bluuebunny/crossref_metadata_2025 huggingface dataset:
Green tea extract reduced myocardial injury in ischemia/reperfusion of rat heart
Authors: Liou Ying‐Ming , Hsieh Shih‐Rong , Wu Tsu‐Juey
Published: June 2021

We recently showed that green tea extract (GTE) can improve the cardiac function by the direct effects on the cardiac contractile apparatus. Since reperfusion injury is one of serious problems in cardiac surgery, we created a rat model of ischemia/reperfusion (IR) by left anterior descending coronary ligation (LAD) for 20 min followed by releasing ligation to determine effects of GTE pretreatment on myocardial protection from IR injury. After LAD ligation, several arrhythmias patterns were defined: GTE significantly suppressed the genesis of arrhythmia after ligation, reperfusion only induced arrhythmias at the initial 5 min with or without GTE. At 24 hr after LAD ligation rats increased serum cTnI levels, while GTE significantly reduced cTnI‐released levels by IR. At 3 days after ligation, echocardiogram showed 26% decrease in Ejection Fraction (EF), and 34% reduction in Fractional shortening (FS), while GTE pretreatment had no changes in EF and FS as compared with controls. In addition, single cardiomyocytes freshly isolated from hearts of control, IR rats with or without GTE at 3 days after ligation showed GTE exerting effects on IR‐induced changes of intracellular Ca2+ and moderating the cellular distribution of contractile, gap junction, and adhesion proteins in cells. Taken together, our results indicated that GTE effectively prevents from IR injury on myocardium. Grant: NSC‐98‐2320‐B‐005‐001‐MY2.
The hypercoagulability of initial intraosseous aspirates
Authors: Polykratis Irene Amy , Rubal Bernard J. , Sondeen Jill L. , DeLorenzo Robert A. , Dubick Michael A.
Published: June 2021

Intraosseous (IO) access is a recognized alternative to intravenous routes for administration of resuscitation drugs and fluid therapy. A rapid saline flush immediately following access is reported to improve intraosseous flow rates. We examine the thrombogenicity of initial IO aspirates to elucidate mechanisms for improved flow with flush procedures. In this study thromboelastographic (TEG, Haemoscope Inc., Niles, IL) profiles of initial IO aspirates were compared with whole blood from arterial and central venous catheters in eight swine (40.8 ± 6.5 Kg). The viscoelastic clotting parameters assessed included: R‐time (minutes), K‐time (minutes), angle α (degrees) and maximum amplitude (MA, mm). Intraosseous access was achieved using EZ‐IO 15 GA needle sets (Vidacare, San Antonio, TX). IO aspirates were obtained from the tibia, femur or humerus. In two animals IO aspirates clotted too rapidly for TEG analysis. Table (mean ± 95% CI) compares TEG profiles of IO aspirates with arterial and central venous sites (ANOVA * P<0.05).

R‐time (min)
K‐time (min)
Angle α (degree)
MA (mm)

Arterial
7.9±1.7*
2.0±0.2*
62.4±4.1*
70±2

Venous
5.7±2.3*
1.4±0.5*
71.6±5.4*
72±3

IO
2.3±1.0
0.9±0.1
78.2±0.9
72±8

Conclusion

This study demonstrates hypercoagulability of initial access IO aspirates. Clots form rapidly without flush at the IO cannulation site and contribute to poor IO flow rates.
PROTEOLYTIC CLEAVAGE OF INSULIN RECEPTOR BY PANCREATIC DIGESTIVE ENZYMES LEADS TO INSULIN RESISTANCE IN HEMORRHAGIC SHOCK
Authors: DeLano Frank A , Schmid‐Schönbein Geert W
Published: June 2021

Hemorrhagic shock is associated with metabolic defects, including hyperglycemia and insulin resistance. The mechanisms are unknown. We recently demonstrated that insulin resistance may involve cleavage of the extracellular domain of the insulin receptor by degrading proteases. In shock, transfer of digestive enzymes into the systemic circulation after breakdown of the intestinal mucosal barrier causes inflammation and organ dysfunction. Suppression of the digestive enzymes in the lumen of the intestine with protease inhibitors was shown in this laboratory to be effective in reducing the level of the inflammatory reactions. To determine what degree blockade of digestive enzymes affects insulin resistance in shock, rats were exposed to acute hemorrhagic shock (mean arterial pressure of 30 mmHg) for 2 hours at which time all shed blood volume was returned. Digestive proteases in the intestine were blocked with a serine protease inhibitor (tranexamic acid, 0.3 mM, in polyethylene glycol‐3350, 60 g/L) and the density of the insulin receptor was measured with immunohistochemistry in the mesentery microcirculation. The untreated rat without enzyme blockade had significantly attenuated levels of insulin receptor density as compared to control and treated rats. Blockade of the digestive proteases after 60 min of hypotension in the lumen of the small intestine lead to a lesser decrease in insulin receptor density compared to controls without protease blockade. These results suggest that in physiologic shock elevated microvascular digestive enzyme activity leads to proteolytic cleavage of the insulin receptor and subsequent insulin resistance

(Supported by an academic gift from Leading Ventures and in part by GM85072).
Hemorrhagic‐induced acidosis does not affect recombinant Factor VIIa (rFVIIa) function in Swine
Authors: Darlington Daniel Norman , Kheirabadi Bijan S. , Martini Wenjun Z. , Dubick Michael A.
Published: June 2021

