bluuebunny/crossref_metadata_2025 huggingface dataset:
Genotype‐phenotype correlation of polymorphisms in cytochrome b5 and NADH cytochrome b5 reductase and hydroxylamine reduction in human breast
Authors: Rhoads Keelia Marie , Trepanier Lauren
Published: June 2021

4‐aminobiphenyl (ABP), found in tobacco smoke, is oxidized to a hydroxylamine (ABP‐NHOH) and further bioactivated into a nitrenium ion that binds DNA. While these DNA adducts are thought to play a role in initiating breast cancer, epidemiological studies looking at the relationship between ABP exposure (via smoking) and breast cancer have been inconclusive. We have previously shown that hydroxylamines can be reduced back to their parent compounds by hepatic cytochrome b5 (cyt b5) and NADH cytochrome b5 reductase (b5R), but no studies have investigated these two enzymes in human breast. The aims of this study are to characterize variability in hydroxylamine reduction in 100 human breast samples, and to determine cDNA genotypes for cyt b5 and b5R in these same samples. Our overall goal is to determine how polymorphisms in these two enzymes may modulate local breast detoxification of ABP‐NHOH. Results to date indicate that the kinetic parameters for ABP‐NHOH reduction in breast tissue was similar to that previously found for human liver (Km =280 µM, Vmax =0.66 nmol/mg/min). cDNA sequencing has been complete for 95 samples. Two individuals were heterozygous for novel non‐synonymous single‐nucleotide polymorphisms in either cyt b5 (H22R) or b5R (A158T). Work is ongoing to determine ABP‐NHOH reduction activities in these and other individual breast samples and to determine the relationship between these non‐synonymous cSNPs and altered reduction activities.

Supported by NIH grant GM61753, NIEHS grant T32 ES007015
Inhibition of anion fluxes in Calu‐3 cells by polystyrene nanoparticles: role of lipid rafts
Authors: McCarthy Joanna , Duszyk Marek , Radomski Marek
Published: June 2021

Introduction

There is growing concern about the potential toxicity of non‐engineered and engineered nanoparticles. Lungs are particularly susceptible to the injury brought about by inhalation of gases and aerosols containing nanoparticles. Aims: We investigated the effects of apical exposure (up to 60 min) of cells to polystyrene nanoparticles (PNPs) (positive, negative, uncharged; 20, 50, 100nm) on transepithelial anion fluxes.

Methods

Calu‐3 cells grown on inserts were mounted into modified Ussing chambers. Anion fluxes (measured as short‐circuit current, Isc) were stimulated with forskolin (10µM) in the presence or absence of PNPs (88‐104 µg/ml). In some experiments lipid rafts were disrupted by removing cell surface cholesterol with cyclodextrin (CD).

Results

Forskolin resulted in mean peak increase in Isc from 29.4 to 108.4µA/cm2. All charged PNPs caused a significant (p<0.05, n.≥3) reduction of forskolin‐stimulated anion fluxes (from 24 to 40‰), an effect undetectable with uncharged particles. The CD treatment led to a significant potentiation of the inhibitory effects of 20 and 50nm PNPs. In contrast, the treatment abolished the inhibitory effects of 100nm PNPs.

Conclusion

Our results suggest that charge, size and interactions with lipid rafts are essential factors in determining the extent of PNPs‐mediated inhibition of forskolin‐stimulated anion fluxes in Calu‐3 cells.
Antibacterial Activity of some New substitued 4‐(2‐Benzothiazolylamino‐2H[1]‐Pyran‐2‐ones
Authors: Vehapi Idriz , Hoti Ramiz , Kurteshi Kemajl , Mulliqi‐Osmani Gjyle , Troni Naser , Ismaili Hamit
Published: June 2021

Some new substitued 4‐(2‐benzithiazolylamino‐2H[1]‐pyran‐2‐ones are synthesized by condensation of 2H[1}‐pyran‐2‐ones and corresponding 2‐aminobenzothiazoles. Condensation of 4‐chloro‐6‐methyl‐2H[1]‐pyran‐2‐one 2 and 2‐aminobenzothiazoles 3(a‐c) gave corresponding 4‐(2‐benzothiazolylamino)‐6‐methyl‐2H[1]‐pyran‐2‐ones 4(a‐c). By condensation of 4‐chloro‐3‐nitro‐6‐methyl‐2H[1]‐pyran‐2‐one 6a and 2‐aminobenzothiazoles 3(ad) were synthesized 4‐(2‐benzothiazolylamino)‐3‐nitro‐6‐methyl‐2H[1]‐pyran‐2‐one 6a and 4‐(6‐ethoxy‐2‐benzothiazolylamino)‐3‐nitro‐6‐methyl‐2H[1]‐pyran‐2‐one 6c . The antibacterial activity of products 4(a‐d) against E. coli S. aureus and Clebsiella were investigated. Compounds 4(a‐d) showed light bactericide activity against E. coli, S. aureus and Clebsiella. Compounds 4b and 6d were more active against E. coli. Emphatic activity against S. aureus exhibited compounds 4c and 6d whereas compounds 6a and 6d exhibited the strongest activity against Clebsiella. In general there was a direct positive correlation between increasing concentration of the compound and antibacterial activity.
Sterile biofilm on intraperitoneal polymers results in local and systemic inflammation in mice
Authors: Flessner Michael F , Gomez‐Sanchez Elise , Potter Rebecca , Li Xiarong , He Zhi , Henegar Jeffrey , Hoskins Glenn
Published: June 2021

