bluuebunny/crossref_metadata_2025 huggingface dataset:
Nox2 mediates HGF induced angiogenesis
Authors: Schröder Katrin , Kohnen Andreas , Schütz Susanne , Brandes Ralf P
Published: June 2021

Hepatocyte growth factor (HGF) promotes angiogenesis and prevents senescence of endothelial cells. Reactive oxygen species (ROS) play an essential role in angiogenesis. One of the most important sources of acutely induced ROS‐production are the members of the NADPH oxidase (Nox) family, in endothelial cells namely Nox2. The angiogenetic potential induced by HGF depends on Nox2. The ability of HGF to induce aortic ring outgrowth was significantly reduced in aortic rigs of Nox2 deficient (Nox2y/‐) mice when compared to wildtype mice. Mobilization of endothelial progenitor cells (EPCs) from the bone marrow into peripheral blood has been suggested to be an essential step in the formation of new vessels. Indeed HGF induced mobilization of EPCs in wildtype mice. This effect of HGF was blocked in Nox2y/‐ mice. Accordingly in wildtype mice HGF induced colony forming unit formation which was absent in Nox2y/‐ mice. In human EPCs HGF acutely induced the production of hydrogen peroxide measured by Amplex Red. After depletion of Nox2‐expression by antisense‐oligonucleotides no HGF inducible ROS production was detectable. Further analyses revealed an HGF inducible phosphorylation of Jak2 and STAT3 which was absent in Nox2 depleted cells.

We conclude that EPC mobilization might contribute to the angiogenetic effects of HGF. Nox2 derived ROS play an essential role in HGF inducible EPC‐mobilization and aortic ring outgrowth.
Impact of fructose feeding on the control of cardiac oxygen consumption by NO‐superoxide interaction during pregnancy
Authors: Kertowidjojo Elizabeth C , Krishnan Sheila , Hintze Thomas H
Published: June 2021

Fructose consumption (FC) has caused much concern because of its prominence in today's diet and its implication in cardiovascular disease. We hypothesized that FC alters cardiac metabolism by generating superoxide, which reduces NO bioavailability. We then extended the study to the heart during pregnancy where NO plays an essential role in its successful completion. Female SD rats (N=16) were fed either a normal or a 60% purified fructose diet for 21 days. Hemodynamic and metabolic measurements showed significant increases in diastolic and mean arterial pressures, and insulin levels (3.10 ± 0.42 to 5.47 ± 0.44 ng/mL) with FC. The ability of bradykinin to reduce myocardial O2 consumption (MVO2) in vitro was impaired by FC (28.46 ± 2.30% to 16.59 ± 1.64%). This effect was reversed by apocynin or tiron, which inhibits NADPH oxidase and scavenge superoxide, respectively. MVO2 decrease by bradykinin with L‐NAME, an eNOS inhibitor, was comparable to that in rats with FC. In pregnant SD rats (N=16), a reduction in the ability of NO to control MVO2 with FC was also exhibited, along with significant increases in diastolic, systolic and mean arterial pressures. We conclude that a high‐fructose diet lowers NO bioavailability through the generation of superoxide, alters the control of arterial pressures, and disrupts the regulation of O2 consumption in the normal myocardium, especially during pregnancy. Supported by HL43023, 50142
Increased Myogenic Reactivity of Resistance‐sized Mesenteric Arteries from Pregnant Rats with Episodic Reductions in Uteroplacental Perfusion
Authors: Reho John J , Peck Jennifer , Novak Jacqueline , Ramirez Rolando J
Published: June 2021

Pregnancy is associated with increased placental blood flow (BF) to the developing fetus. A central component of preeclampsia is thought to be a reduction in placental BF leading to altered vascular reactivity. It is suggested that short term elevations in blood pressure may lead to periodic reductions in uteroplacental blood flow (UBF) and this may play a role in preeclampsia. The purpose of this study was to develop a gravid rat model characterized by episodic reductions in UBF to elucidate the role of periodic decreases in UBF on vascular reactivity. We used pregnant Sprague Dawley rats and subjected them to Sham or aortic occlusion surgery on Day 14 of gestation. Aortic occlusion surgery consisted of implantation of a silastic vascular occluder around the abdominal aorta along with silver clips (0.106mm ID) around the uterine‐ovarian arteries. Aortic occlusion animals were subjected to 5 days of reduced UBF (40% reduction) for 1 hour each session. On Day 21, second order mesenteric arteries were mounted on a pressurized arteriograph. Myogenic reactivity of small mesenteric arteries was increased in Occluder animals compared to Sham (p<0.05). This data suggest that episodic reductions in UBF may mimic some of the characteristics of preeclampsia including altered vascular reactivity toward a constrictive phenotype. Aortic occlusion may be a new model for investigating mechanisms associated with reduced UBF.
Inhibition of glycogen synthase kinase (GSK)‐3β is involved in isoflurane‐induced delayed cardiac protection
Authors: Kawaraguchi Yoshitaka , Panneerselvam Mathivadhani , Tsutsumi Yasuo M. , Kidd Michael W. , Moreno Ana L. , Patel Piyush M. , Roth David M. , Patel Hemal H.
Published: June 2021

Introduction

Volatile anesthetics, such as isoflurane, produce acute and delayed cardiac protection. Recently, glycogen synthase kinase (GSK)‐3β has been reported to play an important role in acute protection from ischemia/reperfusion injury via inhibition of mitochondrial permeability transition pore (mPTP) opening. However, involvement of GSK‐3β in delayed protection has not been well understood. Therefore, we examined the role of GSK‐3β in delayed cardiac protection.

Methods

Mice were exposed to 1.4% isoflurane or 100% oxygen for 30 min under artificial ventilation. After 24 hr recovery, mice were subjected to 30 min coronary artery occlusion followed by 2 h of reperfusion. Infarct size was determined via planimetry. Additionally, after 24 hr recovery, the hearts were excised and lysates of left ventricle were analyzed by immunoblot.

Results

Twenty‐four hours following isoflurane exposure, a reduction in myocardial infarction was observed when compared to control mice (24.0 ± 2.5% vs. 45.1 ± 2.9% of the area at risk; P < 0.05; n = 12, respectively). Western blot analysis showed that phosphorylated GSK‐3β in the isoflurane group significantly increased.

