bluuebunny/crossref_metadata_2025 huggingface dataset:
Age‐related changes in peripheral nociceptor function (659.2)
Authors: Sullivan Laura , Jamshidi Raehannah , Chavera Teresa , LoCoco Peter , Dahlhausen Christine , Berg Kelly , Clarke William
Published: June 2021

Effective and safe pain management in the elderly is a major medical challenge. Furthermore, little is known regarding the effect of aging on nociceptor (pain‐sensing neuron) responsiveness or the function of opioid receptors expressed on nociceptors. Here, we compared the effects of the sensitizing agent, bradykinin (BK), and delta and kappa opioid receptor (DOR, KOR) agonists on nociceptors in young (4‐months‐old) and aged (26‐months‐old) Fisher x Brown Norway rats. Behavioral responses to noxious heat, cold, or mechanical stimulation were determined following intraplantar injections of BK. For all three stimuli, aged rats had a greater allodynic response to BK. In primary cultures, activation of phospholipase C in nociceptors from aged rats was more sensitive to BK than those from young rats. In addition, both opioid agonists, SNC80 (DOR) and Salvinorin A (KOR), produced greater antinociceptive effects in aged rats compared to young rats. Similarly, opioid receptor‐mediated inhibition of adenylyl cyclase activity was greater in nociceptors from aged rats. Overall, our results indicate that aging enhances allodynic effects of the inflammatory mediator, BK, as well as increases antinociceptive effects of opioid agonists. We propose that peripherally‐restricted opioids may be a valuable analgesic strategy for treating pain in the elderly.

Grant Funding Source: Supported by T32DA031115, Baptist Health Foundation, Nathan Shock Center of Excellence AG013319
Mechanisms of long‐term regulation of peripheral kappa opioid receptor function by the antagonist, norBNI (659.4)
Authors: Jamshidi Raehannah , McGuire Blaine , Sullivan Laura , Chavera Teresa , Clarke William , Berg Kelly
Published: June 2021

KOR is known to regulate cellular signaling cascades including c‐Jun N‐terminal Kinase (JNK). We have found that norBNI, a prototypical KOR “antagonist”, has “agonist” activity towards JNK, leading to long‐term reduction in some aspects of KOR function in peripheral pain‐sensing neurons. Using a rodent behavioral model of nociception, we have found that KOR‐mediated peripheral antinociception is abolished 2 and 7 days following a single intraplantar injection of norBNI. This effect was blocked by pre‐injection of a selective JNK inhibitor, SP600125. Treatment of pain‐sensing neurons in culture with norBNI for 1h, followed by washing, eliminated KOR‐mediated inhibition of adenylyl cyclase, measured 24h later, which was blocked by SP600125. However, KOR‐mediated activation of extracellular signal‐regulated kinase (ERK) was unaffected by norBNI treatment.

Since JNK is a well‐known activator of transcription factors, ultimately leading to protein synthesis, we sought to determine if protein synthesis is required for the long‐term effects of norBNI. Prior treatment with the protein synthesis inhibitor, cycloheximide, eliminated effects of norBNI, suggesting that increased synthesis of an unknown protein leads to prolonged reduction in some KOR‐mediated responses. These data provide strong evidence that norBNI is a protean ligand which has powerful capacity to regulate peripheral KOR function.

Grant Funding Source: Supported by: DA024865, T32DA031115, TST128233/151325
Potential use of the nano‐compound fullerenol for the treatment of paraquat‐induced acute lung injury in rats (660.16)
Authors: Aires Rosária , Socarrás Teresa , Ladeira Marina , Guatimosim Silvia , Ladeira Luis , Campos Paula , Cortes Steyner , Lemos Virginia
Published: June 2021

The present work aimed at investigating the effect of fullerenol in the treatment of paraquat‐induced acute lung injury (ALI). Wistar rats were separated in control, ALI and ALI+fullerenol groups. In rats given paraquat there was increase of lipid peroxidation in lung, neutrophilia, accumulation of neutrophils in lung and bronchoalveolar lavage (BAL), associated with an increase in pulmonary levels of TNF‐α, IL‐1β, IL‐6 and CINC‐1, and serum levels of TNF‐α and IL‐6. Histological analysis showed inflammatory infiltrate, perivascular and intra‐alveolar edema, hemorrhage, vascular congestion and atelectasis. There was elevation of the basal production of nitric oxide (NO) and hyporesponsiveness of the aorta to vasoconstrictors. Treatment with fullerenol normalized lipid peroxidation, neutrophils number on BAL, lung and blood, the level of IL‐1β and CINC‐1 in lung, and TNF‐α and IL‐6 on serum. All histological parameters were normalized. Furthermore, treatment with fullerenol normalized the basal production of NO and vascular function. Finally, fullerenol increased the survival of animals with ALI. In conclusion, fullerenol combines antioxidative and antiinflammatory activities, reducing pulmonary and vascular alterations, and mortality induced by paraquat. Hence, fullerenol can be an effective and promising drug for the treatment of paraquat‐induced acute lung injury.

