bluuebunny/crossref_metadata_2025 huggingface dataset:
The identification and characterization of Glucagon‐like Peptide‐1 receptors on rat peripheral nerves
Authors: Zaveri Shachi Dilip , Kiel Dan
Published: June 2021

Peripheral neuropathy, a common neurological disorder and a major cause of neuropathic pain, results in negative symptoms such as thermal hypoalgesia, loss of pain sensation, undetected infections and limb amputations, and positive symptoms such as burning and stabbing pain. The well established sciatic nerve injury model exhibits an equivalent capacity of regeneration in rats compared to subhuman primates. Glucagon‐like peptide 1 (GLP‐1), an incretin hormone secreted from the enteroendocrine L cells in distal ileum and colon, increases circulating postprandial insulin levels. While it has been reported that GLP‐1 exhibits neuroprotection, no studies have yet been undertaken to identify GLP‐1 receptors on peripheral nerves. Therefore, it is unknown whether GLP‐1's beneficial effects are through a direct action on the nerves or indirectly through elevated insulin levels. We performed immunoblotting assays using a rabbit polyclonal antibody to the GLP‐1 receptor. A 34kDa protein band was detected in homogenates of rat peripheral sciatic nerves and rat insulinoma RINm5F cells known to express the GLP‐1 receptor. Future studies will characterize GLP‐1 receptors on sciatic nerve and evaluate the effects of GLP‐1 on neurobehavioral aspects of neuropathy caused by neurotrauma and conditions like diabetes. These findings may aid in the development of potential therapies for peripheral neuropathy.
The Morphological Investigation Of The Mechanism Of Astragalus Polysaccharide Against Endoplasmic Reticulum Stress By Activation Of AMPK In TIID Rats
Authors: Zhao Ming , Ouyang Jingping
Published: June 2021

OBJECTIVE

To investigate the effects of Astragalus polysaccharide on reducing ER stress in a rat Model of Type 2Diabetes.

Methods

44 SD male rats were randomly divided into two groups, group A was fed with high fat forage and injected with small dosage STZ 25mg/kg through caudal vein, successful rat models were randomly divided into two groups: diabetes group DM group,DM +APS group.Group B were fed with normal forage and also randomly divided into two groups: control group (group C), control+APS (group C +APS group) ,C +APS group and DM +APS group were administered with APS 700mg/kg/day , while group C and DM group were administered with the same dose of Sodium Chloride for 8 weeks. The animals were killed at the end of 8 week treatment with APS,the expression of p‐PERK and p‐AMPK in liver tissue was detected by immunohistochemistry.

Results

1, The expression of p‐PERK was significantly increased in DM group as compared with the control group ,but the expression in DM +APS group was markedly lower than DM group 2,The expression of p‐AMPK was significantly decreased in DM group as compared with the control group, but the expression in DM group was markedly lower than DM+APS group.

Conclusion

APS treatment can relieve ER stress through reducing the expression of phosphorylated PERK in a rat model of type 2 diabetes by activation of AMPK.
Differential effects of COX‐2 inhibitors on vascular smooth muscle cell function
Authors: Mackie Alexander R , Brueggemann Lioubov I
Published: June 2021

Selective cyclooxygenase‐2 inhibitors (known as coxibs) are efficacious anti‐inflammatory drugs. Despite their efficacy, reports of off‐target cardiovascular effects of coxibs led us to investigate the effects of these drugs on vascular smooth muscle ion channels. The ability of various coxibs to influence KCNQ K+ and L‐type Ca2+ currents in rat mesenteric artery smooth muscle cells (MASMCs) was evaluated using perforated patch clamp techniques.

Results

Voltage clamp recordings of MASMCs revealed that celecoxib (10 μM) enhanced KCNQ currents two‐fold while rofecoxib and diclofenac (both at 10 μM) were without effect. Celecoxib also decreased L‐type Ca2+ channel maximal conductance three‐fold in MASMCs. Isolated artery pressure myography experiments revealed that celecoxib induced dose‐dependent dilation of rat mesenteric arteries (MAs) pre‐constricted with 100pM vasopressin. Endothelium‐denuded MAs also dilated in response to celecoxib, suggesting that this effect was endothelium‐independent. In separate experiments, celecoxib (20 μM) produced 97.6 ± 1.2% of dilation of MAs pre‐constricted with 100pM vasopressin, whereas rofecoxib and diclofenac (both at 20 μM) produced only 11.5 ± 2.5% and 5.8 ± 2.0% dilation respectively.

Conclusion

coxibs display differential effects on vascular KCNQ and L‐type Ca2+ channels that may account for some off‐target cardiovascular effects. This work was supported by the AHA (0715618Z to ARM).
Sulfenic Acid Modification: a Novel Link Between the Cardiovascular K+ Channel, Kv1.5, and Oxidative Stress
Authors: Svoboda Laurie Kathleen , Reddie Khalilah G. , Richardson Rudy J. , Carroll Kate S. , Martens Jeffrey Randall
Published: June 2021

Atrial fibrillation, a life‐threatening arrhythmia, is strongly associated with oxidative stress. The voltage‐dependent potassium (Kv) channel, Kv1.5 is a prominent cardiovascular K+ channel that is vital for atrial repolarization. Numerous studies suggest that Kv channels are redox‐sensitive; however, a molecular link between oxidative stress and altered Kv1.5 expression/activity has not been established. Kv1.5 possesses intracellular cysteine residues that may be redox‐sensitive. Reversible formation of cysteine sulfenic acid in response to oxidative stress is a potentially important means of posttranslational protein regulation. Using a novel chemical probe, we find that Kv1.5 is modified with sulfenic acid in a redox‐sensitive manner. Mutagenesis to identify the redox‐sensitive cysteine residues reveals that sulfenic acid formation occurs on the COOH terminus. Using immunohistochemistry, we find that acute oxidant treatment decreases cell surface expression of Kv1.5, with no significant effect on Kv1.5 protein expression. Together, these data suggest that cellular redox state can induce formation of sulfenic acid and alter Kv1.5 cell surface density. Redox‐mediated alterations in cell surface density of ion channels may affect cellular excitability, leading to pro‐arrhythmic changes in cardiac action potentials. Research support: NIH Grants HL0270973 and T32ES07062.
role of protein kinase C in 15‐HETE‐induced hypoxic pulmonary vasoconstriction
Authors: Zhu Daling , Guo Lei , Tang Xiaobo , Chu Xiaojie
Published: June 2021