Conclusion

Our findings suggest that the inhibition of GSK‐3β plays an important role for isoflurane‐induced delayed protection.
Effects of Zoledronate in the Repair of Chronically Infarcted Rat Myocardium
Authors: Hwang Hyosook , Hale Sharon L. , Kloner Robert A.
Published: June 2021

Zoledronate (Zol), a class of bisphophonate drugs, is commonly used to treat post‐menopausal osteoporosis. Treatment of liposomal bisphosphonates, a technique that is often used to deplete tissue macrophages, has experimentally shown to worsen myocardial infarct repair. The purpose of this study was to investigate the effect of Zol in the repair of chronically infarcted myocardium without liposomal encapsulation to mimic the clinical setting. Zol (20 μg/kg) was subcutaneously administered to rat 1 day prior to coronary ligation. Rats receiving phosphate‐buffered saline were utilized for experimental comparison (control). Left ventricular function and infarct size and remodeling were studied at 4 weeks post‐infarction. Zol pre‐treatment did not affect left ventricular ejection fraction in hearts with myocardial infarction (49.5 ± 1.4% in Zol; 50.6 ± 2.1% in control). Infarct size was similar in Zol‐treat versus control hearts (34.2 ± 2.9% in Zol; 33.4 ± 2.9% in control). Left ventricular cavity volume and circumference, infarct thickness, and expansion index were comparable between the groups. In conclusion, administration of Zol prior to myocardial infarction had no adverse effects on cardiac function, infarct size, and remodeling. These results suggest that treatment with Zol in patients with an increased risk of myocardial infarction is safe and does not cause worsening of infarct repair.
Effect of Sildenafil on Sympathetic Hyperinnervation in Post‐infarcted Rat Hearts
Authors: Lee Tsung‐Ming , Chang Nen‐Chung
Published: June 2021

Sildenafil has been shown to provide cardioprotection against ischemia/reperfusion injury via activation of mitochondrial ATP‐sensitive potassium (KATP) channels. We assessed whether sildenafil attenuates cardiac sympathetic innervation after myocardial infarction through activation of KATP channels. After ligation of the anterior descending artery, male Wistar rats were randomized to either vehicle, nicorandil, pinacidil, sildenafil, glibenclamide, or a combination of nicorandil and glibenclamide, pinacidil and glibenclamide or sildenafil and glibenclamide for 4 weeks. To elucidate the role of mitochondrial KATP channels in modulating nerve growth factor, 5‐hydroxydecanoate was assessed in an in vitro model. Myocardial norepinephrine levels revealed a significant elevation in vehicle‐treated rats at the remote zone compared with sham. Immunohistochemical analysis for tyrosine hydroxylase, growth associated factor 43 and neurofilament also confirmed the change of myocardial norepinephrine. This was paralleled by a significant upregulation of nerve growth factor protein expression and mRNA in the vehicle‐treated rats, which reduced after administering either nicorandil, pinacidil or sildenafil. Arrhythmic scores during programmed stimulation in the vehicle‐treated rats were significantly higher than those treated with sildenafil. In contrast, beneficial effects of sildenafil were reversed by the addition of either glibenclamide or 5‐hydroxydecanoate. Chronic use of sildenafil after infarction, resulting in attenuated sympathetic innervation by activation of mitochondrial KATP channels, may modify the arrhythmogenic response to programmed electrical stimulation.
O‐GlcNAc agonist treatment attenuates early inflammatory response in the lung after cecal puncture and ligation induced sepsis in rats
Authors: Nöt Laszlo G , White C Roger , Vamhidy Laszlo , Chatham John C
Published: June 2021

Sepsis remains a major clinical problem with a high mortality rate. We have previously shown that increasing levels of O‐linked β‐N‐acetylglucosamine (O‐GlcNAc) on proteins by inhibition of O‐GlcNAcase (OGA) improved outcomes following hypovolemic shock. Therefore, the aim of this study was to determine the effects of OGA inhibitor PUGNAc (PN) on the pro‐inflammatory response to cecal puncture & ligation (CLP)‐induced sepsis. Immediately following either sham surgery (SH) or CLP, rats were treated with 20 μmoles of PUGNAc or with normal saline Control (CON). 5 hrs following CLP, TNF‐α, IL‐10 and IL‐6 were all increased (p<0.05) in CON Vs. SH (TNF‐α: 10±2 Vs 0.7±0.5 IL‐10: 68±9 Vs 0.8±0.7; IL‐6: 1071±221 Vs 192±86 pg/ml). PUGNAc had no effect on TNF‐α or IL‐10; but attenuated IL‐6 (p<0.05). In bronchoalveolar lavage IL‐6 levels were also increased in CON Vs. SH (165±5 Vs 127±9 pg/mL, p<0.05) and this was attenuated by PN. CLP increased ICAM‐1 levels in lung tissue (CON: 126±6 Vs SH: 100±6 %, p<0.05), and this was also attenuated by PN treatment (p<0.05). The PN mediated decrease in CLP‐induced inflammation was associated with a ~2‐fold increase in O‐GlcNAc levels in the lung compared to SH & CON groups (p<0.05). These results suggest that OGA inhibition attenuates the early inflammatory response to CLP and may represent a new therapeutic approach to the treatment of sepsis. Supported by NIH grants HL067464/HL079364.
Inward coronary artery microvessel remodeling in Ossabaw swine with metabolic syndrome
Authors: Trask Aaron J. , Katz Paige S. , Neeb Zachary P. , Alloosh Mouhamad , Sturek Michael , Lucchesi Pamela A.
Published: June 2021