Grant Funding Source: Supported by FAPEMIG/PRONEX BioNC; CNPq and CAPES
Acute aspiration of graphene sheets evokes transient airway hyperreactivity to methacholine in mice (660.4)
Authors: Fedan Jeffrey , Thompson Janet , Sager Tina , Roberts Jenny , Fedan Jeffrey
Published: June 2021

Concern exists that the use of graphene sheets (GS) in composite materials might expose manufacturing workers to an inhalation hazard. Studies have shown that GS are cytotoxic in vitro (PC12 cells, fibroblasts) and in vivo in mice (lung granuloma), and Roberts et al. (2013) found non‐oxidized GS with larger lateral dimensions (蠅 5 μm) and a greater number of layers (~20) produced more lung inflammation up to 7 d after aspiration in mice when compared to smaller GS (<1 μm laterally, ~ 4 layers). The lung toxicity of various forms of GS has not been characterized completely. Here, we investigated the effects of GS on basal lung resistance (RL), basal dynamic compliance (CDyn), and reactivity to inhaled methacholine (MCh) aerosol. Mice were given a non‐oxidized GS (5 μm x 5 μm laterally, 7 nm thick equal to ~ 20 layers; 40 µg) suspended in dispersion medium (DM; Porter et al., 2008) or DM (control) via aspiration. RL and CDyn and reactivity to increasing concentrations of MCh aerosol were measured 4 h ‐ 2 mo after GS exposure. Basal RL was increased 4 h post‐exposure but at no other time; basal CDyn was unaffected at any time. Airway reactivity to MCh (as ΔRL) was increased at 4 h post‐exposure, and ΔCDyn responses were decreased. GS was essentially without effect on RL or CDyn at 1 d, 1 wk, 1 mo and 2 mo after administration. The results indicate that a single exposure to GS increases transiently lung resistance and reactivity to MCh.

Grant Funding Source: Supported by NIOSH
Wheel access in the prior 22 hours is sufficient to reduce d‐methamphetamine self‐administration in rats (661.5)
Authors: Taffe Michael , Aarde Shawn , Creehan Kevin , Vandewater Sophia
Published: June 2021

Initial rat studies appeared to show that an extended, ~6 week interval of activity wheel exposure was needed to reduce intravenous self‐administration of stimulant drugs. Yet a recent study found that ~22hrs of wheel access prior to sessions, without any extended prior exposure, was sufficient to reduce cocaine self‐administration in a between‐groups design. The present study sought to distinguish if the effects of ~22hr wheel access on methamphetamine (MA) self‐administration are an acute or lasting phenomenon using a within subjects design. Groups of male Sprague Dawley rats were trained to stably self‐administer MA (0.05 mg/gk/infusion, i.v.) and then subjected to wheel access/no access conditions when in the vivarium. Intake of MA was significantly lower on sessions following ~22 hrs of access to the wheel compared with sessions which followed ~22 hrs of no‐wheel housing. Effects of the wheel were observed within‐subject in an ABAB design as well as between groups. These data provide evidence that a suppressive effect of wheel activity that had previously been attributed to sustained, multi‐week wheel access is instead due to acute wheel activity in the day prior. Thus, brain plasticity mechanisms that require 6 weeks of chronic activity to produce are unlikely to be related to the suppressive effect of activity on drug self‐administration. This study also has important implications for using exercise as an adjunct to human drug cessation therapy because an extended interval of escalated activity may not be required. Likewise, consistent daily activity may be required for maximum effect.

Grant Funding Source: Supported by USPHS Grant DA024105 and DA024705
KK‐92A, a novel GABAB receptor positive modulator, attenuates the rewarding effects of nicotine in rats (661.9)
Authors: Li Xia , Kaczanowska Katarzyna , Sturchler Emmanuel , Cameron Michael , McDonald Patricia , Finn M. G. , Griffin Patrick , Markou Athina
Published: June 2021

GABAB receptors (GABABR) are thought to be one of the most promising targets for the treatment of drug dependence. GABABR positive allosteric modulators (PAMs) have been shown to exhibit similar effects as agonists in animal models of nicotine dependence with superior side‐effect profiles. This study involved the synthesis of new selective GABABR PAMs, and the assessment of their effects in animal models of nicotine dependence. New derivatives of BHF177, a potent GABABR PAM with efficacy in animal models of nicotine dependence, were synthesized. Nine active molecules were selected based on in vitro microsomal stability and efficacy in multiple cell‐based functional assays, including cAMP accumulation, Ca2+ mobilization, ERK activation and a cellular impedance‐based (CellKey) assay. Among these compounds, KK‐92A showed improved efficacy in the cAMP and Cellkey assays compared to BHF177, and had a similar in vivo drug metabolism and pharmacokinetic profile, thus, was selected for in vivo assessment. We found that KK‐92A decreased intravenous nicotine self‐administration under fixed‐ and progressive‐ratio schedules of reinforcement in rats. The results indicate that KK‐92A, a novel selective GABABR PAM, inhibited both the primary reinforcing and incentive motivational effects of nicotine, suggesting that GABABR PAMs may be useful antismoking medications.

Grant Funding Source: NIH 2U19DA026838
G protein contribution to complex signaling pathways downstream of the dopamine D2L receptor (662.10)
Authors: Brust Tarsis , Hayes Michael , Roman David , Watts Val
Published: June 2021

The dopamine D2 receptor (D2R) is a drug target that activates multiple signaling pathways via G protein‐dependent and independent mechanisms. Different ligands can activate these signaling pathways with distinct potencies and efficacies (i.e. biased ligands) which may provide a path for developing safer and more effective therapies. The present study examined several complex signaling pathways that may require multiple immediate effectors of the D2R. Specifically, we analyzed ERK phosphorylation, heterologous sensitization, AKT phosphorylation, and dynamic mass redistribution in response to a series of D2R ligands. The ligands tested included previously characterized D2R modulators that are used for the treatment of Parkinson’s disease and schizophrenia. The results were compared to previous studies examining Gαi/o, Gβγ, and β‐arrestin signaling in the same model system. Ligand bias was assessed using previously described methods as well as a new Weighted Efficacy Analysis (WEA). The WEA utilizes a straightforward equation incorporating two key pharmacological measures (i.e. pEC50 and relative efficacy) to calculate bias. Additional studies explored the requirement of both Gαi/o and Gβγ activation for these complex signaling pathways. Our results reveal unique bias profiles of several D2R ligands and provide insight regarding the effector requirements for complex signaling pathways of the D2R.