The aim of the present study was to investigate the roles of protein kinase C (PKC) signal transduction pathways in the 15‐hydroxyeicosatetraenoic acid (15‐HETE)‐induced down‐regulated expression of KV1.5, KV2.1 and KV3.4, and pulmonary vasoconstriction under hypoxia. Tension measurements on rat pulmonary artery (PA) rings, Western blots, semi‐quantitative PCR, and whole‐cell patch clamp technique were employed to investigate the effects of 15‐HETE on PKC and KV channels. Hypericin (6.8 μmol/L), a PKC inhibitor, significantly attenuated the constriction of PA rings to 15‐HETE under hypoxia. The down‐regulation of KV1.5, KV2.1 and KV3.4 channel expression, and inhibition of whole‐cell K currents (IKV) induced by 15‐HETE were rescued, and restored, respectively, by hypericin. These studies indicate that the PKC signal transduction pathway is involved in 15‐HETE‐induced rat pulmonary vasoconstriction under hypoxia. 15‐HETE suppresses the expression of KV1.5, KV2.1 and KV3.4 channels and inhibits IKV through the PKC signaling pathway in pulmonary arterial smooth muscle cells.
Regulation of basal cAMP levels in rabbit cardiomyocytes
Authors: Xin Wenkuan , Liu Yanping , Cohen Michael V , Downey James M , Rich Thomas C
Published: June 2021

We recently found that basal cAMP levels critically influence the timecourse of agonist‐induced cAMP signals. We sought to identify which if any phosphodiesterases (PDEs) control basal cAMP levels in isolated rabbit cardiomyocytes. cAMP concentration near the plasma membrane of cardiomyocytes was monitored by measuring current through adenovirus‐expressed cyclic nucleotide‐gated (CNG) channels. In the absence of receptor agonists basal cAMP levels were low. A PDE inhibitor cocktail (PDE types 1, 3, and 4) triggered transient increases in cAMP and subsequent addition of a non‐selective PDE inhibitor caused no further increase. These data revealed that adenylyl cyclase (AC) and PDE are constitutively active in rabbit cardiomyocytes and that basal cAMP levels are primarily controlled by 3 PDE types. Any one of the 3 type‐specific PDE inhibitors caused small rises in cAMP. Addition of any second inhibitor triggered another rise, suggesting that the 3 PDE types work in concert to maintain basal cAMP levels. 1 μM H89 (a PKA inhibitor) reduced the rate of cAMP decline in the presence of PDE inhibitors, indicating that PKA‐mediated feedback contributed to the transient nature of these signals. In conclusion, basal cAMP is highly regulated by a balance between AC and PDE activity and increases in cAMP trigger PKA‐mediated feedback that returns cAMP to its basal level. Supported by HL74278, HL20648.
Modulation of Synaptic Transmission by G Protein βγ Subunits
Authors: Betke Katherine , Wells Christopher , Lindsley Craig , Gao Alice , Marala Ravi , Hamm Heidi
Published: June 2021

Synaptic transmission is characterized by exocytotic events which mediate the release of chemical transmitters to facilitate neuronal communication. Gi/Go coupled GPCRs play an important role in controlling exocytosis. In addition to direct interaction with voltage‐gated calcium channels to inhibit calcium influx, Gβγ subunits bind directly to SNARE proteins in the regulation of fusion events. The current study is aimed at developing small, druglike molecules which target the βγ /SNARE interaction to elucidate details involved in this novel mode of neuromodulation.

Compound libraries were screened using a novel, label free detection method, Corning Epic®, which identifies protein‐protein interactions as changes in the local index of refraction due to binding events. Using these signals, we evaluated the ability of small molecules to modulate the Gβγ /SNARE interaction. Initial screening identified five lead compounds. By further application of an iterative analog library synthesis approach, we will develop analogs with improved physiochemical properties to serve as tools to dissect the therapeutic relevance of the Gβγ /SNARE interaction. This work was supported by the National Institutes of Health (EY010291).
Involvement of 5HT1A receptor‐mediated inhibition of GSK3 beta in the antidepressant‐like phenotype of RGS‐insensitive mice
Authors: Talbot Jeffery N , Jensen Kelly E , Clemans Crystal F , Nicol Melanie R , Huang Xinyan , Neubig Richard R , Traynor John R
Published: June 2021

We have shown that mice lacking Regulators of G Protein Signaling (RGS) activity at Gαi2 (Gαi2(G184S)) exhibit an antidepressant‐like phenotype with enhanced activity in response to SSRIs and 5HT1A agonists. Glycogen synthase kinase‐3β (GSK3β) is a constitutively active, pro‐apoptotic kinase that is inhibited by serotonin, SSRIs and 5HT1 receptor agonists via phosphorylation at Ser‐9. Therefore, we tested whether GSK3β activity was altered in RGS‐insensitive Gαi2(G184S) mice. Heterozygous (GS/+) and homozygous (GS/GS) Gαi2(G184S) animals showed increased basal GSK3β phosphorylation at Ser‐9 in the cortex and hippocampus. WAY 100635, a 5HT1A receptor antagonist, caused a reduction of phospho‐GSK3β in the cortex and hippocampus of both GS/+ and GS/GS but not wild‐type animals. Behaviorally, treatment of Gαi2(G184S) mice with the same dose of WAY 100635 reversed the antidepressant‐like phenotype in the tail suspension test, an established rodent model of antidepressant‐like behavior. These data demonstrate that enhancing Gαi2 activity by the removal of RGS protein regulation at Gαi2 results in an antidepressant‐like phenotype that is manifest both biochemically and behaviorally and is reversed by 5HT1A receptor blockade. Supported by DA04087, GM39561, T32 DA007268, the Bower, Bennet, & Bennet Endowed Chair Research Award, and the AACP New Investigator Program Award.
ACTIVATOR OF G‐PROTEIN SIGNALING 3: THE ROLE OF THE TETRATRICOPEPTIDE REPEAT DOMAIN IN REGULATING THE INTERACTION OF AGS3 WITH G‐PROTEIN.
Authors: An Ningfei , Vural Ali , Oner Sukru S. , Blumer Joe B. , Lanier Stephen M.
Published: June 2021

