bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of recombinant traceable analogs of the parathyroid hormone
Authors: Marceau François , Charest‐Morin Xavier , Bawolak Marie‐Thérèse , Lodge Robert , Allaeys Isabelle , Poubelle Patrice E. , Fortin Jean‐Philippe
Published: June 2021

We hypothesized that parathyroid hormone (PTH) fragment1–34 might be C‐terminally extended with biotechnological cargoes for cell imaging. Firstly, a fusion protein composed of the preproinsulin signal peptide, PTH1–34 and enhanced green fluorescent protein (PTH1–34‐EGFP) was generated. Secondly, prepro‐PTH1–84 C‐terminally fused with the myc epitope (PTH1–84‐ myc) was exploited. Both mature PTH forms were produced as conditioned media (CM) of HEK 293a cells (~1.5 and 187 nM, ELISAs for GFP and PTH, respectively). Both types of CM induced the expression of c‐Fos (1–3 h) and of a reporter gene controlled by cyclic AMP response elements (6 h). In recipient HEK 293a cells expressing PTH1R receptors (PTH1R), PTH1–34‐ EGFP CM or PTH1–84‐myc CM (supplemented with a fluorescent anti‐myc tag antibody [Ab]) labeled endosomes positive for Rab5 and β‐arrestin1 (microscopy, cytofluorometry). MG‐63 osteoblastoid cells also internalized the PTH1–84‐myc‐Ab complex. Ex vivo, murine cartilage took up PTH1–84‐myc in cytosolic inclusions reactive with the anti‐myc Ab (paraffin sections). PTH1–34‐EGFP and PTH1–84‐myc are bona fide agonists that support the feasibility of transporting large cargoes inside cells via PTH1Rs.
Functional selectivity of ligands at the dopamine D2L dopamine receptor
Authors: Brust Tarsis , Watts Val
Published: June 2021

The dopamine D2 receptor (D2R) is an important drug target that exerts its functions through several signaling pathways. It has been previously shown that different ligands can activate these signaling pathways with distinct potencies and efficacies (i.e. functional selective ligands). Additionally, recent studies suggest that selective activation of individual D2R signaling pathways may provide safer and more effective drug therapies. The objective of the present study was to develop a D2R cellular model using a single cell line background to assess the activation of different signaling pathways downstream of the D2R. By using a single cell background to assess activation of distinct signaling pathways, we eliminate potential confounds associated with variability in the expression levels of the D2R or other signal transduction proteins involved in receptor signaling. We used CHO‐D2L cells from DiscoveRx in the PathHunter system to detect β‐arrestin‐2 recruitment by the D2R. Additional protocols were developed to assess Gαi/o, Gβγ, and MAPK signaling. The ligands tested include many previously characterized D2 receptor modulators that are currently used for treatment of Parkinson's disease and schizophrenia (e.g. pramipexole and aripiprazole). Using this approach we have discovered new examples of previously unappreciated functionally selective ligands (supported by MH60397 and BBRF).
Novel Positive Allosteric Modulators Bias Acetylcholine Signaling at Human M4 Muscarinic Receptors
Authors: Klein Michael Thomas , Digby Gregory , Niswender Colleen , Tarr Christopher , Poslusney Michael , Melancon Bruce , Wood Michael , Lindsley Craig , Conn P. Jeffrey
Published: June 2021

M4 muscarinic acetylcholine receptors (mAChRs) are heavily expressed in the CNS, and activation of these receptors is thought to mediate the antipsychotic effects of the M1/M4‐preferring agonist xanomeline. Clinical trials have shown xanomeline to be highly effective in treating the positive symptoms, negative symptoms, and cognitive deficits of schizophrenia as well as Alzheimer's type dementia. As a consequence, much interest has been placed on developing M4‐selective drugs for the treatment of various forms of psychosis. To achieve robust selectivity, our group has identified M4‐selective positive allosteric modulators (PAMs); these compounds have shown robust efficacy in animal models used to predict antipsychotic efficacy. In this study, we examined the ability of M4‐selective PAMs to differentially modulate several intracellular signaling pathways (ERK phosphorylation, GIRK channel activation, arrestin recruitment) and differentially desensitize G‐protein‐mediated responses. Interestingly, multiple PAMs have the ability to bias the signaling of acetylcholine towards specific responses and have profound and differential effects on M4 receptor β‐arrestin recruitment. These data indicate that M4 allosteric ligands engender receptor conformational changes and signaling outcomes not previously described for GPCR orthosteric ligand interactions. Identification of signal bias and differential desensitization of M4 receptors may be important for therapeutic actions of these compounds and will guide future M4 receptor drug development efforts. Future efforts will concentrate on signaling properties that correlate with robust antipsychotic efficacy in vivo.
Insights into Selectivity from Rationally Designed Inhibitors of G protein‐coupled Receptor Kinase 2
Authors: Homan Kristoff , Wilson Michael , Larsen Scott , Tesmer John
Published: June 2021

No small molecule inhibitors selective for G protein‐coupled receptor kinase 2 (GRK2), a validated target for the treatment of heart failure, have advanced to clinical trials. Through the development of a high throughput GRK2‐RNA displacement assay, over 500,000 potential inhibitors have been evaluated. One potential inhibitor of GRK2 of particular interest is the selective serotonin reuptake inhibitor paroxetine. We have shown that paroxetine is capable of inhibiting GRK2 with up to 50‐fold selectivity over other GRKs and mediates increases in contractility in both isolated cardiomyocytes and whole animal studies. Analyses of additional compounds have contributed to the development a structure activity relationship model for compounds based on the paroxetine scaffold. A crystal structure of GRK2 in complex with paroxetine has been used to rationally design derivatives of paroxetine, which effectively decouples the SSRI and kinase inhibition functions of paroxetine without major changes to the scaffold. A newly obtained crystal structure of GRK2 in complex with a paroxetine derivative confirms the feasibility of rational design with the GRK2·paroxetine scaffold. Additional kinetic characterization of derivatives of paroxetine and newly obtained crystallographic complexes containing inhibitors suggest the underlying molecular mechanisms of inhibitor selectivity among AGC family protein kinases.
Differences in β‐arrestin dependent signaling by PAR2 and NK1R are directed by their C terminal tails
Authors: Pal Kasturi , Defea Kathryn
Published: June 2021