Acidosis attenuates the activity of rFVIIa in vitro. However, it is not known if acidosis caused by hemorrhagic shock will also attenuate rFVIIa activity in vivo. The purpose of this study was to determine if acidosis affects rFVIIa function in swine. Swine (n=8) were bled (50–60%blood volume) and held at a MAP of 30 mmHg. Arterial acidosis (pH7.1) was then achieved after decreasing ventilation. Control swine (pH7.4) were subjected to sham operation. rFVIIa was injected after target pH was achieved. Arterial blood samples were taken before and 5 min after injection of rFVIIa (90μg/kg) for measurement of coagulation parameters (PT, Thrombin Generation, TEG). Acidosis led to a significant decrease (P<0.05) in coagulation function as measured by most parameters. rFVIIa injection led to a significant decrease in PT in control and acidotic pigs (−2.9±0.1 and −2.5±0.3 sec), a decrease in lagtime for Thrombin Generation (−0.59±0.12 and −0.22±0.08 min), and an increase in MA (3.7±1.0 and 6.8±3.2mm) and Angle (5.7±2.4 and 15.0±7.2 degrees) for TEG. However, there was no difference between control and acidotic pigs in the change of these parameters, suggesting that acidosis did not affect rFVIIa function. Acidosis caused by hemorrhagic shock and hypoventilation led to mild coagulopathy. This condition did not inhibit the ability of rFVIIa to enhance coagulation.
Role of STIM1 in Coronary Endothelial Cell Dysfunction in Type 1 Diabetic Mice
Authors: Nolta Nicholas Fredrick , Estrada Irene , Han Wenlong , Scott Brian T. , Dillmann Wolfgang H. , Yuan Jason X.‐J. , Makino Ayako
Published: June 2021

Ca2+ handling plays an important role in endothelial functions including regulation of vascular tone. An increase of intracellular [Ca2+] triggers the production of vasodilatative factors in endothelial cells (ECs) and leads to vascular relaxation. The [Ca2+] in the endoplasmic reticulum (ER) determines in great part the generation of important Ca2+ signals, and many proteins contribute to ER Ca2+ refilling after store depletion, most notably, stromal interaction molecule 1/2 (STIM1/2), Orai1/2/3, and sarcoplasmic/endoplasmic reticulum Ca2+‐ATPase 3 (SERCA3) in ECs. Although endothelial dysfunction has been implicated in the diabetic heart, little is known about the contribution of STIM1. Vascular relaxation induced by ACh in coronary arteries (CAs) from diabetic mice was significantly attenuated compared with control CAs. Mouse coronary ECs (MCECs) isolated from diabetic mice exhibited a significant decrease in CPA‐induced Ca2+ release from the ER and had significantly lower protein expression levels of STIM1 than control. Furthermore, STIM1 positive mutant overexpression restored the Ca2+ release from ER in diabetic mice. These data suggest that impaired ER Ca2+ refilling due to downregulation of STIM1 protein expression attenuates CA relaxation in diabetes and leads to diabetic coronary vascular disease. This research was supported by grant DK083506.
Relationship between markers of bone turnover and coronary arterial calcification in newly menopausal women
Authors: Miyabara Yuko , Kearns Ann E. , Miller Virginia M.
Published: June 2021

Bone loss and development of atherosclerosis characterized by coronary arterial calcification (CAC) accelerate in women after menopause. This study evaluated the relationship between blood borne markers of bone turnover and development of CAC in newly menopausal women being screened for the Kronos Early Estrogen Prevention Study. Women (n=137) were 52.3±2.3 years of age and were 18.1±9.2 months past their last menses. CAC was measured using 64‐slice computed tomography. Concentrations of osteocalcin, bone alkaline phosphatase (BAP), tartrate‐resident acid phosphatase 5b and osteopontin as bone matrix protein in serum and plasma were evaluated by ELISA. CAC scores ranged from 0 to 327.6 Agatston Unit (AU); 113 women had scores of 0 AU, 20 had CAC scores of 0<50 and 4 had CAC scores >50. Average levels of all of the markers of bone turnover were within a normal range reported in the literature. There was a positive correlation between BAP and osteocalcin. However, levels of these markers did not correlate with age or months past menopause. None of the markers correlated with CAC scores. These results suggest that blood borne markers of bone turnover do not correlate with the presence of CAC in newly menopausal women. Additional work is needed to determine if this relationship would change with increasing age.

Supported by the Kronos Longevity Research Institute and Mayo Foundation.
Genetic architecture underlying the native collateral circulation
Authors: Wang Shiliang , Zhang Hua , Dai Xuming , Sealock Robert , Faber James
Published: June 2021

Collaterals are arteriole‐to‐arteriole anastomoses that connect adjacent arterial trees. They lessen the severity of ischemic tissue injury by serving as endogenous bypass vessels when one tree becomes occluded. We previously found remarkably wide variation in native collateral number and diameter in inbred mouse strains. These findings suggest that a significant genetic component underlies formation of the native collateral circulation. Here we sought to identify candidate genes and their polymorphisms responsible for this wide variation. To explore the underlying genetic architecture, we created 221 F2 mice intercrossed between C57BL/6J and BALB/cByJ and performed linkage analyses to identify quantitative trait loci (QTL) for native collateral number and diameter. These same traits were quantified in 15 inbred strains with 10 mice from each to permit haplotype association mapping. Our results suggest that collateral number is governed by 4 QTL, including an epistasis between the loci on chromosomes 1 and 3 and collateral diameter by one identical QTL. This common QTL, possessing a large genetic effect (above 30%), is on chromosome 7. Association mapping pinpointed a 172k bp region with high p value (p = 2.2 × E10−5) and 2 candidate genes. We conclude that number and diameter of native collaterals are highly heritable complex traits with one QTL predominantly affecting their variation.
Renal medullary NAD(P)H‐oxidase subunit p67phox as a candidate gene in salt‐sensitive hypertension
Authors: Feng Di , Yang Chun , Kurth Terry , Liang Mingyu , Moreno‐Quinn Carol , Jacob Howard , Cowley Allen W.
Published: June 2021

