bluuebunny/crossref_metadata_2025 huggingface dataset:
Streptococcal collagen‐like protein 1 shields group A Streptococcus from antimicrobial molecules (836.8)
Authors: Shimamoto Jacqueline , Doehrmann Simon , Etesami Neelou , Martin Gary , Nizet Victor , Okumura Cheryl
Published: June 2021

Group A Streptococcus causes a spectrum of diseases ranging from pharyngitis to life‐threatening diseases such as streptococcal toxic shock‐like syndrome. Invasive strains of GAS increase the expression of virulence factors that promote bacterial survival in the face of the human immune system. One such virulence factor is Scl‐1, a surface collagen‐like protein that has been shown in other less virulent strains to mediate attachment and resistance to serum factors. Previous work in our lab demonstrated that Scl‐1 plays a role in bacterial survival against phagocytes, but the molecular mechanism for this enhanced survival has not been determined. In the current work, we determined that the presence of Scl‐1 promotes resistance to phagocytic antimicrobial factors such as peroxide. We then used transmission electron microscopy (TEM) to examine whether Scl‐1 can act as a physical barrier to antimicrobial molecules. Preliminary analysis of wild‐type, scl‐1 mutant, and plasmid‐complemented strains suggests differences in the cell wall density of the scl‐1 mutant strain. TEM analysis of peroxide‐treated strains also suggests that Scl‐1 provides a physical barrier to protect the bacterial membrane from oxidative damage. Thus, Scl‐1 contributes to the virulence of the bacteria by acting as a molecular “shield” to protect the bacteria from antimicrobial molecules produced by phagocytic cells.
Potential role of anxiety and stress in aggressive behavior in rats (839.2)
Authors: Patki Gaurav , Solanki Naimesh , Alkadhi Isam , Allam Farida , Salim Samina
Published: June 2021

The social defeat paradigm involves aggressive encounters by a large Long‐Evans (LE) male rat (resident) toward a smaller Sprague‐Dawley (SD) male rat (intruder). Successful application of chronic social defeat stress to SD rats is dependent upon appropriate selection of LE rats with consistent levels of aggressive behaviors, as determined from the 3‐d screening process. During screening for the aggressor LE rats, we observed that half of all screened LE rats did not reach the criterion for aggression (LE rats performing a defeat, characterized by the intruder surrendering or acquiring a supine position for at least 3 sec). This difference in aggression between age and weight matched LE rats was startling and led us to the present study. Herein, we measured behavioral differences between the two groups of LE rats (aggressors and non‐aggressors). We analyzed their anxiety‐like behavior using open‐field and elevated plus maze tests. We also examined aggressive behavior in the two groups. (Aggression was measured by recording time taken to defeat the intruder SD rat and time taken to attack a novel object. We observed a significant increase in anxiety‐like behavior in aggressor rats when compared to the non‐aggressive group. Furthermore, time taken to defeat the intruder rat and to attack a novel object was significantly lower in aggressive LE rats. Biochemical data suggests occurrence of significant differences in the levels of stress (assessed via corticosterone assay) and oxidative stress (analyzed via 8‐isoprostane and protein carbonylation) in rats associated with aggressive and non‐aggressive behaviors.

Grant Funding Source: NIH‐R15‐G103327
Pentadecapeptide BPC 157 counteracts celecoxib‐induced lesions on gastric mucosa in rats (840.6)
Authors: Drmic Domagoj , Kolenc Danijela , Seiwerth Sven , Sikiric Predrag
Published: June 2021

Aim: We investigated the anti‐ulcer effect of pentadecapeptide BPC 157, after celecoxib overdose. Methods: Gastric mucosal injuries in rats were induced by celecoxib (1g/kg) intraperitoneally. To counteract, BPC 157 (10 µg, 10 ng (/kg)) or saline (5ml/kg) were given intraperitoneally immediately thereafter. Rats were sacrificed after 24 and 48 hours, and stomach mucosal defects were assessed as described (Curr Pharm Des 2013;19(1):76‐83). Results: Celecoxib induced huge gastric lesions, huge confluent lesions (4±1 (48h)), and multitude of punctiforming black hemorrhagic lesions (20±3 (24h), 30±5 (48h)), that were both counteracted in BPC 157 rats (no confluent lesions, no punctiform lesions at 24h, punctiform lesions at 48h: 4±2 (µg);10±3 (ng)). Conclusions: Celecoxib induces in overdose huge gastric lesions. BPC 157 was able to counteract lesion formation. This effect is along with counteraction of the lesions induced by nonselective NSAIDs (Curr Pharm Des 2013;19(1):76‐83).

Grant Funding Source: 108‐1083570‐3635, Ministry of Science, Education and Sports, Croatia
Fibroblast activation protein cleavage of collagen enhances class A scavenger receptor‐mediated macrophage adhesion (842.12)
Authors: Mazur Ania , Vadali Shanthi , Kelly Thomas , Post Steven
Published: June 2021

The importance of interactions between cancer and stromal cells within the tumor microenvironment to tumor progression is well documented. In contrast, how interactions between different stromal cell types, such as fibroblasts and macrophages, support tumor growth is not understood. Increased infiltration and retention of tumor‐associated macrophages (TAMs) in tumors is indicative of poor prognosis in cancer patients. TAMs secrete a variety a factors that activate stromal fibroblasts. Tumor associated fibroblasts (TAFs) differ from quiescent fibroblasts by upregulating the expression of type 1 collagen, the primary component of the tumor extracellular matrix, and by expressing the post‐prolyl serine protease, fibroblast activation protein (FAP). In this study, we used a quenched fluorescent substrate to show that FAP proteolytically cleaves type 1 collagen, and that the adhesion of primary macrophages to collagen‐1 is substantially increased by FAP‐mediated cleavage. Further, using macrophages isolated from knock‐out mice and specific receptor inhibitory approaches, we show that increased macrophage adhesion to FAP‐modified collagen is mediated by the Class A Scavenger Receptor (SR‐A). These results suggest a novel TAF‐TAM axis in which FAP can promote tumor progression by cleaving collagen and increasing the SR‐A‐dependent retention of TAMs.
Molecular mechanisms of phospholipase C‐β autoinhibition (843.10)
Authors: Lyon Angeline , Begley Jessica , Manett Taylor , Tesmer John
Published: June 2021