To address the hypothesis that inflammatory biofilm on an intraperitoneal (ip) polymeric foreign body is associated with tissue and systemic inflammation, we inserted ip into 40 C57Bl/6 mice: 5 sterile loops of catheter made from silicone (SI), polyurethane (PU), or polyethylene (PE); controls had sham operations. After 1‐5 weeks, dis‐membranated biofilm and peritoneal tissue were cultured to demonstrate sterility and studied with electron microscopy (EM) and immunochemistry (IC). Transmission EM demonstrated macrophage, fibroblast, lymphocyte, mesothelial cells but no bacteria. Total biofilm cell density (#/cm2) varied from 1‐4 x 104 in the first week and increased with time. Marked changes were noted in thickness, angiogenesis and peritoneal staining of TGFβ, FGF, VEGF, and αSMA =4 times that of controls. Biofilm cells were identified with IC (antibody) as macrophages (F4/80), lymphocytes (CD8), mesothelial cells (cytokeratin), myofibroblasts (aSMA, vimentin). Biofilm and extraperitoneal tissue IC staining for cytokines paralleled the alterations in the peritoneum. Control animals had minimal staining in all tissues. These data demonstrate a sterile biofilm is associated with marked structural and inflammatory changes in peritoneal and extraperitoneal tissue.
Are Inward Rectifier K+ Channels Responsible for Integrin‐Modulated Oxidative Burst Activity?
Authors: Colden‐Stanfield Margaret
Published: June 2021

Integrin‐dependent modulation of macrophage K+ channels and its impact on macrophage function has only recently been studied (Colden‐Stanfield, 2008). Parallel patch‐clamp and oxidative burst experiments were performed in murine bone marrow‐derived macrophages (BMDMs) with or without clustered very late antigen‐4 (VLA‐4) integrins. BMDMs cultured in suspension that were further incubated on uncoated polystyrene (POLY) for 5 h only possessed inwardly rectifying potassium (Kir) currents with a resting membrane potential of ‐57 mV. Ninety percent of BMDMs with clustered VLA‐4 integrins possessed both Kir and delayed rectifier potassium (Kdr) currents with a more depolarized resting membrane potential (‐47mV). Under identical experimental conditions basal and stimulated intracellular hydrogen peroxide production was measured. Basal and PMA‐stimulated oxidative burst activity was enhanced 5‐and 14‐fold in BMDMs with clustered VLA‐4 integrins, respectively. Although specific blockade of Kdr currents with margatoxin did not alter the VLA‐4‐augmented oxidative burst activity, exposure to the Kir blocker, CsCl, significantly reduced the enhanced response. These data suggest that VLA‐4 integrins modulate K+ currents to possibly enhance the ability of macrophages to mount a respiratory burst in an immune response. This research was supported by NHLBI K01 HL076628.
Role of mast cells in ischemia/reperfusion‐induced arteriolar endothelium‐dependent vasodilatory dysfunction.
Authors: Dai Hongyan , Kalogeris Theodore , Korthuis Ronald
Published: June 2021

The aim of this study was to determine whether ischemia/reperfusion (I/R)‐induced mast cell degranulation plays a role in the development of postischemic arteriolar endothelium‐dependent vasodilator dysfunction. To address this issue, vasodilatory responses to acetylcholine or nitroprusside were monitored in rat mesenteric arterioles (30 um diameter) under control conditions (no ischemia) and following 20 min of ischemia and 60 min of reperfusion using intravital microscopy. The mesentery was superfused during reperfusion with the mast cell stabilizers. Mast cell degranulation was monitored by use of ruthenium red staining. I/R was associated with mast cell degranulation and a marked reduction in vasodilator responses to acetylcholine, but not to nitroprusside. Treatment with mast cell stabilizers prevented mast cell degranulation and improved arteriolar responses to acetylcholine. Moreover, superfusing the mesentery with compound 48/80, at doses that produced a similar degree of mast cell degranulation as noted during I/R, produced a similar deficit in arteriolar endothelium vasodilator responses. Our results are consistent with the concept that postischemic arteriolar endothelium‐dependent vasodilator dysfunction occurs by a mast cell‐dependent mechanism. This work was supported by HL‐082816 from the NIH.
Thymoquinone Decreases Macrophage Cell Viability and Function
Authors: Surber Kirk , Tucci Michelle , Benghuzzi Ham , Cameron Joseph A.
Published: June 2021

Thymoquinone has been established as the major component of the oil extracted from Nigella saliva plant seeds, which is a frequently used herbal medicine. Thymoquinone has been shown to have anti‐inflammatory properties. The objective of the present study was to determine the immunomodulatory role of thymoquinone (TQ) regarding its effect on the production of nitric oxide (NO) by macrophages. Under certain conditions, macrophages and certain other cells can produce high concentrations of NO from its precursor L‐arginine via inducible nitric oxide synthase (iNOS) pathway. Macrophage cells administered 5 μM, 10μM and 50 μM of TQ were evaluated for cell number, cellular glutathione content, cellular membrane damage, and cellular nitric oxide after periods of 24, 48 and 72 hours. Following 24 and 48 hours of TQ administration there were no significant differences in cell numbers, cellular glutathione content, or cellular membrane damage between the control and treatment groups. By 72 hours there was a dose dependent decrease in cell numbers. As the dose increased there was a significant loss in cell numbers. There was a corresponding increase in NO as the dose of TQ increased. This information is important in understanding the potential therapeutic effects of TQ. Supported in part by NIGMS‐NIH Grant R25 GM50117.
Acute Exposure to Conventional Peritoneal Dialysis Fluids Does Not Alter the Hepatic Microcirculation
Authors: Patrick Amanda , Fox‐Robichaud Alison
Published: June 2021

Peritioneal Dialysis (PD) has been associated with the development of structural and functional alterations of the peritoneal membrane and vasculature, including that of the liver. In this study, we investigated the contribution of pH buffers and glucose content of conventional PD fluids on the hepatic microvasculature.

To determine whether acute exposure to a variety of PD solutions altered the hepatic inflammatory response, male C57BL/6 mice received TNF 500 ng intraperitoneally (IP) or 50 μl normal saline. At 3hrs, all mice received 2mL of one of eight PD fluids (IP), which varied in either sugar, concentration of sugar, or buffer. Controls received 2mL of lactated Ringer's. The hepatic microcirculation was examined at 1hr post‐PD by intravital microscopy.

As expected, TNF‐treated mice showed significantly higher leukocyte recruitment as compared to controls in both post‐sinusoidal and sinusoidal venules (P<0.05). There was no significant difference in post‐sinusoidal rolling and adhesion or sinusoidal adhesion between any of the 8 PD fluids tested. White blood cells count did not differ significantly between all solutions (P<0.05).