There is great interest in better defining the genetic basis of salt‐sensitive hypertension (SSHT). We have generated a consomic strain (SS‐13BN/Mcwi) by introgression of the entire chr 13 of the normotensive Brown Norway rat into the Dahl salt‐sensitive SS/JRHsdMcwi (SS) genetic background. We also generated a congenic strain SS.BN‐(D13Rat151‐D13Rat197)/Mcwi introgressing a region of BN chr 13 and found both strains protected from SSHT. We previously found that renal medullary infusion of apocynin, an NAD(P)H oxidase inhibitor, partially blunted SSHT in the SS. p67phox(Ncf2), a gene located in the substituted region, encodes one of the cytosolic subunits of NAD(P)H oxidase. The mRNA and protein expression levels of p67phox and NAD(P)H oxidase activity in the outer medulla were significantly different between the SS and either SS‐13BN congenic or consomic on day 3 of high salt with no differences in mean arterial pressure (MAP) between these three strains. By day 7, MAP was significantly higher in the SS than the consomic or congenic strains and the difference in expression of p67phox was further enhanced. We conclude that the expression difference of p67phox determines the greater levels of NAD(P)H oxidase activity in SS rats compared to this congenic strain, and that p67phox is a viable candidate gene contributing to salt‐sensitive hypertension. Sequence variations are currently being evaluated. (HL‐827980)
Mitochondrial genomic polymorphisms in rat genetic models of hypertension
Authors: Kumarasamy Sivarajan , Gopalakrishnan Kathirvel , Thangavel Jayakumar , Shafton Asher , Nixon Jeremy , Farms Phyllis , Joe Bina
Published: June 2021

Hypertension is a complex trait that has been extensively studied for genetic contributions of the nuclear genome. However, recent studies indicate that naturally occurring variation in the mitochondrial genome contribute to several complex traits. We examined whether there are variations between the mitochondrial genomes of genetic models including the Dahl salt‐sensitive (S) rat, the Dahl salt‐resistant (R) rat, the spontaneously hypertensive rat (SHR) and the Albino Surgery (AS) rat. The results provide evidence to suggest that variations in the mitochondrial genome do not account for observed differences in blood pressure between the S rat and the R rat. However, a total of 105, 99 and 18 variations in mtDNA were observed between the three comparisons‐S and SHR, S and AS and SHR and AS, respectively. Among the 13 protein coding genes of the mtDNA, 8 genes possessed nonsynonymous variations between S, SHR and AS. While the lack of any sequence variants between the mitochondrial genomes of S and R rats provides conclusive evidence that divergence in blood pressure between these two inbred strains is exclusively programmed through their nuclear genomes, the variations detected among the various hypertensive strains provides the basis to construct conplastic strains and further evaluate the effects of these variants on hypertension and associated phenotypes.
Extracorporeal Membrane Oxygenation (ECMO) Does Not Restore Renal Autoregulation in Endotoxin‐Induced Acute Renal Failure
Authors: Uyehara Catherine F T , Sato Aileen K , Hernandez Claudia A , Ichimura Wayne M , Batts Sherreen , Mu Thornton S , Andaya January M
Published: June 2021

Endotoxin‐induced shock may be treated with ECMO for cardiopulmonary support. Renal function during ECMO may be altered due to partial cardiopulmonary bypass and altered regulation of cardiovascular regulating hormones. In anesthetized pigs (n=8), we compared renal function at baseline, after endotoxic shock with acute renal failure was established (via i.v. injection of E coli endotoxin), and after 2 hours of stabilization on venoarterial ECMO. Endotoxin caused a mean arterial pressure (MAP) 33% decrease (p≤0.05) from 88±3 to 59±3 mm Hg. ECMO could maintain blood flow to prevent any further deterioration of MAP, but did not return MAP (56±4 mm Hg) to baseline. Interestingly, the 59% decrease (p≤0.05) in renal microcirculatory blood flow (as measured by microsphere method) caused by endotoxin, was prevented from dropping further with ECMO. However, the endotoxin‐induced 76% decrease in GFR (p≤0.05) continued to decline with ECMO resulting in deterioration of urine flow towards complete renal shut down. The urine flow decrease was due mainly to a decrease in osmotic clearance. Results are consistent with the drop of endogenous vasopressin (VP) levels seen with ECMO that we previously reported. We and others have demonstrated that exogenous VP at high doses is needed to reverse renal shut down. Results suggest that during ECMO, VP replacement therapy may be necessary to restore renal perfusion pressure to maintain urine flow.
High Dietary Protein Exacerbates Hypertension and Renal Damage in Dahl Salt‐Sensitive (SS) Rats by Increasing Infiltrating Immune Cells
Authors: De Miguel Carmen , Lund Hayley , Mattson David
Published: June 2021

Experiments assessed the role of protein intake on salt sensitive hypertension in SS rats fed low (6%), normal (18%) or high (30%) protein diets. Increasing dietary NaCl from 0.4% to 4.0% for 3 weeks significantly increased mean arterial pressure (MAP) and albumin excretion rate (Ualb) in rats (n>6/group) fed the high protein diet (153 ± 7 mmHg and 114 ± 37 mg/day) when compared to rats fed normal (132 ± 3 mmHg and 26 ± 7 mg/day) or low protein diets (132 ± 6 mmHg and 4 ± 1 mg/day). Histological analysis demonstrated that high protein‐fed animals had significantly greater glomerular and tubular damage. Significantly greater numbers of infiltrating T‐lymphocytes were observed in kidneys of SS rats fed the high protein diet (18.9 ± 3 × 105cells/kidney) than in rats fed the low protein diet (9.1 ± 3 × 105 cells/kidney). Furthermore, treatment of SS rats fed the high protein diet with the immunosuppressant agent mycophenolate mofetil (20mg/kg/day) significantly reduced the number of infiltrating T cells in the kidneys by 44% while decreasing salt‐sensitive MAP by 16% and Ualb by 33%. These results indicate that dietary protein content is important for determining the degree of salt‐sensitive hypertension and kidney disease in SS rats; moreover, infiltrating T‐lymphocytes appear to play an important role in mediating the changes in blood pressure and kidney damage in these animals. Supported by HL29587 and DK062803.
PGE2 release from tryptase‐stimulated rabbit ventricular myocytes is mediated by calcium‐independent phospholipase A2γ
Authors: Sharma Janhavi S , McHowat Jane
Published: June 2021

