bluuebunny/crossref_metadata_2025 huggingface dataset:
Nocturnal asthmatic responses in house dust mite (HDM)‐sensitive Cynomolgus macaques detected by respiratory inductance plethysmography (RIP)
Authors: Wang Xiaojia , Reece Shaun , Olmstead Stephen , Van Scott Michael R. , Wardle Robert L.
Published: June 2021

Rationale

Nocturnal bronchoconstriction is a common symptom of asthma. The goal of this study was to document nocturnal bronchoconstriction in a nonhuman primate model of allergic asthma. RIP parameters were investigated as proxies for changes in dynamic compliance (Cdyn) and lung resistance (RL), and then were used to monitor nocturnal asthmatic responses.

Methods

Animals were anesthetized; instrumented for simultaneous measurements of Cdyn, RL, and RIP parameters; and challenged with aerosolized HDM allergen. Multiple linear regression was used to correlate changes in RIP parameters, Cdyn and RL following HDM challenge. RIP parameters of conscious animals were monitored remotely overnight, plotted against time, and quantified as the area under the curve (AUC).

Results

Effort Phase Relations (ePhRL and eBPRL) correlated with changes in Cdyn and RL: ePhRL = 30.2 ‐ 4.4 Cdyn + 1.7 RL (p = 0.001; p < 0.001)

eBPRL = 26.8 ‐ 3.8 Cdyn + 1.7 RL (p = 0.003; p < 0.001)

In 29 nocturnal trials, 19 subjects exhibited increases in ePhRL and eBPRL between midnight and 5 AM, with peak activity at 4 AM. ePhRL and eBPRL during the nocturnal responses differed from that of non‐responders (p < 0.03).

Conclusion

The results indicate that allergic asthmatic macaques exhibit nocturnal bronchoconstriction. Studies are underway to directly measure RL at the peak of the nocturnal response.
Sarcoplasmic Reticulum Ca2+ Reuptake and Airway Smooth Muscle Inflammation
Authors: Venkatachalem Sathish , Thompson Michael , Bailey Jeffery , Pabelick Christina , Prakash Y S , Sieck Gary C
Published: June 2021

Intracellular Ca2+ ([Ca2+]i) levels are increased in inflamed airway smooth muscle (ASM). Sarcoplasmic reticulum (SR) Ca2+ release and reuptake are key components of ASM [Ca2+]i regulation. Ca2+ reuptake occurs via the ATPase SERCA. We hypothesized that inflammation disrupts SERCA function, contributing to increased [Ca2+]i. We examined SERCA expression and regulation in human ASM exposed to pro‐inflammatory cytokines TNFα and IL‐13. Western blot analysis showed significantly decreased SERCA expression following overnight cytokine exposure. In other tissues, SERCA is regulated by the inhibitory protein phospholamban (PLB). We found that human ASM does not express PLB protein (although mRNA is detectable). Cytokine exposure did not induce PLB expression, raising the issue of how SERCA is regulated. We then found that direct SERCA phosphorylation (potentially via CaMKII) occurs in human ASM. In fura‐2 loaded human ASM cells, we examined [Ca2+]i responses to ACh or bradykinin (zero extracellular Ca2+) and found significant reduction in rates of fall of [Ca2+]i transients in cells exposed to TNF‐α or IL‐13. We further found that CaMKII antagonist KN‐93 significantly slowed the fall of [Ca2+]i transients in both controls and cytokine‐exposed human ASM cells. These data suggest that a) SERCA in human ASM is regulated by mechanisms such as CaMKII; and b) airway inflammation decreases SR refilling by blunting SERCA expression and function.

