bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of Potassium‐induced [3H]D‐Aspartate Release from Isolated Bovine Retina by 5‐epi‐5‐F3t‐Isoprostane: Role of Prostanoid Receptors
Authors: Opere Catherine A , Jamil Jamal , Salvi Ankita , Bankhele Pratik , Flowers Arnecia , Floyd Namonique , Durand Thierry , Galano Jean‐Marie , Guy Alexandre , Oger Camille , Njie‐Mbye Ya Fatou , Kulkarni Madhura , Ohia Sunny E
Published: June 2021

Arachidonic acid‐derived F2‐isoprostanes (F2‐IsoPs) can regulate neurotransmitter release in bovine retina (Jamil J. et al, 2012). It is, however, unclear whether eicosapentanoic acid‐derived F3‐IsoPs can produce a similar effect in the retina.

Purpose

To investigate the pharmacological actions of 5‐epi‐5‐F3t‐IsoP on K+‐induced [3H]D‐aspartate release from bovine retina. We also examined the role of prostanoid receptors on the F3‐IsoP‐induced response.

Method

Isolated neural retina were incubated in oxygenated Krebs solution containing 200 nM of [3H]D‐aspartate and then prepared for studies of neurotransmitter release. Release of [3H]D‐aspartate was evoked by K+ (50 mM) stimuli applied at 90 mins (S1) and at 108 mins (S2) after the onset of superfusion.

Results

5‐epi‐5‐F3t‐IsoP (0.1 nM – 0.1 μM) elicited an inhibitory action on K+‐evoked [3H]D‐aspartate release in a concentration‐dependent manner, achieving a maximum inhibition of 46.9% at 0.1 μM (IC30 of 1 nM). The effect of 5‐epi‐5‐F3t‐IsoP (0.01 μM) was partially reversed by the prostanoid receptor antagonists, SC 19220 (1 μM; EP1), SC 51322 (10 μM; EP1) and AH 6809 (10 μM; EP1–3/DP1) while AH 23848 (1 μM; EP4/TP1) and SQ 29548 (10 μM; TP) had no effect on the IsoP.

Conclusion

5‐epi‐5‐F3t‐IsoP can inhibit K+‐evoked [3H]D‐aspartate release in isolated bovine retina, presumably via mechanisms that involve prostanoid receptors.
Characterization of Src and EGFR protein expression in chronic morphine treated SH‐SY5Y cells
Authors: Randell Kesa , Prenus Rose , Goodman Carl B.
Published: June 2021

Scientific advances have been made in various areas of medicine. However, morphine remains the leading opioid analgesic of choice for the treatment of moderate and severe pain. Although morphine has very potent analgesic actions, it also has adverse side effects which include the development of tolerance which is the reason for its limited use. The exact mechanisms underlying the development of opioid tolerance is unclear, but in recent years studies have shown that the phosphorylation of G‐coupled protein receptors by various kinases has been linked to receptor internalization. Preliminary studies in our lab yielded results indicative of a significant decrease in the gene expression of Src and EGFR after chronic 24 hrs morphine treatment in differentiated SH‐SY5Y human neuroblastoma cells. The present study was conducted to validate the expression of Src and EGFR by western blot analysis after chronic morphine exposure (10 μM) in the absence and presence of naloxone (10 μM) for 1 hr. Additional studies will determine the involvement of Src and EGFR in morphine‐induced MOR‐1 gene expression. The overall results from this study will provide an understanding on the molecular mechanisms of opioid tolerance and the role that the AKT pathway plays in the development of morphine tolerance. Research supported by NCRR/RCMI G12 RR03020 and G12 MD007582–28
Opioid‐immune crosstalk: role of microRNA regulation following opioid and cytokine treatment in normal human astrocytes
Authors: Stevens Craig W. , Castoro Ryan J. , Davis Randall L.
Published: June 2021

There is great interest in the interaction of opioid and cytokine ligands and their respective signaling molecules in neural and immune cells. One avenue of research that is largely unexplored focuses on the role of microMNA (miRNA) as a mediator of opioid‐immune crosstalk. This project focused on two miRNA species: miR‐146a and miR‐let7a. The role miRNA‐146a was established as important for the regulation of NFκB pathways in peripheral immune cells as its expression is increased with pro‐inflammatory cytokines and miR‐146a then decreases the expression of key positive protein partners in the NFκB pathway, such as IRAK1 and TRAF6. The role of miR‐let7a is established in the regulation of the mu opioid receptor (MOR) and its increased expression downregulated MOR mRNA at by post‐transcriptional mechanisms. Recent preliminary data from our laboratory shows that unstimulated normal human astrocytes (NHA) express low levels of and that MOR is increased by treatment with the cytokine, interleukin‐1β (IL‐1β). At the same time, IL‐1β treatment increased the expression of miR‐146a, a key miRNA species recognized to play a role in dampening innate immune signaling pathways. Confirmation of the regulatory role of miR‐146a in signaling pathways in NHA cells was done by Western immunoblots of the NFκB protein partners, IRAK1 and TRAF6. Treatment of NHA cells with IL‐1β decreased the protein expression of IRAK1 at 12 and 24h. Treatment of NHA cells with IL‐1β also decreased the protein expression of TRAF6 at 24h. The miRNA, miR‐let7a is also expressed in NHA cells and is regulated by morphine and IL‐1β treatment of NHA cells. These studies suggest that miRNA may play a role in the opioid‐immune crosstalk observed in numerous peripheral immune cell studies and centers the attention on innate immune cells in the brain, namely the astrocytes. Supported in part by Oklahoma Center for Advancement of Science and Technology (OCAST) contract HR10–31 to CWS.
Neuroprotective effects of Fisetin and Diosmetin, alone and in combination, on lipopolysaccharide‐induced neuronal cells
Authors: Bhatt Pankti R , Benzeroual Kenza E
Published: June 2021

Alzheimer's disease (AD) is characterized by the accumulation of amyloid beta (Aβ) peptides and the presence of neurofibrillary tangles. Amyloid precursor protein (APP) processing into Aβ, is considered as a contributor to mitochondrial dysfunction and neurodegeneration in AD. This study investigated the neuroprotective effects of fisetin and Diosmetin, two flavonoids, in the lipopolysaccharide (LPS) injured PC12 cells. PC12 cells were pretreated with different doses of flavonoids for 2 hours prior to LPS stimulation for 48 hours. Results reported that LPS stimulation increases the expression of APP (A4), Cyclophilin D, reactive oxygen species (ROS), and reduces the level of interleukin 10. Fisetin and diosmetin, alone or in combination suppressed the expression of APP, cyclophilin D and ROS production, while increased the expression of interleukin 10 in a dose dependent manner, respectively. The results suggest that an increase of APP processing may trigger mitochondrial dysfunction and cell death leading to neurodegeneration as seen in the presence of LPS, and its prevention with flavonoids. The present study, reports that fisetin and diosmetin alone and in combination may offer therapeutic opportunities for neurodegenerative diseases such as AD.

