on cell membranes
Authors: Perez‐Abadia Gustavo , Goga Ledia , Pushpakumar Sathnur B , Soni Chirag , Olson Paul , Anderson Gary , Barker John , Maldonado Claudio J
Published: June 2021

Excessive complement activation following ischemia and reperfusion (IR) induces endothelial cell injury, altering vascular and endothelial barrier function causing tissue dysfunction. To mitigate IR response various systemic anti‐complement therapies have been tried. Recently, we developed a local therapy that uses biotinylated fusogenic lipid vesicles (BioFLVs) to incorporate biotin tethers onto cell membranes, which then bind to therapeutic fusion proteins containing streptavidin (SA) resulting in decoration of cell membranes. Alteration of formulation, concentration and duration of incubation of BioFLVs were conducted to demonstrate the ability of the system to modulate biotin tether incorporation. A rat hind limb model was used to demonstrate the efficacy of this system for 48 h of reperfusion. The feasibility of a BioFLV‐based anti‐complement therapy was tested in human ovarian carcinoma (SKOV3) cells using SA fused with vaccinia virus complement control protein (SA‐VCP), a C3 convertase inhibitor. To activate complement cells were treated with a SKOV‐3‐specific antibody (trastuzumab) and incubated in human serum. Decoration of cells with SA‐VCP effectively reduced complement deposition. We conclude that BioFLV‐mediated decoration of cell membranes with anti‐complement proteins has potential to reduce IR‐mediated complement injury in transplantated or replanted tissues.
Pyruvate‐fortified Fluid Resuscitation Suppresses Inflammation and Arrhythmogenesis During Hemorrhagic Shock and Hindlimb Ischemia
Authors: Gurji Hunaid , Hoxha Besim , Flaherty Devin , Sun Jie , Schulz Diana , Blaylock Jessica , Olivencia‐Yurvati Albert , Mallet Robert
Published: June 2021

Fluid resuscitation for hemorrhagic shock can elicit inflammatory and metabolic stress. We tested whether resuscitation with pyruvate can mitigate these effects. Goats (~25kg) were hemorrhaged to mean arterial pressure (MAP) of 50 mmHg, subjected to hindlimb ischemia with a tourniquet, and then resuscitated (10 ml/min iv) with lactate‐ (LR; n=4) or pyruvate‐ (PR; n=6) enriched Ringers solution for 90 min; tourniquet was released at 60 min. Time controls (TC, n=5) experienced neither hemorrhage nor hindlimb ischemia. At 4 h recovery, left ventricular myocardium (LV) and ischemic gastrocnemius (IG) were biopsied to measure myeloperoxidase (MPO), a marker of neutrophil infiltration. Peak plasma pyruvate concentrations (mM) were 3.03±0.83 in PR, 0.46±0.15 in LR (p<0.05 vs. PR) and 0.15±0.002 in TC. Systemic lactate/pyruvate redox state was higher in LR (30.3±4.5) vs PR (1.9±0.3; p<0.001) and TC (3.7±0.1; p<0.01). Although MAP fell post‐resuscitation in all groups, pulse pressure, a measure of systemic vasodilation, increased only 15% following PR vs 133% post‐LR and 59% in TC. PR lowered MPO activity by 30% in LV and 23% in IG vs LR. Pro‐arrhythmic QTc dispersion was 25±6 ms post‐LR vs. 7.7±0.9 ms post‐PR (p<0.05). Resuscitation with pyruvate stabilized peripheral resistance and electrocardiographic function and suppressed tissue inflammation ‐ all goals for fluid resuscitation. US Dept Defense W911NF0910086.
Increased protease activity and receptor cleavage in a rat model of diet‐induced obesity
Authors: Mazor Rafi , Schmid‐Schönbein Geert
Published: June 2021

Obesity is a major health problem and leads to increased morbidity and mortality from cardiovascular complications. Leptin acts on the brain to regulate food intake through its interactions with its receptor (ObR). Defects in leptin production and in ObR leads to the development of obesity. In addition previous studies have found leptin resistance concomitantly with high circulating leptin levels in obese patients. We hypothesize that calorie overconsumption can lead to increased proteolytic activity in the brain, promoting cleavage of key receptors governing food intake. Wister rats were fed high‐fat diet (HF) for 10 weeks. A control group was provided with regular chow. At 10 weeks, rats were sacrificed and the brain was removed for determination of proteolytic activity and levels of the intra and extracellular domains of the ObR. In‐situ zymography showed increased proteolytic activity in the HF rat's brain, which could be blocked by EDTA. Gel zymography of whole brain lysate revealed an approximately 70 KDa protein that was highly active compared to controls. Western blot analysis and immunohistochemistry demonstrated lower levels of the ObR extracellular domain in HF brain, while levels of the intracellular domain were not significantly different. We conclude that calorie over‐consumption can promote proteolytic activity and cleavage of key receptors in the brain. The origin of the proteolytic activity in HF rat's brain needs to be further elucidated.
Enhanced calcium sensitivity of endothelial calcium‐activated large conductance potassium channels
Authors: Riddle Melissa Anne , Walker Benjimen R
Published: June 2021