These data suggest that during an acute exposure to the tested PD fluids and with the variation in glucose content, sterilization and buffering methods, hepatic leukocyte‐endothelial cell interactions were not significantly altered. Importantly, these effects were also not seen when the hepatic microcirculation was stimulated with the cytokine TNF. Compared to our corresponding work in the peritoneal microcirculation the liver may require a longer exposure to PD fluids.
Activation of Peroxisome‐Proliferator‐activated receptors alpha and gamma mediates remote ischemic preconditioning against myocardial infarction
Authors: Lotz Christopher , Redel Andreas , Stumpner Jan , Thorsten Smul , Tischer‐Zeitz Tobias , Schmidt Johannes , Wunder Christian , Lange Markus , Roewer Norbert , Kehl Franz
Published: June 2021

Background

Remote ischemic preconditioning (RIPC) protects the heart against myocardial infarction. We tested the hypothesis that remote ischemic preconditioning is mediated by PPARα and PPARγ activation.

Methods

New Zealand white rabbits (n = 78) were subjected to a 30‐min coronary artery occlusion and 3 h of reperfusion. 3 cycles of RIPC consisted of 10 minutes renal ischemia alternating with 10 minutes of reperfusion. In different groups animals received the PPARα‐Antagonist GW6471 or the PPARγ‐Antagonist GW9662 alone or in combination with RIPC. Infarct size (IS) was determined gravimetrically. Myocardial tissue levels of PGJ2 and nitrite/nitrate (NOx), as well as PPAR DNA binding activity were measured using specific assays. Data are mean±SD.

Results

RIPC significantly reduced myocardial infarct size (42.2±4.9%*, p<0.05), compared to control (61±1.9%), and increased PPAR DNA‐binding activity (189.6±19.8RLU*, p<0.05) and tissue levels of PGJ2 (179.7±7.9 pg/ml*, p<0.05). Infarct size reduction and increased PPAR DNA‐binding activity by RIPC was abolished by GW6471 (56.8±4.7% and 61.1±14.2RLU) or GW9662 (57.4±3.3% and 52.7±16.9RLU), in the presence of significantly reduced tissue levels of NOx (5.4±0.3μM* and 4.3±0.2μM*, p<0.05).

Conclusion

The results obtained suggest a pathway whereby RIPC leads to the production of PGJ2, activating PPARα and PPARγ, in turn mediating the production of NO.
Sudden death in severe gut ischemia: a possible role for the autonomic nervous system
Authors: Penn Alexander Hayes , Schmid‐Schönbein Geert W.
Published: June 2021

Occlusion of blood vessels in the gut is a life‐threatening event. For example, splanchnic arterial occlusion (SAO) followed by reperfusion is an established model for physiologic shock. In addition SAO is highly lethal on its own, either associated with gradual drops in mean arterial blood pressure (MABP) over <1 to 3 hours or with a severe rapid drop in MABP (often > 80 mmHg) over 1 to 6 minutes, leading to left ventricular arrest. The rapid drop occurs after ~ 75‐150 min of ischemia. We observed that high levels of glucose in the lumen of the small intestine decrease the ischemic duration before which a rapid drop may be observed (as early as 30 min) and increase its rate of occurrence (from 29% to 63%). As afferent neurons in the gut are capable of sensing ischemia and/or hyperglycemia, our objective was to determine whether a total subdiaphragmatic vagatomy (TSV), which prevents parasympathetic sensing in the gut, could prevent the rapid drop in MABP. We found that survival time was increased by TSV (P<0.05). While not preventing the gradual drops in MABP, TSV significantly reduced the instances of rapid drops in MABP (P<0.008). These results suggest that meals high in glucose and parasympathetic sensing in the gut may greatly affect the amount of time available to treat gut ischemia before catastrophic cardiovascular failure. Supported in part by HL 67825, 76180 and an unrestricted educational gift from Leading Ventures.
Myogenic constriction and dilation of isolated lymphatic vessels
Authors: Davis Michael J , Davis Ann M , Ku Christine W , Gashev Anatoliy A
Published: June 2021

We tested if lymphatic vessels exhibit myogenic constrictions and dilations to intraluminal pressure changes. Collecting lymphatic vessels were isolated from rat mesentery, cannulated and pressurized for in vitro study. Diameters were measured in response to pressure steps of different magnitudes, starting from various baseline pressures. Myogenic constriction, a time‐dependent decrease in end‐diastolic diameter over a 1‐2 min period following pressure elevation, was observed in the majority of vessels (~74%) and occurred over a relatively wide pressure range (1‐15 cmH2O). Myogenic dilation, a rise in end diastolic diameter following pressure reduction, was observed in over half the vessels equilibrated at a low baseline pressure. The inflammatory mediator substance P, which is known to enhance lymphatic contractility, increased the percentage of vessels exhibiting myogenic constriction, the magnitude and rate of that constriction, and the pressure range over which it occurred. Our results demonstrate that myogenic responses occur in lymphatic vessels and suggest the possibility that the response may aid in preventing vessel overdistention during conditions of inflammation/edema. NIH HL‐089784.
Evidence for the Involvment of Endothelial A2A Receptors in Adenosine Induced KATP Channel‐dependent Arteriolar Vasodilation
Authors: Maymon Nir , Sarelius Ingrid H
Published: June 2021

Adenosine (ADO) has an important role in many vascular processes including vasodilation, inflammation, permeability and injury protection. We have shown previously that endothelium is required for ADO mediated arteriolar dilation (Duza et al, AJP 285:H26, 2003). In the present study we investigated the receptor subtype and signaling cascade that mediate the ADO induced vasodilation in mouse cremaster arterioles. We examined which receptor subtype is implicated in the ADO induced vasodilation using specific P1 receptor antagonists; DPCPX, SCH442416 and PSB603 for A1, A2A and A2B receptors respectively. Control ADO dilation of 16.3±1.3 ?m (n=19) was decreased to 3.4±1.2 ?m (n=4, P<0.05) with SCH442416 (A2A antagonist). DPCPX (A1‐ antagonist) and PSB603 (A2B antagonist) had no effect on the ADO induced dilation: 14.8±3.3 ?m (n=5, P>0.05) and 16±4.1 ?m (n=5, P>0.05) respectively. Additionally we used the KATP channel blocker Glibenclamide to test the involvement of KATP channels in A2A receptor mediated dilation using the A2A specific agonist CGS21680. Control dilation was reduced from 10.4±1.8 ?m to 2.3±1.1µm (n=6, P<0.05) with Glibenclamide.