PAR2 and NK1R are 7 transmembrane receptors (7TMRs), which signal by Gαq leading to Ca2+ release and protein kinase C (PKC) activation. Both receptors are desensitized by β‐arrestin binding to their C termini. They can also activate ERK1/2 through β‐arrestin scaffolding complexes. They differ in ERK1/2 mediated physiological outcomes: cell migration versus proliferation. Using β‐arrestins, PAR2 activates cofilin, to promote chemotaxis. We hypothesized that the differences in β‐arrestin dependent signaling by 7TMRs can be attributed to how the molecular scaffolds bind the C‐tail of the receptors. Using wild type and C‐tail chimeras, we showed that the rate of desensitization, internalization, subcellular localization post endocytosis and ERK1/2 mediated responses depend on the nature of interaction of β‐arrestins with the C‐termini. BRET assays showed that PAR2 recruits both β‐arrestin 1/2 faster and with greater affinity than NK1R. We further show that initial Gαq signaling is necessary for β‐arrestin 1/2 recruitment to NK1R. PAR2 recruits β‐arrestin 1/2 even when Gαq pathway is blocked. Assays with C terminal phosphorylation mutants of PAR2 indicated that, phosphorylation of certain residues are necessary for β‐arrestin 1/2 recruitment. Phosphorylation at putative PKC sites determine stability of PAR2‐β‐arrestin 1/2 complex. This in turn ensures downstream cofilin activation and cell migration
Amino acids within intracellular loop 3 of the D2 dopamine receptor are necessary for arrestin binding and receptor internalization
Authors: Clayton Cecilea C , Neve Kim A
Published: June 2021

Arrestins facilitate dopamine D2 receptor internalization and desensitization and are important mediators of dopaminergic behaviors. Using bioluminescence resonance energy transfer (BRET) to measure receptor‐mediated recruitment of arrestin, our objective was to determine the smallest mutation that prevents arrestin binding while retaining signaling through G‐proteins. Mutation of four residues in intracellular loop three (212–215, IYIV) abolished arrestin recruitment and internalization, but impaired receptor expression. Mutation of three (YIV) or two (IV) residues reduced arrestin recruitment by forty and thirty percent respectively, without impairing receptor expression. Importantly, mutating three residues rendered the receptor incapable of internalization, even when over‐expressing arrestin3 and GRK2, while sparing the receptor's ability to signal through G‐proteins. Thus, mutation of the sequence YIV in IC3 of the D2 receptor selectively impaired arrestin recruitment and abolished receptor internalization, suggesting that this mutant will be a useful tool for elucidating cellular and behavioral effects of D2 receptor stimulation that are mediated by arrestins. (VHA, MH045372 and DA007262)
Differential β‐arrestin2 requirements of constitutive and agonist‐induced internalization of the CB1 cannabinoid receptor
Authors: Gyombolai Pál , Boros Eszter , Hunyady László , Turu Gábor
Published: June 2021

Internalization of G protein‐coupled receptors is an important process in the regulation of receptor signaling. CB1 cannabinoid receptor (CB1R) internalizes both constitutively and upon stimulation by receptor agonists, however, the mechanisms underlying these processes and the roles of β‐arrestins in the regulation of CB1R function are not completely understood. In this study, we followed CB1R internalization in confocal microscope and by bioluminescence resonance energy transfer measurements. We found that upon activation CB1R binds β‐arrestin2 (β‐arr2), but not β‐arrestin1. Furthermore, both the expression of dominant‐negative β‐arr2 (β‐arr2‐V54D) and siRNA‐mediated knock‐down of β‐arr2 impaired the agonist‐induced internalization of CB1R. In contrast, neither β‐arr2‐V54D nor β‐arr2‐specific siRNA had a significant effect on the constitutive internalization of CB1R. We conclude that upon activation, CB1R binds to β‐arr2, and this binding is required for its agonist‐induced internalization. In contrast, constitutive CB1R internalization is β‐arr2‐independent, showing that the molecular mechanisms underlying these two processes are different.
A Single Lys Residue on the First Intracellular Loop Modulates the Endoplasmic Reticulum Export and Cell‐Surface Expression of α2A‐Adrenergic Receptor
Authors: Fan Yi , Li Chunman , Wu Guangyu
Published: June 2021

Export from the endoplasmic reticulum (ER) represents an initial step in intracellular trafficking of G protein‐coupled receptors (GPCRs). However, the underlying molecular mechanisms remain poorly understood. We have previously demonstrated that a highly conserved Leu residue on the first intracellular loop (ICL1) is required for exit of several GPCRs from the ER. Here we found that, in addition to Leu64 residue in the ICL1, the neighboring positively charged residue Lys65 also modulates the cell‐surface transport of α2A‐adrenergic receptor (α2A‐AR). Mutation of Lys65 to Ala, Glu and Gln significantly attenuated, whereas mutation of Lys65 to Arg strongly augmented α2A‐AR expression at the cell surface. Consistent with the effects on the cell‐surface expression of α2A‐AR, mutation of Lys65 to Ala and Arg produced opposing effects on α2A‐AR‐mediated ERK1/2 activation. Furthermore, confocal microscopy revealed that the α2A‐AR mutant K65A displayed a strong intracellular expression pattern and was extensively co‐localized with the ER marker DsRed2‐ER, suggestive of ER accumulation. These data provide the first evidence indicating an important function for a single Lys residue on the ICL1 in the ER export and cell‐surface expression of α2A‐AR. These data also suggest that the ICL1 may possess multiple signals that control the cell‐surface targeting of GPCRs via distinct mechanisms (R01GM076167).
Cocaine alters cell cycle activity of rat c6 glial cells by altering the expression cell cycle regulatory protein
Authors: Triplett Jennifer , Badisa Ramesh , Goodman Carl B.
Published: June 2021