Inflammation is associated with several cardiovascular diseases and mast cells have been implicated in many of these, but their precise role remains unclear. We hypothesize that tryptase, a pre formed mast cell mediator, stimulates rabbit ventricular myocyte calcium independent phospholipase A2 (iPLA2) activity, resulting in increased arachidonic acid and prostaglandin E2 (PGE2) release. Tryptase stimulation (20 ng/ml) increased iPLA2 activity after 5 mins (3.56 ± 0.12 to 5.87 ± 0.31 nmol/mg protein/min, n=6, p<0.01). This was accompanied by increased arachidonic acid (1.02 ± 0.12 to 5.02 ± 0.21%, n=6, p<0.01) and PGE2 release (4.50 ± 0.30 to 16.70 ± 0.59 ng/mg protein, n=8, p<0.01) in stimulated myocytes. To distinguish between different myocardial iPLA2 isoforms, we pretreated ventricular myocytes with the R‐ and S‐ enantiomers of bromoenol lactone (BEL, 5 μM, 10 mins) to selectively inhibit iPLA2γ and β respectively. (R)‐BEL pretreatment resulted in complete inhibition of tryptase‐stimulated iPLA2 activity, arachidonic acid and PGE2 release, suggesting the iPLA2γ is activated in response to tryptase. Pretreatment with (S)‐BEL had little effect on tryptase‐stimulated responses. Previous studies suggest that PGE2 may be cardioprotective. Thus tryptase release from activated mast cells may play a protective role in cardiac inflammation via iPLA2γ activation and increased PGE2 release from myocytes.
PP2A attenuates cardiac response to LPS‐induced sepsis
Authors: Gergs Ulrich , Schulz Nico , Grossmann Claudia , Gekle Michael , Neumann Joachim
Published: June 2021

Lipopolysaccharide (LPS), a cell membrane component from Gram‐negative bacteria, is involved in sepsis‐associated cardiac dysfunction. We wanted to know if protein phosphatase 2A (PP2A) influences LPS‐signaling in the heart. Therefore, we used transgenic (TG) mice with heart‐specific overexpression of PP2A. TG and littermate wild type (WT) mice were intraperitoneally injected with 30 μg LPS per g body weight or with NaCl as control (n = 14 – 18 per group). After 3 days, successful induction of LPS‐induced sepsis was monitored by enhanced plasma levels of interleukin‐6. Contractility of WT and TG hearts was analyzed using the isolated work performing heart preparation. Left ventricular systolic pressure (LVP) and the first derivative of developed pressure (+dP/dt and −dP/dt) were decreased in hearts of LPS‐treated WT mice, whereas hearts of TG mice showed no decrease of contractility after LPS‐treatment (Table).

Contractile parameters
Control
LPS

WT
TG
WT
TG

LVP (mmHg)
74±2.2
66±4.3
50±4.3*
61±1.9#

+dP/dt (mmHg/s)
2481±186
2145±254
1306±246*
1901±171#

−dP/dt (mmHg/s)
−1371±114
−1073±139
−720±135*
−1006±115

p<0.05 vs. control,

p<0.05 vs. WT

Quantitative PCR revealed that LPS increased IL‐6 mRNA expression in TG and WT hearts but TNFα mRNA levels only were enhanced in WT. In summary, LPS‐induced myocardial dysfunction was attenuated in TG mice compared to WT. It is tempting to speculate that increased PP2A activity in TG hearts is protective against LPS‐induced sepsis probably via diminished TNFα expression. (Supported by the DFG)
Response gene to complement 32 interacts with Smad3 to promote epithelial‐mesenchymal transition of renal tubule cells
Authors: Guo Xia , Chen Shi‐You
Published: June 2021

Previous studies have shown that response gene to complement 32 (RGC‐32) mediates TGF‐β‐induced EMT of human renal proximal tubule cells. This study is aimed to elucidate the mechanism underlying RGC‐32 function. RGC‐32 function in EMT appears to be associated with Smad3. Co‐expression of RGC‐32 and Smad3, but not Smad2, induced a higher expression of myofibroblast marker α‐SMA as compared to RGC‐32 or Smad3 alone; knockdown of Smad3 by shRNA blocked RGC‐32‐induced α‐SMA expression. These data suggest that RGC‐32 interacts with Smad3 in the regulation of EMT. RGC‐32 colocalized with Smad3 in the nuclei of renal tubule cells upon TGF‐β induction. Co‐immunoprecipitation assay indicates that Smad3, but not Smad2, physically interacts with RGC‐32 in renal tubular cells. Additional studies showed that RGC‐32 and Smad3 synergistically upregulated α‐SMA promoter activity. Blockade of RGC‐32 significantly inhibited Smad3 activity in activating α‐SMA promoter; knockdown of Smad3 inhibited RGC‐32‐indced α‐SMA mRNA expression. Moreover, RGC‐32 and Smad3 synergistically activated fibronectin while downregulated an epithelial marker E‐cadherin. Mechanistically, RGC‐32 and Smad3 coordinate the induction of EMT by regulating the EMT regulators Slug and Snail. Taken together, our data demonstrate that RGC‐32 interacts with Smad3 in mediating TGF‐β‐induced EMT of human renal tubule cells. (Funding source: NIH R01HL093429)
Quantitative synthesis of dynamic baroreflex pressure regulation using baroreflex induced changes in ventricular and vascular properties
Authors: Sakamoto Takafumi , Tsutsumi Takaki , Tanaka Atsushi , Murayama Yoshinori , Tobushi Tomoyuki , Sakamoto Kazuo , Sunagawa Kenji
Published: June 2021

Background

Baroreflex (BR) stabilizes arterial pressure (AP) by changing ventricular and vascular properties. However, how those changes quantitatively determine AP remains unknown. We previously developed a framework of circulatory equilibrium (AJP 2004, 2005) where we can algebraically derive the equilibrium using ventricular end‐systolic elastance (Ees), heart rate (HR), vascular resistance (R) and stressed blood volume (V). Using the framework we investigated if we can quantitatively synthesize AP responses on the basis of BR induced those properties.