The aim of the current study was to determine if coronary microvessels (<150 ìm diameter) in Ossabaw swine with metabolic syndrome (MetS) undergo inward remodeling. Mature Ossabaw were fed either normal (n=3; Lean) or hypercaloric atherogenic diet (n=7; MetS) for 6 months after which coronary microvessels were isolated and mounted on a pressure myograph. Microvascular wall thickness and diameters were measured and recorded over a range of pressures (0–125 mmHg). Coronary microvessels isolated from MetS trended towards decreased luminal diameters (Lean: 123.8 ± 9.2 μm vs. MetS: 109.7 ± 9.6 μm p = 0.08) with 2‐fold thicker microvascular walls (Lean: 14.1 ± 1.2 μm vs. MetS: 29.4 ± 3.1 μm p < 0.05), which significantly increased the wall:lumen ratio (Lean: 11.5 ± 0.8 vs. MetS: 25.9 ± 2.0 μm p < 0.05). External diameters were not different between Lean and MetS (Lean: 152.0 ± 10.7 μm vs. MetS: 168.6 ± 14.6 μm p = 0.05). Young's Modulus of Elasticity was significantly decreased in microvessels isolated from the MetS (Lean: 8.8×10^5 ± 0. 9× 10^5 vs. MetS: 3.2×10^5 ± 0.8×10^5, p < 0.01), and microvascular compliance tended to be increased in MetS (Lean: 0.08 ± 0.02 vs. MetS: 0.21 ± 0.05, p = 0.15). These data suggest that MetS induced by atherogenic diet causes hypertrophic inward remodeling of coronary microvessels. Support: NIH HL056046, Nationwide Children's Hospital to PAL; NIH RR013223, HL062552, UL1 RR025761, Purina TestDiet to MS.
Hypusine‐containing Protein eIF5A Promotes Translation Elongation
Authors: Dever Thomas E , Saini Preeti , Eyler Daniel E , Green Rachel
Published: June 2021

The factor eIF5A, the sole protein in eukaryotes and archaea containing the unusual amino acid hypusine [Nε‐(4‐amino‐2‐hydroxybutyl)lysine], was originally identified based on its ability to stimulate the yield (endpoint) of methionyl‐puromycin synthesis, a model assay for first peptide bond synthesis thought to report on certain aspects of translation initiation. In Saccharomyces cerevisiae, eIF5A is encoded by the genes TIF51A and TIF51B; and both in yeast cells and in model assays of eIF5A function, hypusine formation is essential for eIF5A activity. Using molecular genetic and biochemical assays, we found that eIF5A promotes translation elongation. Depletion or inactivation of eIF5A in yeast impaired protein synthesis and resulted in the accumulation of polysomes and an increase in ribosomal transit times. Addition of recombinant eIF5A from yeast, but not a derivative lacking hypusine, restored translational activity in extracts prepared from eIF5A mutant strains and enhanced the rate of tripeptide synthesis in reconstituted translation assays. Moreover, inactivation of eIF5A mimicked the effects of the eEF2 inhibitor sordarin, indicating that eIF5A might function late in the translation elongation cycle like eEF2. As eIF5A is a structural homolog of the bacterial protein EF‐P, we propose that eIF5A/EF‐P is a universally conserved translation elongation factor.
β2‐adrenergic receptor gene variation and systemic vasodilation during baroreflex inhibition
Authors: Hesse Christiane , Schroeder Darrel R. , Nicholson Wayne T. , Hart Emma C. , Penheiter Alan R. , Turner Stephen T. , Joyner Michael J. , Eisenach John H.
Published: June 2021

β2‐adrenergic receptor (ADRB2) gene polymorphisms influence vasodilation, but studies investigating regional or systemic responses conflict, possibly due to counter‐regulatory baroreflex action during systemic β2‐agonist infusion. We tested the hypothesis that ADRB2 gene variation would influence the SVR response to ADRB2 agonist terbutaline (TERB) during baroreflex inhibition. 40 healthy young adults were recruited according to the double homozygous haplotypes: Arg16+Gln27 (n=13), Gly16+Gln27 (n=6), and Gly16+Glu27 (n=21). Arterial pressure was measured by brachial arterial catheter, cardiac output by acetylene breathing. Lymphocytes were sampled for ex vivo analysis of ADRB2 density and binding conformation. Following baroreflex ablation with trimethaphan (3–7 mg/min), phenylephrine was titrated to restore blood pressure to baseline. TERB was infused i.v. at 33 and 67 ng/kg/min for 15 min/dose. There was partial evidence to suggest a main effect of haplotype on the change in SVR (P = 0.06). For SNP position 16, the highest dose of TERB produced lower SVR in Gly16 vs Arg16 (P = 0.03). Lymphocyte ADRB2 binding conformation was similar but receptor density was greater in Gly16 vs Arg16 (P = 0.05). During baroreflex inhibition, the SVR response to systemic ADRB2 agonist is suggestive of augmented ADRB2 function in Gly16+Glu27 homozygotes, with greater influence from Gly16.

Supported from NIH and DFG.
RNAi Inhibition of Interleukin‐6 Attenuates Cold‐Induced Hypertension
Authors: Crosswhite Patrick , Sun Zhongjie
Published: June 2021

The purpose of this study was to determine the role of the pro‐inflammatory cytokine IL‐6 in cold‐induced hypertension (CIH). We hypothesized that RNAi knockdown of IL‐6 would decrease inflammatory infiltrates and attenuate CIH. Four groups of male rats were used (6 rats/group). After blood pressure (BP) was stabilized, 3 groups were injected intravenously with recombinant AAV carrying IL‐6shRNA, AAV carrying scrambled shRNA (ScrshRNA), and PBS, respectively, prior exposure to a cold environment (5°C). The last group received PBS and was kept at room temperature (25°C, warm) as a control. AAV delivery of IL‐6shRNA significantly attenuated cold‐induced elevation of BP and kept it at the control level. Chronic exposure to cold up‐regulated IL‐6 and TNF‐α expression in aorta, heart and kidneys and increased macrophage and T‐cell infiltration in kidneys, suggesting that cold exposure increases inflammation. RNAi knockdown of IL‐6 abolished cold‐induced up‐regulation of IL‐6 and TNF‐α, leukocyte infiltration, and increases in vascular superoxide production. IL‐6shRNA prevented the cold‐induced increase in collagen deposition in the heart (cardiac remodeling). Cold exposure caused glomerular collapses which could be abolished by knockdown of IL‐6. Conclusions: Cold exposure up‐regulated IL‐6 expression leading to inflammation which plays a critical role in the pathogenesis of CIH and cardiac and renal damage.
Heat shock protein 70 in the prevention of vascular calcification in renal failure
Authors: Lu Tzong‐Shi , Lim Kenneth , Zehnder Daniel , Yang Yun‐Chun , Yiao Szu‐Yu , Hsiao Li‐Li
Published: June 2021

Vascular calcification (VC) is highly correlated with cardiovascular mortality in renal failure. Osteo/chondrocytic transformation of human aortic smooth muscle cells (HA‐SMCs) is important in the initiation and progression of VC. In vitro and in vivo studies have shown this process can be driven under hyperphosphatemic and hypercalcemic conditions seen in renal failure. Several transcription factors, including Cbfa1/Runx2 is essential for induction of osteo/chondrocytic transformation.

