bluuebunny/crossref_metadata_2025 huggingface dataset:
Biocompatibility Assessment of Green Synthesized Cu2O Decorated Graphene Nanosheets for Therapeutic Applications
Authors: Alshatwi Ali A , Athinarayanan Jegan , Periasamy Vaiyapuri Subbarayan , Lei David KY
Published: November 2023

Nanotechnology offers incredible prospective for variety of future applications; specifically, several kind of nanostructures have been broadly applied for biomedical applications owing to their unique physical, chemical and biological properties. Currently, graphene‐based nanohybrids have received a considerable in diagnosis and therapeutic applications due to their tremendous aqueous processability, easy to cellular internalization, biocompatibility and facile surface functionalizability. In this present study, a facile synthetic route to fabricate Cu2O/graphene nanocomposites (Cu2O/G) was developed using green chemistry approach. The copper ions anchored surface of graphene oxide to Cu(OH)2/graphene oxide was prepared using NaOH. Subsequently, Cu(OH)2/graphene oxide was reduced to Cu2O/G using date palm fruit syrup. This Cu2O/G was analyzed adopting field emission scanning electron microscopy, Fourier transform infrared spectroscopy, X‐ray diffraction, and energy dispersive X‐ray spectroscopy. From a typical FESEM images, it is observed that Cu2O nanoparticles with diameters of about 50–70 nm were anchored on the wrinkled G sheets. The XRD pattern of Cu2O/G nanocomposites diffraction peaks can be exclusively indexed to the cubic phase Cu2O on G sheets. The Cu2O/G nanosheets show biocompatibility in human mesenchymal stem cells (hMSCs) up to 100 μg/mL. There is no major changes were observed in cellular and nuclear morphology of nanocomposite treated hMSCs. The Cu2O/G exhibits biocompatibility, and it can be a promising material for biomedical applications.

Support or Funding Information

We gratefully acknowledge the financial support of the Deanship of Scientific Research, King Saud University, Saudi Arabia (Grant No.: RGP‐VPP‐276)
The Role of PAK1 in a Gut Manipulation Model of Ileus
Authors: Uray Karen , Bhattarai Deepa
Published: November 2023

Feeding intolerance is prevalent in surgical, critically ill, and trauma patients leading to prolonged hospital and intensive care unit stays, increased infectious and thromboembolic complications, and increased patient care costs. Ileus is a slowing of intestinal motility which contributes to feeding intolerance. At present there are no effective pharmacological treatments for preventing or treating feeding ileus. In previous studies, we have shown that upregulation of PAK1 activity inhibited intestinal contractility in an intestinal edema model of ileus. The purpose of this study was to determine if PAK1 also contributed to decreased intestinal contractility in a gut manipulation model of ileus. We hypothesized that, after gut manipulation, increased inflammatory mediators cause PAK1 activation leading to decreased intestinal contractility. We utilized both mouse and rat models. Ileus was induced by gently rolling the entire length of the small intestine between two cotton tipped applicators three times. Contractile activity was measured ex vivo in an organ bath system. PAK1 levels and Raf phosphorylation were measured by western blotting. In both mouse and rat models, we found that PAK1 expression was increased after gut manipulation. Inhibition of PAK1, as well as PAK1 genetic ablation, attenuated the decreased contractility induced by gut manipulation. PAK1 inhibition also improved response to carbachol in mice after gut manipulation. Pretreatment with a Rho kinase inhibitor blocked the effects of PAK1 inhibitors on contractile activity. CXCL1 and LIX, both CXCR2 ligands, were significantly upregulated within 1 hour after gut manipulation in intestinal smooth muscle. We found that CXCL1 can increase Raf1 phosphorylation, a known PAK1 target, and decrease MLC phosphorylation in primary human intestinal smooth muscle cells. CXCL1 also reduced the response to carbachol in rats. Taken together, these data suggest that PAK1 mediates the inhibitory effects of several inflammatory cytokines on intestinal contractility after gut manipulation. PAK1 is a potential drug target for the treatment of ileus.
Defining the Regulatory Role of Lipid Raft Microdomains During T cell Receptor Activation
Authors: Osigwe Chinweoke Crystal , Levine Alan D
Published: November 2023

We have previously concluded that the heterogeneity of the lipid composition of the plasma membrane plays a critical role in the T cell receptor (TCR) activation signaling pathway. Our lab has presented that highly‐ordered, densely packed lipids in raft‐like microdomains are required for the activation of TCR complex. In particular, we have also shown that the early, enzymatic events associated with TCR activation are dependent on the lipid raft micro‐domains. While our results support the importance of these lipid raft micro‐domains in TCR signaling, no evidence is available to describe how these rafts are assembled and organized within the plasma membrane. The objective of our study is to define the three dimensional changes on the plasma membrane during TCR signaling pathway from the perspective of lipid rafts. The following techniques have been employed to investigate this objective. From whole blood, peripheral mononuclear cells (PBMCs) were isolated by Ficol‐hypaque density separation. Purified T cell lymphoblast were expanded from PBMC culture by a seven day feeding with interleukin‐2. Both purified T‐cell lymphoblast and Jurkat T cells were activated by cross‐linking the TCR with anti‐CD3/anti‐CD28 co‐stimulatory antibodies. Propagation of the signal was analyzed through immunoblot analysis through a phospho‐ERK, phospho‐LAT, and phospho‐Lck response. Lipid raft composition and structure will be probed with fluorescently labelled phospholipids that selectively associate with or exclude distinct regions of the plasma membrane, thus labeling either a raft or non‐raft domain. Localization and assembly of these fluorescently labelled phospholipids, balanced against the localization of protein components of the TCR signaling complex using fluorescently tagged antibodies, will be analyzed with confocal and deconvolution microscopy. The study's biological implication will address how TCR engagement within the context of a lipid raft may lead to an immune response or immune tolerance.