Methods and Results

In 5 anesthetized with isolated carotid sinuses, we randomly perturbed carotid sinus pressure (CSP) using a servo‐pump while recording hemodynamic variables including ventricular pressure‐volume relations. We estimated transfer functions from CSP to HR, Ees, R and V and predicted time domain values of each variable in response to changes in CSP. We then synthesized changes in AP using the equilibrium framework. Synthesized AP matched well with those measured (r2=0.80~0.91). A sensitivity analysis indicated that V and R accounted for 80±6% and 17±6%, respectively, while Ees and HR less than 2% of the BR induced AP changes.

Conclusion

The BR induced AP changes can be quantitatively predicted on the basis of BR regulated ventricular and vascular properties. V mainly contributes to the BR induced AP changes.
Cycloxygenase‐2 Inhibition in Endothelium Removed Bovine Pulmonary Arteries Causes Attenuation of Hypoxic Pulmonary Vasoconstriction
Authors: Ahmad Mansoor , Kandhi Sharath , Kelly Melissa R. , Malpani Saurabh , Prieto Dolores , Wolin Michael S.
Published: June 2021

Superoxide derived peroxide maintains a basal relaxation in endothelium removed Bovine pulmonary arteries (BPA). Removal of this mechanism during hypoxia results in hypoxic pulmonary vasoconstriction (HPV). Inhibition of cyclooxygenase (Cox) 1 and 2 with 10μM Indomethacin resulted in a significant reduction in 5μM lucigenin‐detectable superoxide. Force development to 25mM KCl was not affected in the presence of indomethacin, whereas, HPV was reduced by 63% (n=9). A Cox‐2 inhibitor (1μM NS‐398) decreased superoxide levels and attenuated HPV by 64% (n=8) without affecting 25mM KCl force. However inhibition of Cox‐1 with 1μM SC‐560 did not result in either a decrease in superoxide levels or attenuation of HPV. Thus, inhibition of Cox‐2 and not Cox‐1 in bovine pulmonary vascular smooth muscle results in a decrease in superoxide associated with a partial attenuation of HPV.

Supported by NIH grants HL31069, HL43023 and HL66331.
Decreased Protein Kinase G Activation by Disulfide‐Mediated Subunit Dimerization is Observed when Hypoxia Promotes Contraction of Bovine Pulmonary Arteries
Authors: Neo Boon Hwa , Kandhi Sharath , Wolin Michael
Published: June 2021

Our laboratory had previously provided evidence that hydrogen peroxide (H2O2) stimulates soluble guanylate cyclase under conditions where it relaxes isolated endothelium‐removed bovine pulmonary arteries (BPA), and that hypoxia promotes BPA contraction through suppressing relaxation by endogenous H2O2. Since it was reported that H2O2 induces coronary relaxation associated with a NO/cGMP‐independent thiol oxidation/subunit dimerization activation of protein kinase G (PKG), we investigated if this mechanism participates in the contraction of BPA to hypoxia. BPA precontracted with 20 mM KCl showed decreased PKG dimerization in arteries exposed to hypoxia (PO2 ~10 torr) versus BPA contracted under aerobic conditions (21% O2). Hypoxia also promoted decreased phosphorylation of vasodilator‐stimulated phosphoprotein (VASP) at the serine‐239 site known to be mediated by PKG. Treatment of BPA with 1 mM dithiothreitol increased potassium contraction and abolished contraction to hypoxia, under conditions where it prevented PKG dimerization. Interestingly, serotonin was observed to increase PKG dimerization under hypoxic conditions in a manner which may be related to its oxidase activation‐associated promotion of pulmonary artery smooth muscle remodeling. Thus, attenuation of PKG activation by disulfide‐mediated dimerization appears to be a contributing factor to the contraction of BPA by hypoxia.
Ability of Hyperoxia to Activate the Unfolded Protein Response (UPR) in the Newborn Lung
Authors: Shenberger Jeffrey S , Konsavage Wesley , Zhang Lianqin
Published: June 2021

Hyperoxia generates reactive species which have the potential to interfere with protein folding in pulmonary secretory cells. Given that hyperoxia is known to suppress protein synthesis and increase eIF2α phosphorylation (p‐eIF2α) in cultured lungs cells, we hypothesized that hyperoxia would activate the UPR in the lung. To test our hypothesis, we studied each of the 3 UPR sensors, PERK, ATF6, and IRE1α/XPB1 in the lungs of 4‐day‐old newborn rats exposed to 95%O2 (Ox) for 72 hrs. We found that exposure inhibited pulmonary mRNA translation. Immunoblot analysis of whole lung lysates found that hyperoxia increased p‐eIF2α without activating PERK and failed to alter ATF6 protein expression despite decreasing the expression of BiP protein. We did find that Ox enhanced IRE1α endonuclease activity as evidenced by increased XBP1 splicing. Fractionation indicated that Ox induced the nuclear accumulation of CHOP/GADD153 and Nrf2, downstream effectors of ATF4 and ATF6 activation. Immunofluorescent microscopy localized enhanced p‐eIF2α to airway epithelial and parenchymal cells while electron microscopy denoted engorged and disordered endoplasmic reticulum within alveolar interstial cells. These findings suggest that hyperoxia does induce global UPR activation within the lung, but that specific cell types undergo either partial UPR activation or UPR‐independent activation of select UPR intermediates.
Hypoxia activates NFκB and HIF1α in an erythrocyte‐dependent manner in lung endothelium
Authors: Huertas Alice , Das Shonit R. , Emin Memet , Inamdar Nilufar M. , Rifkind Joseph M. , Bhattacharya Sunita , Bhattacharya Jahar
Published: June 2021