We previously demonstrated that HSP70 prevents vascular permeability changes in certain acute inflammatory processes. Studies have shown significantly higher circulating HSP70 levels in individuals with no evidence of coronary artery disease (CAD), while lower levels in pre‐dialysis patients, and even lower levels in hemodialysis (HD) patients were found.

Our results have shown for the first time that heat‐shock treatment induced expression of HSP70 and markedly reduced the extent of calcification in HA‐SMCs, in vitro. Cbfa1 was upregulated in calcified HA‐SMCs, while heat‐shock treatment down‐regulates its expression. Furthermore, HSP70 expression was found to be lower in arteries from renal failure patients compared to healthy controls, ex vivo. In conclusion, HSP70 reduces the development of calcification through the inhibition of smooth muscle cell transformation.
Lumbar sympathectomy does not attenuate the development of mild DOCA‐salt hypertension
Authors: Kandlikar Sachin , Osborn John , Fink Gregory
Published: June 2021

Increased sympathetic nervous activity (SNA), responsible for the development and maintenance of hypertension, is more prominent in some vascular beds than others. Previous studies in anesthetized DOCA‐salt hypertensive rats show that elevated hindlimb vascular resistance is mediated in part through higher lumbar SNA. This indicates that increased lumbar SNA could be a cause of hypertension in DOCA‐salt rats. The purpose of this study was to evaluate in conscious rats the importance of lumbar SNA to the development of mild DOCA‐salt hypertension. To accomplish this goal, chronic lumbar sympathectomy was performed in one group of male Sprague Dawley rats (n=8) while another group underwent SHAM operation (n=8). During the same operation, rats were instrumented with a radiotelemeter for blood pressure (BP) and heart rate (HR) measurements. Rats were allowed free access to water containing 1% NaCl and 0.2% KCl. After a 7 day recovery and a 3 day control BP measurement period, a DOCA pellet (50 mg/kg, SC) was implanted in both groups. BP was then measured for another 4 weeks. During the control period, BP and HR were similar in the two groups. Following DOCA treatment, BP increased similarly in both groups. HR also was not different between the two groups during the development of hypertension. We conclude that lumbar SNA is not critical in the development of mild DOCA‐salt hypertension. Supported by PO1‐HL070687.
Role of central angiotensinergic mechanisms on the facilitation of the recovery of hemorrhage‐induced hypotension by noradrenergic A2‐lesions
Authors: Freiria‐Oliveira Andre Henrique , Blanch Graziela T. , De Paula Patricia M. , Colombari Eduardo , Menani Jose V. , Colombari Debora S. A.
Published: June 2021

Lesions of A2 neurons in the commissural nucleus tractus solitarii (cNTS) facilitate the recovery of hemorrhage‐induced hypotension. In the present study we investigated the effects of intracerebroventricular (icv) injections of losartan (AT1 receptor antagonist) on the recovery of arterial pressure (AP) after hemorrhage (4 blood withdrawals, 2 ml/rat, every 10 min) in conscious A2‐lesioned rats. Thirty days before tests, male Holtzman rats (280–320 g) received an injection into the cNTS of anti‐dopamine‐beta‐hydroxilase‐saporin (A2 lesion; 12.6 ng/60 nl, n=6–8) or IgG‐saporin (sham; 12.6 ng/60 nl, n=5–7). Seven and 1 day before tests rats also received an icv cannula and femoral artery and vein catheters, respectively. After the 4th blood withdrawal, hypotension was similar in both A2‐lesioned and sham rats (−62±7 and −73±7 mmHg, respectively). The A2‐lesioned rats treated with saline icv rapidly recovered from hypotension (−7±2 mmHg 20 min later), while sham rats did not completely recover from hypotension (−20±3 mmHg at 60 min). Losartan (100 μg/1 μl) icv did not affect AP in sham rats, however it abolished the recovery of AP in A2‐lesioned rats in the first 30 min of test (−24 ± 6 mmHg). The results suggest that the facilitatory effects of A2 lesions of the cNTS on the recovery of AP after hemorrhage depend, at least partially, on the activation of central angiotensinergic mechanisms.

Funded by CNPq, FAPESP.
A critical role for connexin 40 in hypoxia induced pulmonary vasoconstriction
Authors: Wang Liming , Yin Jun , Isakson Brant , de Wit Cor , Kuebler Wolfgang M.
Published: June 2021

The ideal location for O2 sensor is at the site of gas exchange, i.e. in lung capillaries where pulmonary vascular smooth muscle cells (PASMCs) are absent. We speculated that hypoxic signals may be propagated retrogradely along the endothelial layer from lung capillaries to PASMC‐bearing upstream arterioles via connexin 40 (Cx40), causing hypoxic pulmonary vasoconstriction (HPV). In isolated mouse lungs, we detected an ~60% attenuation of HPV in wild type (WT) mice with nonspecific gap junction blocker 18α–glycyrrhetinic acid or Cx40 specific mimetic peptide Gap2740. HPV was similarly reduced in Cx40−/− mice while the response to Angiotensin II was unmitigated. The physiologic relevance of Cx40 in HPV‐dependent ventilation‐perfusion (V/Q) matching was evaluated in ventilated mice after tracheal instillation of saline. The resulting hypoxemia was prominent in Cx40−/− mice as compared to WT (paO2: 99±8 vs. 76±12 mmHg). By fluorescence imaging of di‐8‐ANEPPS loaded isolated mouse lungs, we detected a hypoxia‐induced membrane depolarization in extra‐alveolar capillary and arteriolar endothelial cells of WT lungs that was attenuated in Cx40−/− mice. Our data identify a critical role for Cx40 in HPV and V/Q matching, suggesting that hypoxia is sensed in lung capillary endothelial cells and propagated as membrane depolarization via Cx40 from capillaries to upstream arterioles.
Role of L‐Citrulline/L‐Arginine Cycle in Hyperoxia‐Induced Decrease of Airway Smooth Muscle Relaxation
Authors: Sopi Ramadan , Gjorgoski Icko , Mladenov Mitko , Lajçi Azem , Jakupaj Muharrem
Published: June 2021