Chronic hypoxia (CH; 48 hr 0.5 atm) causes blunted arterial vasoconstriction in rats. Inhibition of endothelial cell (EC) heme oxygenase (HO) or large conductance Ca2+‐ activated K+ channels (BK) restores reactivity, but is without effect in controls. Further, BK currents in ECs from CH rats are inhibited by HO blockade, suggesting functional coupling of HO with BK channels. Since HO‐derived CO may enhance BK Ca2+ sensitivity, we hypothesized that channels from CH ECs are more sensitive to Ca2+ than BK channels from vascular smooth muscle (VSM) due to the tonic presence of CO. We performed patch clamp experiments on freshly dispersed aortic ECs in whole cell‐cell attached and inside‐out configurations to characterize BK. Small (SK) and intermediate (IK) conductance K+ channels were detected in cells from each group. In contrast, iberiotoxin (IBTX)‐sensitive channels with unitary conductance of 146±10 pS/222±13 pS (asymmetrical/symmetrical K+) were found only in ECs from CH rats. EC BK channels were more sensitive to Ca2+ than SK and IK and had markedly greater Ca2+ sensitivity than VSM BK. This enhanced Ca2+ sensitivity may contribute to the apparent tonic activity of EC BK channels following CH.
Longitudinal investigation of permeability and distribution of macromolecules in mouse tumor development and malignant transformation using PET
Authors: Rygh C B , Seo J W , Qin SP , Mahakian L M , Zhang H , Kukis D L , Chan J Q , Cheng‐Liao J , Reed R K , Ferrara K W
Published: June 2021

Liposomes are widely used as carriers of drugs, genes, or contrast agents while albumin is a commonly used tracer for permeability studies. Here we studied the permeability and distribution of 64Cu‐labeled liposomes and albumin (64Cu‐BAT‐liposomes, Seo et al. Bioconjugate Chem.19: 2577, 2008, and 64Cu‐BAT‐BSA) in a mouse model of ductal carcinoma in situ. Precancer mammary intraepithelial neoplasia outgrowth (MINO, line 4) tissue was transplanted bi‐laterally into gland cleared mammary fat pads in young FVB/N mice (n=8) and were PET scanned (Focus 120, Siemens Medical Solutions, Inc.) at week 3, 5, 7 and 8 after transplantation. Each animal was scanned at multiple time‐points after bolus injection of the tracer to create time‐activity curves (TACs). The apparent permeability of both tracers increased over time as the lesions transformed, and was higher for albumin than for liposomes (from 3.46 ± 0.69 to 12.48 ± 2.94 and 0.81 ± 0.61 to 2.29 ± 0.56 10−8 cm/s for albumin and liposomes at week 3 and 8, respectively). In general, the distribution of liposomes within the tumor was more heterogeneous than with albumin.

In conclusion, longitudinal progression of disease could be detected in vivo with PET imaging, as well as heterogeneity of tracer permeability and distribution within the lesions which is valuable information when evaluating drug‐delivery efficacy.

This project was supported by NIH CA 103828 and CA 134659
Relationship between total peripheral resistance and measures of central artery wave reflection
Authors: Shepherd John RA , Hart Emma C. , Curry Timothy B. , Charkoudian Nisha , Joyner Michael J. , Casey Darren P.
Published: June 2021