One of the key questions for Activator of G‐protein Signaling 3 (AGS3) and related proteins containing G‐protein regulatory (GPR) motifs is what controls their positioning within the cell and their interaction with G‐proteins. AGS3 has 7 tetratricopeptide (TPR) motifs upstream of 4 GPR motifs, each of which bind and stabilize the GDP bound conformation of Gialpha. The TPR domain of AGS3 serves as a key determinant of subcellular positioning through its interaction with specific binding proteins. We addressed the role of individual TPR domains and these binding partners in regulating the interaction of AGS3 with G‐protein in living cells (HEK‐293) by bioluminescence resonance energy transfer (BRET) with the donor‐acceptor pair AGS3‐ Renilla luciferase (RLuc) and Gialpha1‐YFP. Progressive deletion of TPR domains increased net BRET compared to wild type AGS3. Similar increase in net BRET signal occurred upon disruption of the TPR domain such as a single nucleotide polymorphism identified between the 4th and the 5th TPR motif. Co‐transfection of AGS3‐binding partners LKB1, Inscuteable or Frmpd1, which bind to the TPR or linker domains in AGS3, with AGS3‐RLuc and Gialpha1‐YFP differentially regulated the interaction of the two proteins. These data indicate that the TPR domain regulates the interaction of the AGS3 GPR domain with G‐proteins.
Chemotherapy associated memory/learning loss and reversal with supplemental amino acids in a rat model
Authors: Morgan Lee Roy , Thornton Melissa E. , Benes Edmund , Luttrell Melody M , LaHoste Gerald J. , Jursic Branko S.
Published: June 2021

Introduction

Most chemotherapy drugs (5‐FU, penclomedine (PEN), phosphamides) that alter memory are pyrimidines, pyridines or other pseudohetero‐cyclic rings that could interact with/antagonize the CNS N‐methyl‐D‐aspartate (NMDA)receptor complex ‐ a memory transmitter pathway. NMDA has numerous neurotransmitter substrates ‐ glutamate, serine, glycine, N‐methyl D‐spartate, all of which can also exist as pseudo‐pyridine or pyrroles. Agents that reverse the memory/learning deficiencies are described here.

Methods

The Morris water maze was modified to screen agents for behaviorial changes. Adult female rats were dosed IP once with PEN (100 mg/kg or 5‐FU (78 mg/kg) and compared vs. controls for learning/memory over 24 h. The time required for each rat to swim & find a pedestal was compared (6 consecutive swims ‐ avg/SD of the 1st vs 6th were compared). Either N‐acetyl cysteine (NAC)(200 mg/kg) or L‐lysine (50 mg/kg) was administered (PO) x 3 days pre‐chemotherapy. The results were compared with the NMDA binding assay in vitro in rat brain homogenates.

Results

PEN and 5‐FU had significant impacts on memory and learning with 1.5 & 2.5‐fold impairment, resp., after 3 hr., and >85% for PEN & 5‐FU at 24 h.,which continued for 1‐3 days, despite normal appearances. 5‐FU and PEN impairments were reversed 1.5 ‐ 2.3 fold with NAC and L‐lysine, resp.

Conclusion

Drugs that can exist in 5‐6 membered ring configurations could interact antagonize the NMDA receptor complex. NAC is a Lewis acid and L‐lysine a Lewis base, each reversing basic and acidic drugs, resp. The described behavorial screen is easy to use and could be useful in screening anti‐cancer agents for behavioral modification. Supported by NCI/SBIR grant ‐ 5R44CA85021.
Lobeline attenuates behavioral and neurochemical effects of ethanol in a preclinical model of alcoholism
Authors: Sajja Ravi Kiran , Dwivedi Chandradhar , Rahman Shafiqur
Published: June 2021

Lobeline is a plant alkaloid obtained from Lobelia inflata and a brain nicotinic acetylcholine receptor (nAChR) ligand that has been implicated in the behavioral and neurochemical effects of nicotine and amphetamine. Here, we examined the effects of acute lobeline injection on voluntary ethanol (10% v/v) consumption using 24h access two‐bottle free choice (water vs ethanol) drinking paradigm in C57BL/6J mice. Lobeline 4 mg/kg and 10 mg/kg significantly decreased (53%‐59%) and (58%‐68%) ethanol drinking, respectively without altering water consumption at 1 h‐12 h post injections. Using HPLC coupled with electrochemical detection, we determined the effects of lobeline (10 mg/kg) on acute ethanol (2.5 g/kg)‐induced increase in dopamine and dihydroxyphenylacetic acid (DOPAC) content in ventral striatum (VS) and medial prefrontal cortex (mPFC), known brain reward relevant regions for ethanol drinking. Lobeline (10 mg/kg) significantly reduced ethanol‐induced increase in dopamine (146% and 110% of control) and DOPAC (116% and 101% of control) in VS and mPFC, respectively. Overall, these data indicate that lobeline modulates the behavioral and neurochemical effects of ethanol by targeting nAChRs in brain reward relevant regions. Therefore, the preclinical profile suggests that lobeline can be developed as potential pharmacotherapy for alcoholism in humans. (AACP NIP RG (SR), SD 2010 RISG (SR).
Age‐dependent differences in antidepressant response
Authors: Lacayo Liliana Margarita , Farrokhi Catherine , Manalo Charlene M , Reyes Kevin C , Pechnick Robert N
Published: June 2021

