bluuebunny/crossref_metadata_2025 huggingface dataset:
Epigenetic Regulation of OCTN1‐mediated Cytarabine Transport in Acute Myeloid Leukemia
Authors: Anderson Jason T. , Buelow Daelynn R. , Guttke Christina D. , Hu Shuiying , Gibson Alice A. , Sparreboom Alex , Baker Sharyn D.
Published: June 2021

BACKGROUND

Cytarabine is used as first‐line therapy in acute myeloid leukemia (AML) and is reported to be transported into leukemic cells via OCTN1[i]. One hallmark of AML is dysregulation at the genetic and epigenetic level. Currently, clinical trials are underway using methyltransferase inhibitors such as 5‐azacytidine or decitabine to “epigenetically prime” AML patients and reverse these epigenetic dysregulations (NCT03164057). These clinical trials would benefit from a stronger understanding of the mechanism underlying improved patient outcomes. Here, we tested our hypothesis that these improved patient outcomes are due to epigenetic modifications impacting OCTN1 expression, intracellular accumulation of cytarabine and subsequent antileukemic effects in AML cells.

METHODS

Uptake and cytotoxicity studies were performed in AML cell lines with prior 3‐day treatment with hypomethylating agents, 5‐azacytidine or decitabine. Uptake was conducted by using radioactive substrates and quantified by scintillation counts. OCTN1 expression levels were compared between treated and untreated AML cells with normalization to GAPDH. Methylation profiles were determined using bisulfite modification and pyrosequencing (BS‐Seq). Cytarabine cytotoxicity was determined via MTT and developed after 72 hours of treatment.

RESULTS

Cytarabine uptake (1 μM; 15 min) and cytotoxicity (72 hours) varied among the 10 AML cell lines studied and were used to categorize “high” and “low” uptake. AML cell lines that showed low uptake had consistently higher methylation patterns in CpG islands upstream of SLC22A4 (OCTN1) as determined by BS‐Seq. Exposure to 500 nM of hypomethylating agent decitabine for 72 hours increased OCTN1 expression (5.0 fold), uptake of cytarabine (2.9 fold), and subsequent cytotoxicity (7.3 fold) in CHRF cells with low OCTN1 expression. In contrast, lower methylated cell line OCI‐AML3 showed modest to no increases in OCTN1 (1.0 fold), uptake of cytarabine (1.5 fold) and cytotoxicity (2.8 fold) following decitabine treatment. These findings are consistent with the notion that methylation of OCTN1 was inversely related to cytarabine uptake.

CONCLUSIONS

These results identify SLC22A4 (OCTN1) methylation status as a contributor to the expression of OCTN1, cellular uptake and the efficacy of cytarabine; these results have broad implications for the future design of combination chemotherapy regimens.

Support or Funding Information

5R01CA138744‐09

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Connexin43 dephosphorylation at serine 282 is associated with connexin43‐mediated cardiomyocyte apoptosis
Authors: Luo Dali , Yang Yutong , Xue Jingyi
Published: June 2021

Gap junction protein connexin 43 (Cx43), beyond electrical coordination regulation, plays an important role in the regulation of cardiomyocyte survival, and Cx43 dephosphorylation found in severe cardiac pathologies is proposed to be involved in myocardium injury. However, the mechanisms remain unknown. Here, we found that transfection of adenovirus carrying Cx43 mutant gene at serine 282 substituted with alanine (S282A) into neonatal rat ventricular cardiomyocytes (NRVMs) induced cell apoptosis and Ca2+ transient desynchronization, whereas gap junction inhibitor and knocking down Cx43 expression by Cx43‐miRNA caused uncoupled Ca2+ signalling but no cell death. Similarly, while Cx43‐S282A+/+ failed in generation, Cx43‐S282A+/− mice exhibited cardiomyocyte apoptosis and ventricular arrhythmia in a phosphorylated S282 deficiency‐dependent fashion. Further we found via physical interaction with p38 mitogen‐activated protein kinase (p38 MAPK), S282 dephosphorylation in Cx43 activates p38, factor‐associated suicide and caspase 8 apoptotic pathway. Finally, we found that Cx43 dephosphorylation at S282 participates in ischemia‐reperfusion (IR) induced cardiomyocyte death by activation of p38 MAPK/Fas/FADD/caspase 8. These findings uncover a specific phosphorylation residue of Cx43 in the regulation of cardiomyocyte homeostasis. Deficiency in S282 phosphorylation acts as a trigger inducing cardiomyocyte apoptosis and cardiac arrhythmia, contributing to IR‐induced myocardium apoptosis. Thus, this study provides a potential mechanism for Cx43‐mediated myocardium injury during severe cardiac diseases.