We examined the relationship between total peripheral resistance (TPR) and central artery pressure and indices of wave reflection in 16 subjects (10F/6M; 26 ± 1 yr). Non‐invasive aortic pressure waveforms were synthesized from high‐fidelity radial pressure waveforms obtained from applanation tonometry of the radial artery. Radial blood pressure (BP) was calibrated from brachial artery BP (catheter). Central artery pressures, aortic augmentation index (AIa), roundtrip travel time of the reflected wave to and from the periphery (Δtp; inverse of pulse wave velocity), and wasted left ventricular energy (Ew) were calculated from the generated aortic pressure waveform. Cardiac output (CO) was measured using an open‐circuit acetylene uptake technique and used to calculate total peripheral resistance (mean arterial pressure/CO). TPR was positively related to aortic pulse pressure (r = 0.49), augmented pressure of the central artery wave (r = 0.60), pulse pressure amplification (r =0.54), AIa (r = 0.53), and Ew (r = 0.66), (P < 0.05 for all). Additionally, TPR was inversely related to Δtp (r = − 0.51, P < 0.05). Our data suggests that increased levels of TPR are related to measures of central artery pressure, wave reflection characteristics and indices of central artery stiffness.

Supported by NIH K23 DK‐082424 (TBC), AHA 2170087(ECH) and CTSA RR‐024150
Ursolic acid accentuates contractile and secretory functions in beating rabbit atria
Authors: Cui Hao Zhen , Choi Hye Ran , Cho Kyung Woo , Kang Dae Gill , Lee Ho Sub
Published: June 2021

Aim of study

Ursolic acid (UA) is a well known hydroxyl pentacyclic triterpenoic acid and has been found in vegetarian foods and medicinal herbs. However, the effects of UA on cardiac functions have not well been documented. The purpose of the present study was, therefore, to address whether UA has contractile and secretory effects in perfused beating rabbit atria.

Materials and Methods

The UA‐induced changes in atrial mechanical dynamics, pulse pressure and stroke volume, cAMP efflux, and ANP secretion were measured in perfused beating rabbit atria.

Results

UA increased atrial dynamics and ANP secretion in a concentration‐dependent manner without changes in atrial cAMP levels. The UA‐induced increase in atrial dynamics was not changed by inhibition of the L‐type Ca2+ channels and SR system. On the other hand, UA‐induced increase in ANP secretion was attenuated by inhibition of L‐type Ca2+ channels.

Conclusion

These results indicate that UA accentuates atrial ANP secretion concomitantly with increase of atrial dynamics and also suggest that the accentuation of ANP secretion is related with changes in cellular Ca2+ metabolism.
Oxidation of myocardial glutathione increases susceptibility to ventricular arrhythmia in the aged diabetic heart
Authors: Sloan Ruben C , Frasier Chad , Anderson Ethan , Wingard Christopher , Brown David A
Published: June 2021

Recent work from our group using young healthy animals has shown that overwhelming cellular anti‐oxidant defenses can induce ventricular arrhythmia under normoxic conditions. The purpose of this study was to determine whether aged (22 ± 1 months) diabetic hearts were more susceptible to ventricular arrhythmia following treatment with diamide, a thiol‐oxidizing agent. Male Zucker Diabetic Fatty rats had higher resting glucose levels (476 ± 17 mg/dL) than their lean non‐diabetic counterparts (126 ± 9 mg/dL; P < 0.05). Hearts were excised and perfused with Krebs‐Henseleit buffer containing 200 uM diamide. Baseline left ventricular developed pressures were significantly lower in diabetic rats (55 ± 11mmHg) than non‐diabetic counterparts (89 ± 15mmHg; P < 0.05), and this functional difference persisted 10 minutes through diamide treatment (59.97 ± 10.57 mmHg vs. 105.1 ± 19.36 mmHg after 10 minutes of diamide, respectively; P < 0.05). After diamide administration, diabetic hearts transitioned into ventricular arrhythmias much sooner than non‐diabetic hearts. Time to ventricular fibrillation was 21.69 ± 1.456 min in diabetic hearts vs. 30.44 ± 1.711 min in non‐diabetic hearts (P < 0.05). These results demonstrate that the aged diabetic heart is more susceptible to development of fatal arrhythmia when presented with an oxidant challenge, which has broad implications for cardiac disease in diabetics.
AMP‐activated protein kinase activation stimulates heme oxygenase‐1 gene expression to promote human endothelial cell survival
Authors: Liu Xiao‐ming , Peyton Kelly J. , Wang Hong , Korthuis Ronald J. , Durante William
Published: June 2021