Supported by NIH grants HL74309, the Flight Attendants Medical Research Institute (FAMRI), and the Department of Anesthesiology, Mayo Clinic.
Role of Interleukin‐4 on Agonist Induced Airway Contraction in Mouse Lung Slices
Authors: Mukherjee Seema , Perez‐Zoghbi Jose F
Published: June 2021

Asthma is a chronic disorder of the airways characterized by inflammation and airway hyper responsiveness (AHR). Interleukin‐4 (IL‐4) is released by T‐cells and mast cells in the airway and is believe to have an important role in asthma. We hypothesize that IL‐4 has a direct effect on the airway smooth muscle cells (SMCs) by increasing their contractile response to agonist such as acetylcholine (ACh). The effect of IL‐4 on airway contraction was studied in mouse lung slices with phase‐contrast microscopy. We found that the treatment of lung slices with 100 ng/ml of IL‐4 for 12 to 24 hours resulted in an increase in contraction of the airways induced by ACh as compared with the same response measured in slices treated without IL‐4 (control). However, IL‐4 treatment did not alter the sensitivity of airways to agonists as determined by the absence of a change in the concentration of ACh that produced 50% of the maximal contraction. These results suggest that IL‐4 contributes to asthma pathology by increasing airway SMC contractility. We are investigating the mechanisms underlying the increase in airway contraction induced by IL‐4 by exploring possible changes in Ca2+ signaling and other Ca2+ independent mechanisms. This study will provide useful information to develop new specific therapeutics to treat asthma.
Heart‐Specific Inducible Inactivation of ClC‐3 Gene Eliminates Native Volume‐Sensitive Outwardly Rectifying Anion Currents (VSOACs) and Results in Cardiac Hypertrophy and Compromised Heart Function
Authors: Xiong Dazhi , Heyman Nathanael , Airey Judith , Ye Linda , Singer Cherie , Rawat Shanti , Zhang Mi , Evans Rebecca , Burkin Dean , Britton Fiona , Duan Dayue , Hume Joseph
Published: June 2021

To test the hypothesis that chloride channel ClC‐3 short isoform (sClC‐3) comprises a key component of native volume‐sensitive outwardly rectifying anion channels (VSOACs) in mammalian heart and assess its physiological role in cardiac function, we generated cardiac‐specific inducible ClC‐3 knockout (KO) mice, by breeding ClC‐3c/c mice with tetO‐Cre and á‐MHC(á‐myosin heavy chain)‐tTA animals. These transgenic mice were maintained on a doxycycline diet to preserve ClC‐3 expression in the heart. Removal of doxycycline activates Cre recombinase to inactivate the ClC‐3 gene. Examination of VSOACs in isolated KO atrial myocytes 3 weeks off doxycycline revealed a complete elimination of the currents, whereas at 1.5 weeks, the VSOACs were significantly reduced, compared to age‐matched control animals. RT‐PCR from KO atrial myocytes confirmed loss of ClC‐3. Echocardiography revealed dramatically reduced ejection fraction and fractional shortening, and severe signs of myocardial hypertrophy and heart failure in the KO mice at the same time point. The results indicate that sClC‐3 is a key component of native VSOACs and plays a significant cardioprotective role against cardiac hypertrophy and failure.(NIH grants HL‐49254, HL‐063914, and NCRR P20RR1581)
Exercise‐induced angiogenesis: When Murine Double Minute‐2 and Vascular Endothelial Growth factor run together for more capillaries
Authors: ROUDIER Emilie , BIROT Olivier
Published: June 2021

Running exercise has been described as a physiological stimulus for skeletal muscle angiogenesis. Recent data, mostly from tumor cells studies, have evidenced that the Murine Double Minute‐2 (Mdm2) oncoprotein, the main negative regulator of p53, exerts itself some angiogenic effects, mainly by stimulating endothelial cells migration.

Objectives

We investigated whether exercise regulated Mdm2 and whether Mdm2 could thus represent a new actor of exercise‐induced muscle angiogenesis.

Methods

We developed an innovative model of ex vivo muscle explant culture allowing the time‐course study of muscle angiogenesis. Plantaris muscles from sedentary or active (70‐90 min exercise on a running treadmill, 12% slope, 25 m/min) Sprague‐Dawley rats were used for protein measurements and our ex vivo angiogenesis assay.

Results

We showed that 1) Mdm2 protein was strongly activated by phosphorylation on Ser166 in response to exercise, 2) exercise stimulated endothelial cells migration in our ex vivo muscle explant model, 3) such exercise‐induced angiogenic effect is dependant on Mdm2 activation, 4) Mdm2 activation is mediated by the Vascular Endothelial Growth Factor (VEGF) ‐ Extracellular signal‐regulated kinase 1/2 (ERK1/2) pathway.