Immunohistochemistry revealed that both A2A and A2B receptors are located on the endothelium. Thus, our results suggest that the primary pathway for this endothelium‐dependent ADO vasodilation is through A2A receptors acting via KATP channels. (Supported by HL76414)
Uterine distension stimulates axial but not circumferential uterine vessel remodeling
Authors: Barron Carolyn Jane , Osol George
Published: June 2021

During pregnancy, there is a dramatic increase in blood flow to the uterus. This process is facilitated by circumferential remodeling of uterine arteries that involves nitric oxide signaling. Significant axial growth also occurs, although the underlying mechanism is not known. The objective of this study was to test the hypothesis that arterial axial growth is stimulated by uterine myometrial stretch, which occurs secondary to placentation and fetal growth.

To directly evaluate the effects of stretch, we developed a surgical procedure in which one rat uterine horn was injected with 0.3 ml of medical grade silicone. Animals were sacrificed 6‐8 weeks post surgery and uterine and vessel measurements were determined in unstressed conditions and compared to the unoperated horn.

The procedure induced biaxial stretch of the uterine corpus, with increases of 120 and 250% in length and width, respectively. This uterine stretch caused 40‐60% increases in arterial length and a near doubling of the length of the segmental vessels which emanate from the main uterine artery and feed the uterine wall. No appreciable differences were noted in the circumferential remodeling of the main uterine artery, suggesting that: (1) the mechanisms that govern circumferential vs axial remodeling may be distinct, and (2) uterine stretch may specifically induce axial rather than circumferential gestational remodeling of uterine arteries.
ASIC1 Contributes to Store‐Operated Currents in Pulmonary Vascular Smooth Muscle
Authors: Jernigan Nikki L , Paffett Michael L , Walker Benjimen R , Resta Thomas C
Published: June 2021

Store‐operated Ca2+ entry (SOCE) plays a significant role in pulmonary vasoregulatory mechanisms such as hypoxic pulmonary vasoconstriction and receptor‐mediated arterial constriction. Our laboratory has recently suggested that a unique family of non‐selective cation channels, the acid‐sensing ion channel 1 (ASIC1), contributes to SOCE in pulmonary arterial smooth muscle (PASM). To further investigate a role for ASIC1 in SOCE, we hypothesized that ASIC1 contributes to store‐operated Ca2+ currents in PASM cells. To test this hypothesis we measured cyclopiazonic acid (CPA)‐induced Ca2+ and Na+ currents using conventional whole cell patch‐clamp configuration in voltage‐clamp mode. We found that the ASIC inhibitors, amiloride (30 µM) and benzamil (10 µM), largely attenuated both CPA‐induced Ca2+ and Na+ currents in freshly isolated and primary cultured rat PASM cells. Consistent with these results, we additionally found that ASIC1 siRNA inhibited CPA‐induced Ca2+ and Na+ currents. In contrast, ASIC3 siRNA diminished CPA‐induced Na+ currents, but had no effect on Ca2+ currents, whereas ASIC2 siRNA was without effect on either CPA‐induced Ca2+ or Na+ currents. Together, these data provide further evidence for a novel role of ASIC1 in mediating SOCE in PASM. This work is supported by NIH and AHA.
Intermittent Hypoxia Augments NO‐Dependent Pulmonary Vasodilation
Authors: Norton Charles Elbert , Jernigan Nikki , Kanagy Nancy , Walker Benjimen , Resta Thomas
Published: June 2021

Both hypocapnic and eucapnic intermittent hypoxia (H‐IH and E‐IH, respectively) augment endothelium‐derived nitric oxide (NO)‐dependent pulmonary vasodilation in rats, a response that is masked by endogenous superoxide anion (O2−) following H‐IH only. We hypothesized that H‐IH and E‐IH increase pulmonary vascular smooth muscle sensitivity to NO, and that augmented NO‐dependent vasoreactivity after H‐IH is limited by scavenging of NO by endogenous ROS. To test these hypotheses, we examined vasodilatory responses to the NO donor S‐nitroso‐N‐acetylpenicillamine (SNAP), in isolated, U‐46619 constricted, saline perfused lungs from H‐IH (3 min cycles of 5% O2/air flush, 7 hr/day, 4 wk), E‐IH (5% O2, 5% CO2/air flush) or Sham‐treated rats (air/air cycling) in the presence of the NO synthase inhibitor L‐NNA. Parallel experiments were conducted following administration of the superoxide anion (O2−) scavenger tiron to evaluate the contribution of O2− to limit NO‐dependent dilation. Vasodilatory responses to SNAP were augmented in lungs from E‐IH, but not H‐IH, rats compared to Sham controls in the absence of tiron. However, with the addition of tiron, the H‐IH group showed a similar increase in reactivity to NO. We conclude that intermittent hypoxia increases pulmonary vascular smooth muscle sensitivity to NO. Furthermore, endogenous O2− limits NO‐dependent pulmonary vasodilation following H‐IH.
Hyperresponsiveness to serotonin in rat pulmonary arteries from rats with streptozotocin–induced diabetes
Authors: Frazziano Giovanna , Lopez‐Lopez Gustavo , Moral Javier , Gomez‐Villalobos Maria Jesus , Cogolludo Angel , Menendez Carmen , Perez‐Vizcaino Francisco
Published: June 2021