Source of research support: Provided by The Long Island University, Arnold & Marie Schwartz College of Pharmacy and Health Sciences
Intranasal GDNF for Parkinson's Disease: Next steps in preclinical development
Authors: Lee Waszczak Barbara , Wachtman Lynn , Newsome Ginnifer C , Aly Amirah , Silva Nilsa , Westmoreland Susan , Rowlett James , Migliore Mattia M
Published: June 2021

We have shown that glial cell line‐derived neurotrophic factor (GDNF) given by the nasal route of administration provides significant protection of substantia nigra (SN) dopamine neurons in the rat 6‐hydroxydopmaine model of Parkinson's Disease (PD). The next step in preclinical development is to test the efficacy of intranasal GDNF in a primate model of PD. Eighteen common marmosets (Callithrix jacchus) were given three doses of MPTP (2 mg/kg on day 1 and 1 mg/kg on days 2 and 3) to induce a partial lesion of the SN dopamine neurons. Nine animals received 5 daily doses of intranasal GDNF (100 μg in 50 μl saline) and nine received an equivalent volume of saline. Doses were administered on consecutive days with the first given 1 day prior to the start of MPTP, the next three doses given 1 hr prior to each dose of MPTP, and the last dose given 1 day after the last dose of MPTP. Animals recovered for 2 months before sacrifice. Lesions severity was assessed using locomotor activity monitoring, performance on an object retrieval task, motor disability scoring, and by tyrosine hydroxylase (TH) immunohistochemistry (IH) of the SN and striata of each animal. The study concluded in October 2012, and analyses of the behavioral and TH‐IH data were ongoing at the time of abstract submission. Final results will be presented.
Safety evaluation of a second generation enzyme replacement therapy
Authors: D'Angona Alida Louise , Finn Patrick , Murray James , McVie‐Wylie Alison , Andrews Laura
Published: June 2021

Pompe disease (glycogen storage disease type II) is a lysosomal storage disease in which a deficiency of the enzyme acid‐alpha glucosidase (GAA) results in accumulation of glycogen primarily in the cardiac and skeletal muscles. Enzyme replacement therapy (ERT) utilizing recombinant human GAA (rhGAA, Myozyme/Lumizyme) currently represents the most effective therapeutic approach. While treatment with rhGAA has proven effective, it has been demonstrated that increasing mannose‐6‐phosphate (M6P) levels on rhGAA increases targeting to skeletal muscles through the M6P receptor resulting in improved glycogen clearance. The level of M6P on rhGAA has been increased by conjugating synthetic hexasaccaride glycans containing two terminal M6Ps via the sialic acid moieties on rhGAA (neoGAA). From a preclinical development perspective, neoGAA is an interesting molecule because it has components of both a small molecule (synthetic linker) and a biologic. A preclinical program was designed to include acute (pilot) and chronic toxicology studies, safety pharmacology and genetic toxicology studies. The results of these studies demonstrated no genotoxicity or toxicity associated with administration of neoGAA to both rodents and non‐human primates.
A major compound isolated from Undaria Pinnatifida downregulates CYP3A4 and MDR1 expression by antagonizes pregnane X receptor activation
Authors: Lim Yun Ping , Liu Cheng Ling , Hu Miao Lin
Published: June 2021

Pregnane X receptor (PXR) has been reported to regulate the expression of drug‐metabolizing enzymes, such as CYP3A4 and MDR1. A major carotenoid which isolated from brown sea algae, Undaria Pinnatifida, compound A, is a putative chemopreventive agent. In this study, we determined whether compound A may overcome inducer‐drug interaction through attenuation of rifampin‐induced CYP3A4 and MDR1 gene expression by PXR‐mediated pathways in HepG2 hepatoma cells. We found that compound A significantly attenuated rifampin‐induced CYP3A4 and MDR1 mRNA and CYP3A4 protein expression at 24 h of incubation. Mechanistically, compound A strongly attenuated the PXR‐mediated CYP3A4 promoter activity in HepG2 cells. In addition, compound A attenuated CAR‐and rPXR‐mediated CYP3A4 promoter activity in this cell line. Using the mammalian two‐hybrid assay, we found that compound A significantly decreased the interaction between PXR and SRC‐1, a PXR co‐activator. Thus, this compound A can decrease rifampin‐induced CYP3A4 and MDR1 expression through attenuation of PXR‐mediated CYP3A4 promoter activation and interaction between PXR and co‐activator. These findings could lead to potentially important new therapeutic and dietary approaches to reduce the frequency of adverse drug reactions.
MALT Lymphoma Stem Cell and its Niche in Helicobacter heilmannii‐infected Mice Stomach
Authors: Nakamura Masahiko , Matsui Hidenori , Takahashi Tetsufumi , Tsuchimoto Kanji , Takahashi Shinichi
Published: June 2021

Background & Aims

Although cancer and leukemia stem cells are indispensable in the initiation and enlargement of lesions, the gastric MALT lymphoma is thought to be polyclonal with the persistent infection of Helicobacter species and then change to monoclonal with genetic aberrations, and the participation of stem cells in the tumor formation remains to be elucidated. We performed a histochemical analysis in Helicobacter heilmannii‐induced gastric and hepatic MALT lymphoma in the infected alone and eradicated after infection groups.