Support or Funding Information

This work was supported by the National Natural Science Foundation (81370339, 81570206) and Scientific Research Key Program of Beijing Municipal Commission of Education (KZ201710025023).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
GRK5‐mediated inhibitory phosphorylation is essential for inverse agonism at the cardiac mineralocorticoid receptor
Authors: Desimine Victoria Lynn , Parker Barbara Marie , Wertz Shelby Lynn , Duarte Yanelys , Maning Jennifer , McCrink Katie Anne , Pollard Celina Marie , Koch Walter J , Lymperopoulos Anastasios
Published: June 2021

Background

In the heart, aldosterone binds the mineralocorticoid receptor (MR) to exert damaging effects, e.g. fibrosis, apoptosis, oxidative stress, etc. GRK2 and GRK5 are the most abundant cardiac G protein‐coupled receptor (GPCR)‐kinases (GRKs). Both phosphorylate GPCRs but also non‐GPCR substrates. We recently showed that GRK5 blocks the cardio‐toxic MR‐dependent effects of aldosterone in the heart by directly phosphorylating the cardiac MR and inhibiting its transcriptional activity. MR antagonist (MRA) drugs are beneifical in human CHF by blocking the cardiac MR. Novel non‐steroidal MRAs, such as finerenone, may provide better cardio‐protection against aldosterone's cardio‐toxic actions than the classic steroidal MRAs, such as sprironolactone and eplerenone. Indeed, finerenone was very recently shown to be a more potent and efficacious inverse agonist at the MR than eplerenone in terms of cardiac fibrosis/adverse remodeling attenuation. Thus, in the present study, we sought to investigate potential differences between finerenone and eplerenone at engaging GRK5‐dependent cardiac MR phosphorylation and subsequent blockade.

Methods

We used the cardiomyocyte cell line H9c2 and neonatal rat ventricular myocytes (NRVMs). We measured MR phosphorylation via phospho‐serine immunoblotting & transcriptional activity via the luciferase reporter assay.

Results

GRK5 phosphorylates the MR in H9c2 cardiomyocytes in response to finerenone but not to eplerenone. Contrary to eplerenone, finerenone alone potently and efficiently suppresses MR transcriptional activity in H9c2 cardiac myocytes. GRK5‐dependent phosphorylation is necessary for the robust inverse agonism displayed by finerenone at the cardiac MR, since CRISPR‐mediated GRK5 gene deletion renders finerenone incapable of blocking cardiac MR transcriptional activity. Conversely, this non‐steroidal MRA fully suppresses cardiac MR transcriptional activity upon GRK5 overexpression in H9c2 cardiomyocytes. Eplerenone alone cannot suppress cardiac MR basal activity upon GRK5 overexpression or knockout. Finally, finerenone blocks aldosterone's effects more efficiently than eplerenone in control and GRK5‐overexpressing NRVMs.

Conclusions

Finerenone appears a more efficacious cardiac MR blocker than eplerenone, partly via GRK5‐dependent cardiac MR inhibition, which eplerenone does not induce. This non‐canonical effect of GRK5 on the MR is essential for the efficient blockade of aldosterone's deleterious actions in the heart.

Support or Funding Information

1) American Heart Association 2) NSU President's Faculty Research & Development Grant

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
PARTICIPATION OF ESTRADIOL AND NITRIC OXIDE ON THE VASOCONSTRICTOR EFFECT OF BRADIKYNIN
Authors: Campos Maria Elena Hernández , Romero Karla Stephania Dominguez , Hernandez Ignacio Valencia
Published: June 2021

Ovarian hormones privation in menopause can contribute to development of hypertension, which has high morbimortality in the world. Estrogens protect cardiovascular system by reducing angiotensin‐converting enzyme (ACE) activity, preventing bradykinin degradation and promoting a reduction in the vascular tone. These actions of estrogen are related with an increase in the synthesis of nitric oxide. However, bradykinin can produce, under certain conditions, vasoconstriction, a much less studied effect. Therefore, the objective of this work was to study the role of 17‐β estradiol (E2) on the vasoconstrictor effect of bradykinin. We used a group of 24 Wistar ovariectomized rats (OVX) and 6 Sham rats. After 14 days of recovery, a group of 6 OVX rats were administered wit E2 (5 μg/Kg/day s.c. for 5 days) and 6 OVX rats were administered with corn oil (vehicle). At the end of the treatment, rats were sacrificed and thoracic aorta extracted and cut in 5 mm length rings, subsets of aortas were obtained with and others without endothelium. Bradikynin concentration‐response curves were constructed, both in the presence and absence of L‐NAME. All procedures in animals complied with NOM‐062‐ZOO‐1999 and institutional animal care committee. The results showed that ovariectomy or treatment with estradiol did not modify blood pressure values. Treatment with estradiol, however, significantly reduced heart rate in OVX rats. Bradykinin produced a concentration‐dependent contractile effect that was of greater magnitude in rings without endothelium and was blocked by HOE140. Incubation with L‐NAME did not modify the contractile effect of bradykinin in the aortic rings from SHAM rats but inhibited this effect in the OVX treated rat with E2. These results indicate that in the presence of ovarian hormones, where estradiol seems to play an important role, bradykinin produces contraction independently of the endothelium and NO, this contraction seems to be mediated by the activation of B2 receptors.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Ceramide‐Mediated Enhancement of Inflammatory Exosome Secretion from Podocytes during Hyperhomocysteinemia
Authors: Zhang Qinghua , Li Guangbi , Hong Jinni , Lohner Hannah , Li Pin‐Lan
Published: June 2021