Support or Funding Information

American Physiological Society STRIDE Fellowship
Modulation of Lemur Tyrosine Kinase 2 (LMTK2) Expression by TPA Response Elements
Authors: Dey Isha A , Bradbury Neil A.
Published: November 2023

Lemur Tyrosine Kinase 2 (LMTK2), a membrane anchored Serine/Threonine kinase, has been shown to be involved in a number of diseases, including cystic fibrosis and prostate cancer, as well as neuronal development and intracellular endocytic trafficking. Our previous studies have shown that LMTK2 is downregulated in prostate cancer, and that modulation of LMTK2 affects CFTR‐mediated chloride conductance at the plasma membrane of polarized epithelial cells. Moreover, CFTR, the Cl− selective anion channel mutated in cystic fibrosis has been found to a substrate for LMTK2 kinase activity. Based on these observations, we propose that LMTK2 to be a novel therapeutic target for both cystic fibrosis and prostate cancer. However, the precise mechanism to modulate the expression or activity of LMTK2 is not understood.

In our current study, we have investigated the molecular mechanisms regulating LMTK2 gene expression. Since the promoter of a gene regulates its expression, we analyzed the region upstream of LMTK2 start codon for possible regulatory elements. We identified a putative TPA response element (TRE). TPA, a phorbol ester, is a synthetic activator of the classical and novel isoforms of Protein Kinase C, which in turn initiates a plethora of signaling cascades in a cell. We observed that TPA regulates LMTK2 expression in a dose‐ and time‐dependent manner, both at the protein and the mRNA levels. This effect is seen in multiple cell lines. However, TPA does not affect the stability of the LMTK2 mRNA transcript. Moreover, TPA increases the promoter activity of LMTK2. We further deduced that the TPA response on LMTK2 expression is mediated by direct binding of the activator Protein‐1 (AP‐1) complex on LMTK2 promoter. Overall, we have shown that LMTK2 is a TPA‐responsive element‐containing gene, and a novel gene upregulated downstream of PKC activation. Future studies will identify the cellular and extracellular signaling elements causing PKC activation and increased LMTK2 expression.

Support or Funding Information

R01HL102208 to NAB
Oxytocin is involved in cellular signaling within the retina
Authors: York Nathaniel Walter , Halbach Patrick , Chiu Michelle , Bird Ian , Pillers De‐Ann , Pattnaik Bikash
Published: November 2023

Background

Oxytocin (OXT) is a neuropeptide that activates the oxytocin receptor (OXTR), a rhodopsin family G‐protein coupled receptor. We have recently localized OXTR to the retinal pigment epithelium (RPE) and OXT to the adjacent cone photoreceptors.

Objective

We sought to characterize the signaling mechanism of OXTR in the RPE and explore the downstream effects of OXT, focusing on the regulation of inwardly rectifying K+ channel Kir7.1.

Methods

Ca2+ response to OXT was measured in cultured human fetal RPE cells (hfRPE) using Fura‐2 AM in the presence of 2‐APB and nifedipine, pharmacological inhibitors of Ca2+ signaling pathways. HEK‐293 cells where used for the stable expression of human OXTR. Cellular signaling was visualized using live cell fluorescence imaging following transient expression of PH‐GFP and PKC‐GFP, monitors of GPCR metabolites PIP2 and DAG. Whole cell patch clamp electrophysiology was performed on HEK‐OXTR cells transfected with GFP‐fused Kir7.1 as well as freshly isolated mouse RPE cells to study Kir7.1 channel function.

Results

OXT treatment of RPE cells in culture resulted in a transient increase in cytoplasmic Ca2+ that was reduced by 95% in the presence of the IP3R antagonist, 2‐APB (P < 0.0001). Upon bathing the cells in Ca2+‐free extracellular solution or nifedipine, the Ca2+ response to OXT was not inhibited, although amplitude was reduced. Time to the rise in [Ca2+]i was not altered but recovery was slowed down with time constants (τ) of 0.35 ± 0.02 (r2 =.99), 0.34 ± 0.03 (r2 =.98), and 0.20 ± 0.005 (r2=.99) min for Ca2+‐free, nifedipine, and Ringer's solution, respectively. We also demonstrate that OXTR activation blunted Kir7.1 channel current. In isolated mouse RPE, we observed an average 61.81 ± 4.77 % decrease in K+ inward current amplitude at ‐160mV and an average 11.4 ± 3.2 mV depolarization in resting membrane potential by OXT.

Conclusion

We propose that OXTR stimulates a mobilization of intracellular Ca2+, independent of extracellular Ca2+, through 2nd messengers coupled to OXTR/G‐protein in the RPE. This OXT‐OXTR signaling in the RPE cell also integrated the parallel modulation of the Kir7.1 channel. Kir7.1 channel is responsible for controlling RPE resting membrane potential and can influence retinal health and vision. We suggest that novel OXT‐OXTR signaling pathways in the outer retina will be of fundamental importance for eye development, health and visual function.