Materials and Methods

We used a Helicobacter heilmannii sample isolated from the stomach of a cynomolgus monkey and maintained in C57BL/6 mouse stomachs. Mucosal homogenates were used to inoculate C57BL/6 mice, which were then examined over 24 months. Macroscopic observations were carried out, and PCR analysis of the bacteria of the Helicobacter species was performed at intervals over the observation period. Histochemical analysis was performed using the gastric and hepatic MALT lymphoma with monoclonal and polyclonal stem cell‐related antibodies against doublecortin‐like kinase (DCAMKL1), Musashi‐1, proliferating cell nuclear antigen (PCNA), CD44, CD133, as well as myofibroblast, endothelial, and pericyte markers.

Results

DCAMKL1, Musashi‐1, CD144 positivities were not recognized in the early stage of the lymphoma formation but gradually found in the lymphocytes located in the marginal zone of the MALT lymphoma both in the gastric and hepatic MALT lymphoma. The well‐developed microcirculatory network accompanied the stem cell rich area. The myofibroblasts were also distributed in these area, suggesting the formation of stem cell niche similar to that found in epithelial cells.

Conclusions

MALT lymphoma stem cells were found to exist in the marginal zone of the lymphoma, constituting the niche by the myofibroblast and microcirculatory components.
Development of Screening Assays for Two Stem Cell Lineages Using Corning® Epic® Label‐free Technology
Authors: Sherman Hilary , Gitschier Hanna , Welch Elizabeth Z , Rothenberg Mark E , Randle David H
Published: June 2021

Using stem cells for therapeutic assays has gained momentum over the last ten years. Novel technologies, ranging from culture systems to new methods for screening potentially active compounds has opened a range of possibilities for gaining further insight into the physiologic pathways inherent to stem cells. Epic Technology is a label‐free and non‐invasive high‐throughput screening platform incorporating optical biosensors to monitor dynamic mass redistribution (DMR) in living cells following receptor activation by target ligands. Utilizing Epic Technology, screening capabilities were developed for use with human Mesenchymal (hMSC) and Neural (NSC) stem cell lines to study G protein‐coupled receptors (GPCR)using the LOPAC™ library. Previous work utilizing Epic Technology on a number of parental and engineered cell lines has shown comparable responses to those published in the literature using standard screening methodologies focusing on GPCR targets. In conclusion, the combined use of Epic Technology enables screening of two different stem cell lineages.
Disruption of the PI3K axis abrogates ionizing radiation‐induced cell death
Authors: Lazo John S. , Epperly Michael W. , Sharlow Elizabeth R. , Lira Ana , Skoda Erin M. , Wipf Peter , Kagan Valerian E. , Greenberger Joel S.
Published: June 2021

Effective and safe therapies are lacking to abrogate the lethal effects of accidental or intentional public exposure to toxic doses of ionizing radiation. Our recent unbiased phenotypic screen using a sentinel human pluripotent cell line and a small interfering RNA (siRNA) library targeting druggable genes implicated the phosphoinositide‐3‐kinase (PI3K) pathway in cellular death after ionizing radiation. Using a model of embryonic stem cells, we now demonstrate that the prototype PI3K inhibitor LY294002 as well a new PI3K inhibitor class EMF388–021 mitigated the lethal effects of ionizing radiation including the induction of apoptosis. Moreover, a single intraperitoneal dose of LY294002 (30 mg/kg) given 4 or 24 h after 9.25 Gy of total body irradiation significantly reduced death. Similarly, a single intraperitoneal dose of EMF388–021 (10 mg/kg) given 4 h after 9.25 Gy of ionizing radiation markedly reduced lethality. We found PI3K inhibition stimulated radiation‐induced autophagic flux and reduced mitochondrial respiration providing potential mechanisms for the radiation mitigation. This study documents that an unbiased siRNA assay can assist in the identification of pharmacologically useful radiation mitigators and implicates the PI3K pathway in the human radiation response.
Changes in the activity of the Renin Angiotensin System on resistance arteries in isolated perfused heart of hypertensive rats.
Authors: Calderon Emilio Lopez , Villavicencio Nallely Acevedo , Lara‐Padilla Eleazar , Guevara‐Balcazar Gustavo , Chong Enrique Hong , Castillo‐Hernandez Maria Carmen
Published: June 2021

High blood pressure (HBP) is a disease with high morbidity and mortality worldwide and is considered one of the main etiological factors of multiple cardiac pathologies. One of the regulatory mechanisms of blood pressure is the Renin Angiotensin System (RAS). It is important consider adjuvant therapies to improve the outlook and quality of life of patients with HBP. One therapy that has been shown clinically to improve the condition of patients with HBP is hyperbaric oxygenation (HBO), that consists in subjecting a subject at a pressure greater than 2 atmospheres absolute with 100% oxygen. This therapy has been shown to improve vascular smooth muscle relaxation, increasing oxygenation of hypoxic tissues, decrease the degree of fibrosis, among other beneficial effects.

Methodology

We used rats Wystar of 340 ± 20 g. A corresponding groups were induced hypertension of renal origin by a surgical method (cellophane method) at the eighth day after surgery we began the therapy OHB. At the end of treatment, the rats were sacrificed getting heart, this was mounted on a system of isolated organ (Langendorff system). Curves to Angiotensin II (Ang II) were obtain in coronary resistance arteries.

Results

The diastolic pressure measurement was obtained (150 ± 15 mmHg). The HBP group without treatment (control) showed an increase in vascular reactivity in comparison with the healthy group (control) after a dose of 1×10−7M of Ang II (p <0.001). Whereas the study group no presented changes in vascular reactivity to any dose of Ang II, compared to healthy control group (p>; 0.05).
The Difference Between Calculated Functional Ejection Fraction by Using Cardio Ankle Vascular Index Examination and Measured Ejection Fraction by Using Transthoracic Echocardiography
Authors: Obata Yurie , Shigemi Kenji
Published: June 2021

These days some studies report that 40% to 50% of patient with heart failure have preserved ejection fraction and the pathogenesis of that may relate to the ratio of ventricular end‐systolic elastance (Ees) to effective arterial elastance (Ea). Ees/Ea is known to reflect not only ventricular mechanical performance but also energetic performance. Despite these useful features, we don't use it in clinical site because of need invasive technique to estimate it. Hayashi et al developed a framework to estimate Ees/Ea without measuring ventricular volume and pressure. We evaluated the difference between calculated functional ejection fraction (EFe =SV/(Ved‐V0), SV is stroke volume, Ved is end‐diastolic volume, V0 is left ventricle unstressed volume) using by Cardio Ankle Vascular Index (CAVI) examination and measured ejection fraction (EF) by using transthoracic echocardiography.