The efficacy of tricyclic antidepressant treatment in children and adolescents is highly controversial. This investigation examined potential age‐dependent differences of tricyclics in a widely utilized screen for antidepressant activity. Male peripuberal (30 days of age) and adult rats were implanted with osmotic pumps containing saline or desmethylimipramine (DMI; 10 mg/kg/day). An untreated control group was added to rule out potential effects produced by pump implantation. On day 6, locomotor activity was assessed using the open field test (OFT). On day 7, animals were tested in the forced swim test (FST), and floating (i.e., immobility), swimming, and active climbing behaviors were measured. DMI produced a slight reduction of locomotor activity in the OFT in both the peripuberal and adult rats. In the FST, DMI increased climbing in the both the peripuberal and rats. Although DMI decreased floating in adults, floating was not reduced in the peripuberal rats. Further investigation revealed that in the peripuberal rats, implantation of saline‐containing pumps reduced floating compared to untreated rats. Thus, DMI showed antidepressant activity in both age groups. The data also show that osmotic pumps impair the ability of peripuberal rats to float, indicating that minipumps might not be an appropriate approach to deliver drug when peripuberal rats will be tested in the FST.
Temporal and Spatial Activation of Angiogenesis and Expression of FGF‐1/FGF Receptors in the Infarcted Heart
Authors: Zhao Tieqiang , Zhao Wenyuan , Chen Yuanjian , Sun Yao
Published: June 2021

The current study is to determine the temporal and spatial expression of FGF‐1/FGF receptors and their relation to angiogenesis in the infarcted heart. Rat hearts were collected at day 1, 3, 5, 7, 14, 21 and 28 postMI. Normal rats served as controls. Cardiac FGF‐1 gene and protein expression, FGF receptors and vascular density were examined. Pre‐existing vessels underwent necrosis in the infarcted myocardium at day 1 postMI. Newly formed vessels first appeared at the border zone as early as day 3, followed by the infarcted myocardium. Vascular density peaked at day 7 and 14, and gradually declined at day 21 and 28. FGF‐1 immunostaining was strongly positive at the border zone at day 3 and at the infarcted myocardium at day 7 and 14 and then became less evident at day 28. Autoradiographic FGF receptor binding was significantly increased in the infarcted myocardium from day 7 and remained elevated thereafter. The expression of FGF‐1 and FGF receptors remained unchanged in noninfarcted myocardium. Thus, angiogenesis is activated in the infarcted myocardium in the early stage of MI and becomes quiescent thereafter. FGF‐1/FGF receptor expression is elevated in the infarcted myocardium, which is temporally and spatially coincident with angiogenesis. This finding suggests that FGF‐1 is involved in cardiac angiogenesis following MI.
Obesity induced‐insulin resistance causes endothelial dysfunction without reducing the vascular response to hindlimb ischemia.
Authors: Belin de Chantemele Eric J , Ali Mohammed Irfan , Mintz James , Stepp David W
Published: June 2021

Type II diabetes (T2DM) impairs vascular growth but the progression and mechanisms are poorly understood. To determine whether obesity and early T2DM impair endothelium‐dependent relaxation and vascular response to ischemia, acetylcholine‐induced dilatation and angiogenic response to ischemia were assessed in young (C57) and old lean mice (old C57), in obese (db‐C57), and in mice suffering an early (db‐KsJ) and sustained T2DM (old db‐KsJ). Glycemia gradually increases from the db‐C57 to the old db‐KsJ while early and established T2DM significantly reduced the level of insulin which was significantly increased in the obese mice. Endothelial function was assessed in isolated resistance arteries while the angiogenic response induced by unilateral hindlimb ischemia was analyzed, after 28 days, with a laser Doppler and angiography. Aging (‐21%), obesity (‐45%), early (‐58%) and sustained T2DM (‐69%) induced a progressive impairment of the endothelium‐dependent relaxation of the gracilis artery. Early and sustained T2DM only, impaired skin blood flow recovery. Impairment of the vascular collateralization was observed in the old db‐KsJ only. These results demonstrate that endothelial dysfunction induced by aging and obesity is insufficient to alter the angiogenic response to ischemia. Furthermore the development of frank T2DM is required to impair the vascular response to hindlimb ischemia.
Angiogenic potential of isolated microvessel fragments is independent of their tissue origin
Authors: Nunes Sara Santana , Maddie Melissa A. , Benton Richard L. , Hoying James B.
Published: June 2021

Adipose‐derived microvessel fragments (MFs) form a mature, perfused microvessel network when implanted in SCID mice. However, adipose tissue is rich in resident mesenchymal cells that could uniquely drive spontaneous neovascularization from adipose derived MFs. Therefore, we evaluated if a more specialized vascular bed, lacking these precursor cells, would recapitulate MF angiogenesis and neovascularization. Microvessel fragments isolated from rodent brain cortex (BMFs) suspended in collagen gels were either cultured or implanted into host mice. Characterization of BMFs showed the presence of associated mural cells but not astrocytes. In vitro, these fragments form a neovessel network by undergoing sprouting angiogenesis similar to adipose‐derived MFs. When implanted for 28 days, brain‐derived MFs formed a microvasculature similar to adipose‐derived MFs which was functionally connected to the host circulation as demonstrated by FITC‐lectin perfusion. Importantly, implanted BMFs retained staining for Glut1 receptor, considered a marker of brain endothelium, and exhibited reduced macromolecular vessel permeability. This demonstrates that there is an intrinsic ability for isolated microvessel elements to neovascularize which is independent of the tissue origin and degree of vessel specialization. Supported by NIH R01 EB007556.
Insulin‐like growth factor I (IGF‐1) downregulates lipoprotein lipase and suppresses atherosclerotic foam cell formation in vivo and in vitro
Authors: Sukhanov Sergiy , Vaughn Charlotte , Higashi Yusuke , Titterington Jane , Delafontaine Patrick
Published: June 2021