Phospholipase C‐β (PLCβ) enzymes hydrolyze the inner membrane lipid PIP2 to produce the second messengers inositol‐1,4,5‐triphosphate and diacylglycerol. PLCβ enzymes are characterized by low basal activity, due in part to a linker region within catalytic core that physically occludes the active site. We previously identified a highly conserved autoinhibitory helix in the PLCβ C‐terminal regulatory region that makes extensive interactions with the PLCβ catalytic core. Mutations within this interface lead to dramatic increases in basal activity and decreased thermal stability. Furthermore, this autoinhibitory helix docks in close proximity to the PLCβ active site and the regulatory linker. However, the molecular basis for how this inhibitory helix regulates basal activity, and whether or how it works in conjunction with the catalytic core regulatory linker remains unclear. We have used mass spectrometry, site‐directed mutagenesis, and biochemical assays to begin to elucidate the molecular basis of autoinhibition of lipid hydrolysis by these various regulatory elements. A better understanding of the mechanisms governing PLCβ activity are essential for developing novel therapeutic approaches for the treatment of cardiac hypertrophy and heart failure.

Grant Funding Source: NIH grants HL086865 and HL071818 (J.J.G.T.) and AHA Fellowship 13POST1637009 (A.M.L.).
Measuring and modeling dynamics of regulators of G protein signaling to facilitate discovery of allosteric inhibitors of protein‐protein interactions (843.13)
Authors: Higgins Colin , Fuentes Ernesto , Roman David
Published: June 2021

Ischemic stroke morbidity and disability stem from neuronal death largely driven by glutamatergic excitotoxicity following hypoxia in brain tissue deprived of blood flow. Pharmacological inhibition of RGS4 with chemical probes significantly potentiates endogenous neuroprotection against excitotoxicity in vivo. The only known RGS4 inhibitors are unsuitable as clinical leads because they are relatively nonspecific covalent modifiers that inhibit through an uncharacterized, though putatively allosteric, mechanism. We seek to determine the structural mechanism by which current small‐molecule chemical probes allosterically inhibit RGS4. We hypothesize that inhibitors, binding to Cys148, trap a stochastically minor open conformation of RGS4 and disrupt a structurally key helical bundle to both prevent a unique conformational shift in RGS4 required to bind G‐alpha and to shift the population dynamics toward increasingly extended and unstable conformations. Structural changes in RGS proteins upon binding to inhibitory compounds measured with structural biological techniques including nuclear magnetic resonance, circular dichroism, light scattering, small‐angle X‐ray scattering, and atomistic molecular dynamics simulations support our hypothesis. Our investigations further show that RGS8, a homolog of RGS4, differs significantly in biophysical properties and we conclude that that these differences underly specificity of inhibitors for RGS4.

Grant Funding Source: Supported by 5T32GM067795‐08 and pre‐doctoral fellowship from AFPE.
GPCR‐Gs‐mediated cyclic AMP signaling for neuritogenesis, growth arrest, and cell survival is parcellated among the cAMP sensors NCS/Rapgef2, Epac, and PKA (843.7)
Authors: Emery Andrew , Eiden Maribeth , Mustafa Tomris , Eiden Lee
Published: June 2021

Ligands that elevate cAMP through Gs‐coupled GPCR activation have profound effects on the differentiation of neuroendocrine cells. Using the PC12 cell subclone NS‐1, we found that cAMP‐induced neurite extension, cAMP‐induced growth arrest, and cAMP‐dependent cell survival are all dissociable at the level of the cAMP sensor through which signaling occurs. Previously, we showed that the protein product of the Rapgef2 gene (NCS/Rapgef2) is activated by cAMP and is coupled to the activation of the Rap1‐BRAF‐MEK‐ERK signaling cascade necessary for cAMP‐dependent neuritogenesis (Emery and Eiden, FASEB J. 26, 3199, 2012; Emery et al., Sci. Signal. 6, ra51, 2013). Cyclic AMP elevation also causes Epac (Rapgef3/4) activation, which we now find leads to activation of both Rap1 and the MAP kinase p38. Surprisingly, these two downstream effectors of Epac appear to be arranged in separate signaling pathways, since p38‐dependent growth arrest initiated by Epac was not affected by inhibition of Epac‐dependent Rap1 activation. Thus, in NS‐1 cells, cAMP‐dependent growth arrest is wholly dependent on Epac‐p38 signaling and does not require NCS/Rapgef2‐ERK or PKA‐CREB activity. Finally, we found that cAMP protects NS‐1 cells from serum withdrawal‐induced cell death, also by a wholly separable signaling mechanism, which is PKA‐dependent and does not require the activation of either NCS/Rapgef2 or Epac.