AMP‐activated protein kinase (AMPK) is a ubiquitously distributed energy sensing enzyme that is activated by changes in cellular metabolism. Recent studies indicate that AMPK inhibits endothelial cell (EC) activation and apoptosis, but the underlying mechanisms remain unclear. Since heme oxygenase‐1 (HO‐1) is a highly inducible vasoprotective protein, the present study investigated whether HO‐1 contributes to the anti‐apoptotic action of AMPK. Treatment of cultured human umbilical vein ECs with the AMPK activator 5‐aminoimidazole‐4‐carboxamide‐1‐β‐D‐ribofuranoside (AICAR) stimulated a concentration‐ and time‐dependent increase in HO‐1 protein and mRNA that was prevented by the knockdown of AMPKα1/2. AICAR‐mediated HO‐1 expression was associated with a significant increase in Nrf2 protein, and overexpression of a dominant‐negative Nrf2 mutant or silencing of Nrf2 abrogated the induction of HO‐1. Finally, treatment of ECs with tumor necrosis factor‐α stimulated DNA fragmentation and caspase‐3 activation, that was blocked by AICAR. However, the anti‐apoptotic action of AICAR was abolished by the HO inhibitor tin protoporphryin‐IX or by the knockdown of HO‐1. In conclusion, this study demonstrates that AMPK stimulates HO‐1 gene expression in human ECs and that the induction of HO‐1 is required for the anti‐apoptotic effect of AMPK. Supported by NIH.
Inhibition of Nuclear Factor of Activated T‐cells (NFAT) reduces diabetes‐induced atherosclerosis
Authors: Zetterqvist Anna V , Nilsson‐Berglund Lisa M , Nilsson Jan , Bengtsson Eva , Gomez Maria F
Published: June 2021

Diabetes is associated with devastating macrovascular complications including coronary heart disease and stroke. The molecular mechanisms leading to vascular disease are still poorly understood. We have previously shown that hyperglycemia activates the transcription factor NFAT in native arteries. Once activated, NFAT promotes cell proliferation, enhances SMC excitability and the expression of inflammatory markers (i.e. IL‐6, tissue factor, COX‐2). Here we tested whether NFAT is involved in the development of macrovascular disease. Treatment of ApoE KO mice with streptozotocin (STZ) led to hyperglycemia, elevated plasma cholesterol and triglycerides. STZ‐treatment resulted in 80% increased aortic atherosclerosis, enhanced expression of the pro‐atherosclerotic protein osteopontin (OPN) in subvalvular aortic lesions and increased plasma OPN. Four weeks treatment of diabetic mice with the NFAT blocker A‐285222 completely inhibited the hyperglycemia induced aggravation of atherosclerosis, but had no effect on atherosclerosis in non‐diabetic mice. Body weight and levels of inflammatory cytokines were not affected by A‐285222 treatment, suggesting that the reduced atherosclerosis is less likely due to systemic immunosuppression. Our results provide evidence for the involvement of NFAT in the development of macrovascular complications in diabetes. Funding: Swedish Heart & Lung Foundations
Progress and Setbacks in the Study of CNS Axonal Plasticity and Regeneration
Authors: Tuszynski Mark H , Kadoya Ken , Lu Paul , Blesch Armin , Rosenzweig Ephron , Brock John , Loew Karin
Published: June 2021

Understanding of basic mechanisms underlying the refractory state of axonal regeneration in the CNS has advanced remarkably in the last 50 years. Mechanisms that underlie the failure of central regeneration include: 1) an absence of permissive extracellular matrices in lesion sites, 2) insufficient secretion of trophic molecules, 3) the presence of growth‐inhibitory molecules in the CNS, and 4) an intrinsic failure of adult CNS neurons to upregulate extensive sets of growth‐related genes. Addressing several of these mechanisms simultaneously, it is possible to elicit growth of select spinal cord axonal populations into and beyond lesion sites, and to reinnervate normal targets. However, several critical challenges remain in SCI research, including: 1) elicitation of regeneration or corticospinal axonal systems; 2) remyelination of regenerating axons; 3) elicitation of substantial growth at chronic stages after SCI, and 4) translation of these findings to primate systems, given fundamental differences in axonal organization and function between rodent and primate models. This talk will summarize these topics and highlight future directions of research.
Hypoxia inducible factor‐1 protects against nitrate tolerance and stunning in cardiac myocytes
Authors: Weiss Harvey R , Tan Tao
Published: June 2021