Although hypoxia causes lung inflammation, the initiating mechanisms are not understood. We reported that in hypoxic lungs, erythrocyte‐derived H2O2 diffuses to capillaries (Kiefmann, 2008). To determine endothelial signaling, we ventilated isolated mouse lungs, perfused with erythrocyte‐containing (ERY+) or ‐free (ERY−) buffer, for 4 hours with 21 (normoxia) or 8% (hypoxia) O2. Then we infused chilled collagenase and trypsin in the pulmonary artery to obtain freshly isolated lung endothelial cells (FLEC) (Yiming, 2008). For hypoxia alone, immunoblots of FLEC nuclear fractions revealed bands for NF?B subunit p65 and HIF1a. These responses were abrogated both for ERY− perfusion and ERY+ perfusion containing catalase, indicating that the NF?B and HIF1? pathways were activated by erythrocyte‐ and H2O2‐dependent mechanisms. In mice expressing endothelial‐specific, inactive I?Ba, exposure to 8% O2 for 4 hours failed to induce nuclear translocation of p65 or HIF1a, indicating that NF?B is an upstream regulator of HIF1a. Taken together, our findings are the first evidence that in hypoxia, erythrocyte‐derived H2O2 triggers NF?B and HIF1a activation in endothelial cells. Further, our data affirm that hypoxia‐induced endothelial HIF1a stabilization is NF?B dependent. These signaling pathways are likely to be important in the induction of hypoxic lung inflammation. (Support: HL69514, HL36024).
Lysophosphatidic acid (LPA) enhances barrier function in intralobar rat pulmonary artery endothelial cells
Authors: Umesh Anita , Cornfield David N.
Published: June 2021

Lysophosphatidic acid (LPA) is a bioactive serum lipid whose levels are increased in atherosclerotic plaques. In endothelial cells (ECs), LPA promotes migration, adhesion, wound healing, and modulates barrier function, in part, via 6 receptors (LPA1–6). While its sphingolipid homologue sphingosine‐1‐phosphate, enhances PAEC barrier function, the LPA effects on PAECs are unknown. Here we hypothesized that LPA also enhances PAEC barrier function. To test this, we determined the expression of LPA1–6, GPR87 and P2Y10; and examined the LPA effects on PAEC transendothelial resistance (TER), cytosolic calcium ([Ca2+]i ), and [cAMP] in ECs isolated from 3rd/4th generation intralobar rat PAs. Intralobar PAECs express non‐Edg LPA4/p2y9/GPR23 and LPA6/p2y5. Low [LPA] (0.1uM) increased TER by 4.9±0.6 % (t=30m), while 10uM LPA initially decreased TER by 10.4±1.0% (t=10m) followed by a sustained rise of 14.4 ± 2.1 % (t=90m). LPA increased [Ca2+]i dose‐dependently, and [cAMP] in the presence of forskolin that was potentiated by pertussis toxin (PTX). Both entry and release mechanisms contribute to the LPA‐induced Ca transients since absence of extracellular Ca reduced the signal by 70±10% (p<0.05). LPA‐induced Ca entry was PTX‐sensitive, while release was PTX‐insensitive. Based on these data, we conclude that circulating [LPA] enhance PAEC barrier function and modulates [Ca2+]i and [cAMP] via LPA4 activation.
Endothelial Large‐conductance Calcium Activated Potassium (BKCa) Channels And Pulmonary Endothelial Function
Authors: Choudhary Gaurav , Vang Alexander , Newton Julie , Rounds Sharon
Published: June 2021

We hypothesized that endothelial BKCa channel activation can improve pulmonary endothelial function by causing change in membrane potential (Em), [Ca2+]i and NO release.

Methods

Lung microvascular endothelial cell (EC) Em, [Ca2+]i and NO change was measured using fluorescence imaging with DiBAC4(3), Fura‐2AM and DAF‐FM, respectively. Phenylephrine preconstricted pulmonary arterial (PA) rings were used to assay the effect of acetylcholine (Ach) on vessel relaxation. PA pressures were monitored in preconstriced isolated ventilated‐perfused rat lungs (IPL) after BKCa channels activation.

Results

BKCa channels were demonstrated in macro‐ and microvascular endothelium by immunohistochemistry in rat lungs and by immunoblot in EC lysates. BKCa channel activation with 30μM NS1619 led to endothelial hyperpolarization and an increase in [Ca2+]i and NO. Also, NS1619 caused enhanced endothelium‐dependent dilation in PA rings, with no effect on endothelium‐independent dilation using NO donor or endothelium‐denuded rings. K+ channel blocker, TEA, abolished the effect of NS1619 on endothelium dependent dilation. In IPL, BKCa channel activation by NS1619 caused a dose‐dependent reduction in PA pressures that was attenuated by the inhibition of NO production.

Conclusion

Pulmonary EC express BKCa channels. Activation of pulmonary endothelial BKCa channels leads to improvement in endothelial function.
Conditional up‐regulation of Receptors for Advanced Glycation End‐Products (RAGE) in Alveolar Epithelium Causes Respiratory Distress and Perinatal Lethality
Authors: Stogsdill Jeffrey A , Phinney Megan N , Reynolds Paul R
Published: June 2021

Receptors for advanced glycation end‐products (RAGE) are multiligand cell‐surface receptors highly expressed in pulmonary tissue during development and likely function in the transition of alveolar type (AT) II to ATI cells required for normal alveolar formation. However, the precise contribution of RAGE during lung organogenesis remains uncertain. We generated a transgenic mouse that over‐expresses RAGE in ATII cells. Dams were continuously fed doxycycline to induce RAGE prior to conception and the resulting pups were removed at embryonic day (E) 18.5 for analysis. Double transgenic mice experienced high mortality and appeared smaller and cyanotic when compared to single or non‐transgenic control littermates. H&E staining revealed large, vacuous areas in the periphery of lungs from double transgenic mice when compared to the normal airways and dilated air spaces characteristic of the saccular period of pulmonary development. Immunohistochemistry employing specific markers for alveolar epithelium, ciliated and non‐ciliated epithelium proved cell‐specific anomalies when RAGE is up‐regulated in ATII cells. These results demonstrate that RAGE must be specifically regulated and that over‐expression in alveolar epithelial cells perturbs epithelial cell differentiation culminating in respiratory distress and perinatal lethality. Supported by a FAMRI and BYU MEG Award (PRR).
Characterization of a novel mouse model for alveolar epithelial type 2 cell regeneration
Authors: Driscoll Barbara , Lee Jooeun , Reddy Raghava
Published: June 2021