Grant Funding Source: Supported by NIMH‐IRP project ZO1‐MH002386
Ethanol‐induced ROS production is prevented by globular adiponectin via modulation of Nox2 expression in macrophages (844.19)
Authors: Kim Mijin , Nepal Saroj , Shrestha Anup , TiliJa pun Nirmala , Park Pil‐Hoon
Published: June 2021

Adiponectin predominantly secreted from adipose tissue plays a crucial role in lipid metabolism and possesses potent anti‐inflammatory properties. Adipoonectin has been shown to generate protective effects in a number of pathophysiological states. Recently, it has been shown that adiponectin prevents ROS production induced by stimulation with various stimuli. In the present study, we investigated the effect of globular adiponectin (gAcrp) on ethanol‐induced ROS production in macrophages and underlying mechanisms. Herein, we found that gAcrp efficiently suppresses ethanol‐induced ROS production both in RAW 264.7 macrophages and murine peritoneal macrophages. We next investigated the role of NADPH oxidase, comprising a multi‐component enzyme complex, in the suppression of ROS production by gAcrp, since NADPH oxidase plays a critical role in ethanol‐induced ROS production in macrophages. Globular adiponectin prevented ethanol‐induced NAPDH oxidase activation. Particularly, it inhibited ethanol‐induced increase in expression of Nox2 and p22phox. Furthermore, gAcrp restored ethanol‐induced activation of p38MAPK‐NF‐κB pathway, which is critical for Nox2 expression. Taken together, these results suggest that adiponectin prevents ethanol‐induced ROS production via modulation of NADPH oxidase in macrophages.
A “heavy metal sponge” for selective removal and recovery of toxic or precious metals from water (844.8)
Authors: Nguyen Phuc , Madera Paul , Rendon Adrian , Ong Aaron , Mao Herbert , Trujillo Nancy , Acey Roger
Published: June 2021

Metallothionein (MT) is a low molecular weight metal binding protein. It has the unique ability to selective bind trace amounts of toxic heavy metals such as lead and cadmium. It does not bind essential metals such as sodium or calcium. We are developing a novel patented “heavy metal sponge” technology for the removal and recovery of toxic, e.g., lead and mercury, and precious metal, e.g., gold and platinum, from water. MT also has the ability to bind the lanthanides lanthanum and europium. Recombinant MT was cloned into pET vectors with and without a Pel secretory sequence, pPMpel, and pCODAmt, respectively. The protein is expressed in bacteria and purified to near homogeneity from cell lysates by a simple and cost effective method using FPLC anion exchange chromatography. The purity of the preparation was confirmed by SDS‐PAGE. Expression of MT and secretion of the protein using the pPMpel construct is pH dependent. The level of expression from both constructs was the same for IPTG ranging from 0.5 to 2.0 mM. The purified MT was used to determine the potential for removing trace amounts of heavy metal from seawater. The protein was mixed with seawater containing 100 ppt (trillion) of 109Cd. The protein/metal complex was then collected using a membrane filter centrifugal device and the flow through assayed for radioactivity. The efficiency of metal removal was determined from the radioactivity in the flow through. Under these conditions, 88% of the metal was removed from the seawater after a single centrifugation.

Grant Funding Source: Funded by a grant from CSUPERB
Aldehyde dehydrogenase 2 null mice as an oxidative stress‐based model of age‐related cognitive impairment with Alzheimer’s disease‐like pathologies (845.3)
Authors: D'Souza Yohan , Elharram Ahmed , Frith Margaret , Andrew David , Bennett Brian
Published: June 2021

Most current mouse models Alzheimer’s disease (AD) exhibit pathological changes dependent on overexpression of mutant human genes linked to familial AD, and animal models that mirror late‐onset/age‐related AD (95% of AD cases) are lacking. Oxidative stress is considered a causative factor in age‐related AD, and Aldehyde dehydrogenase 2 (ALDH2) is important for the detoxification of endogenous aldehydes, such as 4‐hydroxynonenal (HNE), a lipid peroxidation product formed during periods of oxidative stress that can form protein adducts, altering cell function. HNE adducts accumulate in the brains of AD patients and are associated with AD pathology. Given this linkage, manipulations that increase HNE levels might be expected to result in biochemical and cognitive changes that mirror those found in AD. Accordingly, we followed the age‐related changes of a number of relevant AD markers over a 12 month period in hippocampal homogenates from Aldh2 null mice. Marked increases in HNE adduct formation occurred as early as 3 months, and there were age‐related increases in monomeric amyloid β (Aβ), phospho tau protein, and activated caspases 3 and 6. Also observed were age‐related decreases in PSD95 and synaptophysin (indicating synaptic loss) and of total and phosphorylated cAMP‐response element binding protein (CREB). Aldh2 null mice exhibit a graded, age‐related decrease in performance in recognition and spatial memory tasks beginning at 3 months of age and maximal at 6 months. These mice also exhibit dystrophic neurites and loss of dendritic spines, defects in cholinergic agonist‐induced CREB phosphorylation, endothelial dysfunction, and monomeric and oligomeric Aβ deposition in cerebral microvessels. Aldh2 null mice exhibit a wide spectrum of AD‐like pathological changes that are rarely observed together in current AD animal models, and may prove useful for assessing the efficacy of therapeutic agents for improving memory and for slowing, preventing, or reversing disease progression.

Grant Funding Source: Botterell Foundation
SCM‐198 protects neurons from microglia‐induced neurotoxicity and improves cognitive performances in Alzheimer’s disease models (846.3)
Authors: Hong Zhen , Shin Ru , Zhu Kai , Wu Ting , Zhu Zhun
Published: June 2021

Background: Chronic inflammation in central nervous system is a conspicuous hallmark in Alzheimer’s disease (AD) and microglial activation is now accepted as a key factor in neuroinflammation. SCM‐198 is an alkaloid extracted from Herba leonuri. In the present study, we investigated its direct and indirect neuroprotective effects in neurons and in animal AD models.