We have shown that IGF‐1 decreases atherosclerosis in ApoE‐null mice. Lipoprotein lipase (LPL) increases the uptake of OxLDL by macrophages (MΦ), promoting foam cell formation and atherosclerosis development. To determine a potential effect of IGF‐1 on LPL and foam cell formation, we infused ApoE‐null mice with 1.5 mg/kg IGF‐1 or saline for 12w. IGF‐1 reduced aortic LPL mRNA levels by 31% (RT‐PCR) and serum LPL activity by 66% (fluorescence assay). This effect correlated with a 27% reduction in Oil Red‐positive lesion area and a 36% decrease in plaque MΦ (immunostaining with Mac‐3 a/b) suggesting that IGF‐1 reduced MΦ‐derived foam cell formation.

Human blood monocyte‐derived MΦ were treated with 80 ug/ml OxLDL for 48h to stimulate formation of Oil Red‐positive "foam cells". OxLDL increased MΦ LPL mRNA levels and LPL activity >5‐fold. Pre‐treatment with IGF‐1 (25 ng/ml, 24h) reversed OxLDL‐induced upregulation of LPL activity by 52% and decreased lipid uptake by 88%. Furthermore, adenovirus‐mediated overexpression of IGF1 receptor in MΦ prevented lipid uptake by 82% vs. control Ad‐GFP‐infected MΦ. Conversely, addition of exogenous LPL (5 ug/ml) stimulated DiI‐OxLDL binding by MΦ by 2.3‐fold and increased lipid uptake by 1.9‐fold (Oil Red‐staining). Liver X receptor (LXR) is a nuclear transcription factor regulating MΦ LPL expression. The LXR agonist 22R‐oxycholesterol (50 uM, 16h) increased LPL activity in MΦ >2‐fold and IGF‐1 reversed this effect by 65%. In summary, our data indicate that IGF‐1 downregulates LPL in vivo and in vitro potentially via a LXR‐dependent mechanism and inhibits foam cell formation. This is the first report of an effect of IGF‐1 on cellular lipid internalization and these findings have major relevance for the understanding of mechanisms whereby IGF‐1 reduces atherosclerosis.
Dose effect of shear stress on platelet complement activation
Authors: Yin Wei , Rubenstein David A
Published: June 2021

Altered blood shear stress is one of the risk factors that can induce platelet activation, thrombosis, atherosclerosis and many other cardiovascular diseases. Complement activation, an important contributor in inflammation, has also been reported to be related to blood flow induced platelet activation. In this study, the dose effect of shear stress (magnitude and shear exposure time) on platelet surface complement activation was investigated, using a dynamic cone and plate shearing device. Washed platelets were exposed to uniformly distributed shear stresses at 2.4 or 9 dyne/cm2 for 30 min. Timed samples at 0, 10, 20 and 30 min were taken and assayed for complement activation (C4d, C3b and C5b‐9 deposition) using a solid phase ELISA approach. In parallel, platelet activation was examined using flow cytometry for CD62P expression, and a modified prothrombinase assay for thrombin generation. Results indicate that activated platelets support complement activation to completion, with the production of membrane attack complex (C5b‐9). The generation of complement activation products, C4d, C3b and C5b‐9, increased significantly as shear stress magnitude and shear exposure time increased, correlating to platelet activation levels. Therefore, platelet activation and its associated complement activation may interact and promote cardiovascular disease development under pathological shear conditions.
Development of an animal model of microvascular thrombosis
Authors: Calhoun William R , Kanth Shreya , Torres Filho Ivo
Published: June 2021

We have developed a model of thrombosis to test in vivo the efficacy of antithrombotic agents in a microvascular network. The model involves the induction of a platelet thrombus within venules of the spinotrapezius muscle in anesthetized Sprague Dawley rats using a compound normally employed for microvascular oxygenation measurements (Pd‐tetra‐4‐carboxyphenylporphyrin). The photoactive chemical is injected intravascularly and is excited with a 532 nm pulsed, Er:YAG laser ( 8‐10 ?W, 0.4‐0.6 kHz). As a result, free radicals are formed causing injury to the endothelial wall, subsequent platelet aggregation and thrombus formation without microvascular constriction. Red blood cell (RBC) velocity and intravascular diameter at thrombus site are monitored periodically. After testing venules of different sizes and RBC velocities, the most repeatable data were gathered from venules with diameters ranging from 40 to 60 µm and with RBC velocities ranging from 2 to 4 mm/sec. Upon establishing these control parameters, the model is being used to induce local ischemia and to test the effects of injected anti‐thrombotic agents on platelet thrombus formation and local blood flow.