We tested whether upregulation of hypoxia inducible factor‐1 (HIF‐1) would restore the blunted effects of natriuretic peptides and nitric oxide caused by chronic nitrates and stunning in cardiac myocytes. HIF‐1α was increased with deferoxamine (DFX) and nitrate tolerance was induced with a chronic nitroglycerin patch. We used rabbit ventricular myocytes. Myocytes were subjected to simulated ischemia [15 min 95% N2‐5% CO2] and reperfusion [reoxygenation] to produce stunning. Cell function was measured at baseline and after brain natriuretic peptide (BNP) or S‐nitroso‐N‐acetyl‐penicillamine (SNAP) followed by KT5823 (cGMP protein kinase inhibitor). We also measured cGMP protein kinase protein levels and kinase activity. In control, BNP (−29%) reduced %shortening, while KT5823 partially restored function. DFX treated myocytes responded similarly. In patched myocytes, BNP (−12%) reduced shortening less and KT5823 did not increase function. However, DFX restored the blunted effects of BNP (−21%) and KT5823. In stunned myocytes, BNP (−11%) reduced shortening less and KT5823 did not affect function. DFX increased the effects of BNP (−27%) and KT5823 in stunning. Patch plus stunning also similarly blunted the effects of BNP (−12%) and KT5823, while DFX improved the effects of BNP (−22%) and KT5823. Similar results were seen with SNAP. Stunning reduced cGMP protein kinase activity and DFX restored activity. DFX had no effect on kinase activity in nitrate tolerance. We found that upregulation of HIF‐1 could protect against nitrate tolerance through a cGMP protein kinase‐independent mechanism and through a kinase‐dependent mechanism in stunning.
Phosphoproteomic profile of differentially phosphorylated proteins in human myocardium before and after cardiac surgery utilizing cardioplegia and cardiopulmonary bypass.
Authors: Clements Richard T , Ivanov Alexander R , Smejkal Gary , Bianchi Cesario , Sellke Frank W
Published: June 2021

Objective

Cardiac surgery using cardioplegia and cardiopulmonary bypass(CP/CPB) subjects myocardium to reversible ischemic injury that can impair cardiac function. Changes in myocardial contractile activity are likely regulated in part via acute protein phosphorylation. We performed the following study to determine changes in human myocardial protein phosphorylation following CP/CPB.

Methods

Right atrial appendage was collected from patients pre and post‐CP/CPB and subjected to two‐dimensional electrophoresis, phospho‐protein staining, image analysis, and tandem mass spectrometry.

Results

48 spots were consistently increased in the post CP/CPB group vs pre CP/CPB. There were no consistent changes in the remaining 24 consistently detected proteins post CP/CPB. Some of the identified proteins that changed in phosphorylation included: the cytoskeletal proteins tropomyosin α1, cardiac troponin I, atrial myosin light chain (MLC)‐2, atrial MLC‐4, actin, and heat shock protein 27; the metabolic proteins: ATP‐synthase subunit beta, phosphoglycerate mutase 2, and L‐lactate dehydrogenase B chain; the membrane signaling proteins 14‐3‐3εand annexin A5.

Conclusion

CP/CPB modifies the phosphorylation of specific cytoskeletal, metabolic, signaling, and serum proteins which may have significant implications for CP/CPB‐induced myocardial stunning. (NIH: HL046716,HL093352)
Protection of cardiomyocytes by anesthetic preconditioning involves endothelial cell derived nitric oxide
Authors: Leucker Thorsten Martin , Bienengraeber Martin , Brzezinska Anna K , Pratt Phillip F , Warltier David C
Published: June 2021

NO is involved in the anesthetic preconditioning‐induced protection against cardiac ischemic injury. The goal of this investigation was to test the hypothesis that endothelial cell derived NO enhaces cellular protection against hypoxia and reoxygenation‐induced cardiomyocyte injury.

We studied anesthetic preconditioning in a coculture model of human endothelial cells and HL‐1 cardiomyocytes. Cells were exposed to 0.5 mM isoflurane for 30 min followed by 15 min wash out. Then cells were exposed to 3 h of hypoxia followed by 4 h of reperfusion. To determine cell survival we assessed LDH release and tryptan blue staining.

Our results demonstrate that co‐culturing endothelial cells and HL‐1 cardiomyocytes protects from ischemia‐reperfusion injury. Isoflurane exerts protective effects on HL‐1 cardiomyocytes. However, metabolic interactions between endothelial cells and cardiomyocytes exceed the protective effect of isoflurane alone, adding additional layer of cardioprotection. The protective effect of isoflurane is reversed by the NO synthase inhibitor L‐NMMA given prior to reperfusion, but not when given only during isoflurane treatment.