Methods and Results: (1) Pretreatment of microglia with SCM‐198 could reduce nitric oxide elevation and inhibit iNOS, TNF‐α, IL‐1β mRNA and protein expressions induced by LPS or Aβ1‐40. NF‐κB translocation induced by LPS was effectively mitigated by SCM‐198 in BV‐2 cells. Western blot results showed that IκBα degradation, NF‐κB p65 activation, phosphorylations of JNK1/2 and tau protein were also inhibited in BV‐2 cells. (2) Microglia/neuron co‐culture assay showed that pretreatment of activated microglia with SCM‐198 could significantly increase neuronal survival and alleviate excessive tau phosphorylation and synaptophysin loss in neurons. (3) Pretreatment of primary neurons with SCM‐198 before Aβ1‐40 challenge could reduce lactase dehydrogenase release, inhibit Bax protein elevation, prevent caspase‐3 and ‐9 activations, increase Bcl‐2 protein expressions in neurons. Besides, neuritic morphology was largely preserved under the intervention of SCM‐198. (4) Animal studies demonstrated that SCM‐198 could ameliorate cognitive deficits of both SD rats injected with Aβ1‐40 and APP/PS1 mice in Morris water maze and novel object recognition tests. Suppression of microglial activation could be observed in brain slices of SD rats and less neuronal loss in hippocampus and cortex were found in APP/PS1 mice.

Conclusions: Our findings are the first to report that SCM‐198 could directly protect neurons from apoptosis and indirectly protect neurons by inhibiting microglial overactivation through JNK and NF‐κB pathways. Therefore, SCM‐198 might be a new potential drug candidate for AD therapy in the future.

Grant Funding Source: This study was supported by the National Science fund for Distinguished Young Scholars of China
Cognitive performance and homecage activity in female cynomolgus monkeys: relationship to social rank and cocaine reinforcement (848.1)
Authors: Kromrey Sarah , Czoty Paul , Rowe Michael , Nader Michael
Published: June 2021

Nonhuman primate social groups, which represent a continuum of experiences from environmental enrichment in dominant monkeys to chronic social stress in subordinates, have been used to study susceptibility and resilience to human diseases. We have shown that social rank influences sensitivity to cocaine and other dopaminergic drugs. However, it is not clear what biological and environmental factors determine what rank an individual monkey will attain. In females, novel object reactivity and CSF concentrations of 5‐HIAA predicted social rank; these factors did not change after hierarchies stabilized. The present studies examined whether other variables, including cognitive function (using CANTAB touchscreen software) and activity (using Actical monitors), predict eventual social rank and change after social hierarchies stablized. Prior to social housing 4 monkeys per pen, performance on a cognitive task was not different in eventual dominant and subordinate monkeys. After stable hierarchies were formed, subordinate monkeys demonstrated cognitive impairments. Furthermore, future subordinates had higher activity during the day which remained higher after hierarchies stabilized, and preliminary results suggest rank differences in nighttime activity. Additionally, the relationship between these behaviors and cocaine reinforcement is being investigated. Understanding behavioral phenotypes and individual differences in response to environmental manipulations, such as attainment and occupation of social ranks, and the relationship to cocaine self‐administration will aid in the development of behavioral and pharmacological treatment strategies for drug addiction.

Grant Funding Source: Supported by DA017763
LPS induced LOX‐1 expression via TLR4/MyD88/ROS activated p38MAPK/NF‐κB pathway (851.15)
Authors: Chen Xiuping , Zhao Wenwen , Ma Guixin
Published: June 2021

Lectin‐like receptor for oxidized low density lipoprotein (LOX‐1) plays a key role in endothelial ox‐LDL endocytosis, endothelial dysfunction and atherogenesis. In the present study, the effect of lipopolysaccharide (LPS) on LOX‐1 expression and the underlying molecular pathways were investigated. Human umbilical vein endothelial cells (HUVECs) were treated with LPS and the protein expressions of LOX‐1,TLR4, TLR2, MyD88, Nox4, Nox2, PI3K, p38MAPK, JNK, ERK, Nrf1, Nrf2 and p65 were examined by Western blotting. The intracellular reactive oxygen species (ROS) production was examined by flow cytometry with fluorescence probe DCFH2‐DA. The role of TLR4, MyD88 and Nox4 were determined with specific siRNA. The endothelial ox‐LDL uptake and the endothelial‐monocyte adhesion were evaluated with DiI‐ox‐LDL and Hoechst 33342 respectively. The effect of LPS on LOX‐1 expression in aorta tissue was also studies with Male C57/BL6 mice by intraperitoneal injection of LPS. The results showed that LPS induced LOX‐1 protein expression in a time‐ and concentration‐dependent manner. The mRNA expression of LOX‐1 was also upregulated. The protein expression of LOX‐1 and phosphorylated p38MAPK, p65 was significantly enhanced by LPS both in vitro and in vivo. LPS induced LOX‐1 expression was blocked by siRNA for TLR4, MyD88, and Nox4 and inhibitors for p38MAPK, NF‐κB and NADPH oxidase. Both LPS induced ox‐LDL uptake and endothelial‐monocyte adhesion were significantly inhibited by anti‐LOX‐1 antibody. LPS dramatically induced LOX‐1 protein expression in aorta tissues. In conclusion, our data suggested that LPS induces LOX‐1 expression via TLR4/MyD88/ROS activated p38MAPK/NF‐κB pathway, which put new insight on the role of LPS in atherosclerosis.

Grant Funding Source: Supported by the National Natural Science Foundation of China (No. 81160048)
Comparing the effects of atorvastatin and vitamin E on endothelial function of sildenafil non‐responders (851.8)
Authors: abd Elkawy Khaled , El‐Sisi Alaa , Hegazy Ssahar , Salem Khaled
Published: June 2021

ABSTRACT Endothelial dysfunction causes the failure of sildenafil response due to reduced nitric oxide (NO) . This study aimed to compare the biochemical and clinical effects of atorvastatin and vitamin E on endothelial function of sildenafil non‐responders with investigation to the underlying mechanisms.