Conclusion

Mdm2 is a new key actor of exercise‐induced angiogenesis in rat skeletal muscle. This work was supported by the Natural Sciences and Engineering Research Council of Canada.
VEGF165b inhibits choroidal neovascularization (CNV) in mouse model via intra‐vitreous and subcutaneous delivery
Authors: Hua Jing , Spee Christine , Barron Ernesto , Kase Satoru , Churchill Amanda J , Harper Steven J , Ryan Stephen J , Bates David O , Hinton David R
Published: June 2021

Methods

Four CNV lesions were induced by diode laser in each eye of C57B/L6J mice. Different doses of recombinant VEGF165b or solvent were injected into vitreous (0.01, 0.1, 1, 10ng/eye weekly) or subcutaneously (100µg, twice a week). Two weeks later, fluorescein angiography (FA) was carried out before animals were sacrificed and eyes dissected for sclero‐choroid whole mount or embedded for cross‐sections and samples were assessed by isolectin B4 and H&E staining under confocal and light microscopy.

Results

Intra‐vitreous injection of VEGF165b significantly reduced leakage of lesions in FA (p<0.001 ANOVA, n=5), 3D volume (p<0.0001, ANOVA, n=24, control 5.7, with increasing dose: 1.5, 1.4, 1.2, and 1.5x105 µm3), 2D surface (p<0.0001 ANOVA, n=24, control 6.0, with increasing dose 1.9, 2.1, 1.8, 2.4x105 µm2) and cross‐section surface (p<0.0001 ANOVA, n=5‐10, control 2.9, with increasing dose 1.8, 1.1, 0.9, 1.0x105 µm2). Subcutaneous injection of VEGF165b significantly reduced the leakage (p=0.02 t‐test, n=20), 3D volume (p<0.0001 t‐test, n=25) and 2D surface (p=0.0027, t‐test, n=25) of the CNV lesions.

Conclusion

VEGF165b significantly inhibit the growth of CNV in a mouse model, by either intraocular or systemic application. Considering its reported epithelial and neuroprotective effects, VEGF165b maybe a favorable candidate for choroidal neovascular diseases.
Viral delivery of vascular endothelial growth factor (VEGF) rescues angiogenic imbalance in the BPH/5 mouse model of pre‐eclampsia (PE)
Authors: Butler Scott D , Woods Ashley K , Hoffmann Darren S , Sharma Ram V , Davisson Robin L
Published: June 2021

Pregnant BPH/5 mice exhibit an angiogenic imbalance profile that mirrors women at high risk of developing PE, including diminished levels of circulating VEGF. Serum from pregnant BPH/5 mice (e12.5) shows markedly reduced angiogenic potential in an in vitro angiogenesis assay compared to serum from C57 controls (BPH/5 0.8±0.1 vs. C57 1.3±0.04, normalized to FBS, n= 4‐5, p<.05). Here we hypothesized that by increasing circulating levels of VEGF, we could rescue the angiogenic imbalance in this model. At e7.5, pregnant BPH/5 and C57 were injected via tail vein with an adenovirus encoding VEGF121 or LacZ. VEGF levels were verified using ELISA. A functional angiogenic assay was performed to assess the effect of serum from these mice on tube formation using HUVEC cultures. As expected, serum from LacZ‐treated BPH/5 pregnant mice (e12.5) induced significantly less tube formation than C57 (BPH/5 LacZ 0.9±.01 vs. C57 LacZ 1.3± 0.1, normalized to FBS, p<.05). Importantly, serum from Ad‐VEGF‐treated BPH/5 restored the angiogenic potential to the same levels as C57 (1.3±0.1 normalized to FBS, P<0.05 vs. BPH/5Lacz). Interestingly, Ad‐VEGF therapy had no effect on the angiogenic potential of serum from pregnant C57 mice, indicating an optimal level of circulating angiogenic factors in normal pregnancy. These results highlight the potential for VEGF therapy in high risk pregnancies complicated with angiogenic deficits.
CD31+ T Cells Represent a Functionally Distinct Vascular T Cell Phenotype
Authors: Kushner Erich Joseph , Morgan Richard G. , Van Engelenburg Alexander M. , MacEneaney Owen J. , Van Guilder Gary P. , DeSouza Christopher A.
Published: June 2021