Support: AD Willliams Trust Funds (VCU).
Selective enhanced constriction and attenuated dilation in leptin deficient mice.
Authors: Frame Mary D , Vigilance Jacqueline E
Published: June 2021

We tested responses to endothelin‐1 (ET), interleukin‐6 (IL6) and IL8 in leptin deficient diabetic mice (db/db or ob/ob), in comparison to C57 genetic background mice in the absence or presence of inhibitors. The cremaster muscle of anesthetized (50 mg/kg Nembutal) mice (n=63) was prepared for intravital microscopy. Arteriole(A)/venule(V) pairs were chosen for study; baseline diameters A 26±8 ?m, V 50±13 ?m were not different between strains. Micropipette applied adenosine (10‐4M) was attenuated in A and V from db/db or nitroarginine (LNNA) pre‐treated C57 vs. control C57. Phenylephrine (10‐4M) constriction was unchanged in A, but attenuated in V from db/db, ob/ob or LNNA vs. C57. ET1 dose dependent constriction (C57 logM EC50 ‐10±5 A; ‐5±4 V) was significantly attenuated in either db/db or ob/ob mice, yet enhanced with LNNA pretreatment in C57. Responses to IL6 or IL8 were biphasic and dose dependent in A from C57, while V showed only constriction. Responses to IL6 were blocked by PD142893. IL6 caused only constriction in A and V of ob/ob or db/db. IL8 caused biphasic responses in some diabetics. Leptin deficient diabetes thus shows attenuated constriction to ET1, selective absence of dilation to IL6 and inconsistent absence of dilation to IL8. NIH DK68401
Differential Pro‐inflammatory/Pro‐oxidant Effects of BMP‐4 in Coronary and Pulmonary Arterial Endothelial Cells
Authors: Ungvari Zoltan , Labinskyy Nazar , Jo Hanjoong , Csiszar Anna
Published: June 2021

There is increasing evidence that TGF£] family member cytokine BMP‐4 plays different pathophysiological roles in the pulmonary and systemic circulation. Up‐regulation of BMP‐4 has been linked to atherosclerosis and hypertension in the systemic circulation, whereas disruption of BMP‐4 signaling is associated with the development of pulmonary hypertension. To test the hypothesis that BMP‐4 elicits differential effects in pulmonary and systemic circulation, we compared the pro‐oxidant and pro‐inflammatory effects of BMP‐4 in cultured human coronary arterial endothelial cells (CAECs) and pulmonary arterial endothelial cells (PAECs). We found that BMP‐4 in CAECs increased O2.‐ and H2O2 generation, induced NF‐ f;ÛB activation, up‐regulated ICAM‐1 and induced monocyte adhesiveness. In contrast, BMP‐4 failed to induce oxidative stress or endothelial activation in PAECs. Also, BMP‐4 treatment impaired ACh‐induced relaxation and increased O2.‐ production only in cultured rat carotid (but not pulmonary) arteries. Thus, BMP‐4 exerts pro‐oxidant, pro‐hypertensive and pro‐inflammatory effects only in the systemic circulation, whereas pulmonary arteries are protected from these adverse effects of BMP‐4. The vascular bed‐specific endothelial effects of BMP‐4 are likely to contribute to its differential pathophysiological role in the systemic and pulmonary circulation.
Mitochondria determine TNFα receptor distribution in lung microvessels
Authors: Rowlands David John , Lindert Jens , Bhattacharya Jahar
Published: June 2021

Circulating tumor necrosis factor alpha (TNFα) ligates endothelial (EC) TNFα receptor 1 (TNFR1) to promote inflammation. Although mitochondria are capable of proinflammatory signaling in lung EC (Ichimura, JCI 2003), the mitochondrial role in TNFα‐mediated inflammation remains unknown. To determine mitochondria‐TNFR1 relationships, we viewed ECs of single microvessels in isolated, blood‐perfused rat lungs by live confocal microscopy. To determine spatial expressions of EC mitochondria and TNFR1 by fluorescence quantification, we infused microvessels respectively, with the mitochondria‐specific fluorophore, mitotracker green (MTG), or a fluorophore‐conjugated, anti‐TNFR1 mAb. Buffer microinjections removed unbound dye. Microvascular expressions for both mitochondria and TNFR1 were 3‐fold higher at venular branch points than at septal capillaries (p<0.05, n=4). Fluorescence determinations along the microvascular length indicated that mitochondria and TNFR1 fluorescence intensities correlated at venular branch points (p<0.05 by linear regression), but not in septal capillaries. These findings are the first evidence that in lung microvessels, TNFR1 localizes to mitochondria‐rich regions. Mitochondrial mechanisms may regulate TNFR1‐induced inflammatory responses.
Exercise Training Improves Endothelial Dysfunction in Type 2 Diabetes
Authors: Lee Sewon , Park Yoonjung , Zhang Cuihua
Published: June 2021

Type 2 diabetes (T2D) is a leading risk factor for cardiovascular diseases including peripheral artery disease and coronary heart disease. Exercise training (ET) is thought to have a beneficial effect on these disorders, but the basis for this effect is not fully understood. Because endothelial function (EF) and insulin resistance (IR) play key roles in the pathological events leading to vascular disease in T2D, we hypothesized that the ET will exert its effects on improving EF and IR. To test this hypothesis, we assessed the effects of ET in vascular function of diabetic (db/db, BKS.Cg‐m+/+Leprdb/J) mice by evaluating EF of isolated coronary arterioles of wild‐type (WT, C57B/J) and db/db with/without ET. Although dilation of vessels to the endothelium‐independent vasodilator, sodium nitroprusside was not different between db/db and WT, dilation to the endothelium‐dependent agonist, acetylcholine (ACh) was impaired in db/db (n=12, P<0.01) compare to WT (n=8). However, vasodilation to ACh was restored in db/db with ET (n=8). In addition, IR was improved in the db/db after ET. ET did not change body weight of db/db, but superoxide dismutase (SOD1 and SOD2) protein expression of heart tissue was up‐regulated by over 40% by ET. Although we have not yet linked the exercise‐induced increase in anti‐oxidants to the improvement in EF in T2D, we suggest that this connection may be the basis for the benefit of ET in T2D.
The Cardioprotective Effects of Adiponectin in the ZDF Heart Following Ischemia Are Associated with a Significant Attenuation of Myocardial ICAM‐1 Expression
Authors: Choi Ji‐Eun , Davis‐Gorman Grace , Tsao Tsu‐Shuen , McDonagh Paul
Published: June 2021