Astroglial cells seldom receive as much attention as neurons in drug abuse studies involving the central nervous system. Astroglial cells were previously thought to be passive bystanders reduced to only playing a supportive role to neurons. However, increasing understandings of the myriad functions these glial cells actually possess suggest that glial cells may play a more active role in instances of drug exposure. Furthermore the mechanisms by which cocaine uses to elicit its effects on glial cells have yet to be elucidated. This present study uses rat astroglioma cells to investigate the effects cocaine has on cell viability using a Crystal Violet Dye Uptake Assay; analyze cell cycle activity using flow cytometry; and finally the expression of cell cycle regulatory proteins using an anti‐body microarray. It is expected that cocaine will decrease rat astroglioma viability in a dose‐dependent manner. It is also expected that cocaine will cause alterations the expression of specific cell cycle regulatory proteins which will subsequently result in disruptions in the normal progression of the cell cycle in these astroglioma cells. These results may clarify how cocaine elicits its effects on the cell cycle of glial cells. Research supported by NCRR/RCMI G12 RR03020 and G12 MD007582–28
Involvement of 5‐HT1A receptors into the dorsal hippocampus in the hypophagic effect caused by fluoxetine in rats
Authors: Scopinho América Augusto , de Aguiar Corrêa Fernando Morgan , Resstel Leonardo Barbosa Moraes
Published: June 2021

Aims

Studies showed changes in feeding behavior after lesions of the dorsal hippocampus (DH) suggesting the involvement of this area in the modulation of food intake. Serotonin has been known to play an important role in food intake regulation and the hippocampus receives serotoninergic innervation from the median raphe nucleus. So, the present study examined the effects of the serotonin reuptake inhibitor (fluoxetine) into the DH on food intake and the serotoninergic receptors mediating its effect.

Methods

Fed and fasted (18 h food‐deprived) male Wistar rats were used. Cannulae were implanted into the DH for injection of drugs: 1) vehicle (aCSF, n=6) or fluoxetine (0.2, 2 or 20 nmol/100 nL, n=6 for each dose); 2) 5‐HT1A receptor antagonist (WAY‐100635, 0.3 nmol/100 nL, n=5) or aCSF (n=5) and fifteen minutes later they received fluoxetine (2 nmol/100 nL) or aCSF. Fifteen minutes later they were submitted to the food intake test (1 hour).

Results

Our results showed that microinjection of fluoxetine into the DH evoked hypophagic effects in fasted rats (F3,40=78.5, P<0.0001) with a correlation between the doses and reduction in food consumption (r2=0,94, df=14). The microinjection of WAY‐100635 blocked the hypophagic effect caused by fluoxetine into the DH (F=22,5 P<0.0001).

Conclusion

These results suggest that 5‐HT1A receptors in the DH modulate the hypophagic effect caused by local DH fluoxetine administration.
Elevation of lysosomal pH leads to the release of Ca2+, ATP and cytokines from RPE cells
Authors: Mitchell Claire H , Guha Sonia , Beckel Jonathan M , Laties Alan M
Published: June 2021

The modulation of lysosomal pH can influence the degradation of cellular material delivered through autophagic or phagocytotic pathways. In retinal pigmented epithelial (RPE) cells, lysosomal alkalinization impairs both pathways, while drugs identified to reacidify lysosomes can enhance the clearance of this material. In addition to aiding in degradation, however, lysosomes also serve as a storage site for signaling molecules such as Ca2+, ATP and cytokines. We thus asked whether lysosomal alkalinization led to the release of these compounds in RPE cells. Lysosomal pH was elevated by bafilomycin, chloroquine or the P2X7 receptor agonist BzATP. Cytoplasmic Ca2+ was increased by bafilomycin or chloroquine, but was this rise was lessened by treatment to reacidify lysosomes. Bafilomycin or chloroquine also led to a rapid rise in extracellular levels of ATP, consistent with vesicular release of ATP. While release of cytokine IL‐6 was triggered by bafilomycin or chloroquine, BzATP was more effective than either. IL‐6 release following BzATP stimulation was rapid, sustained, dependent on extracellular Ca2+ and blocked by P2X7R antagonists. In conclusion, elevation of lysosomal pH in RPE cells influences both the degradation of waste material and the release of Ca2+, ATP and cytokines. Whether these release pathways are compromised by chronic elevation of lysosomal pH remains unknown.
Role of the Krebs Cycle Metabolites in Retinal Angiogenesis
Authors: Duhamel François , Zaniolo Karine , Tahiri Houda , Chemtob Sylvain
Published: June 2021

Purpose

Retinopathy of prematurity (ROP), a major cause of blindness in developed countries, occurs in two phases : the cessation of normal eye development after birth and a subsequent abnormal and exaggerated vessel growth. Peripheral hypoxia occur after the first phase in premature eye and induce production of many growth factors implicated in this second destructive neovascularization phase. Our laboratory has previously demonstrated the role of succinate/GPR91 in the modulation of retinal vessel growth. Here, we investigated the role of α‐ketoglutarate (α‐KG) and its cognate receptor GPR99 in retinal angiogenesis.

Methods

Effects of α‐KG on developmental retinal vascularization were assessed following intravitreal injection of Sprague‐Dawley rat pups in development. Moreover, the effects of a siGPR99 knockdown following intravitreal injection were investigated in the neovascular phase of ROP. Endogenous expression of retinal GPR99 was examined in Sprague‐Dawley rat retinas by immunohistochemistry. In vitro, we investigated the expression of GPR99 and pro‐angiogenic factors by Western blot and PCR analysis. The ability of α‐KG to promote vessel sprouting was determined by aortic rings cultured.