Support or Funding Information

NIH EY024995, UnityPoint Health Meriter Foundation, Pediatrics, Graduate School, School of Medicine and Public Health
Follistatin‐like 1 (FSTL1) Increases Transepithelial Resistance in Kidney Epithelial Cells
Authors: Yu Zhang
Published: November 2023

Follistatin‐like 1(FSTL1) is a member of the follistatin protein family, characterized by the FS domain found in follistatin. FSTL1 is a secreted protein. Studies have suggested that FSTL1 regulates tissue formation in embryos and plays a role in heart repair in response to harmful stimuli. However, the role of FSTL1 in kidney function and regeneration is unknown. In this study, we collected kidneys from adult male mice three days after unilateral ureteral obstruction (UUO). FSTL1 expression in interstitial cells was significantly increased in obstructed kidney compared to the contralateral kidney in mice. Our in vitro studies show that FSTL1 increased transepithelial resistance (TER), a measure of tight junction complexity and function, in mouse inner medullary collecting duct (mIMCD3) cells, while it did not have any effect on mIMCD3 cell proliferation, migration and TGF‐β1‐induced epithelial‐to‐mesenchymal transition (EMT). In addition, FSTL1 increased Akt phosphorylation levels in mIMCD3 cells, and inhibition of Akt activity abolished the stimulation of TER by FSTL1. In conclusion, we have shown that UUO stimulated FSTL1 expression in the kidney, and that FSTL1 increased transepithelial resistance through the AKT pathway in mIMCD3 cells. These results suggested that increased FSTL1 expression may play a role in renal tubular regeneration following kidney injury.
MALIGNANT HYPERTHERMIA: The motor unit potential (MUP) predicts susceptibility to developing the MH syndrome.
Authors: Williams Charles H
Published: November 2023

Mating MH positive pigs to MH positive pigs produces an F1generation that is highly stress susceptible. Recording the MUP shows MH + pigs have a higher u voltage than control pigs. Older MH+ pigs have an even higher u voltage than control pigs. The duration of the voltage spike is also increased in MH+ pigs versus control pigs and older MH+ pigs have even a longer duration of the voltage spike.

We can assume that by concentrating the MH genetic defect in the F1 generatioon that the population of defective sodium channels in the acetylcholine receptor was present at a high concentration. Since the acetylcholine receptors are spatially located under the footpiece of the myoneural junction which makes them a bank of receptors that are readily accessible when acetylcholine is released by the nerve, and the action of acetylcholine is very rapid. Therefore, the electromyographic data reflects the genetically defective sodium channels as the major functional component when we recorded the data.

The sodium channels can be likened to a low voltage switch in a telephone circuit that is used to route telephone calls.

We would suggest that the sodium channel at the acetyl choline receptor has been adapted to produce heat as well as muscle contraction and that the ability to produce copious amounts of heat is the biological mechanism that differentiates warm blooded animals from cold blooded animals.
CALCIUM OXALATE (CaOx) CRYSTAL ADHESION/AGGLOMERATION DEPENDENCE OF CLC‐5 EXPRESSION IN RENAL EPITHELIAL CELLS
Authors: Lorsung Rebecca , Harris Peter C , Lieske John C , Chang Minhwang
Published: November 2023

Dent disease I is a rare X‐linked recessive chronic kidney disorder caused by mutations of the CLCN5 gene, which encodes the electrogenic Cl−/H+ exchanger (transporter) ClC‐5. CLC‐5 protein has been localized to the apical membrane and endosomal vesicles of the kidney in the proximal tubule as well as other tubule segments. Clinical manifestations of patients with Dent disease include low molecular weight proteinuria (LMWP), hypercalciuria, nephrocalcinosis, nephrolithiasis, bone disease (rickets), and often progressive renal failure. While it is known that the CLC‐5 protein functions in the formation and maturation of endosomes, the etiology of the nephrocalcinosis and kidney stones often observed in Dent disease patients is not known. Therefore, in this project we investigated the effects of several CLC‐5 mutations identified in Dent disease patients, including S244L, Q629X, and R345W, on cellular processing of calcium oxalate (CaOx) crystals. Monolayers of renal proximal tubular cells (LLC‐PK1) and renal cortical tubular epithelial cells (RCTE) transfected with GFP tagged WT and mutated CLC‐5 were subjected to a CaOx crystal adhesion/agglomeration assay for thirty minutes then imaged by DIC and epifluorescence microscopy. The total area of CaOx crystal formation around each transfected cell within a 35, 55, and 75 μm diameter concentric circle was analyzed using ImageJ, and compared to adjacent un‐transfected cells which served as an internal control. A significant increase in CaOx aggregation was observed around cells expressing CLC‐5 mutants compared to wild type cells, especially for S244L. In LLC‐PK1 cells crystal adhesion/agglomeration decreased in this order: S224L > R345W > Q629X = WT; while in RCTE it decreased in this order: S224L = Q629X > R345W = WT. These findings support the hypothesis that mutated CLC‐5 proteins alter the apical surface of epithelial nephron cells and encourage CaOx aggregation/adhesion. It is also possible that decreased internalization and dissolution of CaOx crystals which form on the cell surface plays a role, as the greatest crystal adhesion/agglomeration was found within the 35 μm diameter circle around cells transfected with mutated CLC‐5 (35 μm > 55 μm > 75 μm). Further investigation into the impact of specific CLC‐5 mutation of protein structure and function, including protein‐protein interactions, is needed.