Ischemic heart disease (IHD) is significantly enhanced in type 2 diabetes (T2D) and inflammation is associated with IHD and T2D. The adipocytokine, adiponectin (APN), which has anti‐inflammatory actions, is chronically reduced in type 2 diabetes. APN may link inflammation, T2D and IHD. We examined if a globular form of adiponectin was cardioprotective in the Zucker Diabetic Fatty (ZDF) rodent model of T2D and if the cardioprotection was associated with myocardial expression of ICAM‐1, a key adhesion molecule and marker of coronary microvascular dysfunction. ZDF (n=14) rats and their lean controls ZLC (n=13) were subjected to 30 minutes of LAD occlusion and 120 minutes of reperfusion. Animals were treated with either PBS or recombinant globular adiponectin (1.0 μg/g). At R120, the extent of myocardial infarction was measured. Myocardial ICAM‐1 expression was also quantified immunohistochemically. We found that adiponectin treatment significantly attenuated the enhanced myocardial infarction in the ZDF model of T2D (p<0.05). The reduced infarction was associated with a significant reduction in myocardial ICAM‐1 expression (p<0.05). These findings suggest that adiponectin treatment protects against I/R injury in the T2D heart. The cardioprotective action may be related to reduced coronary microvascular inflammation enhancing PMN sequestration and PMN‐mediated tissue injury. Supported by NIH HL 58859.
Human thermoregulation and obesity
Authors: de Graaf Albert Arjaan , Freidig Andreas , van Ommen Ben
Published: June 2021

On average 80% of muscular physical activity energy has to be released by the body as heat. Obese people have a thick insulating fat layer. We investigated whether the concomitant thermal isolation can fundamentally limit heat dissipation and therefore exercise capacity in the obese.

To estimate key values of thermoregulatory properties in humans, relevant mechanisms, models, their parametrization and data that were taken from the literature. Investigated thermoregulatory relevant properties and mechanisms included body heat capacity, clothing, sweating, vasodilation, respiratory evaporation, and body thermal resistance.

We found that the most effective mechanisms for heat loss during exercise are vasodilation (increased tissue conductance) and sweating. Thermal insulation by the fat layer is effectively shunted by increased skin blood flow.

Since muscle and skin compete for blood flow for energy supply and heat transport, respectively, we hypothesize that cardiac output and tissue/muscle blood flow are major players in thermoregulation in the obese. The available data suggest that in untrained obese, tissue conductance as well as sweating rate are significantly lower than in lean, fit people, with total heat loss capacity possibly limited to 350 W. It is concluded that thermoregulation may fundamentally restrict exercise performance in unfit obese.
Denitrogenation (Preoxygenation) as a Prophylactic Treatment for “Flier's Bends” During High Altitude Missions in Unpressurized Aircraft of the Allied Air Forces in World War II
Authors: Dean Jay B.
Published: June 2021

Allied bombing missions in unpressurized planes required O2‐breathing at 20,000‐30,000 ft. By using pressure breathing (e.g. photo reconnaissance missions), altitudes of 35,000‐47,000 ft were endured. Any flight > 20,000 ft increased the airman's risk for decompression sickness (DCS); that is, gases dissolved in their tissues at sea level would come out of solution at hypobaric pressure and form bubbles (mostly N2) causing pain in the extremities and, in the worse cases, paralysis or death. DCS‐aka "Caisson's disease" or "Diver's bends"‐had been recognized since the 1840s as a serious problem for men who decompressed too quickly from hyperbaric air pressure. "Flier's bends" was not appreciated until aeronautical engineering developed aircraft capable of flight above 20,000 feet in the 1930s. Beginning in 1934, Harry Armstrong (Wright Field) revealed that DCS occurred in men and animals during high altitude "chamber flights". Armstrong, Randy Lovelace (Mayo Clinic) and others reported in 1940‐41 that 30 min of breathing pure O2 before ascending to altitude (preoxygenation) reduced both the level of N2 in the body (denitrogenation) and the risk of DCS. Breathing O2 for 30‐40 min before flight, or during slow ascent to 20,000 feet, became standard operating procedure for high altitude missions in WWII. Today, astronauts undergo denitrogenation prior to a "space walk" to reduce their risk of DCS.
A model‐based mechanical rationale for mixed strains in the aponeuroses of contracting muscle
Authors: Sinha Shantanu , Chi Sheng‐Wei , Chen Jiun‐Shyan , Hodgson John A , Shin David , Edgerton Reginald V
Published: June 2021

A simple trapezoid muscle in contact with aponeuroses and tendon was modeled in order to investigate strain patterns in skeletal muscle and aponeurosis. Pennation angles of 25° and 47° were modeled in muscle undergoing isometric contractions. Aponeurosis strain was greatest in the region closest to the tendon and decreased with distance from the tendon. Strains were greater at 25° pennation angle and the region of the aponeurosis farthest from the tendon shortened as the muscle contracted. Aponeurosis shortening was not observed at the 45° pennation angle. The results may be explained by recognizing that shortening muscle fibers increase in girth due to incompressibility. The influence of these mixed dimensional changes upon aponeurosis strain depend upon the orientation of the fibers. A rotational transformation of fiber strains onto the aponeurosis indicates variable strains in aponeuroses which depend upon the pennation angle of the muscle fibers and their organization within the muscle. Experimentally, the higher pennation angles which often accompany maximum voluntary contractions favor aponeurosis lengthening at relatively low strains whereas observations at lower forces and correspondingly lower pennation angles favor aponeurosis shortening with greater strain variation along the aponeurosis. Funded by NIAMS Grant RO1 AR‐53343.
Mechanical ventilation induces diaphragmatic mitochondrial oxidant production resulting in mitochondrial damage and respiratory dysfunction
Authors: Kavazis Andreas N , Talbert Erin E , Smuder Ashley J , Hudson Matthew B , Nelson W. Bradley , Powers Scott K
Published: June 2021