Results

α‐KG significantly enhanced developmental vascular densities at different time points. Neovascularization was blocked by a siGPR99 and allowed normal vessel growth in ROP. Moreover, GPR99 was robustly expressed in RGCs as confirmed by co‐labeling with neuron markers and, in vitro, mainly in neuronal cells. Conditioned media from α‐KG stimulated‐neurons induced aortic rings sprouting, in correlation with an up‐regulation of pro‐angiogenic factors expression.

Conclusions

Our results disclose a pro‐angiogenic role for α‐KG and its receptor GPR99, providing additional supports for the involvement of metabolite signalling in ischemic conditions.
Regulation by RhoA of Rod photoreceptor axon retraction induced by retinal detachment
Authors: Wang Weiwei , Townes‐Anderson Ellen
Published: June 2021

Rod photoreceptors, in response to injury, display synaptic plasticity. We are interested in rod axon retraction, which occurs after retinal detachment and results in disconnection of the synapse between photoreceptors and secondary neurons of the visual system.

Our previous studies demonstrated that inhibition of RhoA and its downstream effecter Rho kinase (ROCK) blocks rod axon retraction. Here, we used time‐lapse microscopy to measure axon retraction of isolated salamander rod cells with and without inhibition of another effecter of ROCK, LIM kinase. A LIMK inhibitor (LIMKi) also blocks retraction: 35% decrease of initial rod axon length in controls after 7 hours, compared with only a 15% decrease using 10uM LIMKi.

Since LIMK regulates actin filament turnover through cofilin, we examined cofilin activity in pig retina with western blots. After retinal detachment, phosphorylated cofilin increased over 24 hours; treatment with LIMKi prevented the increases.

Nicardipine (Nc), a Ca channel blocker, also blocked retraction in our previous work, possibly by reducing myosin light chain (MLC) activity. Experiments with isolated rod cells showed that over 3 days LIMKi was no longer effective; however, with Nc, retraction continued to be inhibited.

Thus we suggest that rod axon retraction is a result of both Ca‐MLC regulated actomyosin contraction and ROCK‐LIMK‐cofilin regulated actin filament depolymerization.
Dietary Regulation of Mouse Intestinal P450 Expression and Drug Metabolism
Authors: Zhang Qing‐Yu , Zhang Peng , Jia Kunzhi , Fang Cheng , Zhou Xin , Ding Xinxin
Published: June 2021

To determine the impact of dietary changes on intestinal P450 expression and drug metabolism, we studied wild‐type (WT) and intestinal epithelium (IE)‐specific P450 reductase (CPR) knockout (IE‐Cpr‐null) mice, fed either with a regular chow or with a synthetic diet devoid of phytochemicals. A change from regular chow to the synthetic diet diminished expression of CYP1A, 2B, 2C, and 3A in the small intestine (SI), and of CYP2B, 2C, and 3A in liver, of both WT and IE‐Cpr‐null mice; it also decreased microsomal midazolam‐hydroxylase activity in vitro and first‐pass clearance of midazolam in vivo. However, compensatory increase in SI P450 expression still occurred in IE‐Cpr‐null, compared to WT, mice, on the synthetic diet. The dietary change, but not Cpr deletion, caused large decreases in bile acid (BA) levels in plasma, liver, SI, and intestinal content. Furthermore, the diet change in WT mice led to increases in SI expression of FGF15, an enterohepatic regulator of hepatic BA synthesis, and a decrease in hepatic expression of CYP7A1, a key enzyme in BA synthesis. These results highlight the vital role of diet in maintaining adequate expression of major drug‐metabolizing P450s in the digestive tract, and suggest potential involvement of FGF15 and BA signaling in the regulatory mechanisms. (Supported in part by NIH grant GM082978)
Prostacyclin Release from Stem Cell Derived Endothelial Cells
Authors: Reed Daniel Mark , Foldes Gabor , Kirkby Nicholas S , Gashaw Hime H , Starke Richard D , Paschalaki Koralia E , Randi Anna M , Harding Sian E , Mitchell Jane A
Published: June 2021

Stem cell‐derived endothelial cells are being investigated as potential therapies. Endothelial cells for therapy can be isolated from human embryo (hESC‐EC), circulating progenitors (EPC) or induced pluripotent stem cells (iPS‐EC). Endothelial cells in vessels release the cardioprotective hormone prostacyclin, which is essential for organ homeostasis and a function to be preserved in organ regeneration programs. Prostacyclin is formed from arachidonic acid by the concerted actions of cyclooxygenase (COX) and prostacyclin synthase. Here we have compared the ability of endothelial cells derived from key stem cell phenotypes to release prostacyclin with adult endothelial cells from the lung (HMVEC). hESC‐EC, EPC (late outgrowth), iPS‐EC and HMVEC were treated for 24h +/− LPS (1μg/ml) or IL‐1β (1ng/ml) to activate prostacyclin release. Prostacyclin was measured as its breakdown product 6‐ketoPGF1α by ELISA. Endothelial cells from different phenotypes of stem cells are capable of releasing prostacyclin. The relative efficacy for prostacyclin release was EPC>;HMVEC>;iPS‐EC>;hESC‐EC (Figure 1). These observations show that, of the stem cell progenitors available, EPC‐derived endothelial cells have the greatest propensity to release prostacyclin. These observations may have important relevance in the use of engineered endothelial cells.