Recent data suggest that diabetes is a risk factor for pulmonary hypertension. Several lines of evidence suggest that serotonin is a pathogenetic factor in pulmonary hypertension. We have also recently found pulmonary endothelial dysfunction in diabetic rats. The aim of the present study was to analyze whether diabetes induces changes in the response to serotonin in rat pulmonary arteries and the mechanisms involved. Male Sprague‐Dawley rats were randomly divided into a control (saline) and a diabetic group (70 mg/kg streptozotocin). After 6 weeks, the responses to serotonin were enhanced in pulmonary arteries from diabetic rats. This hyperresponsiveness was endothelium‐independent and unaffected by inhibition of NO synthase but prevented by the cyclooxygenase (COX) inhibitor indomethacin, by superoxide dismutase and by the NADPH oxidase inhibitor apocynin. Lungs from diabetic animals showed an increased expression of COX‐2 as measured by Western blot analysis. In conclusion, as has been described in animal models of pulmonary hypertension, pulmonary arteries from diabetic animals show hyperresposiveness to serotonin. This effect appears to be related to COX‐2 derived metabolites and NADPH oxidase‐induced superoxide production. Supported by Comisi ón Interministerial de Ciencia y Tecnología (CICYT SAF2008‐03948).
Pendrin gene ablation enhances vascular contractility
Authors: Sutliff Roy L. , Walp Erik R. , Bonsall Robert , Ramosevac Semra , Eaton Douglas C. , Kim Young Hee , Pham Truyen D. , Wall Susan M.
Published: June 2021

Pendrin, encoded by Slc26a4, is expressed in the apical regions of a subset of renal intercalated cells, where it mediates Cl− absorption and HCO3− secretion through apical Cl−/HCO3− exchange. Pendrin null mice have reduced blood pressure and a blunted hypertensive response to aldosterone. The purpose of this study was to determine if pendrin is expressed in blood vessels and to determine if the lower blood pressure observed in pendrin null mice occurs from changes in vascular contractility. Mice were pair‐fed a balanced, NaCl‐replete diet for 7 days. Aortas were removed from wild‐type and pendrin null mice, denuded and isometrically mounted. Aortic contraction was studied using potassium chloride (KCl; 0‐80 mM) and phenylephrine (PE; 0.1 nM‐10 ìM). The sensitivity to the contractile agents was unaffected by pendrin gene ablation. However, the maximum contractile responses to KCl (14.6 ± 1.2 and 9.8 ± 1.2; N=5‐6) and PE (18.5 ± 1.3 and 12.6 ± 1.6; N=5‐6) were significantly greater in aortas from pendrin knockout mice when normalized to cross sectional area. However, in aorta Slc26a4 mRNA was detected at very low levels. Thus, pendrin gene ablation enhances vascular contractility, through an indirect effect of pendrin in the vasculature. The increased vascular contractility observed in these mutant mice is likely a compensatory response to the reduced blood pressure, rather than a cause of the blood pressure change.
Time‐dependency of angiotensin II mediated vasomotor dysfunction
Authors: Erkuran Yilmaz Cigdem , Granger D. Neil
Published: June 2021

Angiotensin II (AngII), the main effector of the renin‐angiotensin system, plays an essential role in the regulation of blood pressure and it has been implicated in the pathogenesis of cardiovascular diseases. While AngII is known to alter endothelial function, the time‐course of this response remains poorly understood. The aim of this study was to compare the effects of acute vs chronic angiotensin II treatment on endothelium‐dependent vasomotor function. Wire myography was used to assess vasomotor function of aortic rings obtained from C57BL/6J male mice. Aortic rings were incubated with angiotensin II for either either 15 minutes or 4 hours. In another group, aortic rings were isolated from WT mice subjected to chronic AngII infusion (Alzet pump) at a rate of 2000 ng/kg/min for 14 days. Endothelium‐dependent vasomotor function was assessed by monitoring acetylcholine‐induced vasorelaxation. While AngII exposure for 15 min did not alter endothelium‐dependent vasomotor function, 4 h incubation with AngII significantly (~50%) impaired endothelium dependent vasodilation, compared to untreated rings. A similar level of vasomotor impairment was noted in aortic rings from mice subjected to chronic AngII infusion. These findings reveal a time‐dependency of AngII mediated vasomotor dysfunction and indicates that this response occurs via a mechanism that likely involves new protein synthesis. (HL26441)
LIM Kinase regulation of cofilin activity mediates PDGF‐induced VSMC migration
Authors: Torres Rebecca A , Holland Drury S , Weber David S
Published: June 2021

Platelet derived growth factor (PDGF) is a pro‐migratory factor released in response to vascular injury. Vascular smooth muscle cells (VSMCs) mediate this response and contribute significantly to neointimal formation, thus delineating the molecular mechanisms of VSMC migration is critical to understanding the vascular repair process. Signaling cascades activated by PDGF ultimately converge upon the actin cytoskeleton which is a key regulator of migration. Recent studies report that cofilin, which upon activation depolymerizes actin and it's phosphatase Slingshot (SSH) are essential to PDGF‐induced VSMC migration. In addition to SSH, cofilin activity is also regulated by LIM‐kinase, which attenuates its activity. Thus, in these studies we examined the role of LIMK in PDGF‐induced VSMC migration. Immunoblotting with phosphospecific antibodies confirmed that LIMK activity was increased in a time dependent manner in response to PDGF. Transfection of VSMCs cells with LIMK siRNA attenuated PDGF induced migration by 70% vs. control VSMCs. The knockdown of LIMK expression by siRNA also decreased basal phosphorylation of cofilin and potentiated the cofilin response to PDGF‐induced activation. As PDGF‐induced LIMK regulation appears to mediate VSMC migration, these studies show the importance of further examining the SSH/LIMK balance in mediating cytoskeletal events associated with cofilin activity.
Trichloroethanol relaxes rat carotid arteries by activating BKca channels
Authors: Silswal Neerupma , Parelkar Nikhil , Andresen Jon
Published: June 2021

Thepossible involvement of potassium (K+) channels in trichloroethanol(TCE) induced vasorelaxation was investigated in rat carotid arteries. Theactive metabolite of chloral hydrate, TCE (10−7 M‐10−2 M)elicited concentration‐dependent relaxation of arteries precontracted withphenylephrine (10−7 M) that was minimally altered by indomethacinand L‐NAME, or by endothelial denudation indicating that the TCE response wasprimarily mediated by smooth muscle cells. A cocktail of "classical" K+channel inhibitors [4‐aminopyridine(3 mM), glibenclamide (10 μM), barium chloride (100 μM),and TEA (10 mM) ] impaired relaxation to TCE (‐logEC50 valuedecreased from 3.81±0.04 to 3.26±0.02, P<0.0001). TEA alone at 1 mM similarly impaired the response to TCE (‐logEC50value 3.60±0.04, P< 0.0068). In thepresence of isotonic 60 mM KCl, relaxation to TCE was completely blocked up tothe maximum TCE concentration of 1 mM. We conclude that TCE causes relaxationof carotid arteries by directly activating BKca channels located insmooth muscle cells. This research was supported by an AHA SDG (0735053N) and UMKC start‐up funds.
Characterization of contraction to BzATP, a P2X7 agonist in rat mesenteric arteries
Authors: Chiao Chin‐Wei , Webb R. Clinton
Published: June 2021