Methods: Sixty participants were randomly divided into three groups receiving atorvastatin 80 mg daily, or vitamin E 400 IU daily, or placebo capsules. Participants were examined both before and after six weeks for these biochemical tests; NO, glutathione peroxidase (GPO), interlukin 6 (IL‐6), testosterone, and endothelial nitric oxide synthase (eNOS) and for erectile function tests; international index of erectile function (IIEF ‐5) scores, rigiscan, and duplex l Results: in comparison to control group, the treated groups showed a significant increase in GPO, and a significant decrease in IL‐6. Atorvastatin only showed a significant increase in NO, eNOS, IIEF‐5 scores and rigiscan rigidity parameters. Other rigiscan parameters, testosterone and duplex ultrasound showed no difference in the three groups
Conclusions: Atorvastatin improves endothelial function of sildenafil non‐responders by increasing NO level attributed to enhancing eNOS activity (main mechanism) added to antioxidant and anti‐inflammatory activities. Atorvastatin is a real promising drug for sildenafil non‐responders.
Dynamic regulation of β1 subunits controls vascular contractility (853.7)
Authors: Leo M. , Bannister John , Narayanan Damodaran , Nair Anitha , Jaggar Jonathan
Published: June 2021

Ion channels composed of pore‐forming and auxiliary subunits control physiological functions in virtually all cell types. Whether the multi‐subunit composition of surface channels is fixed following protein synthesis or flexible and open to acute and rapid modulation to control physiological cellular functions is unclear. Arterial myocytes express large‐conductance Ca2+‐activated potassium (BK) channel α and auxiliary β1 subunits that are functionally significant modulators of arterial contractility. Here, we show that native BK α subunits are primarily (~95%) plasma membrane‐localized, whereas only a small fraction (~10%) of total β1 subunits are located at the cell surface in human and rat arterial myocytes. Immuno‐FRET microscopy demonstrated that intracellular β1 subunits are stored within Rab11A‐postive recycling endosomes. Nitric oxide (NO), acting via protein kinase G, stimulated rapid (1 min) anterograde trafficking of β1 subunit‐containing endosomes. These β1 subunits associated with surface‐resident BKα proteins, elevating Ca2+‐sensitivity and inducing activation. Data also show that rapid β1 subunit anterograde trafficking is the primary mechanism by which NO activates myocyte BK channels and induces vasodilation. In summary, we show that rapid surface trafficking of β1 subunits controls functional BK channel activity in arterial myocytes and vascular contractility.

Grant Funding Source: Supported by NIH‐HL67061
The root extract of Angelica gigas Nakai promotes adipogenic differentiation via activation of the insulin signaling pathway in 3T3‐L1 cells (854.6)
Authors: dela Cruz Joseph , Jeong Song‐Ee , Hong Hee Ok , Hwang Seong Gu
Published: June 2021

Type 2 diabetes (T2D) is a complex metabolic disorder characterized by insulin resistance and hyperglycemia. Peroxisome proliferator‐activated receptor gamma (PPARγ) is a key transcription factor and plays an important role in the regulation of genes involved in adipogenic differentiation, glucose metabolism and insulin signal transduction. In this study, the effects of the root extract of Angelica gigas Nakai (AGN) on the differentiation of 3T3‐L1 preadipocytes and the possible mechanism of glucose transport were investigated. Post‐conﬂuent 3T3‐L1 cells were differentiated in the presence or absence of AGN (up to 400μg). Oil red O staining showed that AGN extract significantly enhanced adipocyte differentiation in a dose dependent manner. GPDH activity as well as intracellular lipid accumulation was significantly increased by the AGN treatment. RT‐PCR and Western Blot analysis showed that AGN increased cell differentiation by upregulating the expression of PPARγ dose dependently. AGN also promoted glucose transport by increasing the expression of glucose transporter 4 (GLUT‐4), phosphatidylinositol 3‐kinase (PI3K) and insulin receptor substrates‐1 (IRS‐1). Our results suggest that AGN extract may be a potential therapeutic agent for managing T2D by promoting the differentiation of adipocytes via the upregulation of PPARγ levels and the activation of the insulin signaling pathway.
Effects of calf venous capacitance initiation rate on tolerance to lower body negative pressure in women with and without vasovagal syncope (858.5)
Authors: Skoog Johan , Lindenberger Marcus , Zachrisson Helene , Länne Toste
Published: June 2021

Objective: Lower limb venous blood pooling has been considered an important factor in the pathophysiology of vasovagal syncope (VVS). However, not only the pooled volume, but also the rate by which the hypovolemic stimulus is instituted may be of importance. The aim of this study was to determine the influence of calf venous capacitance (Vcap initiation rate on tolerance to lower body negative pressure (LBNP) in women with and without VVS. Design and Methods: Graded LBNP to presyncope was used to determine LBNP tolerance index (LTI) in 13 young women with VVS and 15 matched controls. During LBNP, Vcap and the initiation rate of Vcap was evaluated with volumetric technique and overall cardiovascular responses were measured. Results: No differences in Vcap were found. In controls, the initiation rate of Vcap was negatively related to LTI (r = ‐0.54, P < 0.05), and a rapid initiation of Vcap was related to a lesser decline in stroke volume (r = ‐0.68, P < 0.05) and pulse pressure (r = ‐0.52, P < 0.05) during LBNP. In VVS, the initiation rate of Vcap was positively related to LTI (r = 0.82, P < 0.01), and a slower initiation was related to lesser decline in stroke volume (r = 0.61, P < 0.05). Conclusions: In healthy women, a rapid institution of Vcap was related to a more effective cardiovascular regulation and a higher LTI, possibly explained by greater baroreceptor deactivation due to a more rapid onset of the hypovolemic stimulus. However, the opposite was presented in VVS women, highlighting fundamental differences in cardiovascular regulation.