In conclusion, during reperfusion, anesthetic induced cardioprotection, in part, depends upon the activity of endothelial NO synthase.
Reactive oxygen species inhibit vascular KATP channel via S‐glutathionylation
Authors: Yang Yang , Shi Weiwei , Cui Ningren , Chen Xianfeng , Zhang Yinwei , Trower Timonthy C , Jiang Chun
Published: June 2021

KATP channel is an important regulator of vascular tones. Its opening and closure associated with vasodilation and vasoconstriction are controlled by intra‐ and extracellular messengers. Such a subtle balance may be disrupted in oxidative stress known to be a major player in a variety of cardiovascular diseases. In the vascular diseases reactive oxygen species (ROS) are produced, which in turn contribute to the development of these diseases. To demonstrate how the vascular KATP channel is affected by ROS we performed the studies. In isolated mesenteric rings, the H2O2 treatment impaired vascular responses to vasodilators. To test whether the KATP channel is involved, we studied the cloned Kir6.1/SUR2B channel in HEK cells. H2O2 had only modest inhibitory effect in excised patches in the absence of cytosolic components. The administration of glutathione (GSH) drastically augmented the channel inhibition by micromolar range of H2O2, suggesting that H2O2 inhibits the channel via S‐glutathionylation. This finding was further supported by a variety of thiol modulating regents as well as biochemical experiments. Using mutagenesis screening, residue Cys176 was found to be critical for the channel inhibition. The mutations of Cys176 disrupted the channel responses to both oxidized glutathione and thiol modulating regents. Taken together, these data demonstrate a novel modulation mechanism of KATP channel by ROS.
Zipper‐Interacting Protein Kinase: Inferring Function In Smooth Muscle Contractility By Identifying Bona Fide Substrates
Authors: Moffat Lori Dawn , Walsh Michael , MacDonald Justin
Published: June 2021

Zipper‐interacting protein kinase (ZIPK) has been implicated in Ca2+‐independent smooth muscle contraction, although its specific role is unknown. To understand the role of ZIPK in smooth muscle contractility, we set out to identify bona fide ZIPK substrates, using a technique that takes advantage of ‘bulky’ ATP analogues (modified at the N6 position) and a bioengineered ZIPK capable of utilizing such substrates. Conserved amino acid residues in ZIPK that come into close contact with the N6 region of ATP were identified and mutated. To identify substrates, 32P‐labeled 6‐Phe‐ATP and ZIPK L93G protein were added to permeabilized rat caudal arterial smooth muscle strips. 32P‐labelled substrate proteins were detected by autoradiography and phosphorimaging following SDS‐PAGE. Mass spectrometry was employed to identify ZIPK substrates and LC20 was confirmed as a direct target. Two other substrates (18 kDa and 40 kDa) were also detected. Experiments were also carried out with rat caudal arterial smooth muscle in the absence of endogenous ATP, but supplemented with 6‐Phe‐ATP and ZIPK L93G, and putative ZIPK substrates identified by western blotting with phosphospecific antibodies. LC20 was confirmed as a direct target of ZIPK by this method; however no phosphorylation of MYPT1 was detected. We conclude that ZIPK is involved in regulation of smooth muscle contraction through direct phosphorylation of LC20.
Deletion of Jak2 Tyrosine Kinase within Vascular Smooth Muscle Cells Attenuates Angiotensin II induced Hypertension in Mice
Authors: Kirabo Annet , Kearns Patrick , Sasser Jennifer , Cardounel Arturo J , Baylis Chris , Wagner Kay‐Uwe , Sayeski Peter P
Published: June 2021

Angiotensin II (Ang II) AT1 receptors in vascular smooth muscle cells (VSMC) are coupled to the Jak2 signaling pathway. However, the importance of this coupling is poorly understood. We hypothesized that VSMC expression of Jak2 is involved in the pathogenesis of Ang II‐induced hypertension. To test this hypothesis, we used the Cre‐loxP system to specifically ablate Jak2 within the VSMC of mice. This strategy was successful in allowing embryonic survival of mice whose VSMC are devoid of Jak2. Phenotypic analysis of these mice revealed that they have a lower heart to body weight ratio (p<0.05) when compared to littermate controls. The VSMC Jak2 null mice also have a lower baseline mean arterial pressure when compared to littermate controls (97 +/− 6 mm Hg vs. 111 +/− 13 mm Hg). Infusion of Ang II increased mean arterial pressure in both groups. However, the increase in VSMC Jak2 null mice was significantly lower than that observed in the controls (124 +/− 6 mm Hg vs. 156 +/− 15 mm Hg). Furthermore, aortic rings obtained from VSMC Jak2 null mice exhibited a decreased contractile ability in response to Ang II when compared to controls (p<0.05). Collectively, these results suggest that Jak2 is involved in the pathogenesis of Ang II‐induced hypertension and may be an important target in the treatment of cardiovascular disease.
Caveolae are necessary for functional ICat activation by IP3Rs in cerebral artery smooth muscle cells
Authors: Adebiyi Adebowale , Jaggar Jonathan H
Published: June 2021