The P2X7 receptor is a uniquely bifunctional molecule through which ATP can open a small cationic channel typical of ionotropic receptors and also induce a large pore permeable to high molecular weight molecules. Until now, the physiological role of P2X7 receptors has not been clearly defined in the vasculature. According to our preliminary data, pretreatment with phenylephrine (PE) increased the contractile response to BzATP in rat mesenteric arteries. Thus we hypothesized that PE contributes to BzATP‐induced vasoconstriction in rat mesenteric arteries.

Vascular reactivity was assessed in first‐order rat mesenteric arteries mounted in a myogragh, with or without endothelium. Arteries were pretreated with or without PE (10 μM), U46619 (0.3 μM) or KCl (60 mM) until a stable response was obtained, followed by treatment with BzATP (0.01 μM‐ 100 μM).

BzATP (10 μM) alone did produced small contractile response (1.53±0.41 mN). Pretreated with PE or U46619 in endothelium‐intact mesenteric arteries, BzATP‐induced contractile responses were significantly increased (PE: 5.01±0.75 mN; U46619: 3.76±0.62 mN), but not in those pretreated with KCl (0.43±0.12 mN) or endothelium‐denuded mesenteric arteries pretreated with PE or U46619 (PE: 1.45±0.38 mN; U46619: 1.08±0.39 mN).

Activation of P2X7 receptor induces vasoconstriction, which is endothelium dependent and may be mediated through a G protein‐coupled receptor pathway.
Dose‐dependence of hydrogen sulfide on vascular endothelial function following ischemia‐reperfusion in perfused rat lung
Authors: Patel Prayash , Russell Michael James
Published: June 2021

Hydrogen sulfide (H2S) modulates vascular tone dose‐dependently in vessels from every vertebrate class, and H2S may protect against ischemia‐reperfusion injury in rat hearts and isolated rat lungs. We have shown that inhibition of H2S reduced acute edema formation following ischemia‐reperfusion while exogenous (200 μM) H2S did not affect edema formation or pulmonary artery (PA) endothelial function in a constant flow, in‐situ rat lung model. In the present study we examined edema formation and endothelial function in isolated rat lungs exposed to high‐dose (10 mM) H2S and 90 min of warm (37°) ischemia. Lungs were ventilated at 3 cm H2O positive end expiratory pressure (PEEP) and perfused at 5.9 ml/min in‐situ for 30 min, exposed to 90 min warm ischemia while inflated to 10 PEEP, then reperfused and ventilated for 30 min at 3 PEEP. Left PA were hung in a myograph. The right lung was dried and weighed for wet/dry ratio. Endothelial function was assessed by acetylcholine (ACh) relaxation of a norepinephrine (NE) contraction. Wet/dry ratio in high‐dose H2S (2.19 ± 0.07; n=5) was not different than either control (2.35 ± 0.12; n=4) or low‐dose H2S (2.26 ± 0.12; n=5) lungs, whereas ACh relaxation of NE was nearly abolished in PA exposed to high‐dose H2S (6.73±6.85%). H2S effects on rat lung following ischemia‐reperfusion are dose‐ and protocol‐dependent. Supported by a Mercer University seed grant.
Aortic microRNA expression is altered with aging in mice
Authors: Lawson Brooke Renae , Magerko Katie A , Lesniewski Lisa A , Rippe Catarina , Seals Douglas R , Donato Anthony J
Published: June 2021

Aging is the major risk factor for cardiovascular diseases (CVD). MicroRNAs (miRNA) are novel posttranslational repressors of protein expression. We determined if miRNAs expressed in CV tissues and implicated in models of CVD change with aging. miRNA expression was determined by RT‐PCR in aortas from 9 old (31 mo) and 11 young (7 mo) B6D2F1 mice. miR‐92, which is predicted to repress translation of cellular stress proteins, was greater in old mice (1.64±0.79 vs. 0.00±0.21 Fold Change [FC], P<0.05) and this was associated with reduced aortic SIRT‐1 (0.9±0.05 vs. 1.1±0.1 au, P=0.05). miR‐222, which may repress nuclear factor κ B (NFκB) pro‐inflammatory signaling, was lower in old mice (0.00±0.19 vs. ‐1.13±0.46 FC, P<0.05) and this was associated with greater aortic NFκB protein (1.9±0.03 vs. 1.1±0.2 au, P<0.01). miR‐133, is predicted to repress several angiogenic factors such as angiopoetin and endothelial differentiation factor, was greater in old mice (1.40±0.41 vs. 0.00±0.31 FC, P=0.01). Expression of miR ‐21, ‐29, ‐125, ‐126, and ‐214 were not different in young and old mice. Our results indicate that expression of selective miRNA, including miR‐92, ‐133, and ‐222, change with age in aorta of mice and may contribute to proteomic and functional phenotypes. Thus, miRNA may represent a molecular target for interventions aimed at the prevention and treatment of arterial aging.

NIH AG006537, AG013038, AG022241, AG015897, AG029337
Myosin heavy chain expression in emphysema induced hypertrophied right ventricles
Authors: Mattson John P , Helwig Bryan G
Published: June 2021

Development of right ventricular hypertrophy (RVH) is a marker of increased morbidity and mortality in chronic obstructive pulmonary disease (COPD) patients. Cor pulmonale leading to RV failure accounts for 20% of heart failure hospital admissions. To this point, we know surprisingly little about the underlying cell signaling pathways activated in this condition.

Myosin heavy chains (MHC) provide structural integrity involved in myocardial contractile and functional properties. Previous reports suggest that changes in either α‐ or β‐MHC lead to alterations in contractility. Furthermore, the proportion of α‐MHC has been demonstrated to decline in heart failure. Given their critical role, a time course of emphysema‐induced alterations in RV MHC expression was measured 4 and 8 months following saline (control) or elastase (emphysema (EMP)) instillation in hamsters.