This work was funded by the Welcome Trust, BHF and an MRC studentship awarded to DMR.
Radioprotective and Radiomitigative Efficacy of Flt3 Ligand in a Murine Model of Acute Radiation Injury.
Authors: Thomas Lawrence J. , Boyer James M. , Round Sarah M. , Naylor Sarah R. , Forsberg Eric M. , Borrelli Kathleen M. , Pilsmaker Catherine D. , Gergel Lauren E. , Marsh Henry C. , Keler Tibor
Published: June 2021

Radiation exposure can occur either intentionally or unintentionally and few drugs have been shown to be effective in mitigating or treating the damaging effects of radiation. Flt3 Ligand (Flt3L, fms‐like tyrosine kinase 3 ligand, CDX‐301) is a hematopoietic cytokine which stimulates the proliferation and differentiation of various blood cell progenitors. We sought to determine the efficacy of Flt3L monotherapy on survival at 30 days following total body irradiation at a LD50 level (LD50/30). C57BL/6 mice were exposed to a single total body gamma radiation dose of approximately 776 cGy from a 137Cs source. Flt3L was administered subcutaneously for 10 sequential daily 25 μg doses beginning 24 hours before irradiation, or 4 or 24 hours after irradiation. Mice were observed twice daily to monitor for morbidity and mortality for 30 days post‐irradiation and animals were euthanized based on criteria approved by the IACUC. As anticipated, 45% of untreated animals did not survive to day 30, where no animals treated with Flt3L s.c. beginning 24 hours before irradiation did not survive to day 30 (p≤0.0001). If Flt3L treatment was started 4 hours after irradiation, only 10% of mice did not survive at day 30 (p≤0.0107) and if treatment was started 24 hours after irradiation 22% did not survive to day 30 (p≤0.0898). In conclusion, data suggest that Flt3L monotherapy can serve as an effective radioprotectant and radiomitigant.
Implementation of a Large‐Scale Arterial System Model to Enhance Collaborative Research in Pulsatile Hemodynamics
Authors: English Ryan C. , Hanna Marina , Mohiuddin Mohammad W. , Quick Christopher M.
Published: June 2021

The large‐scale Westerhof model (1968) has become a standard research tool for investigating how changes in radii and compliances of systemic conductance arteries affect regional pulsatile blood pressures and flows. Its evaluation requires the simultaneous solution of a standard set of 121 second‐order partial differential equations, rendering this classical model only accessible to engineers with expertise in fluid dynamics. This tool, however, is invaluable for clinical investigators studying the manifestation of disease states in various patient populations and for physiologists studying the adaptation of arteries to local pressures and flows. We therefore developed an implementation of the Westerhof model that addresses the competing criteria of computational power, ease of use and customizability. To represent the anatomical arrangement of vessels, we employed Simulink, a powerful computational program with a graphical user interface. The parameters of each vessel can be altered independently, allowing the customizability necessary to simulate responses to localized diseases (e.g., aortic coarctation). Alternatively, sets of parameters can be selected to simulate patient populations (e.g., elderly). By making it possible for engineers, physiologists, clinical investigators, and even students to use the same tool, we aim to enhance both multidisciplinary collaboration and education.
The impact of maternal in utero environment on salt‐induced hypertension in the SS rat
Authors: Geurts Aron M , Skelton Meredith , Kurth Terry , Klotz Jason , Grzybowski Michael , Mattson David , Liang Mingyu , Cowley Allen W.
Published: June 2021

The SS rat is an established model of salt‐sensitive human hypertension. We previously demonstrated that SS animals from parents fed a grain‐based diet are less susceptible to salt‐induced elevations in blood pressure and associated renal damage than SS animals fed a casein‐based protein diet. Others have shown that transfer of SS strain embryos into genetically unrelated recipients can also attenuate salt‐induced blood pressure changes. We hypothesized, therefore, that differences in dietary‐induced susceptibility may be driven by the in utero environment of pregnant females fed these different diets. Using the closely related SS/JrHsdMcwi strain (SS/Mcw), maintained on a purified casein diet, and the SS/JrHsdMcwiCrl (SS/Crl) which is maintained on a grain diet, we addressed this hypothesis by performing embryo transfers between strains and phenotyping the offspring. Compared to control male rats (n=6) generated by the transfer of SS/Mcw embryos into SS/Mcw females, males (n=9) generated by transfer of SS/Mcw embryos into SS/Crl female recipients demonstrated both significantly reduced mean arterial pressure (154 ±7 mmHg vs. 134±6) and lower urinary albumin excretion (200±19 mg/day vs. 74±14 mg/day) following three weeks of a 4.0% NaCl diet. These data demonstrated that the dietary exposure of pregnant SS female rats can impact the susceptibility of their offspring to salt‐induced hypertension.
Decreased thioredoxin reductase activity in endothelial cells exposed to high glucose is associated with protein tyrosine nitration and interaction with caveolin 1
Authors: Lamoke Folami , Bartoli Manuela
Published: June 2021

Thioredoxin reductase 1 (TrxR1) is an important antioxidant enzyme that controls cellular redox homeostasis. Here we wanted to determine the effects of elevated glucose levels on TrxR1activity in human endothelial cells. Human coronary and aortic endothelial cells (HCAEC and HAEC, respectively) were exposed to high glucose conditions (HG= 25mM) for 5 days and/or iso‐osmotic control (L‐glucose). We found that HG diminished TrxR1 activity (30% reduction, p<0.005), stimulated its association with caveolin‐1 and its subcellular localization to caveolae. Immunoprecipitation analysis revealed that HG‐promoted TrxR1 tyrosine nitration. Pre‐treatments of the cells with the peroxynitrite decomposition catalyst FeTPPS (25μM) partially rescued TrxR1 activity and prevented its interaction with caveolin‐1. FeTPPS also attenuated HG‐induced increases in caspase 3 activity and induction of apoptosis in endothelial cells. Our results suggest that inhibition of TrxR1 activity by HG in human endothelial cells is a result of its nitration and interaction with caveolin‐1. Restoration of TrxR1 activity may be a potentially beneficial therapeutic strategy for the prevention of diabetes‐induced vascular complications.
Adipose Angiotensinogen is Upregulated during Insulin Resistance in OLETF Rats
Authors: Araiza David Nicolas , Minas Jacqueline N. , Rodriguez Ruben , Nakano Daisuke , Nishiyama Akira , Ortiz Rudy M.
Published: June 2021