Mechanical ventilation (MV) is a life saving intervention used in patients with acute respiratory failure. Unfortunately, prolonged MV results in diaphragmatic weakness which is an important contributor to the failure to wean patients from MV. We have shown that reactive oxygen species (ROS) play a critical role in diaphragmatic weakness following MV, but the pathways responsible for MV‐induced diaphragmatic ROS production remain unknown. We hypothesized that following MV diaphragmatic mitochondria produce higher rates of ROS resulting in mitochondrial oxidative damage and dysfunction. Our results support this hypothesis as mitochondria isolated from the diaphragm of MV rats possessed a lower respiratory control ratio compared to control. Further, MV diaphragmatic mitochondria produced higher rates of ROS in both state 3 and state 4 respiration. MV diaphragmatic mitochondria also exhibited higher levels of oxidative damage as indicated by increased lipid peroxidation and protein oxidation. Finally, our data also reveal that the activities of electron transport chain complex II, III, and IV are depressed in diaphragmatic mitochondria following MV. In summary, these results are consistent with the concept that diaphragmatic inactivity promotes an increase in mitochondrial ROS production resulting in mitochondrial oxidative damage and respiratory dysfunction.

Supported by an NIH R01 awarded to SKP.
Impact of acute caffeine administration on skeletal muscle oxidative stress and antioxidant status
Authors: DiStasio Thomas J , DeRuisseau Lara R , Cowley Patrick M , Recca Daniel M , DeRuisseau Keith C
Published: June 2021

Caffeine is a pharmacological agent that has been shown to enhance muscle contraction. The stimulatory effects of caffeine may be, in part, due to alterations in the skeletal muscle redox status. Specifically, caffeine administration to rats has been shown to rapidly, but transiently, decrease levels of skeletal muscle nitric oxide (NO) synthase. Decreased levels of NO could lead to elevated oxidative stress, since NO is a potent superoxide radical scavenger. We tested the hypothesis that acute caffeine administration would decrease muscle nitrosative stress, increase oxidative stress, and upregulate the expression of key antioxidant enzymes. Male Sprague Dawley rats received i.p. administration of caffeine (16 mg/kg; n=8) or vehicle (saline; n=8). Thirty minutes post injection the rats were anesthetized and the soleus muscles were harvested for analysis. No differences in oxidative injury (protein carbonyls) or nitrosative injury (3‐nitrotyrosine) were detected (p>0.05). Caffeine administration did not alter manganese superoxide dismutase or copper‐zinc superoxide dismutase protein levels, but resulted in a 50% increase in catalase protein levels (p<0.05). In summary, acute caffeine administration may upregulate skeletal muscle catalase expression by a mechanism independent of oxidative stress. Supported by Syracuse University SOE and APS UGSRF
Mechanically‐induced intercellular calcium waves in renal medullary interstitial cells
Authors: Koo Jahoon John , Toma Ildiko , Vargas Sarah Laurin , Maric Christine , Peti‐Peterdi Janos
Published: June 2021

In addition to providing structural support, renomedullary interstitial cells (RMIC) are thought to play an active physiological role in regulating renal medullary function. RMICs have been shown to have contractile properties, however, the cellular and molecular mechanisms of this action remain unknown. We aimed to identify the existence of molecular elements of an intercellular calcium wave in cultured rat RMICs using Fluo‐4/Fura red and confocal fluorescence microscopy. RMICs expressed connexins (Cx) Cx30 and Cx45 and P2X4 and P2Y2 purinergic receptors measured by RT‐PCR and immunohistochemistry. Mechanical stimulation of a single RMIC caused a 4±0.2‐fold increase in [Ca2+]i (n=45) which was propagated throughout the monolayer (16 ± 1.2 um/s) in a regenerative manner (without decrement of amplitude, kinetics, and speed) over distances >800 um. The gap junction uncoupler 18a‐glycyrrhetinic acid (18a‐GA, 25 uM), breakdown of extracellular ATP (apyrase and hexokinase, 50 U/mL each), or P2 receptor inhibition (suramin, 50 uM) all decreased the speed and D[Ca2+]i of the calcium wave. Physical contact between cells was not required for calcium wave propagation. These studies suggest that mechanosensitive, ATP‐releasing Cx hemichannels and cell‐to‐cell purinergic signaling are key molecular elements of the RMIC calcium wave, which may serve an important role in RMIC‐mediated renal medullary function.
Role of ferroportin‐1 (FPN‐1) in iron (Fe) handling by the kidney proximal tubule (PT)
Authors: Thevenod Frank , Wolff Natascha A. , Lee Wing‐Kee , Liu Wei , Smith Craig P.
Published: June 2021

Previous studies indicate that the kidney PT is involved in body Fe homeostasis: 1) Fe is filtered and reabsorbed by the kidney PT; 2) The divalent metal transporter DMT1 is expressed in lysosomes of PT cells where its expression is responsive to systemic Fe levels. Here we show by immunostaining that the iron exporter ferroportin‐1 (FPN‐1) is expressed in rat kidney cortex and localized to PT cells. FPN‐1 is also expressed in a cell line derived from the S1‐segment of rat PT at the mRNA and protein level. Fe over‐load with ferric ammonium citrate (FAC) or depletion with desferrioxamine (DF) do not change overall FPN‐1 protein expression in cultured PT cells, as evidenced by immunoblotting. However, immunofluorescence staining shows increased cell surface expression of FPN‐1 in FAC treated cells. This was confirmed using cell surface biotinylation and immunoprecipitation of FPN‐1. The results indicate that FPN‐1 is expressed intracellularly and in plasma membranes of kidney PT cells. Fe overload may induce redistribution of intracellular FPN‐1 to the plasma membrane, possibly to increase Fe exit from PT cells. The expression of Fe transport proteins, such as DMT1 and FPN‐1, that are regulated by Fe suggest that the kidney PT is involved in Fe homeostasis. Supported by DFG & Wellcome Trust.