Support or Funding Information

Supported by O'Brien Urology Research Center U54‐DK100227 summer student supplement and the Rare Kidney Stone Consortium (U54‐DK083908)
Defective microglia alters respiratory control and mitochondrial function in mice.
Authors: Rousseau Jean‐Philippe , Laouafa Sofien , Tremblay Marie‐Ève , Joseph Vincent , Kinkead Richard
Published: November 2023

We tested the hypothesis that alteration of microglial function impairs respiratory control and mitochondrial function in the brainstem of newborn mice. Measurements were performed on newborn CX3CR1 knockout (fractalkine receptor deficient) and control (C57Bl6) mice aged of 1 day. Fictive breathing (in‐vitro) was measured by extracting the brainstem with a part of the spinal cord and placing it in a chamber where it was superfused with an artificial cerebrospinal fluid (aCSF). The output signal from the respiratory network was recorded by a suction electrode placed on the fourth ventral root (C4) representing the inspiratory signal sent to the diaphragm via the phrenic nerve. Mitochondrial function was assessed on fresh brainstem tissue (~4–5 mg) permeabilized with saponin using high‐resolution oxygraph (O2k‐Oroboros) allowing to evaluate simultaneously O2 consumption and H2O2 production (reflect of reactive oxygen species). Compared to controls, CX3CR1KO mice showed 41% decreased phrenic burst frequency under baseline conditions (3.0±1.2 vs 7.3±1.0 bursts/min; P<0.05). O2 consumption measurements under basal condition with substrates of complex I (Pyruvate/Malate) and complex II (Succinate) showed no effects between control and CX3CR1KO (CI: 5.8±0.4 vs 3.9±0.5 pmol O2/s/mg; CII: 10±0.3 vs 7.8±0.6 pmol O2/s/mg). However, addition of ADP stimulated mitochondrial respiratory chain and increased O2 consumption in control more than CX3CR1KO mice with substrate of complex I (29.4±2.6 vs 22.3 ±1 pmol O2/s/mg; P<0.01), complex II (39.5±1 vs 31±1.5 pmol O2/s/mg; P<0.005) and complex I+II (55.5±2.1 vs 41.4±1.4 pmol O2/s/mg; P<0.0001). Moreover, H2O2 production tends to increase in CX3CR1KO mice compared to control with substrates of complexes I and II under stimulation by ADP. We can conclude that CX3CR1KO mice exhibit an alteration of respiratory control as well as a mitochondrial dysfunction. These preliminary results provide new hypotheses regarding the origins of respiratory disorders of the newborn involving reduced respiratory drive such as apnea of prematurity.

Support or Funding Information

Founded by NSERC, Région Rhône‐Alpes and consulat de France à Québec.
Effect of circulating angiotensin II on central vascular and inflammatory responses in the rat PVN
Authors: Arocha Rebeca , Zubcevic Jasenka
Published: November 2023

High levels of Angiotensin II (Ang II) are associated with hypertension and blood brain barrier (BBB) disturbance. We have previously shown that neuronal effects of a single systemic injection of Ang II persist for several hours after the blood pressure returns to baseline. Thus, we hypothesize that a single injection of Ang II may cause microglial activation via BBB disturbance. Adult male WKY rats were injected with bolus saline or Ang II (40μg/kg in 0.5ml saline i.p.). At one and 24 hours post‐injection, brains were collected (2% PFA), cryoprotected (30% sucrose) and frozen in OTC. Sections (10μm) were cut in the paraventricular nucleus (PVN) region (1.44–1.92mm caudal to Bregma). Slides were incubated with mouse anti‐rat transferrin (ThermoFisher Scientific Catalog#: 13‐6800; 1:450), rabbit anti‐rat IBA1 (Wako Pure Chemical Industries, Ltd. Catalog#: 019‐19741; 1:500), and rabbit anti‐rat GFAP (Sigma‐Aldrich Co. LLC Catalog#: G4546‐100UG; 1:400) overnight. Images were taken with a Zeiss fluorescent microscope and analyzed with imageJ. Transferrin levels reduced at one hour (−31.5±6.11, P=0.0143), returning to baseline levels at 24 hours post‐Ang II (+20.8±11.9, P=0.202). Similarly, a significant increase in number of activated microglia was present at one hour (+28.2±4.316%, P=0.0196), which remained high at 24 hours post Ang II (+16.2±2.57%, P=0.0143). In contrast, no significant changes in astrocytes were observed at one hour (−22.2±19.6, P=0.429) and 24 hours post Ang II (+4.5±19.1, P=0.849). We propose that even a single systemic injection of Ang II may exert its central effects via disturbance of BBB.

Support or Funding Information

As part of the STRIDE Undergraduate Summer Research Fellowship, the author was funded by the American Physiological Society and a grant from the National Heart, Lung and Blood Institute (NHLBI;1 R25 HL115473‐01). And would like to thank Dr. J Zubcevic for guidance throughout this experiment.
Purinergic signalling in mesenteric vascular smooth muscle in a mice model of essential hypertension
Authors: López‐López José Ramón , Alvarez‐Miguel Inés , Cidad Pilar , Perez‐Garcia M Teresa
Published: November 2023

In vascular smooth muscle cells (VSMCs) non‐selective cation channels such as TRPC3 and TRPC6 have been proposed as the molecular constituents of receptor‐(ROCs) and stretch‐(SOCs) operated channels linking DAG signaling cascade pathway with increased vascular tone. The membrane depolarization caused by TRPCs can increase Ca2+ entry leading to augmented [Ca2+]I and VSMC contraction. Therefore, by influencing membrane potential, TRPC channels could play an important role controlling the increased vascular tone present in several pathologies, such as essential hypertension. We have previously described an increased functional expression of TRPC3 channels in mesenteric VSMCs from hypertensive mice that can contribute to increased vascular tone by favouring depolarization (Alvarez‐Miguel et al., J Physiol, in press). As several mechanosensitive G‐protein coupled receptors (GPCR) are linked to the DAG‐dependent activation of TRPC3/6 channels, we explore here the contribution of this signalling pathway to the increased vascular tone in hypertension.