Grant Funding Source: Supported by MH60397 and BBRF.
Expression of olfactory GPCR signaling genes in the eye (662.15)
Authors: Slepak Vladlen , Pronin Alexey , Levay Konstantin , Velmeshev Dmitry , Faghihi Mohammad , Shestopalov Valery
Published: June 2021

Ocular surface is continuously exposed to particles, microorganisms and other stressors. To advance our understanding how the outer eye interacts with its environment, we asked which cellular receptors are expressed in the cornea, focusing on identification of G protein‐coupled receptors (GPCRs). Total RNA was isolated from the mouse cornea and subjected to next‐generation sequencing using Illumina platform. The data was analyzed by CuffLinks and TopHat software. Gene expression was confirmed by RT‐PCR. Cellular localization of transcripts was analyzed by in situ hybridization using RNAscope technology. The determined mouse cornea transcriptome contained more than 45 million reads representing over 10,000 gene transcripts. We identified more than 150 GPCR gene transcripts including more than 100 were putative olfactory receptors (Olfr’s). Our RT‐PCR analysis confirmed the presence of several Olfrs and related genes, for example, the G protein associated with olfaction, Galpha‐olf, and olfactory marker protein (OMP). In situ hybridization showed that mRNA for Olfr558 was present primarily in vessels of the eye, whereas OMP was found in epithelial cells of the cornea. Thus, our data show that several species of mRNA encoding putative olfactory receptors and related genes are expressed in the mouse cornea and other parts of the eye.

Grant Funding Source: Supported by NIH RO1EY018666 (VZS), R01EY017991 (VIS), P30 EY014801
Quercetin attenuates oxidative testicular damage in STZ‐induced diabetic rats (663.13)
Authors: Alkhamees O , Alroujayee A. , Alarifi N. , Alhusaini K. , Alsharari S. , Alrejaie S.
Published: June 2021

Quercetin (QR) has been shown to reduce oxidative stress in the long‐term treatment of streptozotocin(STZ)‐induced Diabetes mellitus (DM) in animal models. The present study examined the influence of QR on STZ‐induced oxidative stress in rat’s testicular cells. Male Wistar rats (n=36) were allocated into 6 groups: (1) control, (2) QR (25mg/kg), (3) QR (50mg/kg), (4) STZ (65mg/kg), (5) STZ+QR (25mg/kg) and (6) STZ+QR (50mg/kg) for 5 weeks. In testicular tissues, TBARS, T‐GSH, and NP‐SH levels were estimated. SOD, CAT and GST activities were also determined. In STZ group, TBARS levels were significantly increased while T‐GSH and NP‐SH levels were markedly reduced as compared to control rats. QR treatment to diabetic rats showed significant inhibition in increased TBARS levels and decreased T‐GSH and NP‐SH levels when compared to untreated diabetic rats. Enzymatic activities of SOD, CAT and GST were significantly inhibited in diabetic rats. QR treatment enhanced these activities compared to untreated diabetic rats. Histopathological evaluation revealed damage in testicular cells of diabetic rats and the treatment with QR showed protection. These findings suggest QR as a potentially beneficial agent to reduce testicular oxidative damage in diabetic rats, probably by its ability to attenuate free radicals generation and decrease lipid peroxidation.
Death effects of reveromycin A in chondrocytes and synoviocytes (663.14)
Authors: Svrcina Haley , Steinke Ellen , Davitt Kelsey , Sloane Emily , Williams Calli , Jones Dan
Published: June 2021

Osteoporosis is a prevalent disease with several current treatments. However, these treatments can have harmful long‐term side effects on individuals utilizing them. Reveromycin A (Rev A) is a natural compound that specifically causes apoptosis in osteoclasts but not osteoblasts. To further test whether Rev A can be a safe osteoporosis treatment without detrimental effects in the surrounding joint, the compound’s ability to induce apoptosis and necrosis in ATDC5 murine chondrocytes and HIG‐82 rabbit synoviocytes was measured. Inflammatory joint disease is known to accompany osteoporosis; consequently, Rev A‐induced cell death was characterized in each of these two cell lines under both physiological pH and a pH of 6.8 to mimic the pH of an inflamed joint. After treatment, morphology was assessed through phase contrast microscopy, apoptosis was measured through colorimetric caspase 3 activity assay, and necrosis was quantitated by use of a lactate dehydrogenase (LDH) release assay. Six hours of ten micromolar Rev A treatment did not significantly induce chondrocyte apoptosis, chondrocyte necrosis, or synoviocyte necrosis at either pH. Synoviocyte apoptosis was not induced by Rev A at physiological pH, but at a pH of 6.8, Rev A induced apoptosis compared to untreated by 4.9–fold (p<0.005). The results suggest that Rev A may have undesirable apoptotic effects in synoviocytes of the joint under acidic conditions.

Grant Funding Source: Supported by grants from the Indiana Academy of Science and the Hodson Summer Research Institute
Reveromycin A‐induced apoptosis in osteoclasts is accompanied by decreased NF kappa B levels (663.15)
Authors: Jones Dan , Mead Brittany , Morgan Heather , Tedeschi Laura , Sloan Chris , Lang Spenser , Hines Cory , Gragg Megan , Stofer Jonathan , Riemann Kaitlin , Derr Tyler , Heller Emily , Collins David , Landis Paul , Linna Nathan
Published: June 2021

Reveromycin A (Rev A), a small natural product isolated from Streptomyces bacteria, is a potential osteoporosis therapeutic in that it specifically induces apoptosis in osteoclasts but not osteoblasts. The purpose of the study presented here was to further elucidate the intracellular mechanisms of Rev A death effects in mature osteoclasts. RAW264.7 murine macrophages were differentiated in the presence of receptor activator of nuclear factor kappa B ligand (RANKL) and stained for tartrate‐resistant acid phosphatase to confirm their osteoclast character. These osteoclasts were treated with ten micromolar Rev A for 2, 4, and 6 hours at a pH of 5.5. Peak apoptosis induction occurred at 4 hours (5.1‐fold, p<0.05) as measured by caspase 3 activity. Lactate dehydrogenase release assay revealed no significant Rev A‐induced necrosis. Western blot analysis of cytoplasmic extracts demonstrated activation of caspase 9 and a mild decrease of deamidated Bcl‐XL. In both cytoplasmic and nuclear extracts, NF kappa B levels increased on differentiation with RANKL but then decreased at 2 hours of treatment (2.4‐fold for cytoplasmic and 1.7‐fold for nuclear levels). Rev A‐induced apoptosis in osteoclasts is not accompanied by necrosis, suggesting that Rev A would likely have limited effects on neighboring bone cell types, and Rev A decreases NF kappa B, a factor needed for differentiation and survival of the mature osteoclast.