CD3+/CD31+ T cells aid in endothelial repair and revascularization. In contrast, CD3+/CD31‐ cells show no vascular effects. There is limited data regarding the functional differences between circulating CD3+/CD31+ and CD3+/CD31‐ cells that may contribute to their divergent cardiovascular effects. The aim of the present study was to characterize functional differences between CD3+/CD31+ and CD3+/CD31‐ cells. To address this aim, migratory capacity, proangiogenic cytokine release and apoptotic tendency of CD31+ and CD31‐ T cells were determined. Human CD31+ and CD31‐ T cells from peripheral blood were isolated using magnetic‐activated cell sorting. CD31+ T cells demonstrated higher (~60%, P<0.01) migratory capacity to the chemokines VEGF (618±99 vs 259±57) and SDF‐1 (655±99 vs 273±54) vs CD31‐ cells. Release of angiogenic cytokines VEGF, interleukin‐8, and matrix metalloprotease‐9 were also ~100% higher (P<0.05) in CD31+ T cells. CD31+ cells exhibited significantly higher intracellular concentrations of active caspase‐3 (2.61±0.60 vs 0.34±0.09 ng/ml) and cytochrome‐c (21.8± 1.4 vs 13.7±0.96 ng/mL). In summary, CD31+ T cells have greater migratory and angiogenic cytokine release capacity but are more susceptible to apoptosis compared with CD31‐ T cells. Enhanced migratory capacity and angiogenic cytokine release may contribute to the vasculogenic properties of this unique T cell subpopulation.
VEGF mediates age‐dependent remodeling of ovine carotids by chronic hypoxia
Authors: Butler Stacy Michelle , Abrassart Jenna Marie , Williams James Michael , Pearce William Julian
Published: June 2021

Chronic hypoxia alters arterial structure and contractility through poorly defined mechanisms. Chronic hypoxia also increases expression of the transcription factor HIF that promotes expression of VEGF. This study explores the hypothesis that hypoxic increases in VEGF exert a direct effect on vascular smooth muscle that modulates artery structure and contractility. Carotid arteries from normoxic term fetal (FN), hypoxic (110 days @ 3820 m altitude) term fetal (FH), and hypoxic non‐pregnant adult sheep (SH), were denuded of endothelium and placed in organ culture. After 24 hr serum starvation, segments were treated another 24 hr with vehicle or 30 ng/ml VEGF. Next, stress‐strain relations were determined to functionally define responses to treatment. After VEGF, maximum contractile responses to myogenic stretch and 120 mM K+ were increased in SH, were largely unaffected in FH, and were depressed in FN. Similarly, after VEGF optimal length was increased in SH, was unchanged in FH, and was decreased in FN; the L0 results suggest group specific remodeling of the extracellular matrix. H&E staining confirmed that changes in contractility and L0 were not attributable to cell death. These results support the hypothesis that direct effects of VEGF on vascular smooth muscle contribute to the age‐dependent arterial remodeling associated with chronic hypoxia. Supported by USPHS Grants HL54120, HD31266, & HL64867.
Platelet neuropeptide Y (NPY) and the collateral and capillary networks in skeletal muscle ischemia
Authors: Tilan Jason Ulip , Abe Ken , Chalothorn Dan , Faber James E. , Zukowska Zofia
Published: June 2021