Using a mouse model of essential hypertension (BPH mice) and its normotensive control (BPN mice) we explored changes in the molecular components of the mechanotransduction pathway in hypertension. We analized the expression of mechanosensitive ion channels and some VSMCs sensor elements, including GPCRs, channels and transporters with qPCR. Their functional contribution was studied in isolated VSMCs (using electrophysiology) and in intact mesenteric arteries with pressure myography.

Real‐time PCR showed an increased expression of some of the elements of the purinergic signalling pathway, including purinergic receptors P2Y2 and P2Y6, TRPC3 channels and the Ca2+‐activated Cl− channel Clca1. Pressure myography of mesenteric arteries after endothelium removal showed a vasoconstriction response to ATP, UTP and UDP. ATP‐induced vasoconstriction was not different between BPN and BPH arteries. The EC50 of the effect was around 7–10 μM in both preparations, and it could be blocked by α‐β‐methilene‐ATP. UTP‐induced vasoconstriction was not affected by α‐β‐methilene‐ATP and was significantly larger in BPH arteries: 50 μM UTP‐elicited vasoconstriction represented 40% of the maximal response to ATP in BPN arteries and 150% in BPH arteries. Our data indicate that mesenteric vascular reactivity to ATP is not altered in our hypertension model, but there is an increased contribution of the purinergic GPCR signalling pathway to the increased vascular tone. Together with the observed upregulation of P2YR receptors in BPH VSMCs, the augmented functional expression of TRPC3 channels could participate in the increased vasoconstriction observed in response to uridine nucleotides in BPH mesenteric arterie

Support or Funding Information

Supported by grants ISCIII‐FEDER RD12/0042/0006, (Heracles Program), BFU2013‐45867‐R (MINECO) and by a Junta de Castilla y Leon fellowship to IAM
Evaluation of biological and mechanical variations in hypertensive animal models
Authors: Lue Sheng‐I , Ho Lilun , Fu Yaw‐Syan , Lu Tzongshi
Published: November 2023

Background

Hypertension (HT) is a multifactorial cardiovascular disorder that involves a variety of genetic and environmental factors. To properly study the pathophysiology of hypertension, robust and reproducible experimental animal models are required. The ideal HT animal model should have human‐like cardiovascular anatomy, hemodynamics, and physiology that includes genetic and non‐genetic models. Among these HT animal models, Spontaneously Hypertensive Rats (SHR) with both neural and vascular alterations is the most commonly used genetic HT animal model. In this study, we investigated methodical variations among current HT animal models.

Methods

Histamine, Capsaicin, C48/80 or Substance P were administered into the femoral vein of Sprague Dawley rats or SHR to induce endothelial dysfunction. We analyzed mean arterial pressure and analyze plasma extravasation using Evans Blue dye. One way ANOVA with Bonferroni correction, and linear regression were used for statistics. Statistical significances were set at a P< 0.05.

Results

Our data showed significant negative linear regression on the degree of plasma extravasation with blood pressure changes in neurogenic inflammation (Substance P) but positive linear regression in immunogenic inflammation (Histamine, Capsaicin, C48/80). In addition, Evans blue dye inhibited C48/80‐induccd extensive plasma extravasation, mast cell degranulation, and edema significantly.

Conclusions

We have observed significant variations among neurogenic and immunogenic inflammation induced blood pressure changes. Our data shows for the first time that methodical variations observed among animal models, can potentially translate into differential blood pressure patterns, which can be falsely taken to be biologically significant. Our study therefore highlights the pivotal need to carefully consider methodical approaches to animal models that report quantitative differences.
A Slow Pressor Dose of Angiotensin II Decreases Sodium Transporters Expression in the Kidney Cortex and is Independent of PPAR‐alpha
Authors: Khundmiri Syed J , Hardin David , Ecelbarger Carolyn M , Lee Dexter L
Published: November 2023

A slow pressor dose of Angiotensin II (Ang II) has been shown to increase the expression of sodium transporters in the proximal tubules (NHE3), TALH (NKCC2) and distal nephrons (NCC) of Sprague Dawley rats before an increase in blood pressure. Peroxisome Proliferator Activated Receptor ‐ alpha (PPAR‐alpha) has been shown to be involved in pressure natriuresis through changes in sodium transport via ameloride and thiazide‐dependent mechanisms. We hypothesized that the changes in expression of the sodium transporters during Ang II hypertension were dependent upon PPAR‐alpha expression. To address this hypothesis, we treated wild‐type (WT) and PPAR‐alpha knockout (KO) mice with a slow pressor dose of Ang II (400 ng/kg/min) for 12 days. Mean arterial pressure (MAP) was measured by radiotelemetry. Control MAP was not different between WT (110 ± 8 mmHg) and PPAR‐alpha KO mice (112 ± 12 mmHg). On day 12 of Ang II, MAP for PPAR‐alpha KO (156 ± 16) mice was significantly higher than WT (138 ± 11 mmHg) mice. The expression of NHE3, NHERF1, NKA‐α1 subunit, NKCC2, and NCC was detected in kidney cortical homogenates by western blotting. Kidneys were homogenized and 25 μg of supernatant proteins were separated by 10% SDS‐PAGE, transferred to nitrocellulose paper, and blotted against antibodies to NHE3, NHERF1, NKA α1 subunit, NKCC2, and NCC. The slow pressor dose of Ang II decreased the expression of NHE3 in WT + Ang II (0.14 ± 0.02 ODU) and PPAR‐alpha KO + Ang II (0.10 ± 0.02 ODU), when compared to WT (2.61 ± 0.93 ODU) and PPAR‐alpha KO (2.20 ± 0.58 ODU) controls. Ang II‐treatment also decreased NKCC2 in both WT (0.31 ± 0.10 ODU) and PPAR‐alpha KO (0.22 ± 0.03 ODU). Ang II hypertension caused similar decreases in NCC and NHERF1 expression in WT and PPAR‐α KO mice. NKA alpha1 subunit expression was increased during Ang II hypertension in both WT (1.06 ± 0.26 ODU) and PPAR‐α KO (1.64 ± 0.26 ODU) mice. Our results suggest that the effects of a slow pressor dose of Ang II on expression of sodium transporters are independent of PPAR‐alpha expression. Future studies are needed to determine the effects of decreasing NHE3, NKCC2, NCC and NHERF1 expression in the kidney on urinary salt excretion during a slow pressor dose of Ang II.