Exosome secretion has been shown increased during podocyte injury and associated glomerular diseases, and this increased exosome secretion may be important in mediating release of inflammasome products to activate glomerular inflammatory response leading to chronic glomerular disease. Given the crucial role of ceramide in the control of exosome biogenesis and release, the present study used a podocyte‐specific sphingomyelin phosphodiesterase 1 (Smpd1) transgenic mouse strain (Smpd1trg/Podocre mice) to test whether ceramide production enhanced by Smpd1 gene overexpression promotes inflammatory exosome release from podocyte leading to glomerular inflammation during Hcys. We demonstrated that in Smpd1trg/Podocre mice, hHcy (induced by the FF diet) produced significant podocyte inflammasome activation accompanied by large increases in urinary podocyte‐derived exosomes. By immunohistochemical detection, we found that the glomerular inflammatory responses was more remarkably activated by hHcy in Smpd1trg/Podocre mice than their littermates, as shown by increased staining of lymphocyte and T‐cell markers such as CD45 and CD43. Under electronic microscopy, we observed that hHcy induced more severe podocyte injury and exosome release around these podocytes in Smpd1trg/Podocre mice than their littermates. Using confocal microscopy, about 2.8 folds increase colocalization of multivesicle body marker (MVB) Vps16 and NLRP3 inflammasome product IL‐1β, but about 0.4 folds decrease interaction of Vps16 with lysosome (Lamp‐1) (a process determining MVB fate and exosome release) were found in the glomeruli of Smpd1trg/Podocre mice with hHcy compared to their littermates. In cultured podocytes, the nanoparticle tracking analysis confirmed that L‐Hcy treatment significantly increased microparticle release into supernatants especially from 50nm to 150nm and they were characterized as podocytes‐derived IL‐1β‐containing exosomes as shown by detection of synaptopodin, CD63 and IL‐1β via Western blot analysis. This L‐Hcy‐induced podocyte release of IL‐1β exosomes was mimicked by AC inhibitor carmofur, but attenuated by AC inducer, genistein. It was also found that L‐Hcy‐induced decreases in Vps16 and Lamp‐1 interaction could be simulated by carmofur and inhibited by genistein. All these results together suggest that increased ceramide in podocytes enhances release of inflammatory exosomes during hHcy by reduction of lysosome‐MVBs interaction and prolongation of MVBs to facilitate exosome secretion.

Support or Funding Information

supported by NIH grant DK54927, DK120491 and HL057244

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
FACTORS ASSOCIATED TO THERAPEUTIC FAILURE OF LOSARTAN IN PATIENTS WITH HEART FAILURE. PHARMACOKINETIC MODEL
Authors: De León Díaz de León Edgar Alejandro , Almendra‐Pegueros Rafael Antonio , Gonzalez‐Correa Jose Antonio , Sanchez Teresa Tejerina , Moreno Ursula Fabiolo Medina , Moscoso Antonio Augusto Gordillo
Published: June 2021

Background

Decompensated heart failure is a common cause of hospitalization. Therapeutic use of ARA‐II has demonstrated safety in this patients. Circulating drug concentrations can be affected by some factors (age, sex, body surface, formulation), and variations in levels of plasma can be observed, causing a therapeutic failure and therefore, greater hemodynamic instability.

Aim

To Identify factors that affect plasma drug concentration (Losartan patent vs generic) and its active metabolite (E‐3174) as well as possible factors that affect the therapeutic failure in patients with heart failure.

Methods

A double‐blind randomized controlled clinical trial in a single dose (n = 60), comparing Losartan potassium (50 mg) patent Vs generic, This study was approved by the ethics committee, registration number 98‐15. Inclusion criteria: both sexes, stable patients with FEVI less than 40% and informed consent signed. To determine the pharmacokinetic profile, the method was validated to show compliance with international requirements ICHQ2 (R1). After oral administration of the drug, a 12‐hour follow‐up was performed. The data were analyzed by means of multiple linear regression and t‐test in RStudio.

Results

24/60 patients have been analyzed. Mean age 56.2 ± 13.8 years, time of evolution of HF of 1–3 years. The developed and validated analytical method proved to be sensitive, linear, accurate, precise and with fewer run‐time (5 min Vs 15 min) than the standard method. The area under the curve (AUC) of Losartan patent and generic was 3.28 ± 0.77 and 3.12 ± 0.90 (p = 0.64) respectively and for its metabolite E‐3174 patent and generic was 5.27 ± 0.89 and 4.51 ± 0.88 (p = 0.56) respectively.

The maximum concentration (Cmax), was greater in the patent formulation, the metabolite E3174, was lower in male and patients with higher BMI, these data aren't a statistical difference.

Conclusions

the differences in AUC and Cmax between both formulations aren't statistically significant, however, when analyzing the metabolite E‐3174 we see that after 6 hours, the patent formulation shows an increase in the concentration that is not observed in the generic, which can affect the pharmacological effect in the patient. The therapeutic effect (blood pressure reduction) was better for patent formulation Vs generic, even without statistically significant. For these reasons is essential to complete the sample size to establish a more accurate conclusion.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Chalcone derivative L6H21 reduces EtOH‐LPS‐induced liver injury through inhibition of NLRP3 inflammasome activation
Authors: Gu Zelin , McClain Craig , Feng Wenke
Published: June 2021

Chronic alcohol intake increases circulating endotoxin levels causing excessive inflammation that aggravates the liver injury. L6H21, a derivative of chalcone, has been found to inhibit inflammation in cardiac diseases and non‐alcoholic fatty liver disease. However, use of L6H21 in alcoholic liver disease (ALD) to inhibit exotoxin‐associated inflammation has not been explored. In this study, we examined the effects of L6H21 on EtOH‐LPS‐induced hepatic inflammation, steatosis and liver injury, and investigated the underlying mechanisms. C57BL6 mice were treated with 5% ethanol for ten days, and LPS was applied on the last day. L6H21 was given with alcohol. EtOH‐LPS treatment significantly increased hepatic steatosis and serum ALT and AST levels, which were reduced by L6H21 treatment. L6H21 treatment significantly reduced hepatic protein levels of TLR4, p‐P65 and p‐IκB, and inhibited p65 translocation into the nucleus. In addition, L6H21 treatment markedly reduced hepatic protein levels of NLRP3, cleaved caspase‐1 and cleaved‐IL‐1β, which were increased by EtOH‐LPS exposure. Moreover, L6H21 treatment effectively inhibited caspase‐1 associate apoptosis. In vitro study using Raw264.7 cells confirmed the effects of L6H21 on macrophage activation. Taken together, our results demonstrate that L6H21 suppresses the LPS/TLR4/NF‐κB‐mediated inflammation and NLRP3 inflammasome activation in ethanol and LPS‐induced liver injury. L6H21 might be used an alternative strategy for ALD prevention/treatment.