It was shown that inhibitory nonadrenergic noncholinergic (iNANC) nerve–induced nitric oxide (NO) production and airway smooth muscle (ASM) relaxation are impared after exposure to hyperoxia due to decrease of L‐arginine bioavailability to NO synthase (NOS). Therefore, we tested the hypothesis that L‐citrulline/L‐arginine cycle is active in rat pup airways and supplementation of tissues with L‐citrulline, restores impaired relaxation of ASM after exposure to hyperoxia. Tracheal rings were excised from rat pups 12 days old, exposed to hyperoxia or room air for seven days. In precontracted tracheal smooth muscle (TSM), electrical field stimulation (EFS) was applied to induce relaxation. In hyperoxic group, relaxation was reduced as compare to control group. Incubation of tissues with NOS inhibitor (L‐NAME), reduced relaxant responses in control group. In vitro supplementation of tissues with L‐citrulline reversed EFS‐induced relaxation of TSM of hyperoxic group at control level, but did not have any effect in control group under basal condition. Inhibition of enzymes involved in L‐citrulline recycling, prevented this effect of L‐citrulline.

The data demonstrate that L‐citrulline/L‐arginine cycle is active in rat pup TSM and is effective under conditions of reduced bioavailability of L‐arginine to NOS. Presence of L‐citrulline restores hyperoxia‐induced decrease of ASM relaxation.
PI3Kinase/Akt activation trigger ROS production in a model of pulmonary ischemia
Authors: Chatterjee Shampa , Browning Elizabeth , Hong NanKang , DeBolt Kris , Sorokina Elena , Liu Weidong , Fisher Aron B
Published: June 2021

We reported earlier that ischemia or loss of shear in the lung endothelium causes endothelial membrane depolarization (via KATP channel closure) that leads to NADPH oxidase activation and reactive oxygen species (ROS) production. ROS production was abolished in the presence of PI3Kinase/Akt inhibitor, wortmanin. We thus hypothesized that PI3K/Akt activation is the trigger for ROS production with pulmonary ischemia. We used pulmonary microvascular endothelial cells, either wild type (WT), KATP null (KIR6.2−/−), NADPH oxidase null (gp91phox−/−) or WT infected with dominant negative (DN) PI3K, and isolated WT and Akt‐1 null lungs to study signal transduction with ischemia. In WT lungs, the PI3K inhibitor wortmannin had no effect on ischemia mediated depolarization. Cells with DN PI3K and lungs with Akt1−/− depolarized with ischemia but failed to show NADPH oxidase assembly and ROS generation. Depolarization in WT and Akt−/− lungs was blocked upon addition of cromakalim (KATP channel agonist). In WT cells, Akt phosphorylation that was observed with ischemia and was abolished in KIR6.2−/− but not in gp91phox−/− cells. These results indicate that KATP induced depolarization leads to PI3K/Akt activation that triggers NADPH oxidase assembly and eventual ROS generation. Thus, ROS which has hitherto been shown to activate PI3K/Akt in other systems lies downstream of PI3K/Akt in our model of pulmonary ischemia.
Comparison of glutathione peroxidase 1 (GPx1) and peroxiredoxin 6 (Prdx6) in protection against lung oxidative stress in a perfused lung model
Authors: Liu Geng , Dodia Chandra , Feinstein Sheldon I. , Fisher Aron B.
Published: June 2021

Prdx6 plays an important role in lung anti‐oxidant defense, presumably due to its unique ability to reduce phospholipid hydroperoxides. Our previous studies with intact mice and isolated endothelial cells showed greater sensitivity to oxidant stress of Prdx6 null compared to wild type and GPx1 null. We now used isolated perfused mouse lungs to compare their response to 1 hr treatment with t‐butyl hydroperoxide (t‐BOOH, 5 mM) or paraquat (PQ, 1 mM) added to the perfusate. Lung injury as indicated by protein in the broncoalveolar lavage fluid (BALF), LDH in the lung perfusate, and protein carbonyls in the lung homogenate was increased significantly in Prdx6 null compared to GPx1 null lungs. Lipid peroxidation analyzed by TBARS and 8‐isoprostanes in the BALF and lung homogenate as well as by 235nm fluorescence (conjugated dienes), Fe2+‐xylenol orange, and DPPP assays was significantly greater in Prdx6 null compared to GPx1 null lungs. Analysis of expression levels of oxidant related genes using a PCR‐based Superarray (SA Biosciences) indicated no significant differences between the Prdx6 and the GPx1 null, except for the target genes. We conclude that Prdx6 null are significantly more sensitive to oxidant stress as compared to GPx1 null, suggesting that the ability of Prdx6 to scavenge phospholipid hydroperoxides and to repair membrane lipid oxidation can account for its major role in antioxidant defense.
Pulmonary vein endothelial cells (PVECs) exhibit characteristics of multiple lung endothelial cell phenotypes
Authors: Creighton Judy , Buford Anna , Ayers Linn , Stevens Troy
Published: June 2021