Activation of the renin‐angiotensin system (RAS) has been shown to contribute to metabolic syndrome. Hyptertension is a key component of metabolic syndrome and is regulated by RAS. The Otsuka Long‐Evans Tokushima Fatty (OLETF) rat is a relatively new and highly relevant model because its pathogenesis of insulin resistance, metabolic syndrome, and type 2 diabetes closely resembles that of the progression of the human condition. However, the contribution of adipose RAS to metabolic syndrome is not well defined. To address the hypothesis that adipose RAS activation is increased with insulin resistance, rats were divided into two groups (n=10–12/group): 1) lean strain‐control LETO and 2) obese insulin resistant OLETF. Systolic blood pressure (SBP) was measured weekly for 24 weeks and insulin resistance index (IRI) was determined at 9 and 24 weeks. Tissue samples were collected from each group (n=5–6/group) at 15 and 24 weeks. Mean SBP from OLETF was 28% and 27% greater compared to that of the LETO after 15 and 24 wk respectively. Mean IRI was greater at both time periods and was exacerbated in 24 wk OLETF compared to 15 wk. By 24 wk, adipose angiotensinogen (angiotensin precursor) expression had increased in 24 wk OLETF suggesting that activation of adipose RAS was increased. The data suggest that increased activation of adipose RAS may contribute to the increased metabolic disorders (SBP and IRI) commonly associated with metabolic syndrome.
A1 Adenosine Receptor Negatively Modulates Coronary Reactive Hyperemia via Counteracting A2A‐mediated H2O2 Production and Opening of KATP Channel in Isolated Mice Hearts
Authors: Zhou Xueping , Teng Bunyen , Mustafa S. Jamal
Published: June 2021

We previously demonstrated (Sanjani et.al. FASEB J 26:863.6, 2012) that A2A but not A2B adenosine receptor (AR) mediates coronary reactive hyperemia (RH). This study aims to investigate the role of A1 and A3 ARs in coronary RH using A1, A3 KO and A1/A3 double KO (DKO) mice. Coronary flow (CF) of isolated hearts was measured using Langendorff system. No change in baseline CF, HR, LVDP, and dP/dt was observed between the KO and wide type (WT) mice. However, A1 KO or A1/A3 DKO, but not A3 KO mice showed a higher CF repayment (9.4 ± 0.24 and 9.1 ± 0.45 vs. 7.1 ± 0.22 ml/g, p<0.05) and longer repayment duration (2.7 ± 0.10 and 2.8 ± 0.20 vs. 1.7 ± 0.1 min, p<0.05) than WT mice. SCH58261 (a selective A2A antagonist, 1 μM) significantly decreased the baseline CF in all groups of mice and attenuated the augmented RH in A1 KO and A1/A3 DKO mice to a similar level as WT. Scavenging H2O2 by catalase (2500 U/ml, Sigma) also eliminated the enhanced coronary RH in A1 KO and A1/A3 DKO mice without additive effect of SCH58261. Moreover, blocking KATP channels by glibenclamide (5 μM) significantly attenuated the baseline CF and abolished the enhanced RH in A1 KO and A1/A3 DKO mice, but to a greater extent than SCH58261 and catalase. Our data suggest that A1 but not A3AR counteracts the A2AAR‐mediated CF increase and deletion of A1AR results in enhanced A2AAR‐mediated H2O2 production, opening of KATP channel, and relaxation of coronary artery during RH. (HL 027339 and 094447)
Paradoxical vasoconstrictive effects of H2S on shear stress‐mediated vasodilation
Authors: Lee Hon‐Chi , Chai Qiang , Lu Tong , Wang Xiao‐Li
Published: June 2021

We examined the role of H2S on shear stress‐mediated dilation of isolated mouse coronary arteries. Na2S produced concentration‐dependent dilation in these vessels, which was significantly inhibited by iberiotoxin. BK currents in mouse coronary smooth muscle cells were directly activated by Na2S, suggesting H2S produced vasodilation through BK channel activation. Using a pressure servo controller system, freshly isolated mouse coronary arteries were subjected to physiological levels of shear stress (1 to 25 dynes/cm2), producing graded dilatory responses, but such effects were diminished in the presence of 100 μM Na2S. Pre‐incubation with the cystathionine γ‐lyase inhibitor, D,L‐propargylglycine (PPG), resulted in a paradoxical augmentation of shear stress‐mediated vasodilation. However, in the presence of L‐NAME or in coronary arteries from eNOS knockout mice, PPG inhibited shear stress‐mediated vasodilation, suggesting an interaction between NO and H2S signaling. Using cultured bovine aortic endothelial cells, eNOS activity was inhibited by Na2S. These results suggest that both NO and H2S are important shear stress‐mediated vasodilators in mouse coronary arteries but there is a complex interaction between these two signaling pathways that results in paradoxical vasoconstrictive effects of H2S through inhibition of NO generation. (Supported by grants from NIH)
Obesity and statins are predictive of enhanced coronary resistance artery dilation in patients undergoing cardiac surgery
Authors: Cassuto James , Feher Attila , Bagi Zsolt
Published: June 2021