First we confirmed that correlation between measured and calculated Ees/Ea by using Hayashi's original dog data. We used Newton's method to calculate equations. Second we applied this equation to CAVI examination data to calculate human EFe. Afterwards we made scatter diagram about calculated EFe and EF measured by echocardiography in 24 patients who examined CAVI and echocardiography within a period of July 2006 to June 2011 at the Hospital, Fukui University, Japan. The first analysis results showed a correlation coefficient of 0.78 (p < 0.0001, 95% confidence limits of 0.687 – 0.844).Then revised Ees/Ea was calculated by using an approximate equation(revised Ees/Ea=Ees/Ea×0.4301+0.2712). Final scatter diagram about human EF showed that patients have similar measured EF were not similar calculated EFe.

There is possibility of calculate noninvasive human Ees/Ea, and it may correlate heart failure with preserved ejection fraction.
Independent modulation of contractile function by cardiac troponin I Ser43 or Ser45
Authors: Lang Sarah Elizabeth , Stevenson Tamara Kato , Westfall Margaret V.
Published: June 2021

Phosphorylation of Ser43/45 within cardiac troponin I (cTnI) increases with protein kinase C activation during cardiac stress, but the functional role of this cluster and the individual residues is unclear. Our goal is to define the functional impact of cTnI Ser43 and Ser45 phosphorylation in intact myocytes. Wild‐type cTnI, cTnISer43Asp, cTnISer45Asp, or cTnISer43/45Asp constructs increasingly replaced endogenous cTnI over time following viral‐mediated gene transfer. At day 2, cTnISer43Asp and cTnISer45Asp expression diminished peak shortening amplitude while shortening in myocytes expressing cTnISer43/45Asp was similar to wildtype cTnI. The shortening rate also slowed in myocytes expressing cTnISer43Asp or cTnISer43/45Asp while cTnISer45Asp slowed both shortening and re‐lengthening rates. Expression of each phosphomimetic construct similarly reduced peak shortening amplitude by day 4, indicating cTnI Ser43 and Ser45 independently modulate contractile performance. Shortening and re‐lengthening rates were similar to control values though other measures indicated an accelerated shortening and re‐lengthening. Expression of these phosphomimetics also accelerated the time to peak Ca2+ (TTP), the time to 50% Ca2+ decay (TTD50%), and Western detection of cTnISer23/24 phosphorylation was elevated compared to controls. Together, the Ca2+ transient and Western analysis provide evidence phosphorylation of cTnI Ser43 or Ser45 produces compensatory adaptations within the myocyte. Funding: NIH HL067524 (MVW) and the AHA 12PRE8830022 (SEL).
Relationship between arterial stiffness and cerebral vascular reactivity in college‐aged african americans
Authors: Hurr Chansol , Kim Kiyoung , Harrison Michelle L. , Lee Joshua F. , Christmas Kevin M. , Brothers R. Matthew
Published: June 2021

African Americans (AA) have increased risk for cardio and cerebral vascular disease relative to Caucasians (CA). While is it generally accepted that arteries become stiffer at a younger age in AA; less is known regarding cerebral vascular function/reactivity (CVMR) to hypercapnia in AAs. Furthermore, to our knowledge, little is known regarding the relationship between arterial stiffness and CVMR, particularly in young healthy adults. We tested the hypothesis that AAs have elevated arterial stiffness and reduced CVMR during hypercapnia relative to CAs. Furthermore, we hypothesized that there would be a negative relationship between arterial stiffness and CVMR. In 7 AA and 13 CA subjects central arterial stiffness was indexed from carotid‐femoral pulse wave velocity (PWV). CVMR was assessed by the cerebral vascular conductance (CVC) response to rebreathing induced hypercapnia. PWV was elevated in the AAs (AA: 564±54 cm/ms vs. CA: 482±73 cm/ms; P=0.02). CVMR was also significantly reduced during hypercapnic rebreathing in the AAs (AA: 2.7±1.0 %/Torr vs. CA: 4.0±1.1 %/Torr; P=0.01). When data from all subjects was included there was a negative relationship between PWV and CVMR such those with elevated stiffness had an attenuated increase in CVC during hypercapnia (P=0.04). These data indicate that AAs have impaired cerebral vascular responses to hypercapnia and that this might be related to stiffer arteries.
Cardiac alterations induced by moderate zinc deficiency during fetal life and growth
Authors: Arranz Cristina , Juriol Lorena , Veiras Luciana , Garrido Facundo Mendes , Gobetto Natalia , Corrales Andrea , Zilberman Judith , Elesgaray Rosana , Tomat Analia , Costa Angeles
Published: June 2021

Aim

To evaluate the cardiac function, morphology and nitric oxide synthase (NOS) activity at 81 days of life in response to fetal and postnatal growth moderate zinc deficiency. Female Wistar rats received from the beginning of pregnancy up to weaning low (L:8 ppm) or control (C:30 ppm) zinc diet. After weaning, male offspring of each group of mothers fed low (l) or control (c) zinc diet during 60 days (Cc, Ll, Lc). We determined systolic blood pressure (SBP) by indirect tail‐cuff technique, cardiac NOS activity (pmol 14C L‐citrulline/g.tissue. min), myocytes diameter (MD, μm) and TGF‐β (% total area) by immunohistochemistry in heart. Left ventricular (LV) function was assessed by echocardiography, studying diastolic (d) and systolic (s) LV wall (LVW, mm), septum (LVS, mm) and ejection fraction (EF %).

Statistics

t‐test.