Grant Funding Source: Supported by the Swedish Heart and Lung Foundation
Do kappa opioid receptors regulate cardiovascular responses to severe haemorrhage in conscious newborn lambs? (859.3)
Authors: Smith Francine , Samhan Mohamed , Qi Wei
Published: June 2021

Previously, we showed that activation of kappa opioid receptors (KORs) influences systemic haemodynamics and the baroreflex control of heart rate (HR) in conscious lambs. The endogenous ligand for KORs, Dynorphin, is increased in response to severe blood loss however, any role for KORs in the haemodynamic responses to haemorrhage in the newborn is not known. In the present study, mean, systolic and diastolic arterial pressures (MAP, SAP, DAP) as well as HR were measured for 30 min before (Control) and 60 min after 30% haemorrhage in conscious lambs (aged ~one week, N=7) after pretreatment with vehicle (experiment one) or the selective KOR antagonist, GNTI (experiment two). After vehicle, MAP fell from 75±5 (Control) to 48±9 mmHg by 10 min after haemorrhage and remained decreased. This response resulted from a decrease in both SAP and DAP and was not altered by pretreatment with GNTI. HR decreased from 189±29 (Control) to 139±30 beats/min 10 min after haemorrhage; this response was attenuated by GNTI. Therefore, KORs do not appear to influence the blood pressure response to severe blood loss early in life but do abrogate the HR response to haemorrhage.

Grant Funding Source: Supported by the CIHR.
Transient receptor potential vanilloid 2 is a mediator of left ventricular hypertrophy (862.5)
Authors: Rubinstein Jack , Naticchioni Mindi , Gao Xu , Koch Sheryl , Lasko Valerie , Lorenz John
Published: June 2021

Background: Impaired calcium handling has been implicated in the development of left ventricular hypertrophy (LVH) and subsequent development of heart failure (HF). We have recently shown that the calcium channel Transient Receptor Potential Vanilloid 2 (TRPV2) is present in cardiomyocytes and regulates contractility on a beat to beat basis. This study was performed to elucidate if TRPV2 also modulates the development of LVH in a pressure overload mouse model via transverse aortic constriction (TAC).

Methods: WT and TRPV2‐/‐ mice were subjected to pressure overload via TAC and followed echocardiographically for 8 weeks. A subgroup of WT mice were treated with a nonspecific TRPV2 antagonist (tranilast) for the same amount of time. At the conclusion, the hearts were collected for molecular and histological analysis of hypertrophy.

Results: There was significantly less hypertrophy in TRPV2‐/‐ and in the WT mice treated with tranilast (n=5 and 2) in comparison to untreated WT mice (n=7) as noted via heart weight: body weight ratio and LV mass via echocardiography (P<0.05; TRPV2 vs. untreated WT). Furthermore, there was a trend towards improved contractility and less LV cavity dilation in the TRPV2‐/‐ and the WT treated mice in comparison to untreated mice.

Conclusions: We have shown that TRPV2 modulates the development of LVH and is a potential target for the prevention and treatment of LVH and development of HF.
Changes in mTOR/4‐EBP1 pathway induced by a novel mutation in PRKAG2 gene (864.7)
Authors: Xu Yanchun , Tseng Yi‐Tang , Padbury James , Kobashigawa Laura C. , Phornphutkul Chanika , Hardie D. Grahame
Published: June 2021

Objective: The genetics of hypertrophic cardiomyopathy (HCM) is heterogeneous. Mutations in PRKAG2 have been implicated in some previous cases of HCM. PRKAG2 encodes the γ2 isoform of AMPK, a heterotrimeric enzyme with major roles in regulation of energy metabolism in response to cellular stress. We have identified a K475E mutation which results in a neonatal HCM phenotype. We have demonstrated that overexpressing the K475E mutant in HEK‐293 cells leads to 1) a markedly increase in the basal phosphorylation of T172 and associated kinase activity; 2) a reduction in the sensitivity to AMP in allosteric activation. The aim of this study was to investigate changes in downstream signaling pathways induced by the mutation.

Methods: Primary dermal fibroblast cultures of control and a patient with the K475E mutation were maintained under standard cell culture condition. Cell lysates were subject to Western blotting for downstream factors, including Akt, p70S6K, and 4E‐BP1.

Results: K475E mutation results in an increase in the activation of p‐P70S6K as evidenced by increased phosphorylation at both the critical phosphorylation sites (T389 and T421/S424). Fibroblasts from the K475E mutation also show increases in phosphorylation of 4E‐BP1 (S65 and T70).

Conclusions: Our study shows K475E mutation leads to activation of a signaling pathway that favors protein synthesis/growth, providing a logical mechanism for the HCM phenotype observed.

Grant Funding Source: NIH
Novel BK channel regulatory mechanism by protein kinase C in guinea pig urinary bladder smooth muscle (865.1)
Authors: Hristov Kiril , Parajuli Shankar , Smith Amy , Malysz John , Petkov Georgi
Published: June 2021

Large conductance Ca2+‐activated K+ (BK) channels are critical regulators of urinary bladder smooth muscle (UBSM) function. Here, we provide a novel mechanistic insight into BK channel regulation by protein kinase C (PKC) in UBSM. Voltage‐clamp experiments showed that pharmacological activation of PKC with phorbol 12‐myristate 13‐acetate (PMA), inhibited the spontaneous transient BK currents (TBKCs) in native freshly‐isolated guinea pig UBSM cells. Current‐clamp recordings revealed that PMA significantly depolarized UBSM membrane potential, and reduced the spontaneous transient hyperpolarizations in UBSM cells. The PMA inhibitory effects on UBSM membrane potential were abolished by the selective BK channel inhibitor paxilline and were not observed with its inactive analog, 4‐alpha‐PMA. PMA did not affect the amplitude of the whole cell steady‐state BK current or single BK channel open probability (recorded in cell‐attached mode) upon inhibition of all major Ca2+ sources for BK channel activation with thapsigargin, ryanodine, and nifedipine. PMA elevated the intracellular Ca2+ levels in UBSM cells, and increased spontaneous phasic and nerve‐evoked contractions of UBSM isolated strips. The results support the concept that PKC activation leads to a reduction in BK channel activity in UBSM via a Ca2+‐dependent mechanism, thus increasing UBSM contractility.