A lentiviral vector in which surfactant protein‐C (SP‐C) promoter drives a mutated Herpes Simplex Virus thymidine kinase gene (TKsr39) with enhanced affinity for ganciclovir (GCV) was used to create “SPCTK” transgenic mice on a Rosa R26R background. Descendants from one founder were analyzed for the impact of targeted alveolar epithelial type 2 cell (AEC2) depletion. R26R‐SPCTK mice dosed with 10mg/kg GCV over 7 days become lethargic and exhibit labored breathing. Quantitative immunohistological analysis of lung tissue shows a significant decrease in AEC2 and some decrease in bronchioalveolar stem cells (BASC) by day 7. No notable disruption of Clara cell and AEC1 populations due to bystander effects was observed but a moderate cellular infiltrate appeared in the latter stages of treatment. Compared to GCV‐treated R26R AEC2, GCV‐treated R26R‐SPCTK+/− AEC2 show negligible levels of pro‐SP‐C and minimal mature SP‐C protein present by Western blotting after 7 days GCV treatment. When R26R‐SPCTK mice are treated with 50mg/kg GCV for 2 days, more than 95% of AEC2 are eliminated by day 3 following treatment. By day 5, the number of AEC2 and level of SP‐C expression begins to increase in surviving animals, indicating the de novo appearance of an AEC2 population. Characterization of this regeneration is ongoing.
A Stochastic Model of Breathing Embedded with Fractal‐like Scaling
Authors: BuSha Brett Francis
Published: June 2021

Integration of central drive and respiratory‐related feedback results in the combination of stochastic and temporally‐correlated behavior of breathing. The objective of this study was to design a computational model that replicated the random and fractal‐like pattern of breathing. Breath to breath interval (BBI) was recorded for 256 sequential breaths from 4 male and 4 female subjects while seated motionless on a recumbent bicycle, or pedaling sufficiently to produce a 20% increase in heart rate. Eight model sequences of resting and exercise breathing were created, where stochastic behavior was modeled using a random selection of values from the resting or exercise‐based BBI distributions, and temporal correlations were embedded with a memory function optimized using the autocorrelation of resting or exercise breathing data. The model produced BBI breathing sequences with similar distributions, standard deviations and temporal scaling coefficients during rest and exercise as compared to human‐generated breathing sequences. In conclusion, a model that replicated the normal variability, stochastic randomness, and fractal‐like behavior of human breathing patterns was successfully implemented.
Urethane dose‐dependently inhibits genioglossal long‐term facilitation in un‐paralyzed anesthetized rats
Authors: Cao Ying , Liu Chun , Ling Liming
Published: June 2021

For 3 decades, urethane has been successfully used as an anesthetic in numerous long‐term facilitation (LTF) studies in several species of animals, which were anesthetized, paralyzed, vagotomized and artificially ventilated. However, acute intermittent hypoxia (AIH) failed to induce LTF in a study using urethane‐anesthetized spontaneously breathing rats. But LTF was observed in spontaneously breathing animals when urethane was totally or partly replaced by other anesthetics. The objective of this study was to test whether urethane inhibits AIH‐induced LTF in un‐paralyzed rats under normocapnia, and if yes, whether reducing urethane dose can restore LTF. Three groups of adult male Sprague‐Dawley rats were anesthetized (Group 1: 1.6 mg/kg urethane; Group 2: 50 mg/kg α‐chloralose + 0.9–1.2 mg/kg urethane; Group 3: 0.9 mg/kg urethane + 200–400 μg/kg/min alphaxalone), vagotomized and ventilated. Integrated genioglossal activity was measured before, during and after AIH (5 episodes of 3‐min isocapnic 12% O2 with 3‐min 50% O2 intervals). The AIH‐induced genioglossal LTF was absent in Group 1 rats (success rate was only ~1/7), but the LTF was present in Group 2 (in 8/10 rats) and Group 3 (in 10/10) rats. The hypoxic genioglossal response was not different among the 3 groups. These data demonstrated that urethane inhibits LTF in un‐paralyzed anesthetized rats in a dose‐dependent way. (Supported by NIH HL64912)
Enhanced Phrenic Long‐Term Facilitation (pLTF) Following Repetitive Acute Intermittent Hypoxia
Authors: Vinit Stéphane , MacFarlane Peter M , Satriotomo Irawan , Mitchell Gordon S
Published: June 2021

Two well‐studied models of respiratory plasticity are phrenic and XII long‐term facilitation (LTF) following acute intermittent hypoxia (AIH). Since pretreatment with chronic intermittent hypoxia (72, 5 min episodes/day, 7 days) enhances LTF, we tested the hypothesis that pre‐treatment with a less severe protocol similarly enhances LTF, potentially with fewer adverse side effects. Lewis rats were exposed to repetitive AIH (3xwAIH: 10.5% O2; 10, 5min episodes/day, 3 days per week for 4 weeks) and then anaesthetized and ventilated to enable phrenic and XII nerve recordings. After baseline measurements, rats were exposed to AIH (3, 5 min episodes) to elicit LTF. 3xwAIH enhanced pLTF versus sham, normoxia treated rats (176±42% vs 55±8% baseline 60 min post‐AIH, respectively; p<0.01; n=10 per group). However, XII LTF was unaffected by 3xwAIH (99±27% vs 135±30% baseline, respectively; p>0.05; n=10 per group). Enhanced pLTF is associated with increased expression of proteins necessary for pLTF within phrenic motor neurons (Satriotomo et al., ibid) and is modulated by glycolytic flux (MacFarlane et al., ibid). Repetitive AIH may be a useful therapeutic tool to increase respiratory motor output without adverse side effects in conditions of limited ventilatory capacity, such as cervical spinal injury or neurodegenerative disease. (NIH HL69064, NS057778, HL080209 and Craig H. Neilsen Foundation).
Repetitive Acute Intermittent Hypoxia Increases BDNF and TrkB Expression in Respiratory Motor Neurons: Dose Effects
Authors: Satriotomo Irawan , Vinit Stéphane , Flom Amy L , Mitchell Gordon S
Published: June 2021