Support or Funding Information

Howard University Advancing Diversity in Aging Research – National Institute on Aging 1R25AG047843
The effect of Peroxisome Proliferator‐Activated Receptor‐α on NOX‐4 expression in the heart during a slow‐pressor dose of Angiotensin‐II induced hypertension
Authors: Amponsem Joycemary , Lee Dexter L
Published: November 2023

Aging decreases myocardial peroxisome proliferator activated receptor –alpha (PPAR‐α) expression and activation of PPAR‐α reduces hypertension and cellular oxidative damage. Previous results from our laboratory suggest that PPAR‐α attenuates hypertension and oxidative damage by reducing NOX‐2 expression in the heart during a slow‐pressor dose of Angiotensin II (Ang II). The goal of this project is to determine if PPAR‐α also reduces hypertension and cellular oxidative damage through a NOX‐4 dependent mechanism. We hypothesize that PPAR‐α plays an important role in attenuating the pressor response to Ang II by negatively regulating NOX‐4 expression in the heart. To address this hypothesis, male (48 weeks old) PPAR‐α knockout (KO) mice and wild type (WT) mice were implanted with biotelemetry devices and infused with a slow pressor dose of Ang II (400 ng/kg/min) for 12 days. On day 12 of Ang II, MAP was increased in KO when compared to WT, 167 ± 6 and 145 ± 3 mmHg, respectively. Hearts were homogenized and 25 μg of supernatant proteins were separated by 10% SDS‐PAGE, transferred to nitrocellulose paper, and blotted against antibodies to NOX‐4. The expression of NOX‐4 in the heart was not significantly different between KO and WT controls. NOX‐4 expression was also not different between Ang II‐treated KO (10 ± 10 ODU) and Ang II‐treated WT (12 ± 8 ODU) mice. Despite no differences in NOX‐4 expression in the heart, TBARS, an index of oxidative stress, concentration was increased in PPAR‐α KO + Ang II (12.4 ± 1 μM) when compared to WT + Ang II (10 ± 1 μM). Our results suggest that the differences in the blood pressure response to a slow pressor dose of Ang II between PPAR‐α KO and WT mice are not due to increased NOX‐4 expression in the heart. Previous results from our laboratory suggest that increased NOX‐2 expression in the heart and plasma levels of Interleukin‐17 are mechanisms that may cause a significant increase in the Ang II‐induced slow‐pressor response in PPAR‐α KO mice when compared to WT + Ang II. Future studies are needed to determine if increased NOX‐2 expression alone accounts for the signicant increase in blood pressure and oxidative stress in PPAR‐α KO + Ang II mice. Studies are also needed to determine if increased plasma levels of Interleukin‐17 contributes to the increased blood pressure and oxidative stress in PPAR‐α KO + Ang II mice.

Support or Funding Information

Howard University Advancing Diversity in Aging Research – National Institute on Aging 1R25AG047843
INFLUENCE OF DIETARY LARD INTAKE ON THE PROGRESSION OF CARDIOMYOPATHY AND SURVIVAL IN A MURINE DIET INDUCED OBESITY MODEL
Authors: Zemljic‐Harpf Alice E. , Nguyen Alexander H. , Joshi Mughda A. , Alas Basheer F. , Schilling Jan M. , Brenner Sara B. , Nguyen Uyen P. , Roth David M. , Jones Carter W. , Patel Hemal H.
Published: November 2023

Introduction

Obesity poses an enormous healthcare burden and leads to impaired quality of life. We studied effects of a high fat diet (HFD) on the development of obesity related diseases. We hypothesized that increased dietary lard content will reduce survival in a diet induced Type II diabetes model.

Methods

Three month‐old C57BL/6J male mice were fed different percentages (%) of kilocalories by fat (4%, 10%, 45%, and 60% lard per kcal) and observed for 31 months, by monitoring 1) food intake 2) body weight, 3) glucose tolerance, 4) cardiac function and remodeling, 5) blood pressure 6) behavior, and 7) survival.

Results

Three months post diet induction, mice on 45% and 60% HFD were overweight, glucose intolerant, normotensive, and presented cardiac hypertrophy with preserved systolic dysfunction. 12 months post diet induction, balance beam, inverted grid, and open field behavioral tests showed correlation between obesity and reduced voluntary physical activity. At 21 months post, 45% and 60% HFD mice compared to animals on 4% and 10% fat diets were hyposensitive to mechanical stimulation but demonstrated evidence of spontaneous pain, showing preference for gabapentin‐paired environments in the conditioned place preference test. Mice on 45% and 60% HFD developed severe dermatitis, presented weight loss and died prematurely without overt signs of heart failure. Given the average lifespan of male C57BL/6J mice between 24 and 30 months, Kaplan‐Meier survival analysis at 24 months after diet induction revealed a mortality of 100% in 60%‐HFD, 75% in 45%‐HFD, 50% in 10%‐group, and 20% in 4%‐group.