Grant Funding Source: Supported by an Indiana Academy of Science grant and a Hodson Summer Research Institute grant
The potent inhibitor of poly(ADP‐ribose) polymerase‐1, 5‐aminoisoquinolinone, promotes autophagy and reduces neural tissue damage and locomotor impairment after spinal cord injury in mice (663.4)
Authors: Esposito Emanuela , Paterniti Irene , Impellizzeri Daniela , Campolo Michela , Cordaro Marika , Siracusa Rosalba , Bruschetta Giuseppe , Cuzzocrea Salvatore
Published: June 2021

Evidence indicate that Poly (ADP‐ribose) Polymerase (PARP) plays an important role in cerebral ischemia/reperfusion, stroke and neurotrauma. Overactivation of PARP consumes NAD+ and ATP culminating in cell dysfunction and necrosis. PARP has also been shown to associate with and regulate the function of several transcription factors. PARP is involved in the up‐regulation of numerous pro‐inflammatory genes that play a pathogenetic role in the later stage of neurotrauma. The aim of this study was to investigate the effects of a potent PARP‐1 inhibitor, 5‐aminoisoquinolinone (5‐AIQ), on secondary inflammatory damage associated with an experimental model of spinal cord injury (SCI). SCI was induced in mice through a spinal cord compression by the application of vascular clips (force of 24 g) to the dura via a four‐level T5 to T8 laminectomy, and 5‐AIQ (3 mg/kg, intraperitoneally, 1 and 6 hours after SCI) was injected into mice and once daily thereafter for 10 days. Our results demonstrated that the administration of 5‐AIQ significantly decreased the phosphorylation of the p70S6K protein and led to higher expression levels of LC3 and Beclin 1 in the injured spinal cord. In addition, neuronal loss and cell death in the injured spinal cord were significantly reduced in the 5‐AIQ‐treated mice compared to the vehicle‐treated mice. Furthermore, the 5‐AIQ‐treated mice showed significantly higher locomotor function in Basso Mouse Scale scores than did the vehicle‐treated mice. These results indicate that 5‐AIQ promoted autophagy, and reduced neural tissue damage and locomotor impairment after SCI.
Role of adenosine A2A receptor in ischemia reperfusion injury: signaling to extracellular signal‐regulated protein kinase (663.6)
Authors: Atef Reham , Agha Azza , Nassar Noha
Published: June 2021

Adenosine is a potent biological mediator, the concentration of which increases dramatically following brain ischemia. Out of the four known adenosine receptor subtypes (A1, A2A, A2B and A3), the A2A is of critical importance in stroke. Although, both A2A agonists and antagonists are proven protective during ischemia reperfusion (IR) injury, however, ,the mechanisms by which A2A receptors are noxious during IR remain elusive. The objective of the present study was to investigate the role of central A2A receptor in modulating cellular as well as behavioral responses to IR injury and the possible involvement of ERK signalling pathway. Hence, we hereby report that bilateral carotid occlusion for 45 min followed by 24h reperfusion period resulted in a significant memory impairment, motor incoordination as well as increased locomotor activity that were not affected by unilateral intrahippocampal injection of a selective A2A agonist CGS21680, but conversely mitigated by the selective A2A antagonist SCH58261. On the cellular level, IR increased the pERK, NFkB, cytochrome c, BDNF, Nrf2 and TNF‐α content in the hippocampus. With the exception of Nrf2 level, treatment with SCH58261 significantly reversed these effects, however, CGS21680 treatment resulted in a non‐significant change from IR insult. Accordingly these results suggest a possible protective role of the central A2A receptor blockade in IR injury with a prospective involvement of p42/44 pathway.
MAGI1 signaling in cerebral cavernous malformations (664.11)
Authors: Sanchez Atlee , Stockton Rebecca
Published: June 2021

Cerebral Cavernous Malformations (CCMs) are vascular brain lesions affecting 0.5% of humans. Late stage lesions are clusters of grossly dilated capillaries lacking smooth muscle cell or pericyte support, and can cause hemorrhagic stroke. No treatment exists other than surgical removal of symptomatic lesions. CCMs occur sporadically through spontaneous mutations, or by familial autosomal dominant transmission of mutations in three genes : KRIT1, CCM2 or CCM3 (PDCD10). Our lab’s previous work showed that KRIT1 and CCM2 normally regulate endothelial permeability through regulation of RhoA and ROCK1/2. We now used proteomics and CHO cell co‐immunoprecipitations to identify a signalosome consisting of a unique brain microvascular endothelial cell‐specific splice variant of Rap1‐interacting junctional protein MAGI, “MAGI1C‐beta2”, that binds to KRIT1 and CCM2 through a PDZ domain to mediate translocation of two CCM proteins to endothelial junctions, and is required to maintain barrier function. We further identified a MAGI‐1 interacting protein , AMOT (angiomotin) as a convergence point for all three CCM protein signaling pathways, by showing that PDCD10 interacts directly with AMOT at junctional complex , while KRIT1 and CCM2 are localized there through MAGI1C‐beta2. Further, this intact CCM1‐2‐3/AMOT complex suppresses RhoA activation, providing a mechanistic explanation for some aspects of CCM lesion development.