Support or Funding Information

NIH

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Asparaginase Immune Complexes Induce FcγRIII‐Dependent Basophil Activation and Concentration‐Dependent Hypersensitivity Reactions
Authors: Rathod Sanjay , Ramsey Manda , Finkelman Fred D , Fernandez Christian A
Published: June 2021

Background

Asparaginase (ASNase) is an important drug for treatment of pediatric acute lymphoblastic leukemia. However, antibody responses to ASNase increase the risk of leukemia relapse by mediating ASNase‐induced hypersensitivity and blocking ASNase's therapeutic effects. Previously, we have demonstrated two mechanism of ASNase hypersensitivity: one involving an anti‐ASNase IgG response, and another involving cell‐associated anti‐ASNase IgE antibodies. These studies suggested that formation of ASNase immune complexes (ICs) is sufficient to induce ASNase hypersensitivity.

Objective

To detect ASNase ICs in mice after the onset of ASNase hypersensitivity and determine whether FcγRIII engagement by ASNase ICs can lead to immune cell activation.

Methods

Mice were sensitized to native E. coli ASNase using aluminum hydroxide adjuvant. Six μm protein G‐coated polystyrene beads were used to detect plasma ASNase ICs by flow cytometry after the onset of ASNase‐induced hypersensitivity. Fluorochrome‐labeled ASNase was used to detect ASNase IC binding to immune cells. The basophil activation test (BAT) was used to evaluate ASNase IC effects on basophils.

Results

ASNase ICs were detected after the onset of murine ASNase hypersensitivity reactions and ASNase IC concentration strongly correlated with reaction severity (r2= 0.796, P=0.0005). ASNase ICs formed ex vivo at high anti‐ASNase IgG1 antibody concentrations. ASNase IC binding to naïve and sensitized peripheral blood neutrophils, macrophages/monocytes, and basophils was FcγRIII‐dependent. Consistent with these results, the BAT demonstrated that ASNase IC activation of naïve and sensitized basophils was FcγRIII‐dependent.

Conclusion

ASNase‐sensitized mice form ICs after ASNase administration, which activate murine basophils, and likely mast cells, via FcγRIII. This suggests that ASNase ICs can contribute to the onset and severity of ASNase hypersensitivity.

Support or Funding Information

University of Pittsburgh School of Pharmacy and NIH Grants RO1 CA216815 to CAF

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Transmission electron microscopic analysis of mitochondria changes in pulmonary arterial hypertension
Authors: Rybka Vladyslava , Shults Nataliia
Published: June 2021

Mitochondria are dynamic organelles, which play crucial roles in many cellular processes. Their severe damage results in alterations of other ultrastructural components and leads to loss of cellular function. In pulmonary hypertension (PH), increasing of the pulmonary vascular resistance puts the strain on the right ventricle, leading to the demand for ATP production by mitochondria to ensure cardiomyocyte function. The failure of cardiac pump increases the peripheral resistance by inducing the neurohormonal activation and influences the skeletal muscle structure and function, leading to the muscle weakness. In a rat PH model, our previous morphometric study reported changes in the mitochondrial types. Type I–II mitochondria are defined as mitochondria with the preserved ultrastructure and Type III–IV mitochondria are defined as mitochondria with the severe cristae damage that are not able to produce the required amount of ATP for energy‐depended processes in cardiomyocytes. Fischer rats had 100% mortality because of right heart failure in PH. In their RV cardiomyocytes, the types III–IV of mitochondria were predominant, along with a drastic decrease in coefficient of energy efficiency of mitochondria (CEEM), while in LV types II–III of mitochondria were present. Whereas the SD rats that do not die of PH, mostly had mitochondria Type I–II in RV and LV, along with slight decrease in CEEM in RV. In both strains of rats with PH, the treatment with a Bcl‐2 inhibitor, Navitoclax (ABT‐263), lead to severe damage of the mitochondria structure, causing rats to die because of hearth failure. The transmission electron microscopy (TEM) analysis of mitochondria in skeletal muscle cells in PH revealed the decrease in mitochondria volume and the damage to the mitochondria structure. In skeletal muscle cells, Type III mitochondria were present in rats, which correlates with the reduction in aerobic capacities. These results suggest that the ultrastructural defects in mitochondria in both cardiac and skeletal muscle in PH impact the development of the hearth failure and death, thus PH can be described as a systemic disease.