Conclusion

Although 45% and 60% HFD induced the same degree of obesity and hyperglycemia, mortality rate was significantly higher in mice on 60% HFD when compared to 45% HFD. These observations in obese and diabetic mice show a correlation between increased dietary‐lard content and higher mortality.

Support or Funding Information

VA Merit Grant; BX001963
Downregulation of AMPKα Contributes to Cold‐induced Vascular Endothelial Dysfunction
Authors: Xing Yu , Ali Quaisar , Xu Yuechi , Sun Zhongjie
Published: November 2023

Vascular endothelial dysfunction plays a critical role in the initiation and development of hypertension. AMPK, a ubiquitously expressed serine/threonine protein kinase, acts as an energy sensor for both cellular and whole body metabolism homeostasis. Recent studies suggest AMPK is implicated in regulation of vascular endothelial function. We found that AMPKα1, a catalytic subunit of AMPK, was decreased in arteries of cold‐exposed rats. The objective of this study is to investigate whether endothelial cell‐specific overexpression of AMPKα1, a catalytic subunit of AMPK, protects endothelial cells and attenuate the cold‐induce hypertension in rats. Our study showed that endothelial cell‐specific overexpression of AMPKα1 abolished cold‐induced hypertension by rescuing endothelial‐dependent vascular dysfunction of small resistant arteries. Sirt1, and eNOS, as well as coupling‐factor 6 (CF‐6) and mitochondrial open reading frame of the 12S rRNA‐c (MOTS‐c) expression in vascular tissues were significantly down‐regulated by chronic cold exposure, which were abolished by AMPKα1 overexpression. Our study suggests that downregulation of AMPKα plays an important role in cold‐induced endothelial dysfunction and hypertension likely via critical links with Sirt1, eNOS, MOTS‐c, and CF‐6 in an orchestrated network. Interestingly, this study demonstrated for the first time that, CF‐6 and MOTS‐c were down‐regulated in vessels in cold‐induced hypertension, suggesting that CF‐6 and MOTS‐c may be involved in cold‐induced endothelial dysfunction.
Hypoxia‐Induced Changes in the Ubiquitin Proteasome System in a Mouse Model of Pulmonary Hypertension
Authors: Wade Brandy E , Zhao Jingru , Ma Jing , Hart C. Michael , Sutliff Roy L.
Published: November 2023

Pulmonary hypertension(PH) involves a sustained increase in pulmonary arterial pressure and resistance that result in right ventricular(RV) hypertrophy and can ultimately lead to RV failure and death. The mechanisms leading to vascular cell proliferation and vascular remodeling in PH are incompletely defined. The majority of therapeutic approaches target vasodilation but do not address the cellular proliferation and vascular remodeling that underlie PH pathogenesis. The objective of this study is to examine the role of the ubiquitin proteasome system (UPS), an important regulator of protein stability, inhypoxia‐induced PH. Current evidence demonstrates changes in UPS activity in PH; however, the pathways and proteins influenced by these changes are poorly defined. Mice exposed to normoxic or hypoxic (10% O2) conditions for 3 weeks were used to identify proteins with altered ubiquitination in response to hypoxia exposure. Lungs were collected, and the PTMScan® Ubiquitin Remnant Motif (K‐ɛ‐GG) Kit was used to precipitate ubiquitinated proteins for analysis by mass spectrometry (MS). This unbiased MS technique allows identification of changes in ubiquitination of individual peptides as well as global changes in ubiquitin linkage. 198uniquely modified peptides with significant changes in ubiquitination were identified using a fold change threshold of ≥±1.5 (p<0.05). Further analysis revealed a significant decrease in total available ubiquitin though no significant change in total number of ubiquitinated substrates was observed. Hypoxia significantly decreases K11 ubiquitination indicating changes in UPS‐mediated cellcycle regulation. Hypoxia significantly increases K48 ubiquitination suggesting changes in UPS‐mediated protein stability. Increased K48 ubiquitination combined with hypoxia‐induced increases in proteasome and autophagy activity also suggest that ubiquitin‐mediated protein stability may play an important role in PH. These data point to a hypoxia‐induced shift in activity or expression of ubiquitin ligases, the proteins responsible for covalent addition of a ubiquitin moiety to a protein target, and indicate that other shifts in UPS activity may play a significant role in PH pathogenesis.

Support or Funding Information

NIH RO1HL102167 (CMH and RLS), Merit Review from VA Office of Research and Development (1I01BX001910 to CMH), and AHA postdoctoral fellowship (16POST30930007 to BEW), SIRE (to JZ)
Anatomic Characterization of Intrapulmonary Bronchopulmonary Anastomoses in Healthy Rat Lungs
Authors: Murphy Austin , Hoover Michael , Galambos Csaba , Bates Melissa
Published: November 2023

Many neonates with pulmonary hypertension die from right‐to‐left shunt and hypoxemia soon after birth. Intrapulmonary bronchopulmonary anastomotic pathways (IBAPs) have been observed in the lungs of these infants. We indirectly characterized right‐to‐left shunt recruitment by hypoxia (12% O2) in healthy humans and animals that can be reversed by 100% O2. We hypothesized that IBAPs are a normal lung feature whose recruitment is induced by hypoxia. Anesthetized rats were ventilated with 21% or 12% O2 for 20 minutes. Lungs were then immediately removed, airway fixed with 10% formalin, sectioned, and stained for the histological evaluation of IBAPs. The direction of blood flow through these pathways was evaluated by injecting red and green fluorescent microspheres into the right (RV) and left (LV) ventricles of rats ventilated with 12% or 100% O2 and determining their abundance in the lung. Histological sections demonstrated abundant IBAPs in 12% O2‐ventilated lungs, connecting the pulmonary and bronchial arterial trees, but not 21% O2‐ventilated lungs. Consistent with right‐to‐left shunt through these pathways, microspheres injected into the right ventricle were found in the pleural and bronchial vascular beds, but LV‐injected spheres were not found in the pleura. In 100% O2‐ventilated lungs, both RV‐ and LV‐injected microspheres were found coincidentally in pleural vessels, suggesting a possible left‐to‐right shunt. Future studies will aim to anatomically determine the vessel type containing co‐localized red and green microspheres and further characterize IBAPs, including describing their receptor expression.