Excised lung volume (>140%) and RV mass (4‐month = 18%; 8‐month = 42%) increased with EMP at both time points. There were no differences in the protein expression of either α‐ or β‐MHC in the RVs of EMP animals at any time point. However, there was a trend for increased expression of both α‐ and β‐MHC in the RVs of 8‐month compared to 4‐month EMP animals. Furthermore, the ratio of β/α ‐ MHC was decreased in the RVs of 8‐month EMP animals. These data suggest α‐ and β‐MHC may have a progressive role in the RV pathogenesis of COPD patients.
Improvement in exercise hyperemia via ascorbic acid in older healthy adults: role for nitric oxide?
Authors: Crecelius Anne R , Voyles Wyatt F , Dumke Breanna R , Davis Julia A , Kirby Brett S , Dinenno Frank A
Published: June 2021

Acute ascorbic acid (AA) administration increases muscle blood flow during dynamic exercise in older adults, and this is associated with improved endothelium‐dependent vasodilation. Here, we test the hypothesis that the increase in muscle blood flow during AA administration is independently mediated via the endothelium‐dependent vasodilators nitric oxide (NO) and prostaglandins (PGs). In 7 healthy older adults (64±3 yrs), we measured forearm blood flow (FBF; Doppler ultrasound) during rhythmic handgrip exercise at 10% maximum voluntary contraction. After 5 minutes of steady‐state exercise with saline, AA was infused via brachial artery catheter for 10 minutes during continued exercise, and this increased FBF ~23% from 126±20 to 154±22 ml/min (P<0.05). AA was infused for the remainder of the study. Next, subjects performed a 15 minute exercise bout where AA + saline was infused for 5 minutes, followed by 5 minutes of the NO synthase inhibitor L‐NMMA, and then 5 minutes of the cyclooxygenase inhibitor ketorolac. NOS inhibition reduced steady‐state FBF by ~19% (P <0.05) and FBF was no longer different than control exercise (118±16 ml/min). PG inhibition did not change FBF (Δ = 4±6%). Our preliminary data indicate that the increase in muscle blood flow during dynamic exercise during acute AA administration in older adults is mediated primarily via an endothelial NO‐dependent mechanism.

Supported by AG‐027150
Rho‐kinase mediated vasoconstriction is upregulated in aged skin
Authors: Lang James A , Jennings John D , Holowatz Lacy A , Kenney W Larry
Published: June 2021

Primary human aging is associated with augmented pro‐constrictor vascular signaling mechanisms, which may be mediated by activation of Rho‐kinase (ROCK). We hypothesized that the contribution of ROCK to vasoconstriction (VC) is greater in aged skin. Cutaneous VC was elicited by 1) whole‐body cooling (Tsk=30.5oC) and 2) local norepinephrine (NE) infusion (10−6M). Four microdialysis fibers were placed in the forearm skin of 8 young (Y) and 8 older (O) subjects for infusion of 1) Ringers solution (control), 2) 3mM fasudil (ROCK inhibition), 3) 20mM L‐NAME, and 4) both ROCK + NOS inhibitors. Red blood cell flux (LDF) was measured by laser Doppler flowmetry over each site. Cutaneous vascular conductance was calculated (CVC = LDF/MAP) and normalized to baseline CVC (%ΔCVCbase). VC was lower at the control site in O during cooling (Y: ‐34 ± 3, O: ‐23 ± 4 %ΔCVCbase; P<0.05) and NE infusion (Y: ‐52 ± 5, O: ‐39 ± 9 %ΔCVCbase; P<0.05). Fasudil infusion attenuated VC in O but not Y during both cooling (Y: ‐29 ± 5, O: ‐8 ± 1 %ΔCVCbase; P<0.01) and NE (Y: ‐22 ± 4, O: ‐8 ± 4 %ΔCVCbase; P<0.01) suggesting that ROCK mediated 15% and 53% of the total reflex VC response in Y and O, respectively. NOS inhibition did not alter ROCK‐mediated VC (P<0.05). ROCK inhibition significantly attenuated cutaneous VC in aged but not young skin suggesting that downstream vascular mechanisms of VC differ with age, and ROCK is upregulated in aged skin.

Supported by NIH RO1‐AG‐07004‐18
The effects of pyrene on tissue water and plasma osmolyte homeostasis in Rana pipiens and Rana catesbeiana
Authors: Little Ashley L , Anderson Kristy A , Stabenau Erich K
Published: June 2021

The global decline in amphibian populations has been linked to polycyclic aromatic hydrocarbon (PAH) exposure. Pyrene, a PAH, significantly decreases the skin ionic conductance in R. pipiens. This implies that pyrene may alter the R. pipiens osmoregulatory ability. The present study examined plasma osmolality and ion concentrations, as well as tissue H2O content, in control and pyrene‐exposed R. pipiens. Significantly larger sized R. catesbeiana were used for comparison. The results indicate that pyrene‐exposed R. pipiens had a significantly higher hematocrit, and a significantly lower plasma osmolality and skin H2O content. There were no differences in red blood cell H2O content, plasma Na+, K+, Cl− concentrations, plasma protein levels, hemoglobin concentration, or in the liver, muscle, and lung H2O content in pyrene‐exposed R. pipiens. It is noteworthy, however, that plasma protein levels decreased 11% in pyrene‐exposed leopard frogs. In contrast, pyrene did not alter any of the variables in R. catesbeiana. The results of these studies suggest that pyrene causes a reduction in plasma volume, and perhaps, an increase in plasma protein‐pyrene binding. Thus, pyrene‐exposed leopard frogs may have long‐term osmoregulatory problems that compromise long‐term survival. Funding for this research was provided, in part, by a Research Excellence grant to EKS from Bradley University and a NSF‐REU grant.
Non‐invasive characterization of red blood cell population under normoxic and hypoxic conditions in developing zebrafish (Danio rerio)
Authors: Schwerte Thorsten , Yaqoob Nadeem , Prem Caroline , Kopp Renate , Fiechtner Birgit
Published: June 2021