Grant Funding Source: Supported by R01 DK084284 to G. V. Petkov
PKC‐dependent inhibition of MAP kinase activity and caldesmon phosphorylation in rabbit bladder smooth muscle (865.14)
Authors: Wei Dongyu , Sivilich Sarah , Moreland Robert
Published: June 2021

Smooth muscle contraction is primarily regulated by myosin light chain (MLC) phosphorylation although thin filament proteins, specifically caldesmon, may also be involved. Our previous studies have shown that in vascular smooth muscle, p42 mitogen activated protein kinase (MAPK) but not p44 MAPK activity was enhanced in the presence of an inhibitor of PKC, bisindolylmaleimide‐1 (Bis). The goal of this study was to determine if this is a vascular‐specific pathway or a pathway common to all smooth muscles. Experiments were performed using carbachol‐stimulated rabbit bladder smooth muscle in the presence and absence of Bis; p42 and p44 MAPK activity and caldesmon phosphorylation were measured at different time points during the contraction. Our results showed that carbachol‐dependent p42 MAPK activity is enhanced in the presence of Bis with a concomitant increase in caldesmon phosphorylation in bladder smooth muscle. Carbachol dependent force generation increased concurrently with p42 MAPK phosphorylation which was reduced by inhibition of PKC during the entire contraction; a result not found in vascular smooth muscle. Together with our previous results, this novel signaling pathway appears to be general to both vascular and non‐vascular smooth muscles, however the effects on force are different. Future studies will be aimed at determining the mechanism by which inhibition of PKC increases p42 MAPK activity.

Grant Funding Source: NIH DK 85734
Olfactomedin 2, a novel regulator for transforming growth factor‐β‐induced smooth muscle differentiation (866.1)
Authors: Shi Ning , Chen Shi‐You
Published: June 2021

Olfactomedin 2 (Olfm2) is a developmentally regulated gene and mediates the early nervous system development. The role of Olfm2 in smooth muscle cell (SMC) differentiation, however, remains elusive. In this study, we found that Olfm2 was induced in TGF‐β‐stimulated SMC differentiation of human embryonic mesenchymal cells. Olfm2 knockdown suppressed TGF‐β‐induced expression of SMC markers including smooth muscle α‐actin (α‐SMA), SM22α and smooth muscle myosin heavy chain (SMMHC). Olfm2 overexpression, on the other hand, promoted SMC marker expression. TGF‐β induced Olfm2 nuclear accumulation, indicating that Olfm2 may function as a nuclear factor and be involved in transcriptional activation of SMC marker genes. Indeed, Olfm2 regulated SMC marker promoter activity in a serum response factor (SRF)‐CArG dependent manner. Mechanistically, Olfm2 physically and functionally interacted with SRF, but did not alter its binding affinity to CArG elements. Instead, Olfm2 facilitated the interaction between myocardin and SRF, which is required for SMC marker gene transcription. Taken together, our data demonstrate that Olfm2 is a novel regulator for SMC differentiation and regulates SMC differentiation through enhancing the interaction of myocardin with SRF.
Effects of S1P on endothelial cell reattachment on de‐cellularized HUV scaffold for vascular tissue engineering (867.13)
Authors: Chen Wei‐Min , Lee Hsinyu , Lu Jen‐Her , Yao Chao‐Ling
Published: June 2021

Cardiovascular disease (CVD) is the leading cause of mortality and morbidity. Choosing a proper material for bypass grafting is critical. In this study, we use de‐cellularized human umbilical veins (HUV) as natural allograft scaffolds. Cells were de‐cellularized by using sodium dodecyl sulfate (SDS) and incubated the veins with medium containing 12% FBS to remove residual nucleic acids. After de‐cellularization, human umbilical vein endothelial cells (HUVECs) and endothelial progenitor cells (EPCs) were then seeded on HUV in the presence of 1μM sphingosine 1‐phosphate (S1P) in rotational cultural conditions. S1P enhances cells attachment and reduces extracellular matrix exposure, which may cause blood clotting. Coagulation tests also showed that S1P enhanced re‐cellularized HUV scaffolds are potentially suitable for vascular grafts.
Periaortic adipose tissue promotes arterial stiffness in chow fed low‐density lipoprotein receptor deficient mice (867.4)
Authors: Fleenor Bradley , Du Bing , Eng Jason , Wimsatt Jeffrey
Published: June 2021

We tested the hypothesis that young chow fed low‐density lipoprotein receptor deficient (LDLr‐/‐) mice would have periaortic adipose (PAT) related arterial stiffening, which would be associated with greater aortic remodeling. Compared with wild‐type WT controls (6mo, n=7), LDLr‐/‐ (6mo, n=7) mice had increased arterial stiffness assessed by aortic pulse wave velocity (407 ± 18 vs. 353 ± 13 cm/s, P<0.05) and intrinsic mechanical stiffness (5308 ± 623 vs. 3355 ± 330 kPa, P<0.05). Systolic (144 ± 3 vs. 146 ± 3) and diastolic (113 ± 3 vs. 115 ± 2) blood pressure did not differ between groups. Descending thoracic aortic segments from WT and LDLr‐/‐ mice were cultured with (+) or without (‐) PAT for 72 hours followed by intrinsic mechanical testing. Aortas from LDLr‐/‐ mice cultured (+) PAT (6092 ± 480 kPa, P < 0.05) had greater intrinsic mechanical stiffness compared with control (+) PAT, control (‐) PAT and LDLr‐/‐ (‐) PAT (3710 ±316, 3794 ± 496, 3473 ± 577 kPa, respectively). LDLr‐/‐ mice had reduced aortic α‐Elastin protein expression (0.6 ± 0.1 vs. 1.0 ± 0.1 AU, P<0.05), and greater advanced glycation endproducts (1.8 ± 0.2 vs. 1.0 ± 0.1 AU, P<0.05) and collagen I expressions (2.3 ± 0.2 vs. 1.0 ± 0.2 AU, P<0.05) compared with WT animals. Collectively, these data provide initial evidence for PAT in arterial stiffening, which is associated with greater aortic remodeling in chow fed LDLr‐/‐ mice.
Interleukin‐8 stimulates CFTR‐mediated mucus secretion by submucosal glands in swine (869.10)
Authors: Hansvall Haylee , Ianowski Juan
Published: June 2021