Previously, we showed that sympathetically‐derived or exogenous NPY promotes ischemic revascularization after femoral artery ligation (FAL) in rodents by activating Y2 receptors and dipeptidyl peptidase IV (DPPIV), which forms a Y2/Y5‐preferring agonist. FAL also induced early up‐regulation of Y2 and DPPIV mRNA in adductor (collateral zone, CZ) and gastrocnemius (capillary angiogenesis zone, AZ) muscles and elevated plasma NPY levels, followed by increased platelet NPY levels at 7 and 14 days post‐ligation. The latter findings suggested platelet NPY may play a role in angiogenesis/collateralization after FAL, which is evaluated in this study. In 129/SvlmJ/NPY+/+ mice, FAL induced an increase in capillary density (CD31+ vessels) in the AZ by 14 days (vs. sham), which in NPY‐/‐ remained unchanged (no difference in basal capillary density between NPY+/+ and NPY‐/‐ mice.). Transfer of NPY‐/‐ platelets into platelet‐depleted (Busulfan 25 mg/kg, i.p.) NPY+/+ mice attenuated the angiogenic response in the AZ, while NPY+/+ platelet transfer into NPY‐/‐ mice significantly increased the density of CD31+ vessels in this region. In a separate group of mice, adductor and plantar perfusion were decreased in NPY‐/‐ mice after FAL. These data suggest that NPY's effect on both the collateral and capillary networks promote ischemic revascularization and is, at least in part, mediated by platelet NPY.
Role of TLR2 in lung angiogenesis
Authors: Moldobaeva Aigul , Nijmeh Julie , Jenkins John , Wagner Elizabeth
Published: June 2021

After chronic left pulmonary artery ligation (LPAL) in mice, there is rapid neovascularization of the ischemic lung. We showed increased macrophage‐derived growth factors and a positive correlation between the number of lavaged macrophages and the magnitude of angiogenesis. In the current study, we questioned whether macrophage subpopulations with specific surface markers contributed differentially to angiogenesis. Using flow cytometry, we showed an increase in total macrophages based on forward/side scatter characteristics over the first 48 hrs in left lung homogenates after LPAL (n=4). Specifically, we saw a 70% increase in the number of Cd11b+/CD11c‐ macrophages 48 hrs after LPAL. With no inflow to the lung early after LPAL, changes in cell numbers represent proliferation and/or differentiation. Within this same cell population, toll‐like receptor 2 (TLR‐2) expression ranged from 80‐90%. To confirm that TLR‐2 plays a role in angiogenesis, we measured neovascularization 14 days after LPAL in TLR‐2 deficient mice. Angiogenic outcome (microspheres) decreased by 48% of the wild type response (n=4). These data suggest that TLR‐2 plays a role in macrophage activation, important for angiogenesis.
Aging blunts NFκB activation and HSP expression in response to estrogen in isolated female rat cardiomyocytes
Authors: Stice James Patrick , Knowlton Anne A
Published: June 2021

Objective

We investigated the role of aging vs. estrogen loss on the cardioprotective heat shock protein (HSP) response in a rat model. We hypothesized that aging itself would lead to a blunted HSP response, while 17β‐estradiol (E2) replacement in aging rats would confer protection against cellular injury through restoration of the HSP response.

Methods

Cardiomyocytes were isolated from ovariectomized adult (5 mos.) and aged (22 mos.) rats with and without 9 weeks of E2 replacement forming four groups: adult ovx, adult op, aged ovx, and aged op. Myocytes were treated in culture with E2 (5nM and 100nM) and analyzed for NFκB activation at 15 minutes and HSP expression at 12 h. Some cells were subjected to 6 hours of hypoxic injury followed by 24 hours of reoxygenation (H/R).

Results

100nM E2 activated NFκB and increased HSP 72 and 90 expression in adult ovx and adult op (p<0.05), while both aged ovx and aged op show no changes in response to E2. Levels of HSP 27, heme oxygenase‐1, and estrogen receptor (ER) did not did not differ among groups or after E2 treatment. All groups had increased LDH release in response to H/R injury; however, aged ovx had higher LDH release before and after H/R compared to all other groups.

Conclusions

Aging leads to a loss of NFκB activation and HSP expression in response to E2 and this is not due to a change in ER expression.