The obesity paradox, a poorly understood inverse relationship between body mass index (BMI) and morbidity and mortality, has been described in patients with ischemic heart disease. Bradykinin induced endothelium‐dependent dilation was assessed by videomicroscopy in ex vivo coronary arterioles from 64 patients undergoing heart surgery. Linear and multivariable logistic regressions were used to investigate the effects of obesity (BMI ≥ 30 kg/M2) and the influences of medications on vessel responses. The diameter of isolated coronary arterioles was similar between obese and non‐obese patients. Responses to bradykinin exhibited a trend towards enhanced dilation in obese patients (55±25 % vs. 46±27 %, P=0.20), with a 1.1% increase in dilator response per BMI unit. In multivariable analysis, obesity was determined to be a predictor of enhanced dilation (HR=5.00, 95% CI=1.18–21.13) adjusted for other risk factors. Statin administration independently predicted a greater coronary dilation (HR=7.98, CI=1.76–36.19), whereas other medications, such as ACE inhibitors, ARBs and diuretics were without significant effects. Thus, obesity and statin therapy are independently associated with enhanced dilator function of coronary arterioles in patients undergoing heart surgery, which may offer a potential mechanism for the better cardiovascular outcome described as the obesity paradox.
Dipeptidyl peptidase IV inhibition attenuates ventricular vulnerability via cAMP/PKA/CREB pathway
Authors: Lee Tsung‐Ming , Hsu Yu‐Jung
Published: June 2021

We investigated whether sitagliptin attenuates arrhythmias through inhibiting nerve growth factor (NGF) expression in postinfarcted normoglycemic rats, focusing on cAMP downstream molecules such as PKA and Epac. Male Wistar rats after ligating coronary artery were randomized to either vehicle or sitagliptin for 4 weeks. Post‐infarction was associated with increased oxidative stress and myocardial norepinephrine levels. Compared with vehicle, sitagliptin‐treated rats significantly increased cAMP levels and attenuated oxidative stress and NGF levels. Immunofluorescent studies confirmed that sympathetic hyperinnervation was blunted after sitagliptin. Arrhythmic scores in the sitagliptin‐treated rats were significantly lower than vehicle. Ex vivo studies showed that sitagliptin increased phosphorylated cAMP response element binding protein (CREB), which can be reversed by H‐89 (selective PKA inhibitor), not brefeldin A (selective Epac inhibitor). Heme oxygenase‐1 (HO‐1) expression was significantly increased by a PKA agonist but not by an Epac agonist. HO‐1 expression was attenuated in KG‐501 (a CREB inhibitor)‐treated rats in the presence of a PKA agonist. In conclusions, sitagliptin protects arrhythmias by attenuating NGF‐induced sympathetic innervation via upregulation of HO‐1 expression in a cAMP/PKA/CREB‐dependent antioxidant pathway in the non‐diabetic infarcted rats.
Rap1a mediates extracellular matrix (ECM) remodeling through AGE/RAGE signaling in diabetes mellitus
Authors: Randive Rushil , Jeyaraj Selvi C. , Chotani Maqsood A. , Stewart James A.
Published: June 2021

Rap1a is a small monomeric G‐protein in the Ras‐GTPase superfamily that acts as a molecular switch coupling extracellular events to intracellular signaling. There are very few attributed roles of Rap1a in ECM remodeling. We hypothesized that Rap1a signaling intersects hyperglycemia‐mediated AGE/RAGE signaling to alter fibroblast phenotype increasing ECM production and accumulation leading to LV structural remodeling. Rap1a gene ablation and knock‐down were used to determine ECM and AGE accumulation, RAGE expression and fibroblast differentiation to determine the molecular interplay between Rap1a‐ and AGE/RAGE‐dependent signaling in isolated diabetic cardiac fibroblasts from Db/db and db/db. We observed left ventricular (LV) collagen levels were significantly decreased by Rap1a gene ablation. Silencing Rap1a mRNA in diabetic (db/db) fibroblasts returned collagen and RAGE expression to non‐diabetic (Db/db) levels. These studies are the first of its kind to provide unique targets for therapeutic strategies aimed at reducing hyperglycemia/AGE‐mediated ECM production and accumulation in diabetic patieAnts.
AGE/RAGE and PKC‐zeta interplay in 3D matrix mediated fibroblast differentiation
Authors: Cerovsky Chris M. , Syed Zehra N. , Stewart James A.
Published: June 2021

This purpose of this study was to determine whether increases in advanced glycation endproducts (AGEs) in a 3D diabetic collagen matrix will differentiate wild type (WT) cardiac fibroblasts to a profibrotic phenotype. 3D collagen matrices were prepared from collagen extracts from non‐diabetic (Db/db) and leptin receptor deficient, diabetic (db/db) mouse tails. Primary cardiac fibroblasts isolated from WT and AGE receptor deficient (R−/−) mice which were seeded onto both Db/db and db/db 3D collagen matrices for 7 days (chronic exposure). In addition, on day 6, these cells were then treated with inhibitors UO126 (ERK 1/2 inhibitor; 10μM) and PKC‐zeta Pseudosubstrate (PKC‐zeta inhibitor; 1μg/ml) and with a RAGE ligand‐ glycated albumin (AGE‐BSA; 0.5mg/ml) to induce RAGE activation. Chronically exposed R−/− cells were unchanged, however WT cells exhibited functional and phenotypical markers for fibroblast differentiation, such as increased alpha‐smooth muscle actin and RAGE expression. Additionally, blockade of ERK1/2 and PKC‐zeta restored WT expression to non‐diabetic levels. Therefore, chronic exposure to AGE‐crosslinked diabetic ECM resulted in phenotypic alterations in WT fibroblasts. These changes were mediated through AGE/RAGE interactions leading to a profibrotic cell phenotype.
Role of estrogen receptor α in estradiol‐induced cardiac growth
Authors: Kararigas Georgios , Nguyen Ba Tiep , Jarry Hubertus
Published: June 2021