Cystic Fibrosis (CF) is an autosomal recessive condition caused by mutations in the gene encoding the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) anion channel. As a result epithelial fluid and ion transport is compromised. Many organs are affected in CF patients, but most of the mortality and morbidity arise from airway disease characterized by chronic infection and inflammation. However, the events leading from CFTR mutation to airway disease are not fully understood. Recent data suggest that abnormal bacteria clearance due to abnormal mucus secretion is the pathogenic event that triggers airway disease in CF. Thus we studied the response of airway epithelia to proinflammatory cytokines, such as Interleukin‐8 (IL‐8), that are released in response to bacterial infection and are elevated in CF airways.

Secretion assays and electrophysiological techniques were used to measure the response of submucosal glands, the principal mucus source, to IL‐8. The mucus secretion rate increased from 0.052 to 0.156 nl/min upon the addition of IL‐8 (50 ng/ml). IL‐8 stimulated Na+ flux across the serous acini of the submucosal glands. Both effects were blocked by treatment with the CFTR inhibitor, CFTRinh172. Exposure to CFTRinh172 reduced IL‐8‐stimulated mucus secretion to 0.062 nl/min and abolished Na+ transport across the serous acini.

Our results suggest that, in normal lungs, bacteria‐activated secretion increases mucus and bactericidal compounds in the airways, thereby facilitating bacterial removal by mucociliary clearance and reducing the proinflammatory stimulus. In CF airways, bacteria would activate proinflammatory signalling to trigger innate immune responses, but CFTR‐mediated mucus secretion and the resulting “bacterial flush” would be missing, leading to sustained inflammation that may permanently alter the physiology and anatomy of the airways.

Grant Funding Source: Supported by APS, University of Saskatchewan, NSERC, Cystic Fibrosis Canada
Neuroepithelial bodies as airway hypoxia sensors: re‐evaluation of the concept in different mouse live cell imaging models (869.6)
Authors: Schnorbusch Kathy , Brouns Inge , Lembrechts Robrecht , Pintelon Isabel , Kemp Paul , Timmermans Jean‐Pierre , Adriaensen Dirk
Published: June 2021

In pulmonary neuroepithelial bodies (NEBs), acute hypoxia has been suggested to close O2‐sensitive K+ channels, resulting in membrane depolarization, voltage‐gated Ca2+ entry and subsequent transmitter release.

To visualize this mechanism in NEBs, our established live cell imaging (LCI) model, based on lung vibratome slices, was applied. Real‐time changes in different cellular processes were monitored during hypoxic challenge in NEBs of postnatal, prenatal and GAD67‐GFP mice.

As a proof of concept, LCI experiments were performed on isolated mouse carotid body, proving that our LCI set up is indeed capable of visualizing hypoxia‐induced changes. However, in none of the NEBs studied in the lung slice models, severe (2% O2), mild (12% O2) or intermittent hypoxia, resulted in a rise in intracellular Ca2+ ([Ca2+]i) or subsequent neurotransmitter release. Moreover, low O2 levels appeared to decrease NEB cell excitability. The observed hypoxic mitochondrial membrane depolarization and increased ROS production rather seemed to be part of a general cellular defense mechanism, as supported by the non‐selective and non‐functional expression of the key O2 sensing protein NADPH oxidase.

All together, our data argue for a reconsideration of the proposed straightforward hypoxic signal transduction pathway in pulmonary NEBs.

Grants: FWO (G.0589.11), Hercules Foundation (AUAH‐09‐001); UA (GOA BOF 2007)
In vitro elastogenesis (87.1)
Authors: Hinderer Svenja , Shen Nian , Ringuette Léa‐Jeanne , Hansmann Jan , Reinhardt Dieter , Elaine Davis , Schenke‐Layland Katja
Published: June 2021

Background: Elastic fibers are one of the main components of the extracellular matrix (ECM), providing dynamic tissues such as lungs, skin, blood vessels, heart valve leaflets and ligaments with flexibility and elasticity. However, the complex process of elastic fiber assembly is poorly understood und the generation of elastic fibers on a human base is a missing link in tissue engineering.

Methods: A constant shear stress providing, fluid flow bioreactor system was designed and evaluated. For in vitro studies, human vascular smooth muscle cells (VSMCs) were seeded onto three‐dimensional (3D) electropsun scaffolds and cultured in the bioreactor system. After 6 days dynamic culture, the scaffolds were harvested and analyzed regarding elastin gene and protein expression. The produced ECM was investigated applying immunostaining of elastogenesis‐associated proteins and transmission electron microscopy (TEM).

Results: After a 6 day dynamic culture of VSMCs on 3D scaffolds, a significant increase of elastin gene and protein expression was detected when comparing to the 2D cultures and static controls. Moreover, TEM confirmed a proper developed ECM similar to native fetal tissue, including long bundels of microfibrils, collagen and elastin. This is the first report showing developing elastic fibers as well as the availability of elastogenesis‐associated proteins on a human‐based tissue engineered construct.