Cc
Ll

SBP (mmHg)
122±2
141±5*

dLVS
2.3±0.1
1.8±0.1*

sLVS
3.3±0.1
2.7±0.03*

dLVW
2.7±0.2
2.1±0.1*

sLVW
3.5±0.3
2.6±0.1*

EF%
79±7
66±4*

NOS
204±6
164±10*

TGF‐β
40.2±1.5
49.9±1.5*

Major MD
31.8±0.6
26.5±0.8*

Minor MD
18.2±0.3
14.1±0.5*

Values are means±SEM; p<0.05 vs Cc; n=8 rats/group.

Adequate zinc diet in low offpring after weaning (Lc) was unable to avoid alterations induced during fetal life. Zinc restriction during fetal life and growth may program heart diseases in adult life. Supported by: UBA‐Conicet.
Aldosterone Activates Autophagy To Increase Fibroblast Collagen Synthesis and Vascular Stiffness
Authors: Torquato Samantha , Maron Bradley A , Aghamehammadzadeh Reza , Loscalzo Joseph , Leopold Jane A
Published: June 2021

Hyperaldosteronism is associated with increased arterial stiffness; however, the contribution of fibroblasts to this response remains unknown. Accordingly, we treated human aortic fibroblasts (HAoF) with aldosterone (ALDO) (10−7 mol/L) for up to 72 h or vehicle control (V). Compared to V, ALDO increased transforming growth factor‐β (TGFβ) expression by 2.5 ± 0.2‐fold (p<0.01) and collagen III levels by 252% (p<0.01) in a time‐dependent manner. Increased TGFβ expression was associated with activation of autophagy as shown by lipation of LC3BI to LC3BII (p<0.01). To demonstrate that autophagy modulated collagen expression, autophagy was inhibited by transfecting cells with beclin1 siRNA. Inhibition of autophagy led to a 78% decrease (p<0.01) in collagen III expression without influencing TGFβ levels. To confirm these findings in vivo, C57Bl/6 mice were infused with ALDO (50 μg/kg/d) or V for 21 days. In ALDO‐treated mice, aorta TGFβ and LC3BII expression were increased with a concomitant 3.5 ± 0.5 ‐ fold (p<0.01) increase in perivascular collagen deposition. This was associated with an increase in pulse wave velocity (288.4 ± 22.4 vs. 389.6 ± 19.8 cm/sec, p<0.01), a marker of arterial stiffness, in ALDO‐treated mice. These data indicate that ALDO increases perivascular collagen deposition and vascular stiffness through a mechanism that involves activation of autophagy in fibroblasts.
High altitude long‐term hypoxia: Gene expression in adult ovine carotid arteries
Authors: GOYAL RAVI , MATEI NATHANAEL , LONGO LAWRENCE D
Published: June 2021

In humans and other species, exposure to long‐term high altitude (>;2500 m) is known to result in dysregulation of cerebral blood flow (CBF) as a consequence of low O2 tension. To elucidate the signal transduction pathways involved in successful acclimatization of sheep to high altitude, we tested the hypothesis that high‐altitude associated long‐term hypoxia (LTH) results in changes in gene expression of critical signaling pathways in ovine carotid arteries. To test this, we acclimatized non‐pregnant adult sheep to 3801 m altitude for about 100 days and conducted oligonucleotide microarray experiments on carotid arteries. A total of 60 and 58 genes were significantly (>; 2‐fold; P < 0.05) upregulated and downregulated, respectively. Major genes upregulated included suprabasin, myelin basic protein, apoliprotein B; whereas major downregulated genes included protein kinase C delta, endonucleas V, BCL6. Several of these genes belonged to ubiquonone canonical pathway. A number of upregulated genes had STAT3 and USP transcription factor binding sites. Similarly, a number of downregulated genes had a common binding sites for TOD6/DOT6 and ZBTB3 transcription repressor sites. In conclusion, the present study demonstrates novel signal transduction molecules involved in LTH acclimatization of sheep carotid arteries. The study was supported by PHS PO1 HD031226 to LDL.
Female murine Egr‐1 homozygous negative estrus cycle characterization
Authors: Baldwin Aparna Kadam , Gersch Robert P , Rosengart Todd K , Frame Mary D
Published: June 2021

Early growth response protein‐1 (Egr‐1)−/− (null) male mice are a powerful model for studying vessel formation in pathological conditions. We characterized the estrus cycle of female Egr‐1 null mice for use in these studies in relation to estrogen levels. Estrus cycle in Egr‐1 null and control C57BL/6 wild‐type (WT) mice was monitored by daily vaginal smear for two weeks and analyzed after staining. WT estrus cycle is 4–5 days with 4 distinct phases: proestrus, estrus, metaestrus, and diestrus, identified by cell population morphology. Egr‐1 null mice have 4 less distinct phases with a persistent leukocyte presence. This is striking in the estrus phase, where WT displays an absence of leukocytes, but Egr‐1 nulls show a strong presence of leukocytes. Egr‐1 null mice are known to have a luteinizing hormone‐β deficiency that impacts estrogen and progesterone regulation, and may not be allowing the mice to fully enter estrus. With this type of cycling, we suggest that the Egr‐1 null female mice (and WT controls) be used during the metaestrus/diestrus phase of the cycle characterized by lowest estrogen levels and longest window of access. Studies using female mice attest that large animal numbers account for variability of female cycling. However, the changes in hormone balance at any given stage for mice with altered cycles may skew results, making a strong case to selectively study them in a specific cycle phase.
Glutathione Peroxidase‐4 plays a critical role in protection from metabolic syndrome and cardiac remodeling caused by diet‐induced obesity
Authors: Katunga Lalage Adalaide , Mattox Taylor A , Abernathy Scott M , Darden Timothy M , Virag Jitka , Anderson Ethan J
Published: June 2021