Support or Funding Information

NIH

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sigma‐1 receptor agonists elicit arteriolar dilation via Akt/eNOS activation, and also have endothelial barrier protective properties
Authors: Motawe Zeinab Y. , Katnik Christopher P. , Trujillo Andrea N. , Cuevas Javier , Breslin Jerome W.
Published: June 2021

Sigma‐1 receptors (σ1) have previously gained considerable attention pertaining to neuroprotection through its actions on calcium homeostasis, and the mitochondria‐associated ER membrane domain. Our laboratory recently showed that the σ1 agonist afobazole causes an endothelial NO‐dependent decrease in contraction amplitude. However, little is known about the mechanisms and functions of σ1 receptor in endothelial cells. In the current study, we investigated how σ1 agonists impact brain arteriolar tone and the Akt/eNOS pathway. Parenchymal arterioles were isolated from brains of Sprague‐Dawley rats and mounted onto glass micropipettes in a 37 °C bath containing albumin‐physiological salt solution. The nonselective σ agonist afobazole was applied to the bath at concentrations of 50, 100, 150, 200, and 250 μM in the presence or absence of the σ1 antagonist BD1047. The selective σ1 agonist, PRE‐084 was used to stimulate human umbilical vein endothelial cells (HUVEC), in which phosphorylation of endothelial NO synthase (eNOS) or Akt phosphorylation on their activation sites (S1177 and S473, respectively) were evaluated by western blotting. NO production was measured by fluorescence microscopy in cells loaded with the indicator dye DAF‐FM. Trans‐endothelial electrical resistance (TER) was used to determine the ability of PRE‐084 to attenuate disruption of endothelial barrier function caused by the the mitochondrial function inhibitors CCCP and antimycin‐A, or the inflammatory cytokine IL‐1β. The results show that afobazole significantly dilated rat brain arterioles, which was inhibited by the σ1 antagonist BD‐1047. Selective activation of σ1 with PRE‐084 significantly increased eNOS phosphorylation on S1177 and this was diminished in the presence of BD‐1047. Akt phosphorylation on S473 following PRE‐084 treatment was also enhanced. In addition, afobazole and PRE‐084 enhanced NO production in endothelial cells. PRE‐084 significantly attenuated decreases in TER caused by IL‐1β or CCCP, but not antimycin‐A, suggesting a potential barrier‐protective role for σ1 receptor, with some limitations that may be related to the source of mitochondrial dysfunction during inflammation. Collectively, our results suggest that σ1 receptor activation can cause vasodilation via the Akt/eNOS pathway in endothelial cells, and that σ1 may enhance endothelial barrier function under inflammatory conditions.

Support or Funding Information

Supported by NIH grant R01GM120774.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Prolonged Administration of the KCa Channel Activator SKA‐31 Improves Cardiac Function and Blood Pressure in Type 2 Diabetic Goto‐Kakizaki Rats
Authors: Mishra Ramesh C , Belke Darrell , John Cini Mathew , Wulff Heike , Braun Andrew P
Published: June 2021

Endothelial dysfunction represents an independent risk factor contributing to the onset and progression of cardiovascular complications (e.g. hypertension, cardiomyopathy, stroke) in patients with Type 2 Diabetes (T2D). It is further predicted that reduction/reversal of endothelial dysfunction will mitigate T2D‐associated cardiovascular deficits. Small‐ and intermediate‐conductance, Ca2+‐activated K+ channels (KCa2.3 and KCa3.1, respectively) are prominently expressed in the vascular endothelium, and pharmacologic activators of these channels (e.g. SKA‐31) induce robust vasodilation upon acute application to isolated resistance arteries and intact animals. However, the effects of prolonged, in vivo administration of a KCa activator on the cardiovascular system have not been examined to date. In our current study, we have hypothesized that daily administration of SKA‐31 (10 and 30 mg/kg, I.P. injection) to male, non‐obese Type 2 Diabetic Goto‐Kakizaki rats (T2D GK, 14 weeks of age) for 12 weeks would oppose the development of diabetes‐related cardiac dysfunction. Echocardiographic analyses revealed that left ventricular (LV) ejection fraction, stroke volume and fractional shortening all significantly declined over time in vehicle‐treated rats, but remained at pre‐trial levels in animals administered either 10 or 30 mg/kg SKA‐31 for 12 weeks (n = 5–6/group). Chronic blood pressure measurements over the same period via radiotelemetry further revealed that SKA‐31 treatment lowered mean arterial pressure, compared with vehicle treated animals, with effects observed on both systolic and diastolic pressures (n = 5–6/group). To determine if the observed improvements in cardiac performance may be due to direct changes in the responsiveness of the coronary vasculature, isolated hearts from vehicle and SKA‐31 treated T2D GK rats were mounted in a Langendorff perfusion system under constant pressure (n ≥ 3/group). We observed that endothelium‐dependent, agonist‐evoked increases in coronary flow were similar in hearts from vehicle and SKA‐31 treated animals, as was baseline LV developed pressure. Histological staining of isolated hearts revealed differing levels of LV collagen content in the 3 treatment groups. In summary, these results indicate that prolonged SKA‐31 administration in T2D GK rats can improve cardiac function, which likely occur via both direct and indirect effects.