Endothelial cells lining the lungs' pulmonary artery, capillary, and venous blood vessels exhibit vastly different morphologies and functions. We have previously shown that segment specific attributes of pulmonary artery endothelial cells (PAECs) and pulmonary microvascular endothelial cells (PMVECs) are retained in culture. However, fundamental information regarding the characteristics of endothelial cells obtained from the pulmonary vein is lacking. PMVECs are known to possess a higher growth rate than PAECs. We determined that PVECs have a growth rate (doubling time) that is only slightly higher than PAECs (29hrs vs. 33 hrs). PMVECs develop a tighter barrier than do PAECs (2000 ohms vs. 1100 ohms, respectively). Interestingly, PVEC barrier resistance is only slightly less than PMVECs (1800 ohms). All three phenotypes express typical endothelial markers, yet display distinct lectin binding patterns as determined by Fluorescence Activated Cell Sorting (FACS) and immunocytochemical analysis. Taken together these data demonstrate functional heterogeneity among lung endothelial cell phenotypes including those derived from the pulmonary vein. Supported by HL66299.
Lung endothelial dysfunction in heart failure: Role of impaired Ca2+ signaling and cytoskeletal reorganization
Authors: Yin Jun , Kerem Alexander , Kaestle Stephanie M. , Hoffmann Julia , Kuebler Wolfgang M.
Published: June 2021

Congestive heart failure (CHF) causes lung endothelial (EC) dysfunction, which in turn aggravates lung vascular remodeling and right ventricular failure. Here, we elucidated the mechanisms underlying lung EC dysfunction in a rat model of CHF induced by supracoronary aortic banding. Lung EC dysfunction was evident in CHF as impaired EC‐dependent vasodilation and NO synthesis in response to mechanical stress, acetylcholine or histamine, although expression of endothelial NO synthase was unmitigated. Reconstitution of cytosolic Ca2+ ([Ca2+]i) signaling by Ca2+ ionophore restored NO production. Impaired lung EC [Ca2+]i homeostasis and signaling in CHF in response to mechanical stress, histamine, acetylcholine or thapsigargin was confirmed by real‐time fluorescence imaging, yet was not attributable to downregulation of EC Ca2+ influx channels. Rather, we identified a massive remodeling of the endothelial cytoskeleton by increased β‐actin expression and F‐actin formation, which contributed critically to endothelial dysfunction in CHF, since cytoskeletal disruption by cytochalasin reconstituted endothelial [Ca2+]i signaling and NO production. Our findings show that lung EC dysfunction in CHF results from impaired EC [Ca2+]i signaling, and identify the endothelial cytoskeleton as critical regulator of EC [Ca2+]i homeostasis.

Sponsored by EU IP Pulmotension and Kaiserin‐Friedrich Foundation Berlin.
Elevated Wnt–Related Tracheal Aspirate Proteins LEF‐1 and ‐βcatenin Predicts the Development of Bronchopulmonary Dysplasia in Very‐Low‐Birth‐Weight Infants
Authors: Rehan Virender K. , Sakurai Reiko , Dhar Vijay , Torday John
Published: June 2021

Taking a basic biologic approach, and using a wide range of models, we have demonstrated alveolar interstitial fibroblast‐to‐myofibroblast transdifferentiation, characterized by the down‐regulation of Parathyroid Hormone‐related Protein (PTHrP) signaling, and up‐regulation of Wnt signaling as a key event in the pathogenesis of Bronchopulmonary Dysplasia (BPD). Though decreased tracheal aspirate (TA) PTHrP levels in ventilated Very‐Low‐Birth‐Weight Infants (VLBWIs) during the first wk of life predict the later development of BPD, there is no information on the levels of Wnt‐related proteins in the TA of these infants. Twenty ventilated VLBWIs (birth weight 943 ± 302 g (mean ± SD); GA 27 ± 2weeks; 10M:10F) were studied. The TAs were collected once daily during the first 3 days of life, or until extubation, whichever was earlier, and immediately frozen for subsequent analysis by ELISA and Western blot for LEF‐1 and β‐catenin, two key markers of Wnt activation. LEF‐1 and β‐catenin levels, as determined by both ELISA and Western blotting, were significantly higher in infants who continued to be ventilated at 1 wk of age, and who later went on to develop BPD, compared to infants who were extubated by 1 wk of age and did not develop BPD. We conclude that in combination with the lower TA PTHrP content, higher Wnt‐related proteins can be developed into sensitive and specific biomarkers to predict, diagnose, and monitor BPD in ventilated VLBWIs. Disclosure: Supported by grants from the NIH (HL75405, HD51857, HD058948) and the TRDRP (14RT‐0073, 15IT‐0250, 17RT‐0170).
Changes in CO2 during acute hypoxia in immature and adult rabbits and the development of apnea
Authors: Stucke Astrid G , Stuth Eckehard A , Mustapic Sanda , Radocaj Tomislav , Zuperku Edward J
Published: June 2021

Hypoxia can lead to apnea in immature and adult rabbits. Also, a decrease in expiratory CO2 (etCO2) is routinely observed during controlled ventilation. This study evaluated the relationship between etCO2 and development of apnea during acute hypoxia (FiO2 0.08–0.1, 5–8 min). Hypoxic ventilatory responses (HVRs, n=124), as determined from the phrenic neurogram, were observed in 25 immature (< 30d) and 17 adult anesthetized or decerebrate, paralyzed, ventilated, and isothermic rabbits. EtCO2 correlated well with arterial PaCO2. 27% of the HVRs included apneas while 73% showed a marked decrease in rate. EtCO2 during control conditions (52–59 mmHg) and hypoxia (43–46 mmHg) was similar in all animals. There was no difference in the etCO2 decrease in immatures whether apneic or not (−16±3% vs. −17±1%), while the etCO2 decrease in adults was greater with apnea (−26±4% vs. −14±1%). Following termination of hypoxia, all animals showed a marked, temporary increase (7–14%) in etCO2 above prehypoxia control. The results suggest that hypoxia‐induced apnea in adult rabbits correlates with a greater decrease in etCO2. The marked increase in etCO2, posthypoxia, suggests that this may be mediated by a decrease in cardiac output or change in organ blood flow pattern. The lack of a correlation between apnea and drop in etCO2 in immature rabbits may be due to developmental age. Supported by departmental funds.
Hypoventilation in awake goats following unilateral lesions in the rostral ventrolateral medulla (RVLM) targeted by surface cooling‐induced apnea.
Authors: Miller Justin , Neumueller Suzanne , Marshall Brooke , Baogang Qian , Mouradian Gary , Hodges Matthew , Pan Lawrence , Forster Hubert
Published: June 2021