Supported by the American Heart Association Western States Affiliate (JPS), the NIA AG019327 (AAK), and the Department of Veterans Affairs (AAK)
MAS‐Knockout Mice Produces Altered Metabolic Responses in Adipocytes
Authors: Mario Érica Guilhen , Menezes Zélia , Ferreira Adaliene , Santos Sérgio Henrique S. , Pôrto Laura Cristina J. , Santos Robson A.S. , Botion Leida M.
Published: June 2021

This study investigated the adipocyte metabolism in wild type (WT) and FVB/N Mas‐deficient male mice (KO). Plasma triglyceride (TG) and free fatty acid (FFA) were assayed by enzymatic methods. Epididymal adipocytes were incubated in the presence or absence of isoproterenol and the effects of insulin on isoproterenol‐stimulated lipolysis were determined. Quantification of hormone‐sensitive lipase (HSL) protein was measured by Western blotting. Epididymal adipocytes were incubated in the presence or absence of insulin and uptake of 2‐deoxy‐[3H] glucose was estimated. Gene expression of PPARγ was measured by real time RT‐PCR. KO mice showed greater levels of plasma TG and FFA compared to WT mice (p<0.05). The lipolitic activity as basal as induced by isoproterenol was the same in both groups. The insulin decreased 41% the lipolitic activities stimulated by isoproterenol in a WT group (p<0.05), while no effect of the insulin action was observed in KO group. The amount of HSL enzyme was similar in WT and KO groups. The glucose uptake in the basal condition was the same in both groups. However, adipocytes of KO group showed a decreasing of 39% of glucose uptake stimulated by insulin compared to adipocytes of WT group (p<0.05). The expression of PPARγ is reduced in 66% in KO animals (p<0.05). Our data suggested that the lack of Ang‐(1‐7) action through Mas receptor alters the response of adipocytes of insulin action.
Increase in cardiac levels of Angiotensin‐(1‐7) attenuates cardiac collagen deposition and dysfunction in DOCA‐Salt hypertensive rats
Authors: Santiago Nivia M , Guimarães Priscila S , Sirvente Raquel , Irigoyen Maria Cláudia , Santos Robson AS , Campagnole‐Santos Maria J
Published: June 2021

We have evaluated the cardiac function and remodeling after DOCA‐Salt hypertension (DOCA) in transgenic rats that overproduce circulating Ang‐(1‐7), TGR(A1‐7)3292 (TG). TG and Sprague‐Dawley (SD) rats were submitted to DOCA treatment. After 30 days, the systolic and diastolic ventricular function by echocardiography, collagen type I and III mRNA expression in left ventricle (LV) and angiotensins levels in LV and plasma were evaluated. DOCA‐SD presented left ventricle hypertrophy and an increase in the ejection fraction and fractional shortening with signs of diastolic dysfunction observed by a lower aorta/ left atrium ratio and an increase in isovolumetric relaxation time. DOCA‐TG did not develop cardiac hypertrophy or changes in the ventricular function. The preserved cardiac function in TG was accompanied by attenuation in mRNA expression for collagen I and III (1.9 ± 0.5 AU and 1.0 ± 0.3 AU, respectively) as compared to the increased levels of DOCA‐SD (3.3 ± 0.7 AU and 1.4 ± 0.2 AU, respectively). Ang II plasma levels were reduced in SD and TG after hypertension (p<0.05). DOCA in TG induced a reduction in cardiac Ang II and a large increase in Ang‐(1‐7) in the LV (4.2 ± 0.8 vs 1.5 ± 0.1 pg/ mg protein, in Sham‐TG; p<0.05). In conclusion, life‐time increase in Ang‐(1‐7) levels attenuates cardiac collagen deposition and dysfunction after DOCA‐Salt treatment probably due to an increase in cardiac levels of Ang‐(1‐7).
Deiodinase Expression in a Rodent Model of Myocardial Infarction
Authors: McElligott Andrew , Paul Jennifer T , Chu Miensheng , Samarel Allen M , Danzi Sara , Klein Irwin , Henderson Kyle K.
Published: June 2021