For erythroid cell lineage development in vertebrates, GATA‐1 transcription factor is essential. We demonstrated that the approximate developmental status of red blood cells and the progression of blood formation can be studied non‐invasively in GATA‐1:DsRed transgenic zebrafish larvae by characterization of fluorescence luminance spectra. Study was carried out for the animals maintained under normoxic and hypoxic (5.8 and 0.8mg of O2 l−1) conditions up to 10 days post‐fertilization (dpf). The blood cells were classified into five Intensity Stages (IS) on the basis of their fluorescence intensity. We evaluated Epo‐expression by carrying out RT‐PCR of wild type zebrafish up to 15 dpf old larvae in order to determine its correlation with the luminance data. Significant relationship was observed between Epo‐mRNA production, GATA‐1 expression and blood cell concentration. In contrast to normoxic animals 1‐3 Epo mRNA copies that was maintained throughout the development, the hypoxic animals showed remarkable fluctuation of 1 to 12 Epo‐mRNA copies per ng RNA during animal development. Fluorescent cells with medium intensities (IS3) in normoxic animals were found throughout 2 to 10 dpf although in lower quantity while in hypoxic group they appeared from 5 dpf till 10 dpf showing a maximum of 15% of the total luminescent cells at 8 dpf. The fluorescent cells concentration in normoxic group was generally higher as compared to the hypoxic one. A major observable feature of the hypoxic group was its high blood volume and increased dorsal aorta vessel diameter compared to controls.
Role of neutral sphingomyelinase‐derived ceramide in the response to oxygen in the chicken ductus arteriosus
Authors: Cogolludo Angel L. , Villamor Eduardo , Frazziano Giovanna , Moral Javier , Menendez Carmen , Perez‐Vizcaino Francisco
Published: June 2021

Ceramide is a sphingolipid‐derived second messenger modulating different signalling pathways and playing an important role in numerous physiological processes such as cell growth, apoptosis and differentiation. In the present study we have analysed the role of ceramide in the vasoactive effects of oxygen in the chicken ductus arteriosus (DA). Experiments were performed in the pulmonary or aortic sides of the DA isolated from chicken fetuses incubated for 15 or 20 days (total incubation: 21‐d) and in myocytes isolated from these tissues. Exposure to oxygen (21%) induced a contractile response in the pulmonary side of 20‐d DA but not in the other preparations. Similarly, oxygen (21%) increased the ceramide content measured by immunocytochemistry in the pulmonary side of 20‐d DA but not in the other preparations. The contractile response induced by oxygen was partially prevented by inhibitors of neutral sphingomyelinases and PCKzeta but not by the classic PKC inhibitor Go6976. Finally, the addition of exogenous C6‐ceramide contracted the pulmonary side of 20‐d DA but had negligible effects in the pulmonary side of 15‐d DA or in the aortic side of 20‐d DA. In conclusion, neutral sphingomyelinase‐derived ceramide plays a role in the oxygen induced contraction of chicken DA. Supported by "Fundación de Investigación Médica Mutua Madrileña"
Effects of PI(3,5)P2 on the Skeletal Muscle Ryanodine Receptor
Authors: Brotto Marco A. P. , Sherman Joseph A. , Nosek Thomas M , Qu Cheng Kui , Valdivia Héctor H
Published: June 2021

An increase of intracellular PI(3,5)P2 in MIP KO mice is associated with a loss of calcium (Ca) from the SR. Is this loss of Ca due to Ca leakage through the RyR Ca release channel (RyR1)? Using PIP array analyses, we found that PI(3,5)P2 binds to RyR1.Direct physical interaction between PI(3,5)P2 and RyR1 was verified by the PI(3,5)P2‐agarose beads pull‐down assay and anti‐ PI(3,5)P2 co‐immunoprecipitation analyses. We determined functional interactions of PI(3,5)P2 with RyR1 on purified SR microsomes using the [3H]Ryanodine binding assay. The dose‐response curve at near‐optimal free Ca showed that of all PIs tested, PI(3,5)P2 was the most effective activator of RyR1. The functional significance of the interaction between PI(3,5)P2 and RyR1 was tested by adding PI(3,5)P2 to RyR1 reconstituted in lipid bilayers. At nominally‐free Ca solution,30 and 60 micromolar PI(3,5)P2 induced a dose dependent increase in open events with unchanged unitary conductance. These analyses suggest that PI(3,5)P2 binds to RyR1 and directly increases channel activity at contracting (pCa 5) and resting (pCa 7) levels of Ca. The enhanced Ca leak observed in MIP cells may be due, at least in part, to increased PI(3,5)P2 levels, which can explain the phenotypic changes observed in the MIP KO. (Support: AHA 0535555N to MB, NIH HL55438 to HHV, TREC Pilot Grant to TMN, MB, and CKQ, and NIH HL068212 and HL082670 to CKQ)
Urotensin II Induced Signal Involved in Proliferation of Vascular Smooth Muscle Cells
Authors: Iglewski Myriam , Grant Stephen R
Published: June 2021

Urotensin II receptor (UIIR), bound by the ligand Urotensin II (UII), generates second messengers IP3 and DAG, which stimulates the subsequent release of calcium (Ca2+) in vascular smooth muscle cells (VSMC). Ca2+ influx leads to the activation of Ca2+ dependent kinases via calmodulin binding, resulting in cellular proliferation. We hypothesize that UII signaling in pulmonary arterial vascular smooth muscle cells (Pac1) and primary aortic vascular smooth muscle cells (PAVSMC) results in phosphorylation of Ca2+/ CaM dependent kinases leading to cellular proliferation. UII exposure to Pac1 cell cultures increased intracellular Ca2+, which subsequently activated Ca2+/CaM dependent kinases (CaMKK & CaMKI), ERK 1/ 2, and PKD (PKC‐µ). Pac1 and PAVSMC cells treated with UII resulted in increased proliferation as measured by 3H thymidine uptake. UII‐induced increase of 3H thymidine incorporation was inhibited by a CaMKK inhibitor. Taken together, our results demonstrate that UII stimulation of smooth muscle cells leads to cellular proliferation, utilizing signaling mechanisms involving Ca2+/CaM dependent kinases.