Peroxidation of polyunsaturated fatty acids (PUFAs) generates a number of highly reactive lipid peroxidation products (LPPs), and the seleno‐enzyme glutathione peroxidase‐4 (GPx4) is an antioxidant enzyme that selectively neutralizes LPPs. To determine the contribution of LPPs to the pathogenesis of cardiomyopathy precipitated by diet‐induced obesity, male WT and litter‐mate GPx4‐deficient (GPx4+/−) mice were placed on a n‐6 PUFA enriched high fat high sucrose (HFHS) diet for 24 weeks, along with a control group of WT mice (CTL) fed low‐fat standard chow. At termination of diet, adipose tissue mass and glucose intolerance were markedly higher in the GPx4+/− HFHS group compared to WT‐HFHS group, although both HFHS groups were significantly more than CTL. Blood pressure and cardiac function were not significantly different between groups, although left ventricular size and cardiomyocyte diameter was increased in WTHFHS group compared to CTL. Cardiac fibrosis was also markedly higher in both HFHS groups, but substantially more so in GPx4+/− compared to WT. Mitochondrial O2 consumption was higher in WT‐HFHS but not GPx4+/− HFHS, compared to CTL. Surprisingly, the HFHS diet reduced mitochondrial H2O2 by ~3fold in WT vs. CTL, but stayed similar between CTL and GPx4+/−. These findings implicate GPx4 as a critical enzyme necessary for the heart to positively adapt to the oxidative stress induced by obesity.
Decreased Mitochondrial Respiratory Capacity does not correlate with Worsened LV Function in Heart Failure
Authors: OConnell Kelly Anne , Cox James , Xu Wenhong , Shekar Kadambari Chandra , Gamble Dionna , Woodman Bradley , Stanley William C
Published: June 2021

Nutritional therapies for heart failure (HF) are a potential adjunct to pharmaceutical interventions. High fat intake improves cardiac function in rodent models of HF. We compared a standard low fat diet (STD, 15% energy as fat) to diets high in monounsaturated fatty acids (MUFA, 18:1), n6‐polyunsaturated fatty acids (PUFA, 18:2n6) and long chain saturated fat (LCSat, 16:0 and 18:0) which contained 40% energy from fat. Rats were subjected to transverse aortic constriction (TAC) or sham surgery and randomized to dietary treatment after 10 weeks (n=16–17/group). After 16 weeks on diets, cardiac mitochondria were isolated. TAC increased LV mass by ~50% in all groups. LV ejection fraction decreased from 83±1% in sham to 48±5% with TAC on STD diet, 58±4% with MUFA, 56±3% with PUFA, but was prevented by LCSat (67±5%, P<0.05). Maximal mitochondrial respiration with palmitoylcarnitine was decreased from 194±23 to 113±13 nA O*mg−1min−1 with TAC on STD diet and on PUFA (116±19 with TAC), but was prevented with MUFA and LCSat (169±19 and 168±19 nA O*mg−1min−1, respectively), consistent with PPARα activation and improved mitochondrial function. This suggests that increased capacity of fat oxidation does not cause an obligatory increase in LV function. LV function and the decline in mitochondrial capacity for fat oxidation are not causally linked, and impaired mitochondrial respiration does not predict LV remodeling in HF.
Cardiac stroke volume predicts progressive central hypovolemia by three different non‐invasive methods
Authors: Holme Nathalie Linn Anikken , Rein Erling Bekkestad , Walløe Lars , Thoresen Marianne , Elstad Maja
Published: June 2021

Background

Cardiac stroke volume (SV) has been proposed as a clinical tool to predict the magnitude of acute hemorrhage. Multiple non‐invasive measurement methods of SV exist, without a standard method. We aimed to investigate the development of SV during simulated hemorrhage, and examine differences between three non‐invasive methods of SV measurements.

Methods

10 healthy subjects underwent experimental central hypovolemia through lower body negative pressure (LBNP) until −60 mmHg or onset of presyncope (7/10 subjects). SV beat‐to‐beat was calculated by Ultrasound Doppler (SV_usd, SD 100), finger arterial blood pressure curve (SV_bpc, Finometer) and cardiothoracic impedance (SV_imp, Task Force). The non‐parametric median and 95% confidence intervals (CI) were calculated from epochs of 60 or 120 seconds for SV in 5 different LBNP levels.

Results

Baseline absolute values for the three methods were different: SV_usd 78 ml (95% CI: 68,93), SV_bpc 88 ml (77,99), SV_imp 103 ml (89,151). SV_usd decreased with 45% (52%, 37%), SV_bpc decreased with 39% (45%, 27%), while SV_imp decreased with 33% (48%, 21%). Differences between methods were not significant.

Conclusion

SV clearly reflects the development of a simulated hemorrhage. The three methods for non‐invasive SV measurements provide different absolute values of SV during LBNP, but they decrease in a comparable way. Financial support: University of Oslo.
Effect of a calcineurin inhibitor on Lipopolysaccharide‐induced loss of vascular tone and blood pressure decrease in conscious mice
Authors: Staehr Mette , Khatam‐Lashgari Apameh , Hansen Pernille B , Jensen Boye L
Published: June 2021

The immunosuppressive drug cyclosporin A (CsA) inhibits the production of nitric oxide (NO) through inhibition of the NO synthases. Especially iNOS is involved in the synthesis of large amounts of NO seen in sepsis, which leads to hypotension and multiple organ dysfunction. We hypothesized that CsA counteracts the LPS‐induced blood pressure decrease and reduced vasoreactivity in isolated murine aorta rings in a model of septic shock.

To test this, blood pressure and heart rate was measured in conscious mice with indwelling catheters given a bolus (20 or 40 mg/kg) of CsA followed by continuous infusion (20 or 40 mg/kg/day) for one day before an LPS bolus was given (2 mg/kg). In vitro, studies were performed with aortic rings incubated with LPS (50 μg/ml) w/wo CsA, with CsA alone or vehicle.