In cerebral artery smooth muscle cells, inositol 1,4,5‐trisphophate (IP3) activates a non‐selective cation current (ICat), leading to vasoconstriction. IP3‐induced ICat activation occurs independently of intracellular Ca2+ release and via type 1 IP3 receptor (IP3R1) and canonical transient receptor potential (TRPC) 3 channel activation. However, signaling mechanisms mediating this effect are poorly understood. Caveolae are plasma membrane microdomains that compartmentalize signal transduction molecules, including TRPC channels. Here, we explored the physiological function of caveolae in mediating IP3‐induced ICat activation and vasoconstriction. A synthetic peptide corresponding to the caveolin‐1scaffolding domain (CSD) sequence (amino acids 82–101 of caveolin‐1) attenuated IP3‐induced ICat activation in cerebral artery myocytes. Methyl‐β‐cyclodextrin (MβCD), which disassembles caveolae, inhibited IP3‐induced ICat. In pressurized cerebral arteries, MβCD attenuated vasoconstriction induced by Bt‐IP3, a cell permeable IP3 analog. Cholesterol replenishment reversed the MβCD‐induced decrease in IP3‐induced ICat activation and vasoconstriction. Taken together, data suggest that caveolae are necessary for IP3R‐induced ICat activation and vasoconstriction.
Tea, coffee and caffeinated drink consumption and risk of subclinical brain abnormalities on MRI
Authors: Arab Lenore , Lonstreth William , O'Meara Ellen , Mukamal Kenneth , Liebeskind David
Published: June 2021

To further substantiate the published relationship between tea and stroke, we evaluated the associations of subclinical MRI abnormalities of the brain with consumption of tea, coffee, and caffeine. Cranial MRI was assessed by a population‐based cohort study of men and women age 65 years and older within the Cardiovascular Health Study (CHS). Tea, coffee, and total caffeine intake from food frequency was associated with subclinical brain infarcts 3 mm or larger and sulcal, ventricular, and white matter abnormality grades. 42.6% drank coffee daily, and 23.8 % drank tea daily. Tea and coffee consumption were associated with race, gender and each other. No significant dose response relationships were seen between coffee or tea intake and white matter, ventricular, or sulcal grades or presence of infarcts assessed two years later Individuals in the highest categories of coffee use had an odds ratio for infarcts of 0.61 compared with those in the lowest categories (CI= 0.38–0.98). Analysis showed no dose‐response relationships between coffee, tea, or caffeine consumption and white matter, ventricular, or sulcal grades. There was little evidence of an association with lower risk of infarct. Although no consistent associations were seen, the infrequent use of tea in biologically plausible doses in this population limits the inferences that can be drawn about the effects of tea on MRI‐assessed brain abnormalities.
Transfection of parasympathetic ganglionic neurons with AAV2‐nNOScDNA protects rat brain from stroke
Authors: Talman William T. , Easker David , Dragon Deidre Nitschke , Lin Li‐Hsien
Published: June 2021

Previous studies show that interruption of parasympathetic nerves from the pterygopalatine ganglion (PPG) increases stroke size, while electrical stimulation reduces it in an animal model. Because nitric oxide (NO) is a putative transmitter of those nerves we hypothesized that upregulating neuronal NO synthase (nNOS) in the PPG will protect brain during permanent occlusion of the middle cerebral artery (MCAO). In anesthetized rats, we injected vehicle (phosphate buffered saline, PBS; N= 8), adeno‐associated virus type 2 vector (AAV2) with enhanced green fluorescent protein (AAV2‐eGFP; N= 10), or AAV2 with nNOScDNA (AAV2‐nNOScDNA, N = 9) into the PPG 8 to 13 days prior to MCAO. In 16 additional animals no injection was made into the PPG prior to MCAO. Animals were evaluated for clinical signs of stroke and euthanized 24 hours after MCAO. Sections of forebrain were stained with 2,3,5 triphenyltetrazolium chloride and analyzed with ImageJ to quantify stroke areas. Infarcts in untreated control animals involved 37 ± 1 % (mean ± SEM) of the brain at the level of the MCA. Infarct size in animals treated with PBS or with AAV2‐eGFP was 34 ± 2% and 33 ± 2% respectively. In animals treated with AAV2‐nNOScDNA infarcts were significantly smaller (24 ± 4 %) than in each of the other groups. Therefore, upregulation of nNOS in PPG may protect the brain from ischemic injury. Support: VA Merit Review, NIH R01 HL59593 and R01 HL088090.
Effect of COX‐2 inhibition on sodium excretion and ENaC expression in Angiotensin II induced hypertensive rats
Authors: Gonzalez Alexis A , Cespedes Carlos , Villanueva Sandra , Vio Carlos P
Published: June 2021