Support or Funding Information

This study was supported by research funding to APB from the Canadian Institutes of Health Research (MOP‐142467) and the Natural Sciences and Engineering Research Council of Canada (RGPIN‐2017‐04116), and to HW from the National Institutes of Health (R21 NS101876).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The role of enzyme and substrate dependence in NO‐mediated vascular dysfunction with aging
Authors: Bisconti Angela Valentina , Broxterman Ryan Michael , Jarrett Catherine Lee , Shields Katherine Lori , Park Soung Hun , Thurston Taylor Scott , Vidal Daniela , Richardson Russell S
Published: June 2021

The typical aging process results in arterial dysfunction, often attributed to a fall in nitric oxide (NO) bioavailability. The diminished efficacy of the enzyme responsible for NO production, endothelial nitric oxide synthase (eNOS), and the required substrate, extracellular L‐arginine, may play a role in age‐related vascular dysfunction. Supplementation of tetrahydrobiopterin (BH4), a cofactor which recouples eNOS, and chronic supplementation of L‐Citrulline (L‐Cit), which increases plasma L‐arginine, may both increase NO bioavailability. Therefore, this study sought to evaluate the role of increased eNOS activity or L‐arginine concentration alone, and the combination of both, in the NO‐mediated vascular dysfunction associated with advancing age. Eight old subjects (73 ± 6 yr) received BH4 (100 mg per kg wt) acutely and underwent a 7 day supplementation of L‐Citrulline (6 g per day). Vascular function was assessed with: brachial artery flow mediated dilation (FMD: %) and passive leg movement (PLM: blood flow ΔPeak and AUC). Assessments were performed with and without BH4, both before and after L‐Citrulline supplementation (Ctrl; BH4; L‐Cit; L‐Cit + BH4). PLM ΔPeak increased ≈35% and ≈27% in the BH4 and L‐Cit + BH4 conditions, respectively (p< 0.05). PLM AUC increased ≈114% (p< 0.05) in the L‐Cit + BH4 condition. No significant intervention‐induced changes in FMD were detected. Thus, age‐induced vascular dysfunction, as measured by PLM which is likely more NO mediated than FMD, appears to belimited to a greater degree by eNOS function than eNOS substrate. However, the combination of improved eNOS coupling with increased eNOS substrate results in the greatest enhancement in vascular function in this population.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Modeling the Effect of Pulmonary Vascular Flow Regulation on Oxygen Uptake
Authors: Roy Tuhin K , Secomb Timothy W
Published: June 2021

Pulmonary oxygen uptake is affected by the transport characteristics of the alveolar‐capillary membrane as well as heterogeneity of pulmonary blood flow, both of which can limit the lung's capacity for oxygen transport as characterized by its effective diffusing capacity. In previous work, we showed that while under resting conditions the healthy lung has a high degree of tolerance for pulmonary flow heterogeneity, under conditions of exercise such flow heterogeneity can preclude the high levels of oxygen uptake necessary to meet tissue demand. The goal of the present study is to test the ability of hypoxic pulmonary vasoconstriction (HPV) to mitigate flow heterogeneity and enable adequate oxygen transport levels in the lung to meet tissue needs under conditions of exercise. A mathematical model of pulmonary oxygen uptake is used, in which flow heterogeneity is characterized by the coefficient of variation (CV) of pulmonary capillary blood flow. Assuming an initial lognormal distribution of blood flow with a high degree of heterogeneity, we simulate flow regulation in the cases of rest and exercise by adjusting pulmonary flow resistance in each vessel in accordance with previously published experimental data. The effect of HPV on vessel flow resistance is assumed to be characterized by a sigmoidal response dependent on the capillary oxygen tension, which is approximated as the mean of the venous and arterial values in each capillary. The results show a reduction in the effective CV from a baseline of 2.73 to 1.83 under resting conditions and to 0.66 in the case of exercise. The stronger reduction in heterogeneity under conditions of exercise corresponds to the need for greater flow regulation to minimize heterogeneity under conditions of physiologic stress. The predicted level of heterogeneity in exercise is in general agreement with our previous prediction of the upper limit of CV consistent with the levels of oxygen uptake in literature data for exercising humans. The results demonstrate the essential role of HPV to regulate flow and minimize heterogeneity under conditions of exercise and highlight the need for a functional pulmonary flow regulation mechanism under stressful or pathophysiological conditions.

Support or Funding Information

Supported by NIH U01 HL133362.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Integrative Effects of Autophagy and Telomerase on Arteriolar Flow‐Mediated Dilation in Health and Coronary Artery Disease
Authors: Hughes William E , Beyer Andreas M , Gutterman David D
Published: June 2021