CsA (20 and 40 mg/kg) induced a significant increase in blood pressure (≈11 mmHg), with no effect on heart rate. After an LPS bolus, blood pressure and heart rate decreased, but there was no difference in delta blood pressure and heart rate compared to LPS alone. iNOS mRNA and protein expression was significantly reduced by CsA as well as plasma NOx excretion. CsA significantly attenuated the LPS‐induced decrease in vasoreactivity. It is concluded that CsA improve vasoreactivity in vitro while in a murine model of septic shock in vivo, CsA at 20 and 40 mg/kg did not counteract the effect of LPS on blood pressure.
Algebraic Approximations for Blood, Interstitial, and Lymph Pressures, Volumes, and Flows During Hemorrhagic Shock and Fluid Resuscitation
Authors: Knezek Sarah , Dongaonkar Ranjeet M. , Quick Christopher M
Published: June 2021

The tolerance to hemorrhage, effectiveness of fluid resuscitation, and the transport of inflammatory mediators, all depend on how fluid redistributes between vascular and interstitial compartments. Fluid redistribution depends on the complex interaction of cardiac function, venous and interstitial capacitance, arterial and venous resistances, microvascular filtration and lymphatic function. The promise of mathematical models to elucidate the physiology of shock has suffered from the very complexity they aim to address, leading to numerical solutions that only apply for a particular animal or patient population, are sensitive to assumed parameters, and are difficult to interpret. We therefore applied simplifying assumptions to the standard hemodynamic and fluid balance equations typically used in shock research. The result is the first set of algebraic approximations for the pressure, volumes, and flow of blood, interstitial fluid and lymph during shock and subsequent fluid resuscitation. We thus provide a new tool to address the traditional goals of determining the optimal composition, amount, and timing of fluid resuscitation to restore perfusion and limit extravascular redistribution, as well as the newer goal of preventing acute respiratory distress syndrome by limiting the transport of inflammatory mediators transported in mesenteric lymph.
Dietary rapamycin selectively improves arterial function in old mice
Authors: Reihl Kelly D , Seals Douglas R. , Henson Grant D , LaRocca Thomas J , Magerko Katherine , Bosshardt Gary C. , Lesniewski Lisa A , Donato Anthony J
Published: June 2021

Dietary rapamycin inhibits mTOR, reduces age‐related pathologies and extends lifespan, but it is unknown if it can reverse age‐related arterial dysfunction. The effects of mTOR inhibition on endothelial function and arterial stiffness were evaluated in young (Y: 6±2 mo) and old (O: 30±2 mo) male B6D2F1 mice fed control (C) or rapamycin‐supplemented (RAP: 14mg/kg encapsulated chow) diet ad libitum (6 wk). Aortic mTOR signaling, assessed by Western blot, was higher in aged mice (1.0±0.5 v. 2.59±0.8 AU, P<0.05) and lower in rapamycin‐treated old mice (1.5±0.3 AU, P<0.05). Rapamycin selectively increased NO bioavailability (OC v. ORAP, P<0.05) and augmented endothelium‐dependent dilation to acetylcholine (YC: 96±2% v. OC: 73±4%, P<0.05; v. ORAP: 95±1%, P<0.05) in carotid arteries of old mice. RAP also reduced aortic stiffness, assessed by pulse wave velocity, in old animals (Post‐Pre: −43±19 cm/s, P<0.05). These improvements coincided with lower aortic nitrotryosine (YC: 1.0±0.2 v. OC: 4.7 ±1.6, P<0.05; v ORAP: 1.5±0.3, P<0.05) and collagen expression (YC: 37±9% v. OC: 78±5%, P<0.05; v. ORAP: 50±9%, P<0.05). In young mice, RAP impaired endothelial function (P<0.05) and did not affect arterial stiffness. In conclusion, rapamycin improves arterial function in old mice with enhanced mTOR activity, suggesting that mTOR may be an effective therapeutic target in older adults.
Aortic stiffening as a result of reduced elastin content leads to cerebral artery dysfunction
Authors: Walker Ashley E , Henson Grant D , Reihl Kelly D , Bramwell Colton , Li Dean Y , Lesniewski Lisa A , Donato Anthony J
Published: June 2021

With advancing age, large artery stiffening is linked to reduced elastin content and is thought to result in resistance artery dysfunction, but this has not been empirically tested. To isolate the effects of large artery stiffness without other age‐related changes, we compared elastin haploinsufficient (ELN+/−) and wildtype (ELN+/+) mice (4–6 mo, n=4–10/group). ELN+/− had greater aortic stiffness (pulse wave velocity, 406±45 vs 306±29 cm/s, p=0.04) and blood pressure (p<0.05) compared with ELN+/+. In the middle cerebral artery (MCA), endothelium‐dependent dilation (EDD) to acetylcholine was impaired in ELN+/− vs ELN+/+ (max: 37±6 vs 54±4%, p=0.03). L‐NAME pretreatment abolished group differences in MCA EDD (p=0.54), indicating less nitric oxide mediated dilation in ELN+/−. MCA vasoconstrictor responses were greater in ELN+/− vs ELN+/+ for angiotensin II (−53±4 vs −20±6%, p=0.002) and norepinephrine (−62±3 vs −41±2%, p=0.002). In contrast, EDD and vasoconstrictor responses were similar between groups in gastrocnemius feed arteries (P>;0.05). Endothelium‐independent dilation was similar for groups in each artery (P>;0.05). In conclusion, aortic stiffness as a result of reduced elastin leads to impaired dilator and increased constrictor responses in small cerebral, but not skeletal muscle, arteries. Thus, large artery stiffening likely contributes to cerebral artery dysfunction with aging.

Supported by AG029337, AG040297, AG033196, HL007576
LECTINIC BINDING OF CANDIDA GLABRATA TO OLIGOSACCHARIDES ON THE CORONARY LUMINAL MEMBRANE ALTERS FLOW‐INDUCED CARDIAC RESPONSES.
Authors: Torres‐Tirado David , Ramirez‐Zavaleta Candy , De Las Peñas Alejandro , Castaño Irene , Rubio Rafael
Published: June 2021

Candida Glabrata (CG) is a yeast that may invade humans blood stream causing dead. CG surface is lectinic and binds with high affinity to oligosaccharides (O) present in cell membranes, as example, coronary endothelial luminal membrane (CELM).

Conversely, coronary flow (CF) stimulates auricular‐ventricular transmission (A‐Vt). CF acts on CELM lectins (L) and O, causing a reversible L‐O complexing necessary for CF stimulation of A‐Vt. We hypothesized that CG upon binding to CELM's O prevents CF‐induced L‐O complexing; as a result inhibiting CF‐stimulation of A‐Vt.