Acute intermittent hypoxia (AIH) initiates respiratory plasticity, including phrenic long‐term facilitation (LTF). pLTF requires new BDNF synthesis and TrkB activation. Repetitive AIH enhances pLTF (Vinit et al., ibid) via mechanisms of transcriptional regulation modulated by glycolytic flux (MacFarlane et al., ibid). Distinct models of repetitive AIH elicit pLTF meta‐plasticity, including daily AIH (dAIH: 10 episodes/day, 7 days; 10.5% O2) and thrice weekly AIH (3xwAIH: 10, 5min episodes/day, 3 days per week for 4 or 10 weeks). Here, we tested the hypothesis that these repetitive AIH protocols elicit differential BDNF and TrkB expression in phrenic motor neurons. Semi‐quantitative immunohistochemistry was used to compare BDNF and TrkB expression in presumptive phrenic motor neurons (C4) from rats exposed to dAIH and 3xwAIH for 4 or 10 weeks. BDNF expression was increased by all treatments, although dAIH had less effect vs 3xwAIH; there was no apparent difference between 4 vs 10 weeks 3xwAIH. TrkB expression was also increased, with the larger but equal increases after 4 and 10 weeks of 3xwAIH, and a lesser increase after dAIH. A detailed understanding of repetitive AIH protocols and their relative impact on respiratory plasticity may have important implications in the development of therapeutic strategies for the treatment of clinical disorders with limited ventilatory capacity (NIH NS057778 and HL69064).
Influence of the vagal afferent on supraspinal and spinal respiratory activity following cervical spinal cord injury (SCI) in rats
Authors: Lee Kun‐Ze , Sandhu Milapjit S , Dougherty Brendan J , Reier Paul J , Fuller David D
Published: June 2021

Vagal afferents influence respiratory pattern after SCI, but their influence on spinal and supraspinal respiratory motor output is unclear. We tested the following hypotheses using rats with C2 spinal hemisection (C2HS): 1) vagal afferents attenuate ipsilateral (IL) phrenic bursting, 2) contralateral (CL) phrenic output becomes resistant to vagal inhibition after injury, and 3) vagal inhibition of C.N. XII output is altered following injury. Efferent discharge was recorded from IL and CL phrenic nerves and the CL XII nerve in uninjured rats and C2HS rats at 2 or 8 weeks post‐injury. Vagal inhibition associated with lung inflation was varied by manipulating positive‐end expired pressure (PEEP; 3–9 cmH2O). Bilateral vagotomy greatly enhanced IL phrenic inspiratory bursting which was weak or absent in vagally intact rats at both 2 and 8 weeks post‐C2HS. PEEP‐induced inhibition of CL phrenic and XII activity was attenuated at 2 but not 8 weeks post‐C2HS. We conclude that that 1) vagal afferents have a robust inhibitory effect on eupneic IL phrenic bursting, and 2) compensatory increases in both CL spinal (phrenic) and supraspinal (XII) output after C2HS attenuate the inhibitory influence of vagal afferents. Our vagotomy data appear to explain the variability regarding the onset of the spontaneous crossed phrenic phenomenon in spontaneously breathing (vagal intact) vs. ventilated (vagotomized) preparations.
Hypoxic ventilatory responses of rats and mice after perinatal hyperoxia: does the level of hypoxia matter?
Authors: Bavis Ryan W. , Dmitrieff Elizabeth F. , Young Kristen M. , Piro Samantha E.
Published: June 2021

Developmental hyperoxia attenuates the hypoxic ventilatory response (HVR) of rats into adulthood despite return to normoxia. Since most previous studies have assessed the HVR with a single hypoxic gas mixture (~12% O2), we examined the HVR of hyperoxia‐treated rats to several levels of hypoxia (14, 12 and 10% O2). Rats were exposed to 60% O2 for the first two postnatal weeks and their HVR was measured by whole‐body plethysmography as adults. Ventilatory responses to 14% and 12% O2 were significantly reduced, but ventilatory responses to 10% O2 were normal. Interestingly, the only study on the effects of hyperoxia on HVR in mice, which found no lasting effect, used 10% O2 as the test gas (Dauger et al., Chest 123:530‐8, 2003). Therefore, in a second experiment, mice were exposed to 60% O2 for the first two postnatal weeks and their HVR to 12% O2 and carotid body volumes were measured as adults. As in rats, hyperoxia‐treated mice exhibited reduced HVR and smaller carotid bodies. We conclude that hyperoxia impairs the HVR in both rats and mice, but this effect is most evident at moderate levels of hypoxia. Supported by NIH grant P20 RR‐016463 (Maine INBRE).
Hypothyroidism affects dopaminergic D1 receptor expression and modulation of breathing in hamsters
Authors: Schlenker Evelyn , Amor Mitch , Schultz Harold
Published: June 2021

In hamsters, dopamine D1 receptors stimulate ventilation (MV) during exposure to hypoxia. Hypothyroidism modifies dopaminergic receptor function. We hypothesized that ventilation would be decreased in hypothyroid (H) hamsters and D1 receptor modulation of breathing would be altered in euthyroid (E) compared to H hamsters. Adult male hamsters received tap water (TW) or TW containing 0.4% propylthiouracil for 3 months to induce hypothyroidism Ventilation was evaluated in conscious hamsters exposed to air, 10% O2 in N2 (hypoxia) and 5% CO2 in O2 (hypercapnia). The D1 receptor antagonist SCH23390 (SCH, 0.25mg/Kg) and saline (S) were administered subcutaneously. Following S, tidal volume (VT) and frequency of breathing (F) were significantly lower in the H compared to E animals exposed to air or to hypoxia. During exposure to hypercapnia, VT was significantly lower in the H hamsters. Relative to S, SCH significantly decreased MV and F in E hamsters exposed to air, hypoxia and hypercapnia. In H hamsters, SCH significantly increased MV and VT during air exposure and had no effect on MV during hypoxic or hypercapnic exposures. In hamsters hypothyroidism depresses ventilation and alters D1 receptor modulation of breathing. Moreover, in H hamsters D1 receptor protein expression was significantly decreased in the carotid body and brain areas associated with control of breathing. Funding: USD U.Discover & NIH P20 RR‐015567