Flow‐mediated dilation, (FMD) is manifest by arteriolar vasodilation in response to shear stress in an endothelium‐dependent manner. Nitric oxide (NO) is the primary mechanism for FMD in health, while hydrogen peroxide (H2O2) is the dominant mechanisms of dilation in subjects with coronary artery disease (CAD). TERT, the catalytic subunit of telomerase, is responsible for maintenance of telomere length, and telomerase activity (TA) and is necessary to preserve physiological mitochondrial function under conditions of acute stress. Autophagy is a basic cellular recycling process, responding to unfolded proteins and damaged organelles. Both autophagy and TERT levels are downregulated in subjects with CAD. Inhibition of TERT in non‐CAD vessels reduces autophagy (Fig 1A), and switches the mechanism of dilation from NO to H2O2. Conversely, activation of TERT switches the mechanism of dilation from H2O2 to NO in CAD (Fig 1B.). We sought to examine whether autophagy and TA are part of the same pathway regulating microvascular FMD in healthy (non‐CAD) and CAD vessels. Human arterioles from discarded adipose tissue were isolated and prepared for videomicroscopy. Non‐CAD vessels were co‐incubated overnight with TA inhibitor (BIBR 1532; 10 μM) and an activator of autophagy (Trichostatin A, TSA; 100 nM). CAD vessels were co‐incubated with an inhibitor of autophagy (Bafilomycin A1, BAFA1; 10nM) and the transcriptional activator of TERT (AGS 499; 20 nM). Internal diameters were measured after graded increases in intraluminal pressure gradients (flow) in the presence/absence of a NOS inhibitor (L‐NAME; 100μM) or H2O2 scavenger (Peg‐Catalase, Peg‐Cat; 500 units/mL). Data are expressed as % maximal dilation. In vehicle treated vessels, the magnitude of FMD was similar between non‐CAD and CAD. Within CAD vessels, inhibition of autophagy prevented the activator of TERT (AGS) from switching the mediator of FMD from H2O2 to NO (AGS 499 + BAFA1: 84.4±9.4 vs. AGS499 + BAFA1 + L‐NAME: 92.6±6.1 vs. AGS499 + BAFA1 + Peg‐Cat: 19.1±14.0; Fig 2A, n = 3–5; P<0.05). Conversely, in non‐CAD vessels, activation of autophagy prevented the switch from NO to H2O2 typically seen during inhibition of TERT (BIBR + TSA: 86.4±9.2 vs. BIBR + TSA + L‐NAME: 36.3±12 vs. BIBR + TSA + Peg‐Cat: 73.8.9±9; Fig 2B, n = 3–5; P < 0.05,). Our data demonstrate that autophagy is distal to TERT in regulating the mechanism of FMD in human adipose arterioles and may serve as a final common pathway for determining the mechanism of FMD in human arterioles.

Support or Funding Information

Funded by T32GM089586‐09 (WEH), R01‐HL133029‐02 (AMB), and R01‐HL135901‐02 (DDG)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Endothelial Hba1 Regulates Exercise Fitness in Mice
Authors: Keller Alexander S , Brooks Steven , Islam Aditi , Keller T.C. Stevenson , Best Angela K , Murphy Sara , Page Henry Askew , Cortese‐Krott Miriam M , Yan Zhen , Ackerman Hans , Isakson Brant E
Published: June 2021

Hemoglobin alpha (Hbα) is expressed from the Hba1 and Hba2 genes in the endothelium of resistance arteries, where it plays a vital role in regulating NO signaling by endothelial nitric oxide synthase (eNOS). We have previously shown that pharmacologic inhibition of the eNOS‐Hbα interaction reduces blood pressure, and that siRNA knockdown of endothelial Hba1 increases NO signaling in resistance arteries. We have created a genetically modified mouse model in which loxP sites flank the second and third exons of the Hba1 gene (Hba1fl/fl), and crossed these Hba1fl/fl mice with the Cdh5‐Cre/ERT2+line to produce an endothelial‐specific, tamoxifen‐inducible genetic model of Hba1 deletion (EC Hba1Δ/Δ). Similar to mice with global deletion of Hba1 (Hba1−/−), EC Hba1Δ/Δ mice display reduced Hbα expression in the endothelium of resistance arteries; however, EC Hba1‐Δ/Δ mice display normal red blood cell parameters in contrast to the anemic phenotype of Hba1−/− mice. EC Hba1Δ/Δ mice also display unaltered expression of other globins in the vascular wall of resistance arteries, as well as normal capillary density in fast‐twitch and slow‐twitch hindlimb muscles. We used a treadmill running test as a measure of acute endurance capacity to examine the role of endothelial Hbα in exercise hyperemia. EC Hba1Δ/Δ mice showed a significant reduction in endurance capacity as compared to Hba1fl/fl controls, equal to half the effect seen in Hba1−/− mice with constitutive loss of both vascular and RBC Hba1. We conclude that endothelial Hba1 plays an important role in vascular function and exercise performance.

Support or Funding Information

AHA Pre‐Doctoral Award (ASK, TCSK); NIH NRSA (ASK, TCSK), NIH 5T32 GM008715 (ASK); NIH HL088554 (BEI); German Research Council DFG CO 1305/2‐1 (MMCK); NIH R01‐AR050429 (ZY), Support from NHLBI Intramural Funding (SB, HA)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Human Coronary Micro‐artery Dysfunction is Associated with Structural and Smooth Muscle Cell Phenotypic Alterations
Authors: Borysova Lyudmyla , Ye Xi , Smart Nicola , Ascione Raimondo , Dora Kim A
Published: June 2021

Autoregulation is compromised in patients with coronary heart disease. A lack of myogenic reactivity and endothelial dysfunction each contribute to a reduced ability to control blood flow within the coronary microcirculation. Little is known how coronary arterial smooth muscle structure and phenotype links to vascular reactivity.

Right atrial appendage biopsies from consented patients undergoing valve replacement surgery, and from pigs were collected at clinical standards under general anesthesia. Human and porcine arteries from right atrial appendage samples were isolated and mounted for pressure myography. Confocal imaging established vasomotor responses, cellular viability, and immunohistochemical localization of proteins. The human study complies with the Helsinki Declaration and the animal study complies with the UK Animal (Scientific Procedures) Act, 1986.

Here, we compared the structural features of human patient intra‐myocardial coronary arteries (IMCAs) with equivalently handled porcine IMCAs, representing healthy, juvenile controls. More than half the human IMCAs failed to develop ≥10% myogenic tone, even those in which constituent smooth muscle cells (SMCs) could adequately mobilize intracellular Ca2+required for contraction. 3D confocal revealed a novel, distinct layer of longitudinally‐arranged sub‐intimal SMCs (l‐SMCs) between the endothelium and the expected circumferentially‐arranged SMCs responsible for radial contraction (r‐SMC) in the majority of human and porcine IMCAs.