Grant Funding Source: NIH
Differential expression of phosphodiesterase 1‐5 in skeletal muscle microvessels: vessel types, sex, and reproductive maturation (664.4)
Authors: Wang Jianjie , Huxley Virginia
Published: June 2021

Intracellular cyclic nucleotides (cAMP and cGMP) are critical second messengers to mediate various endogenous and exogenous factor‐induced vasomotion, angiogenesis, and vascular barrier function. Phosphodiesterases (PDEs), sole intracellular enzymes, hydrolyze cyclic nucleotides, and modulate cAMP‐ and cGMP‐dependent signaling events and corresponding functions. The objective of the study was to determine PDE1‐5 transcripts in skeletal muscle arterioles and venules and the role of sex and reproductive maturation in PDE1‐5 expression. Abdominal wall arterioles and venules were isolated from 4 groups of rats, adult males and females, juvenile males and females. Total RNA was extracted from arterioles and venules and reversely transcribed into first cDNA which was amplified by quantitative real‐time polymerase chain reaction using specific Taqman primers (real‐time RT‐PCR). Expression levels of PDE1‐5 were PDE5>PDE4>PDE1蠅PDE2>PDE3 in both arterioles and venules of 4 group rats (n=3‐7/gene/group, P<0.05) except PDE expression in venules of adult females was PDE4 greater than PDE5 (PDE4>PDE5>PDE1蠅PDE2>PDE3). In arterioles, greater expression of PDE3A and PDE3B were observed in adult males compared to adult females and juvenile males. In venules, PDE3BmRNA level was greater in adults than juveniles regardless of gender. In general, the findings demonstrate that PDE5, cGMP‐specific hydrolyzing enzyme, is predominantly expressed and the second most expression form is PDE4, cAMP‐specific hydrolyzing enzyme, in both arterioles and venules of 4 groups of rats except adult female venules (PDE4>PDE5). PDE3A and PDE3B exhibit sex‐specific and reproductive maturation‐specific difference.

Grant Funding Source: NIH RO1HL078816
Pericyte‐endothelial junctional communication and calcium coupling (664.8)
Authors: Osborne Mathew , Bearden Shawn
Published: June 2021

Pericytes (PCs) initiate and maintain the blood‐brain barrier phenotype of cerebral endothelium. Despite many studies referencing pericyte‐endothelial junctional (PEJ) communication, little is known of this signaling pathway. The purpose of this study was to determine if human brain endothelial cells (ECs) and PCs are functionally coupled. ECs and PCs were loaded with the calcium indicator fluo‐4 AM (Invitrogen) and treated with different reagents of varying concentrations (10‐4 to 10‐8 M). ECs and PCs both responded to acetylcholine, caffeine, and bradykinin (BK). Neither cell type responded to phenylephrine. Only PCs were stimulated by low concentrations (10‐7 M) of angiotensin 2. The magnitudes of responses to BK were higher in PCs than ECs at all concentrations tested (p< 0.05). To determine whether the PEJ mediates calcium coupling between cells, respective cell types were cultured on opposite sides of membrane inserts and loaded with fluo‐4 AM (0.4 µm pores; Transwell, Corning). BK concentrations ranging from 10‐5 to 10‐7 M elicited robust EC responses when applied to the PC side but were absent when PCs were removed. Immuno‐positive labeling of connexin 45 (Cx45), but not connexins 37, 40, or 43, in membrane pores is consistent with the presence of Cx45‐based gap junctional coupling at the PEJ. Human brain ECs and PCs form functional cell‐cell junctions, PEJs, which facilitate intercellular calcium signaling. This pathway may play a role in the regulation of blood‐brain barrier properties, possibly through Cx45‐dependent signaling.

Grant Funding Source: NIH HL10654801
Obesity in aging exacerbates blood brain barrier disruption, neuroinflammation and oxidative stress in the mouse hippocampus: effects on expression of genes involved in beta‐amyloid generation and Alzheimer’s disease (665.1)
Authors: Tucsek Zsuzsanna , Toth Peter , Sosnowska Danuta , Gautam Tripti , Koller Akos , Sonntag William , Ungvari Zoltan , Csiszar Anna
Published: June 2021

There is growing evidence that obesity has deleterious effects on the brain and cognitive function in the elderly. However, the specific mechanisms through which aging and obesity interact to promote cognitive decline remain unclear. To test the hypothesis that aging exacerbates obesityinduced cerebromicrovascular damage and neuroinflammation, we compared young (7 mo) and aged (24 mo) high fat diet‐fed obese C57BL/6 mice. Aging exacerbated obesity‐induced systemic inflammation and BBB disruption, as indicated by the increased circulating levels of pro‐inflammatory cytokines and increased presence of extravasated IgG in the hippocampus, respectively. Obesityinduced BBB damage was associated with microglia activation, up‐regulation of activating FcγRs and pro‐inflammatory cytokines and increased oxidative stress. Treatment of cultured primary microglia with sera derived from aged obese mice resulted in significantly more pronounced microglia activation and oxidative stress, as compared to treatment with young sera. Serum‐induced activation and oxidative stress were also exacerbated in primary microglia derived from aged animals. Hippocampal expression of genes involved in regulation of the cellular APP‐dependent signaling pathways, beta‐amyloid generation and the pathogenesis of tauopathy were largely unaffected by obesity in aged mice. Collectively, obesity in aging is associated with a heightened state of systemic inflammation, which exacerbates BBB disruption. The resulting neuroinflammation and oxidative stress in the mouse hippocampus likely contribute to the significant cognitive decline observed in aged obese animals.