Support or Funding Information

University of Iowa Start‐Up Fund
Monocyte chemoattractant protein‐1 expression in the lung and kidney is a marker of severity during heat stroke recovery in rats
Authors: Plamper Mark L , Ward Jermaine A , Leon Lisa R , Audet Gerald N
Published: November 2023

Heat Stroke (HS) is a common environmental threat to both civilian and war fighter populations. Building on previous studies, our aim is to determine the course of HS associated organ damage and find new molecular pathways that will help in HS treatment and diagnosis. Monocyte chemoattractant protein‐1 (MCP‐1) is one of the key chemokines that regulate migration and infiltration of monocyte/macrophages, into organ tissues, which may be a key component of HS pathology. In this study, we used conscious passively heated Fisher 344 rats (Tc 42 ±0.1°C) with implantable radio telemetric devices that could gather data before, during and after heating. Data obtained included core temperature (Tc), heart rate (HR) and mean arterial blood pressure. Tissues and blood samples were collected at 24h for molecular and histological analysis. Using results from physiological data and histological sectioning, rats were divided into the categories of mild (MLD), moderate (MOD) and severe (SEV). In the lung, MCP‐1 showed significantly (P<0.001) higher content in the SEV rats (mean 28.51 ±2.34 pg/ml), with proportionate decreasing amounts in MOD (12.03 ±1.56 pg/ml) and MLD (6.66 ±2.34 pg/ml) rats. The control animals showed a lack of response of MCP‐1 (0.0 pg/ml). In the kidney, MCP‐1 results suggest a useful general marker of HS, where SEV (mean 85.54 ±12.60 pg/ml), MOD (89.05 ±10.62 pg/ml) and MLD (97.34 ±12.74 pg/ml) rats were equally elevated compared to the control animals (28.27 ±12.74 pg/ml). Protease‐activated receptor 1 (PAR1), was also tested for a potential marker for HS. Western blot results in lung samples showed significantly (P<0.017) increased intensity of SEV (mean 0.49 ±0.12 au) rats compared with control animals (0.20 ±0.01 au). However, PAR1 was unable to differentiate between MOD and MLD rats versus control animals. Several other molecular markers were also examined, including Matrix metalloproteinases (MMP9), Tissue factor (TF) and Vascular endothelial growth factor (VEGF); none showed elevated levels in lung or kidney tissues at 24h of recovery. In conclusion, the increase in MCP‐1 suggests immune cell infiltration into the lung and kidney and points to MCP‐1 as potential marker for determining overall HS severity during recovery. Author views not official US Army or DoD policy
Dystrophin Insufficiency Causes Locomotor Dysfunction in a Spontaneously Occurring Pig Model
Authors: Spaulding Hannah , Kaiser Adrianne , Hess Andrew , Stalder Ken , Nonneman Dan , Ross Jason , Selsby Joshua
Published: November 2023

Dystrophinopathies are caused by mutations in the dystrophin gene leading to a dystrophin deficiency (Duchenne muscular dystrophy) or insufficiency (Becker muscular dystrophy). These diseases are commonly modeled by the mdx mouse or other related mouse models as well as the Golden Retriever Muscular Dystrophy model. While these models have been useful, inherent limitations necessitate continued development of novel large animal models. The purpose of this investigation was to determine the extent to which a spontaneous dystrophin mutation would impair locomotion in a pig dystrophinopathy model. We performed a monthly gait analysis from 3–12 months of age in male pigs with a dystrophin mutation causing an insufficiency and healthy male littermates using a Zeno gait analysis system. This system consists of a 5 m pad with approximately 50,000 integrated force transducers, which allows sensitive detection of pressure during the gait cycle as the pig walks across the pad. Pigs were euthanized at 3 and 12 months of age and tissues collected for histological analyses. Histological quantifications revealed a 2‐fold increase (p<0.05) in centralized nuclei in affected pigs at 12 months of age and a 2‐fold increase (p<0.05) in centralized nuclei in affected pigs over time at 12 months compared to 3 months of age suggesting progressive disease‐related injury. By 9 months of age affected pigs developed a distinct walking gait. When considering movement in the front limbs, stride length was shortened by 6–10 cm (p<0.05) and by 12 months of age stride width was increased by 7 cm (p<0.05) in affected pigs compared to healthy pigs. In addition to a wider and shorter gait, the swing phase was decreased by 14% and the mean single support center of pressure distance was also decreased at 9 months of age by 36%. Similar results were discovered when measuring movement in the hind limbs and when comparing same side limbs and contralateral limbs. In total, these pigs appear to be moving with a shorter, wider gait with a slower march‐like movement pattern. This unique gait shares similarities with patients affected by dystrophinopathies who also have notable gait changes as the disease progresses. These data suggest that pigs affected with a spontaneously occurring dystrophin insufficiency develop a progressive muscle pathology that ultimately impacts locomotion, consistent with changes in dystrophinopathy patients. These data also contribute to a growing body of literature supporting the utility of this human‐sized model that accurately recapitulates the human disease.

Support or Funding Information

This work supported by NIH grant R21 NS079603.