Contractile function strongly correlated with SMC phenotype, with the synthetic markers L‐caldesmon and vimentin highly expressed in SMCs with poor contractile function, namely the human r‐SMCs and the porcine l‐SMCs. Thus, we propose that l‐SMCs retain an immature, synthetic phenotype in healthy vessels with functional r‐SMCs but, with loss of r‐SMC function in aged patients, l‐SMCs may play a reparative role and can adopt a contractile phenotype in an attempt to restore function and control myocardial perfusion. The role of l‐SMCs in maintaining the integrity of arterial function warrants further investigation.

Support or Funding Information

This work was supported by the British Heart Foundation (grant numbers FS/08/033/25111, FS/13/16/30199, IG/13/5/30431, PG/18/11/33552) and by the Oxford BHF Centre of Research Excellence (RE/13/1/30181).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Loss of endothelial surface molecules in the central retinal artery and retina with methamphetamine
Authors: Lee Minsup , Leskova Wendy , Carter Patsy , Eshaq Randa , Harris Norman
Published: June 2021

Objective

The purpose of this study is to investigate the retinal effects of methamphetamine.

Introduction

METH, a highly addictive stimulant of neurotransmitters is associated with cardiovascular and neurological diseases. METH‐induced ophthalmic complications are also present but have been insufficiently investigated.

Materials and methods

C57BL/6J mice were administrated progressively increasing doses of METH (0 to 6 mg/kg) by repetitive intraperitoneal injections for 5 days (4 times per day). Norepinephrine levels in plasma were measured by ELISA. Cross‐sections of the central retinal artery (CRA) and retina were stained with hematoxylin and eosin. Expression of various proteins was determined by immunostain and immunoblot in the CRA and retina.

Results

Elevated norepinephrine levels in plasma were induced by METH (6.49±1.72 ng/mL, n=5), compared with saline treatment (1.38±0.48, P<0.001, n=5). After treatment of METH, the ocular pupil was dilated and retinal thicknesses of the outer nuclear layer, outer plexiform layer, inner nuclear layer, and inner plexiform layer were decreased. Platelet endothelial cell adhesion molecule‐1 (PECAM‐1) protein expression level was decreased in the CRA and retina of METH‐treated mice, along with the endothelial proteoglycans glypican‐1 and syndecan‐1. Moreover, a regulator of the extracellular matrix, membrane type 1 matrix metalloproteinase (MMP‐14), but not MMP‐2 and MMP‐9, was increased in the retina by METH treatment.

Conclusion

These results suggest that METH administration is involved in retinal degeneration with a vascular loss of PECAM‐1 and the glycocalyx, and an increase of MMP‐14 in the CRA and retina.

Support or Funding Information

This work was supported by funding from the National Institute of Health (NIH) EY025632.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Novel Methodology to Assess Retinal Vascular Permeability in a Rodent Model of Diabetes
Authors: Allen Claire , Malhi Naseeb , Bates David , Arkill Kenton
Published: June 2021

Purpose

Diabetic retinopathy (DR) is a major complication of diabetes and is one of the leading causes of blindness worldwide. Treatment of DR can only be achieved through an enhanced understanding of disease pathogenesis. Structural, functional and biochemical studies cannot be carried out in human subjects and chemically induced diabetic rodent models that mimic the early developmental changes in DR and have become the gold standard. In combination with fundus fluorescein angiography (FFA) retinal abnormalities, non‐perfusion and vascular permeability can be assessed and in response to treatments over time.

Methods

FFA was carried out in non‐diabetic and diabetic Norway Brown rats (250–300g) on days 0, 14, 21 and 28 using the Micron IV retinal imaging microscope (Phoenix Research Labs, CA). Animals received a single dose of PBS (n=6) or streptozotocin (STZ) i.p., 50mg/kg (n=6) and blood glucose levels were measured throughout. Video angiograms were quantitatively analysed for suboptical resolution microvascular permeability by using the arterioles as the measure for luminal concentration. In a separate subset of experiments primary human retinal endothelial cells (pHREC) were exposed to normo‐(D‐Mannitol, 30mM) or hyperglycaemia (D‐Glucose, 30mM) and probed for glycosaminoglycans (GAGs) heparan sulfate (HS), chondroitin sulfate (CS), and the tight junction marker ZO‐1.

Results

Diabetes was successfully induced in Norway Brown rats following a single dose of STZ (Blood glucose 28.4±2.6 mmol/L, day 28). By adapting Fick's Law retinal permeability (Ps, um.s^−1) was calculated and shown to significantly increase (p<0.05) for each time point, over the 28‐day period. To understand the mechanisms that may account for this increase in solute permeability across the retinal vessel wall we observed a significant decrease in sulphated GAG expression, more specifically HS (p<0.05) in pHREC exposed to hyperglycaemia versus normoglycaemia. In addition, the expression of the tight junction protein ZO‐1 significantly decreased in pHREC when exposed to hyperglycaemic conditions.

Conclusions

Retinal vascular solute permeability can be accurately quantified in a rodent model of diabetes using the outlined methodology and mathematical quantification. Advanced quantitative assessments such as this could substantially contribute to a better understanding of the pathogenesis of DR and identify potential drugs for treatment.

Support or Funding Information

MRC

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.