Grant Funding Source: supported by the American Heart Association and the American Federation for Aging Research
Aging exacerbates obesity‐induced impairment of neurovascular coupling and cerebromicrovascular rarefaction: implications for the pathomechanism of vascular cognitive impairment (665.2)
Authors: Tucsek Zsuzsanna , Toth Peter , Sosnowska Danuta , Gautam Tripti , Koller Akos , Ballabh Praveen , Sonntag William , Ungvari Zoltan , Csiszar Anna
Published: June 2021

Obesity has deleterious effects on the brain and cognitive function in the elderly, however the mechanisms through which aging and obesity interact to promote cognitive decline remain unclear. We hypothesized that aging exacerbates obesity‐induced disruption of both functional and structural integrity of the cerebral microcirculation, contributing to a significant decline in higher brain function. To test our hypothesis we compared neurovascular coupling measured by CBF response above the barrel cortex to whisker stimulation, and cerebral capillary density by CD 31‐labeling of cerebral vessels in young (7 mo) and aged (24 mo) high fat diet‐fed obese C57BL/6 mice. High fat diet in young mice severely impaired CBF response to whisker stimulation (~50 % decrease), which was significantly exacerbated by aging. The high‐fat diet‐induced decrease in neurovascular coupling was significantly improved in both young and aged mice in the present of the NADPH oxidase inhibitor apocynin. Aged animals exhibited a significant decrease in cerebral capillary density, and the aging‐induced rarefaction was significantly exacerbated by high‐fat diet. These functional and structural pathological changes were associated with a decreased hippocampal‐dependent cognitive function in aged obese mice.

In conclusion, aging exacerbates high fat diet‐induced decrease in neurovascular coupling via increased NADPH‐oxidase derived ROS production, and significantly enhances capillary rarefaction in the brain probably contributing to cognitive decline observed in aged obese animals.

Grant Funding Source: Supported by the Oklahoma Center for the Advancement of Science and Technology
KCNQ and ERG channels control the rate of diastolic depolarization and electrical pacemaking frequency in lymphatic muscle (666.3)
Authors: Gui Peichun , Li Min , Hill Michael , Davis Michael
Published: June 2021

The spontaneous contractions of lymphatic muscle cells are critical for propulsion of lymph. We investigated the ionic basis of pacemaking in the muscle layer of pressurized rat mesentery and mouse popliteal lymphatics using sharp electrodes and patch clamp methods. At very low pressures (<0.5 cmH2O) membrane potential was stable at ~ ‐42 mV, with infrequent action potentials (APs). At higher pressures APs fired at regular intervals, interspersed with slow diastolic depolarizations (DD) to a threshold of ~ ‐35 mV. Each AP consisted of a single 200‐ms spike, peaking at ~ +5 mV, followed by a 1.5‐sec plateau and after‐hyperpolarization to ~ ‐45 mV. Graded increases in pressure to 5 cmH2O led to graded increases in DD slope and AP frequency. The KCNQ inhibitors XE991 and linopirdine (1‐10 μM) increased DD slope without significantly changing AP height or duration. The selective Kv7.1 activator flupirtine (10 μM) decreased DD slope and AP frequency. In contrast, the ERG‐1 inhibitor E‐4031 (2‐6 μM) had little effect on frequency but prolonged AP duration due to multiple spikes with each AP. Based on PCR, the predominant KCNQ channel isoform was Kv7.1, with lesser expression of Kv7.4 (but not 7.2, 3 or 5). ERG‐1a and 1b (but not ERG‐2 or 3) channels were detected. We conclude that these K+ channel isoforms, which are typical of cardiac nodal cells, are important regulators of electrical pacemaking in lymphatic muscle.

Grant Funding Source: NIH‐HL089784
Angiotensin II and ischemic preconditioning synergize to improve mitochondrial function while showing additive effects on ventricular post‐ischemic recovery (667.2)
Authors: Nuñez Rebeca , Castro Miriam , Javadov Sabzali , Escobales Nelson
Published: June 2021

Recent studies indicate that the cardioprotective effects of ischemic preconditioning (IPC) against sustained ischemia/reperfusion (IR) can be replicated by angiotensin II (Ang II). However, it is not clear whether IPC and Ang II‐induced preconditioning (APC) act through similar mechanisms or synergize to enhance cardioprotection. In this study, Langendorff‐perfused rat hearts were subjected to IPC, APC or their combination (IPC/APC) followed by IR. IPC and less potently APC, induced cardioprotection as determined by significant increases in the percent recovery of the left ventricular developed‐pressure (LVDP), the first derivative of developed pressure (+dP/dt), and the rate pressure product (RPP) compared to controls. In absolute values, however, the post‐ischemic increase in +dP/dt and RPP were significantly higher for IPC/APC compared to IPC or APC. These effects were associated with normalization of ischemic contracture and lowered LDH release. IPC and/or APC also reduced infarct size and improved mitochondrial respiration and reduced mPTP opening. Notably, IPC/APC markedly increase the respiratory control index of mitochondria compared to IPC or APC. These effects were abrogated by pretreatment with losartan or chelerythrine. Therefore, our results demonstrate that IPC in combination with APC exerts additional cardioprotective effects on post‐ischemic recovery of cardiac function that are associated with improved mitochondrial respiration and attenuation of mitochondria‐mediated cell death.