Contrary to its effects in the diseased heart, we recently discovered that estradiol (E2) unexpectedly exerts pro‐hypertrophic effects in the healthy heart of ovariectomized (OVX) mice. Our aim was to investigate the mechanism(s) involved in the regulation of cardiac growth by E2 testing the hypothesis that estrogen receptor α (ERα) mediates these E2 effects. Female wild‐type (WT) C57Bl/6J and estrogen receptor α knockout (ERKO) mice were ovariectomized at two months old and were fed an E2‐containing or soy‐free (Ctrl) diet for three months (n = 5–6/group). E2 supplementation in OVX mice led to a lower body weight (15% decrease; P < 0.05) and a higher uterus‐to‐body‐weight ratio (5‐fold increase; P < 0.001) compared with Ctrl mice, but these E2 effects were abrogated in ERKO mice. E2 led to a higher heart‐to‐body‐weight ratio compared with WT Ctrl mice (20% increase; P < 0.05). Deletion of ERα abolished this cardiac effect of E2. The expression of three E2 target genes in the heart was significantly induced by E2 in WT mice compared with Ctrl, whereas in ERKO mice E2 had no effect on Act2, Igf1 and Myocd expression. In conclusion, our study demonstrates that E2 regulates body weight and exerts significant effects on cardiac growth and gene expression via an ERα‐dependent mechanism. Funding: EUGeneHeart (LSHM‐CT‐2005–018833)
BKCa++ channel inhibition improves myocardial stunning following hyperkalemic cardioplegic arrest (CP) but not reversible ischemia. Potential role of ROS and GSK3B.
Authors: Cordeiro Brenda , Clements Richard T
Published: June 2021

We previously demonstrated, in contrast to pure ischemic injury, that inhibition of BKCa channels improves cardiac contractile recovery following CP. We now investigate the mechanism via which BKCa blockade may be protective during CP. Isolated rat hearts and myocytes were subjected to either in vitro ischemia (30 mins) or hyperkalemic CP (2hrs) followed by reperfusion (30 min). Mitochondrial membrane potential (mMP), ROS generation (n=12), and BKCa‐mediated signaling (n=3) were measured in H9c2 cells with or without CP and the BKCa blocker paxilline (pax). CP and ischemia reduced cardiac contractile function in hearts (n>;6) and myocytes (n>;=15). BKCa blockade increased whole heart recovery and myocyte shortening in CP, but not in ischemic groups. Pax did not affect Ca++ transients in myocytes, or indicators of Ca++ signaling and survival in hearts and H9c2 cells (phospho‐PKA, p‐cTnI, PKA, pAkt, pPLB, p‐sHSP). In H9c2 cells, CP reduced mMP and increased ROS, which was partially blocked by pax. Pax increased phosphorylation of GSK3B in both hearts and cells, independent of changes in Akt phosphorylation. In contrast to protective effects of BKCa openers in the setting of ischemic injury, BKCa channel blockers may improve cardiac recovery in the context of hyperkalemic CP. Cardioprotective mechanisms associated with BKCa blockade include normalized ROS and mMP, and increased GSK3B phosphorylation.
NF‐kB dependent mechanisms of high fat mediated cardioprotection.
Authors: Haar Lauren , Ren Xiaoping , Bertaux‐Skeirik Nina , Tee Trisha , Tranter Michael , Jones WK
Published: June 2021

Clinical and epidemiological studies show that increases in serum cholesterol decrease morbidity in patients following myocardial infarction (MI). Serum cholesterol increases as a result of dietary intake of fat and is known to increase risk for primary myocardial events and associated co‐morbidies such as diabetes, myocardial infarction, athlerosclerotic legions, and some cancers.

The effects of acute high fat feeding were assessed with a murine model of myocardial ischemia reperfusion injury. Mice fed on a high fat diet for 24hrs – 2 wks prior to ischemia/reperfusion (30 min coronary ligation) exhibited a profound cardioprotection against resulting MI relative to mice fed on a control diet (~70% reduction of infarct size P ≤ 0.05). This cardioprotective effect is not apparent until 12–24 hrs after initial feeding and also remained for 24–48 hrs following cessation of feeding, indicating a delayed or prolonged phase of cardioprotection. NF‐κB‐dependent gene expression is a well‐established mediator of the late‐phase of preconditioning cardioprotection. Our results show abrogation of this high‐fat diet mediated cardioprotection in a mouse model with cardiac‐specific blockade of NF‐κB. We then assessed the effect of high fat diet on downstream NF‐κB‐dependent genes. Results suggest that expression of NF‐κB‐dependent genes in the myocardium is triggered by the high fat diet, perhaps by the effects of lipid loading, changes in myocardial fat composition, neurohormonal, and/or cytokine/chemokine responses. Studies will be directed towards understanding the mechanism of this activation.
Canonical correlation analysis identifies key diet macronutrients related with metabolic and cardiovascular disorders in rodents submitted to hypercaloric diet
Authors: Junior Silvio Assis Oliveira , Padovani CR , Silva NR , Martinez PF , Campos DHS , Okoshi MP , Okoshi K , Cicogna AC
Published: June 2021

This study analyzed the correlation between diet macronutrients and cardiovascular parameters in rats subjected to hypercaloric diet (HD).

Methods

Wistar‐Kyoto rats (n=20) were fed a standard control (C; 3.2Kcal/g) or hypercaloric diet (HD; 4.6Kcal/g) for 20 weeks. Afterward, glycemia and insulin were evaluated in serum samples. Systolic blood pressure (SBP) was analyzed by tail pletismography. Echocardiography was used to analyze structure and performance of the heart. Morphometric analysis was assessed in myocardial histological sections. Myocardial myosin heavy chain (MyHC) isoforms were analyzed by electrophoresis.

Statistical analysis

Student's t‐test and canonical correlation.

Results

HD reduced glycemic tolerance and induced cardiac hypertrophy combined with improved systolic performance; additionally, HD increased collagen interstitial fraction (C: 3.7±1.2; HD: 5.4±1.8%; p<0.05) and β/α‐MyHC ratio (C: 0.87±0.27; HD: 1.43±0.67; p<0.05). Saturated lipid intake significantly correlated with glycemia, and multiple myocardial parameters of structure and performance (r=0.99; p<0.05).

Conclusion

Saturated lipids intake is directly correlated with glycemic changes and cardiac remodeling in rodents submitted to HD.