Thirty seconds of cooling the RVLM induces apnea and reduced breathing in anesthetized and awake goats, respectively (J Appl Physiol 78: 247–257, 1995). We recently studied the effects on breathing in two goats of unilateral ibotenic acid (IA: 1 μl) induced RVLM lesions 1 mm below the medullary surface at a critical site identified by cooling under anesthesia. Upon recovery from anesthesia, both goats hyperventilated at rest for 5 days, but thereafter hypoventilated (PaCO2 8–10 mmHg above control) for several days due to decreased tidal volume and breathing frequency. In one goat CO2 sensitivity was attenuated nearly 50% during the period of hypoventilation, but the ventilatory responses to hypoxia and exercise, and breathing during NREM sleep were unaffected. In this goat resting PaCO2 and CO2 sensitivity returned to control about 15 days post‐lesion. Both goats were in excellent health throughout the post surgery period. Post‐mortem histology indicated the lesioned sites were in an area of high density neurokinin‐1 receptor expressing neurons, near or including the retrotrapezoid nucleus. These data suggest that neurons near the RVLM surface are necessary to maintain normal levels of eupneic breathing, and contribute to respiratory CO2 chemoreception.
Systemic inflammation suppresses hypoxic and hypercapnic ventilatory responses in rats.
Authors: Windelborn James A , Mitchell Gordon S
Published: June 2021

Chemoreflex responses to hypoxia and hypercapnia are critical elements of ventilatory control. However, chemoreflexes may be undermined by factors frequently associated with lung disease, such as systemic inflammation. Here, we tested the hypothesis that systemic inflammation suppresses ventilatory responses to hypoxia and hypercapnia in rats. Ventilatory responses were assessed in unanesthetized adult rats via whole‐body plethysmography 1 day after lipopolysaccharide administration (LPS: 1 mg in saline, i.p.). In Lewis rats, LPS suppressed both hypoxic (10.5% O2; vehicle: 90±19%; LPS: 37±11%; p < 0.05) and hypercapnic ventilatory responses (7% CO2; vehicle: 223±22%; LPS: 66±26%; p < 0.05). Splenic iNOS mRNA increased 6.5‐fold (qPCR) in LPS‐treated Lewis rats, demonstrating systemic inflammation. In contrast, there was no evidence for suppressed hypoxic (vehicle: 127±14%; LPS: 92±21%) or hypercapnic ventilatory responses (vehicle: 216±19%; LPS: 239±27%) in LPS‐treated Sprague Dawley rats, nor were splenic iNOS mRNA levels increased. Thus, although LPS sensitivity varies among rat strains, systemic inflammation suppressed chemoreflex responses to hypoxia and hypercapnia. Suppressed chemoreflex responses may impair the ability to respond appropriately to ventilatory challenges in disease states associated with inflammation, including many lung diseases (NIH HL69064 and HL007654).

Grant Funding Source: NIH HL69064 and HL007654
Isometric force of isolated extraocular muscles of wild cowbirds
Authors: Allawzi Ayed Mamdouh , Grelle Eve Nicole , Teclaw Mary Ellen , Moore Bret , Fernández‐Juricic Esteban , Pelaez Nancy
Published: June 2021

Recent studies show that the degree of eye movements varies substantially between different bird species. This variation is important from an evolutionary perspective, because species with a high degree of eye movement can change substantially the configuration of their visual fields, and consequently, visual coverage. More studies are necessary to understand the physiological basis of eye movements from a comparative perspective. Extraocular muscles of brown‐headed cowbirds (a species that shows a high degree of eye movement) were isolated and placed in a muscle bath, and isometric force was recorded in response to Acetylcholine (ACh) or KCl depolarization. With both treatments, stimulation caused a decline in muscle force, suggesting that the isolated muscle was pre‐contracted. Relaxation could be due to stimulation of inhibitory pre‐synaptic neurons. As a next step, experiments with cowbird extraocular muscle will be conducted in the presence of tubocurarine to induce relaxation so that experiments can be conducted to establish a normalized length‐tension curve for cowbird extraocular muscles.
Functionnal properties of skeletal muscles are preserved in erythropoietin deficient mice exposed to hypoxia
Authors: LAUNAY Thierry , Hagstrom Luciana , Canon Francis , Agbulut Onnik , Serrurier Bernard , Marchant Dominique , Richalet Jean‐Paul , Bigard Xavier , Beaudry Michèle
Published: June 2021

Epo‐induced polycythemia is the main factor of adaptation to hypoxia. In this study we analyzed the effects of Epo‐deficiency on intrinsic functional properties of slow and fast‐twitch muscles in a model of erythropoietin deficient mice (Epo‐TAgh) exposed to hypoxia. We hypothesized that Epo‐deficiency would be deleterious for skeletal muscle development, angiogenesis and maintenance of its structure when exposed to ambient hypoxia. Wild‐type (WT) and Epo‐TAghmice were left in hypobaric chamber at 420 mmHg pressure for 14 days. Soleus (SOL) and extensor digitorum longus (EDL) were analyzed in vitro by mechanical measurements, immuno‐histological and biochemical analyses. The results were compared to those obtained in corresponding muscles of aged matched normoxic groups. Our data did not show any difference between the groups whatever the Epo‐deficiency and/or hypoxic conditions for twitch force, tetanic force, fatigue, typology and myosin heavy chain composition. Normoxic Epo‐TAghmice exhibit improved capillary‐to‐fibre ratio compared to WT mice in both SOL and EDL whereas no angiogenic effects of hypoxia or combined Epo‐deficency/hypoxia were observed. These results suggest that skeletal muscles possess a great capacity of adaptation to Epo‐deficiency. Then Epo‐deficiency is not a sufficient factor to induce muscle deconditioning observed in patients suffering from severe renal failure
Skeletal Muscle Oxidative Capacity in Cachectic Mice
Authors: Velazquez Kandy T , Sato Shuichi , White James P , Puppa Melissa J , Baynes John W , Carson James A
Published: June 2021