Grant Funding Source: Supported by NIH Grant SCIHL118669‐01A1 and NIH RCMI Program Grant G12RR‐03051
The preterm pig as a model for acute lung disease after preterm birth (668.3)
Authors: van der Merwe Marie , Ewing Holly , Caminita Frank , Buddington Randy
Published: June 2021

Respiratory distresssyndrome and the more severe sequel bronchopulmonary dysplasia remain the leading causes of morbidity, mortality, and lifelong disability after preterm birth, with inadequate surfactant secretion a key contributor. We present the preterm pig as a translational model for lung disease after preterm birth. Preterm pigs were delivered by caesarian section at gestation days (GD) 98, 100, 102,and 104 (comparable to human fetuses delivered at 25, 28, 30, and 32 wks) and were provided intensive care following newborn neonatal intensive care procedures, including mechanical ventilation (MV) or supplemental oxygen and nutritional support. Expression of Surfactant Protein B (SP‐B),a surfactant‐associated protein absolutely required for postnatal lung function, was determined by western analysis using lung tissue harvested 24 h after delivery or earlier for pigs that became moribund. Pigs delivered at GD 98 and 100, had low SP‐B and MV is required for survival. SP‐B increases with gestational age. Moreover, provision of MV to preterm piglets delivered at GD 102 causes a dramatic increase in SP‐B expression and suggests MV can induce SP‐B secretion and reduce dependency on surfactant administration.Based on SP‐B expression, lung histology and pathology, and the responses to MV, the preterm pig is a translational large animal model for studying acute lung disease after preterm birth and for preclinical trials to evaluate interventions and improve MV protocols. The large litters of pigs reduce the numbers of pregnant animals needed, the attributes of the preterm pig allow investigators to refine experimental approaches, and the preterm pig replaces the baboon as a large animal model to study acute lung disease and injury after preterm birth.
NFKB and CCR2 gene expression reflect long‐term but not acute inflammatory activation of peripheral blood mononuclear cells in patients with type 2 diabetes (669.3)
Authors: Horvath Peter , Radom‐Aizik Shlomit , Oliver Stacy , Zaldivar Frank , Galassetti Pietro
Published: June 2021

In type 2 diabetes (T2D) cardiovascular complications are linked to chronic inflammation, including activated granulocytes, Gc, and monocytes, Mc, present in atherosclerotic lesions. In T2D, hyperglycemia and hyperlipidemia are common, and can acutely activate Gc/Mc surface markers in healthy, obese, and T2D subjects. T2D also displays marked variability of inflammatory status, possibly reflecting long‐term modulation (often involving gene expression). We therefore compared, during 4‐h i.v. infusions of glucose, lipids, or glucose +lipids, acute leukocyte surface marker activation and peripheral blood mononuclear cell (PBMC’s) expression of the key inflammatory genes NFKB and CCR2 (by RT‐PCR) in 5 healthy, 5 obese, and 5 T2D subjects. Unlike cell surface markers, NFKB and CCR2 expression was not affected by acute plasma metabolite changes in any group, but levels of gene expression paralleled overall systemic inflammatory activation, which in T2D varied considerably within subjects across study visits, likely reflecting long‐term inflammatory modulation. Our data suggests a two‐tiered inflammatory mechanisms in T2D, with acute metabolic changes immediately reflected on surface marker expression, and more prolonged stimuli affecting levels of leukocyte inflammatory gene expression.

Grant Funding Source: Supported by NIH Grants P01HD048721 and UL1RR031985
Role of neutrophils in pulmonary vaso‐occlusion during sickle cell disease acute chest syndrome (669.6)
Authors: Bennewitz Margaret , Gladwin Mark , Sundd Prithu
Published: June 2021

Sickle cell disease (SCD) is a hematological disorder that leads to sickling of red blood cells within the microcirculation. The sickle red blood cells (sRBCs) are not only less deformable, but also express α4β1, ICAM‐4, Sialyl Lewis X and glycolipids not expressed on non SCD‐RBCs. These rigid sRBCs can get sequestered in the microcirculation to cause painful vaso‐occlusive crisis (VOC) in SCD patients. In the lung, VOC can contribute to acute chest syndrome, which is a form of acute lung injury. Several factors including the pro‐adhesive sRBCs, the large marginated pool of neutrophils, and the hyper‐inflammatory state present in SCD can contribute to pulmonary VOC. Although neutrophil‐RBC‐endothelium interactions are known to play a role in systemic VOC, the cellular mechanism that drives pulmonary VOC remains unknown. Intravital study of the lung microcirculation of live BERK or Townes SCD mice is conducted using two‐photon microscopy. Prior to observation, mice are challenged with lipopolysaccharide or PBS to cause lung inflammation and administered (iv) with fluorescent anti‐mouse Gr‐1 and Ter‐119 antibodies to label circulating neutrophils and RBCs, respectively. Vaso‐occlusion is defined as cellular aggregation and stasis of blood flow within the lumen of pulmonary capillaries. Preliminary results reveal that circulating macro‐aggregates of neutrophils and RBCs may play a role in pulmonary VOC.

Grant Funding Source: Supported by AHA, VMI startup account, and NIH Pulmonary T32 training grant
Strategical steering of the immune system: induction of leukocyte vascular chaperoning increases functional blood flow responses in ischemic muscle (670.6)
Authors: Vagesjo Evelina , Christoffersson Gustaf , Korsgren Olle , Essand Magnus , Holm Lena , Phillipson Mia
Published: June 2021

BACKGROUND: The role of leukocytes in angiogenesis is emerging. This study investigates leukocytes recruited to ischemic muscle and if vascular repair can be accelerated by induction of local chemokine production.

METHODS: Plasmids encoding CCL2 or CXCL12 were expressed in the cremaster muscle or in ischemic hindlimb muscle in wild‐type, CX3CR1‐GFP and RAGE‐/‐ mice. Functional tissue perfusion was measured during heat‐induced hyperemia in the hindlimb using Laser Doppler Flowmetry or in foot pads using Laser Speckle Contrast Analysis. The role of macrophages was challenged by clodronate liposome depletion. Vascular function, leukocyte phenotypes and position were studied in single vessels of ischemic muscle using intravital confocal microscopy. Effects of chemokine overexpression in the injured muscle on leukocyte subsets, perfused vessels and vascular densities were quantified.