Experimental evidence demonstrates that Prostaglandin E2 (PGE2) production promotes salt excretion in the renal medulla in a setting of activation of the renin‐angiotensin‐system (RAS). We hypothesized that cyclooxygenase‐2 (COX‐2) inhibition in Angiotensin II (Ang II)‐induced hypertensive rats causes Na+ retention by altering the expression of the Epithelial Sodium Channel (ENaC). Sham‐operated male Sprague‐Dawley rats (n=8) were compared to Ang II infused rats (0.4μg/kg/min, n=6) and Ang II infused + COX‐2 inhibitor; Celecoxib (30 mg/kg/d, n=6). After 5 days, Na+ excretion was increased in Ang II (1.37 ± 0.19 vs. 0.74 ± 0.03 mEq/day; P<0.05) and Ang II + Celecoxib (1.34 ± 0.14 vs. 0.74 ± 0.03 mEq/day; P<0.05) groups. PGE2 urinary excretion was augmented in Ang II infused rats (1,261 ± 147 vs. 643 ± 31 pg/h; P<0.05), while Celecoxib suppressed this effect (596 ± 39 vs. 643 ± 31 pg/h; P=NS). COX‐2 protein abundance was increased in the renal medulla of Ang II and Ang II + Celecoxib groups. The mRNA and protein levels of αENaC subunit were up‐regulated in cortex and medulla. No difference in αENaC expression was found between Ang II and Ang II plus Celecoxib groups. These results demonstrate the induction of αENaC expression in Ang II‐induced hypertensive rats and suggest that the up‐regulation of medullary COX‐2 is not involved in ENaC regulation and Na+ handling during chronic RAS activation.
Kidney microRNA expression profile in Ang II‐dependent Hypertension
Authors: Gonzalez‐Villalobos Romer A , Kim Catherine , Neff Ryan M , Satou Ryousuke , Navar L. Gabriel
Published: June 2021

This study was conducted to determine if there are changes in microRNAs expression in the kidneys during angiotensin (Ang)‐II dependent hypertension. microRNAs are small non‐coding RNAs that cause posttranscriptional repression of their targets with emerging recognition as key players in kidney disease. C57BL/6J male mice (8–9 week) were subjected to sham operation (n=3) or Ang II infusions (400 ng/kg/min, n=3) for 12 days during which systolic blood pressure (SBP) was monitored by tail‐cuff method. RNA was isolated from the kidneys and microRNA expression was determined by microarray. SBP was increased by Ang II (151±3 mmHg) vs. controls (119±0.2, p<0.05). When compared to controls, the expression of 5 microRNAs was reduced in the kidneys of Ang II‐infused mice (p<0.05, results expressed as AU): miR‐22 (4,734 ± 1,327 vs. 2,144 ± 637), miR‐133a (34±5 vs. 22±2), miR‐1187 (619±227 vs. 140±66), miR‐574‐5p (501±193 vs. 148±53), and miR‐669f (110 ±43 vs. 25±13). In turn, the expression of 8 microRNAS was increased: miR‐155 (91±17 vs. 151±28), miR‐128 (145±15 vs. 196±4), miR‐183 (49±6 vs. 75±8), miR‐139‐5p (194±9 vs. 294±33), miR‐132 (84±5 vs. 187±39), miR‐200C (804±147 vs. 1,385±280), miR‐25 (895+89 vs. 1,223±103), and miR‐676 (54±6 vs. 98±11). In conclusion, these results demonstrate that there are significant changes in the microRNA expression profile of the mouse kidney during Ang II‐dependent hypertension.

Supported by NIH grants P20RR017659, HL26371, and 1K99DK083455.