Cachexia is often defined as a complex metabolic syndrome associated with underlying illness and characterized by the loss of muscle with or without loss of fat mass. These changes are often attributed to chronic inflammation that can enhance protein degradation in skeletal muscle and lypolysis in adipose tissue. The purpose of this study was to examine if the enhanced metabolic demands during cachexia alter oxidative capacity in red and white portions of the gastrocnemius muscle from cachectic ApcMin/+ mice. Twenty week ApcMin/+ mice were stratified by cachexia severity determined by body weight, muscle, and fat mass. The gastrocnemius muscle was separated into red and white portions to determine phenotype specific changes. Muscle mitochondrial content and succinate dehydrogenase activity were significantly reduced in mice with severe cachexia. Gene expression related to mitochondrial fission and fusion were quantified in the muscle. In glycolytic muscle there was a significant decrease in mfn1 and mfn2 expression, and an increase in fis1 mRNA expression. Nitrotyrosine expression, a marker of oxidative stress was significantly decreased in both oxidative and glycolytic muscle from severely cachectic mice. These results suggest that during severe cachexia skeletal muscle mitochondria number and function are altered in red and white portions of rapidly wasting skeletal muscle. RO1CA121249‐01A2
Alpha Adrenergic Sensitivity and Temperature in Human Arteries: The Role of Nitric Oxide
Authors: Ives Stephen James , Zhao Jia , Noyes R. Dirk , Andtbacka Robert , McDaniel John , Symons J. David , Hayman Melissa , Wray D. Walter , Richardson Russell
Published: June 2021

Previously, we have found that increased local temperature exerts a sympatholytic effect in vitro. We hypothesized that this may be due to upregulation in the activity of endothelial nitric oxide synthase (eNOS), reducing the impact of the α‐1 adrenergic receptor agonist phenylephrine (PE).

METHODS

Human arteries (350–700uM) supplying skeletal muscle were obtained during pre‐scheduled surgeries. Vessels were dissected into 2mm rings and mounted on a wire myograph. All vessels underwent a stepwise length tension procedure to determine LMAX (≤10% increase in mg developed tension (mgΔ) to 100mM Potassium Chloride; KCL). Vessel function was characterized using KCL, Sodium Nitroprusside, PE, & Acetylcholine dose response curves (DRCs) to determine non‐receptor and receptor mediated vasoconstriction and vasorelaxation. PE DRCs were performed both at 37 and 39°C, which were repeated in the presence of the NOS inhibitor L‐NG‐monomethyl‐arginine (L‐NMMA). Data are mean ± SE.

RESULTS

PEMAX at 39°C was lower than 37°C (85±32 vs. 241±173 mgΔ). Nitric oxide blockade enhanced vasoconstriction at 37° and 39°C (537±226 and 256±81 mgΔ, respectively).

CONCLUSION

These findings reveal that NO blockade can restore the attenuated PE‐induced vasoconstriction at 39°C to that of 37°C, but also highlight the important role that vasodilators play in the vascular response to vasoconstrictors.

Support: NIH grant PO1 HL‐091830
Contribution of nitric oxide to the blood pressure and arterial responses to incremental maximal exercise in humans
Authors: Fisher James P , Campbell Ross , Sharman James E , McDonnell Barry J , Frenneaux Michael P
Published: June 2021

We sought to determine the contribution of nitric oxide (NO) to the blood pressure (BP) responses to exercise and post‐exercise alterations in arterial stiffness. In 10 healthy subjects (31±5 years), BP, heart rate (HR) and oxygen consumption were measured before, during and after an incremental cycling exercise test. Trials were performed with placebo (saline) or following acute inhibition of NO synthase with NG‐monomethyl‐L‐arginine (L‐NMMA) on separate days, in a randomized, double blind, cross‐over design. Central (aortic) and peripheral (femoral) arterial stiffness was assessed with pulse wave velocity (PWV). BP was increased with L‐NMMA at rest and during sub‐maximal exercise, but not at maximal exercise (mean BP 118±8 vs. 117±5 mmHg, saline vs. L‐NMMA, P>0.05). L‐NMMA had no influence on exercising HR or maximal oxygen consumption (P=ns). Post‐exercise reductions in femoral PWV were attenuated with L‐NMMA (P<0.05), while aortic PWV was increased similarly with either saline or L‐NMMA (P=ns). Our findings suggest that reductions in NO bioavailability cause augmented BP responses to sub‐maximal exercise, but do not lead to exaggerated BP responses to maximal exercise or reduce maximal oxygen consumption. Furthermore, NO is an important contributor to reductions in femoral artery stiffness following maximal exercise in healthy individuals.

This research was supported by the BHF.
Cardiovascular responses during acute inspiratory muscle exercise in hypertensive or chagasic cardiomiopathy
Authors: Borile Suellen , Ferreira‐Filho Julio César Aires , Sousa Silvia Beatriz Cavasin , Mello Priscila , Irigoyen Maria Claudia , Krieger Eduardo Moacyr , Consolim‐Colombo Fernanda Marciano
Published: June 2021

Aim

To analyze systolic (SBP) and diastolic (DBP) blood pressure, heart rate (HR), total peripheral resistance (TPR), cardiac output (CO) and derivative of ventricular pressure (dp/dt) during inspiratory exercise.

Methods

Cardiovascular parameters were continuously recorded by an indirect blood pressure register device (Finometer) at baseline and during exercise (10 min) with a Threshold® device in 27 hypertensive cardiomiopathy (HCM) and 9 chagasic cardiomiopathy (CCM) patients (LVEF < 45% and inspiratory muscle weakness).

Results

26% of HCM patients did not finish exercise protocol due to exacerbated increase in blood pressure (ΔSBP > 20mmHg). On the other hand, all patients of CCM group completed the exercise protocol; nevertheless, they showed intense fatigue. There were significant differences when comparing baseline vs. exercise for HCM and CCM groups, respectively, in: SBP (Δ= 9±2 and Δ= 7.6±3 mmHg), DBP (Δ= 4.8±1 and Δ= 4.2±1 mmHg) and HR (Δ= 5.5±1,2 and Δ= 6.6±3 bpm). Only HCM group presented increase in CO (Δ= 0.36 ±0.1 l/min) and dp/dt (Δ= 118±35 mmHg/s). There was no significant difference between HCM and CCM.

Conclusion

Acute inspiratory muscle exercise caused important hemodynamic alterations, mainly in hypertensive patients. Therefore, before indication of unsupervised inspiratory training, it is necessary to perform one cardiac monitorized session to recommend this type of exercise.