RESULTS: Plasmid expression peaked at day 2 and was detected for 4 weeks following injection. Ischemic limbs expressing pCXCL12 showed improved heat‐induced hyperemia response and higher density of perfused capillaries 3 and 7 days post ischemia induction. pCXCL12‐expressing ischemic muscles contained increased number of macrophages (CX3CR1+F4/80+), which were polarized towards the pro‐angiogenic phenotype (F4/80+MRC1+) and found to chaperone perfused vessels. The importance of this observation was demonstrated in mice depleted of macrophages, in which functional blood flow response was not improved by pCXCL12. The blood flow effect of pCXCL12 was blunted in RAGE‐/‐ mice despite increased M2 macrophage density, indicating downstream RAGE involvement. Overexpression of pCCL2 did not expand the leukocyte population or improve vascular functional blood flow in this system.

CONCLUSION: Overexpression of pCXCL12 but not pCCL2 in ischemic muscle accelerates restoration of functional blood flow responses by retaining macrophages chaperoning the vasculature.
Role of neutrophils and β2 integrins in platelet extravasation in corneal limbal venules following epithelial wound injury (671.10)
Authors: Li Zhijie , Smith C , Gagen Debjani , Lam Fong , Burns Alan , Rumbaut Rolando
Published: June 2021

Corneal epithelial wound injury in the mouse results in an acute inflammatory response in the surrounding peripheral limbal microvessels. We showed previously that in wild‐type mice neutrophils (PMNs) and platelets accumulate at the inflamed limbus in a co‐dependent manner and approximately 50% of platelets are evident in the extravascular space; the mechanisms by which platelets extravasate are unclear. To determine the role of PMNs and β2 integrins on platelet extravasation, we quantified platelet recruitment in corneal vessels in mice after antibody‐induced depletion of neutrophils (anti‐Ly6G); control mice received isotype control antibody. We also compared extravascular platelet numbers in wild‐type mice and mice deficient in CD18. Anti‐Ly6G antibodies decreased total platelet numbers in corneal limbal vessels 24 hours after epithelial injury (from 141 ± 15 to 66 ± 16 platelets per field, p<0.05) and reduced the relative proportion of extravascular platelets (from 56 ± 5 % to 9 ± 1%, p< 0.001). Similarly, mice deficient in CD18 had a substantial decrease in extravascular platelets following corneal injury. Platelet‐PMN contact was evident in the intravascular and extravascular space by immunofluorescence and electron microscopy. These studies demonstrate that PMNs and β2 integrins contribute to platelet extravasation from corneal limbal vessels following epithelial wound injury.

Grant Funding Source: Supported by R01 HL‐ 116524, and a Merit Review Grant from The Department of Veterans Affairs
A mouse model to assess neointimal formation following endothelial denudation and bioresorbable stent implantation (671.12)
Authors: Nehzati Ashkon , Davalian Dariush , Magana Jesus , Consigny Paul , Cardinal Trevor
Published: June 2021

Restenosis is a common response to bare‐metal stent implantation occurring in 16‐44% of patients. Drug‐eluting stents can reduce restenosis, but late‐stent thrombosis occurs in 0.4‐0.6% of patients following cessation of anti‐platelet therapy. Bioresorbable stents may limit both restenosis and late thrombosis. The goal of this work is to develop a mouse model that can be used to assess neointimal formation in response to injury and/or bioresorbable stent implantation.

The endothelium was denuded in the abdominal aorta of C57Bl/6 mice using a nitinol device inserted into the femoral artery and confirmed using Evans Blue staining and histopathology. Bioresorbable stent implantation was performed by placing a stent (polymeric coil) in a PE tube, and advancing it into the abdominal aorta. Using a wire advancer, the stent was placed while withdrawing the tube simultaneously.

Staining and histopathology revealed endothelial denudation of the abdominal aorta between the renal arteries and iliac bifurcation during acute procedures and increases in wall thickness and cellularity over 28 days. Direct visualization revealed that the polymeric stent can be delivered to area of endothelial denudation with no migration or distal embolization.

In summary, we created a mouse model to assess neointimal formation following endothelial denudation and/or bioresorbable stent deployment. In the future, this model can be used to characterize gene expression and assess specific molecular pathways that contribute to restenosis and thrombosis.
Increased oxidative stress enhances endocannabinoid tone (671.13)
Authors: Matthews Anberitha , Borazjani Abdolsamad , Wang Ran , Ross Matthew
Published: June 2021

Cardiovascular disease (CVD) has been characterized as a chronic inflammatory disease that has become prevalent in industrialized societies. NADPH oxidase contributes to atherosclerosis through the activation of macrophages leading to the internalization of oxidized low‐density lipoproteins (oxLDL). Chronic inflammation is caused in part by monocytes invading the vasculature leading to the formation of lipid‐laden macrophage foam cells with increased flux of oxygen/nitrogen radicals and subsequent chemical modification of extracellular LDL. Endogenous cannabinoids (eCB), such as 2‐arachidonoylglycerol (2‐AG), may be a link between oxidative stress and atherosclerosis. We hypothesize that 2‐AG biosynthesis is enhanced following CD36 ligation by oxLDL and subsequent activation of diacylglycerol lipase β (DAGLβ), the key biosynthetic enzyme of 2‐AG, via upregulated NADPH oxidase activity. Treatment of murine J774 macrophages and human THP1 macrophages/monocytes with either extracellular xanthine/xanthine oxidase or phorbol 12‐myristate 13‐acetate (PMA) caused an increase in superoxide (O2•−) levels and enhanced 2‐AG biosynthesis (2.3‐fold and 2.8‐fold, respectively) compared to vehicle controls. These treatments were not cytotoxic. These data suggest a significant positive correlation between oxygen radical flux and 2‐AG biosynthesis in macrophages. Increased 2‐AG biosynthesis may be an adaptive response to elevated oxidative stress because of antioxidant and anti‐inflammatory actions associated with this bioactive lipid. Therefore, the pathogenesis of atherosclerosis within the vessel wall intima may be reduced by enhancing eCB tone.

Grant Funding Source: Supported in part by NIH R15ES015348‐02