In Langendorff perfused isolated guinea pig hearts A‐Vt was determined at different CF. As CF (ml/min) increase A‐Vt (msec) decrease. (control curve). Thereafter, to study the effects of CG bound to the CELM, CG (CGM35 sir3Δ, 3.7×103 cells/ml), was intracoronary given for 5 min and wash at length for 5 min and an experimental CF‐A‐Vt curve determined. The experimental curve was on top of the control curve i. e. CG upon binding to CELM increases the A‐Vt value at any CF. To test the effect was due to CG bound to L, CG was displaced by perfusing sugars components of O; mannose or Galactose. Perfusion of any of these sugars after CG they reverted its inhibitory effects on CF‐induced A‐Vt stimulation. Furthermore, simultaneous perfusion of CG and any of these sugars prevented the CG‐inhibitory effects, likely competing with CELM's O for CG binding. Thus, CG upon binding to CELM's O competes with L preventing CF‐induced L‐O complexing; as a result inhibits CF‐stimulation of A‐Vt. In other experiments using CG like affinity beads and mixing with CELM proteins, we found that CG bind to angiotensin type 1 receptor, alpha 1 adrenergic receptor, endothelin receptor and vascular cell adhesion protein 1, by western blot. This mean that CG could be altered the response of its receptors by the only interaction L‐O.
Nitroclycerin (GTN) but not the new nitric oxide donor Ru(bpy) 2 (py)(NO2)](PF6) (RuBPY) induced in vitro cross‐tolerance with acetylcholine
Authors: Paulo Michele , Grando Marcella Daruge , Silva Roberto Santana , Bendhack Lusiane Maria
Published: June 2021

This work aimed to verify if RuBPY induces in vitro tolerance and cross‐tolerance with acetylcholine (ACh) in rat cava vein and to compare this effect with GTN. We have analyzed the maximum effect (ME) induced by GNT, RuBPY and ACh in concentration‐effect curves in veins contracted with endothelin‐1. P values ≤0.05 were considered significant. In vitro tolerance was induced by 60 min‐incubation with GTN (0.1mM or 4 μM) or Rubpy (0.1μM or 2 μM). In vitro cross‐tolerance for ACh was induced by incubating vein with GTN or Rubpy (4 μM) for 60 min. The protein expression of phosphorylated eNOS on Ser1177 and total eNOs was accessed by Western Blot. Our results demonstrate that after incubation with GTN (EC50,EC100) the ME was reduced from 75.3±2.2%, n=6 to 45.4±2.2%, n= 6 and to 39.2±1.4%, n= 6, respectively. Incubation with RuBPY (EC50,EC100) reduced the ME from 92.8±4.2%, n=7 to 48.0±2.3%, n=7 and 30.1±1.2%, n=7, respectively. GTN but not RuBPY, reduced ACh‐induced relaxation (from 100.3±5.3% to 75.1±4.2%, n=7). GTN, but not RuBPY induced eNOS phosphorylation. In conclusion, RuBPy and GTN induce tolerance in both EC50, EC100. Only GTN induces cross‐tolerance to ACh and increased eNOs phosphorylation. Supported by FAPESP and CNP
Deficiency in CCR5 protects against vascular smooth muscle cell proliferation in mice fed with high fat diet
Authors: Tsai Min Chien , Tu Yun‐Chin , Lee Yen‐Hsien , Hsieh Po‐Shiuan
Published: June 2021

Chemokines play a crucial role in innate and inflammatory responses and in the pathophysiology of atherosclerosis. Chemokine‐chemokine receptors (CCR) are important modulators of inflammation although they have been shown to be involved in atherosclerosis progression. Previous studies have shown that vascular smooth muscle cells (VSMC) express functional chemokine‐chemokine receptors 5 (CCR5). However, little is known about how CCR5 is regulated in vascular smooth muscle cells. In this study, we analyzed the role of the CCR5 by genetic deletion in mice with a hyperlipidemia‐induced by high fat diet. Deficiency in CCR5 significantly reduced hyperplasia of the arterial wall after fed with a high fat diet for 12 weeks, associated with a decrease VSMC DNA synthase by proliferating cell nuclear antigen (PCNA) staining. Our data also show that deficiency of CCR5 inhibited VSMC proliferation by blocking G1 to S phase progression and repressed the phosphorylation of retinoblastoma protein (Rb). Furthermore, CCR5 knockout mice inhibited tumor necrosis factor‐alpha (TNF‐α)‐induced expression of cell cycle regulatory proteins such as cyclin E and CDK2. Collectively, this data provide an important role to characterize specific functions of the CCR5 in vascular pathology.
The COP9 signalosome subunit 8 (CSN8) hypomorphism impairs deneddylation and exacerbates desmin‐related cardiomyopathy (DRC)
Authors: Su Huabo , Li Jie , Wei Ning , Wang Xuejun
Published: June 2021

Background

Inadequate protein quality control (PQC) contributes to the development of cardiac diseases. We previously identified the COP9 signalosome (CSN), a deneddylase that removes ubiquitin‐like protein Nedd8 from target proteins, is essential to proteasomal and autophagic proteolysis in the heart. The present study determines the impact of CSN8 hypomorphism (CSN8hypo) on the removal of misfolded proteins in mouse hearts.

Methods and results

CSN8hypo accumulated the neddylated forms of cullins and non‐cullin proteins in mouse hearts. Using a proteasome functional reporter mouse, we found the proteasomal function was compromised in CSN8hypo mouse hearts. When crossed with transgenic mice overexpressing CryABR120G, a bona fide misfolded protein, CSN8hypo reduced ubiquitinated proteins but increased protein aggregates in the heart, which eventually aggravated the CryABR120G‐induced cardiomyopathy and shortened the lifespan of the mice. In cultured cardiomyocytes, CSN8 knockdown suppressed the ubiquitination and degradation of CryABR120G, leading to accumulation of protein aggregates and exacerbation of CryABR120G‐mediated proteotoxicity.

Conclusion

The CSN is required for the ubiquitination and clearance of misfolded proteins in cardiomyocytes, and therefore is critical to cardiac PQC. (Funding agencies: NIH and AHA)