Serum triiodothyronine (T3) concentration is reduced in patients with congestive heart failure which may contribute to altered T3 dependent gene expression and cell signaling. This effect could be due to an increase in type III deiodinase (D3) activity. D3 is a fetal enzyme that inactivates thyroxine as well as T3. We hypothesized that myocardial D3 expression would increase in a rodent model of myocardial infarction (MI) and attenuate activation of serine/threonine‐specific protein kinases (Akt) and endothelial nitric oxide synthase (eNOS). Echocardiography was used to assess left ventricular (LV) structure and function in male Sprague Dawley rats before and after coronary artery ligation. One week post‐MI, D3 expression was significantly increased while serum T3 and T4 were attenuated, (90.1±5.7 vs. 74.4±2.2ng/dL) and (4.54±0.15 vs. 4.17±0.10ug/dL), respectively. Additionally, Akt and eNOS activation were reduced as early as 3‐days post‐MI. By 7wks, serum T3 and T4 concentrations were no longer significantly different; however, LV systolic and diastolic volumes progressively increased over the 9wk study. Importantly, D3 expression remained elevated at 9wks. These findings support the hypothesis that myocardial D3 expression is increased and downstream targets of T3 may be attenuated after a myocardial infarction. Funding: Ralph and Marian Falk Medical Research Trust and King Pharmaceuticals.
The genomic action of 17 β‐estradiol accounts for altering calcium homeostasis in human endothelial cells
Authors: Thor Der , Rahimian Roshanak
Published: June 2021

Our preliminary studies showed 17 β‐estradiol (E2)‐regulated Ca2+ homeostasis may play a role in enhancing nitric oxide production in human endothelial cells, EA.hy926. Here, we investigated the genomic and nongenomic actions of E2 on intracellular calcium concentration ([Ca2+]i) in endothelial cells. Cells were treated with either a) E2 (1 μM) in the presence or absence of the transcription inhibitor actinomycin D (1 μg/mL) for 24 h or b) E2 (1 μM) for 5 minutes prior to the analysis of [Ca2+]i. Using a spectrofluorometer, [Ca2+]i from cells loaded with the Ca2+‐sensitive dye, Fura 2‐AM, was monitored in the absence of extracellular Ca2+. The sarco/endoplasmic reticulum Ca2+ ATPase inhibitor, thapsigargin (TG, 1 μM), was used to induce Ca2+ release from the endoplasmic reticulum. TG‐induced [Ca2+]i increase from cells treated with actinomycin D plus E2 was not significantly different from cells treated with vehicle (ethanol). However, the extent of TG‐induced [Ca2+]i increase was significantly lower in vehicle‐ or actinomycin D‐ plus E2‐treated cells as compared with that observed in E2‐treated cells, indicating that gene transcription is required for E2‐mediated alteration in Ca2+ response. Five minutes of E2 treatment did not alter TG‐induced [Ca2+]i increase as compared to vehicle‐treated cells. Our results suggest that E2 regulates Ca2+ homeostasis in EA.hy926 via a genomic mechanism. Supported by NHLBI.
Acute vascular effect of aldosterone on mesenteric artery from normal and heart failure rats
Authors: Ru Xiaochen , Qian Lingbo , Cui Jie , Li Yanfang , Xia Qiang
Published: June 2021

Numerous evidences have emerged in resent years to support the direct role of aldosterone in heart failure. This study was designed to test our hypothesis that, the vascular response to aldosterone alters in a situation of heart failure. Acute heart failure of rat was induced by coronary artery ligation for 30 min. Segments of third‐order branches of the mesenteric artery were isolated for isometric tension recording. We found that in normal rats, aldosterone (10−9 M ‐ 10−6 M) caused further contraction in mesenteric artery precontracted by phenylephrine (PE, 10−6 mol/L), but aldosterone did not cause vasocontraction in the artery from heart failure rats. In the mesenteric artery from normal rats, pre‐incubation of aldosterone (3×10−7 M) for 10 min decreased the contractile response to low concentration of PE (1×10−7 ‐ 1×10−6 M), but enhanced the contractile response to high concentration of PE (3×10−6 ‐ 3×10−5 M). This effect was abolished by eplerenone (2×10−6 M), an inhibitor of aldosterone receptor. However, in the artery from heart failure rats, aldosterone (3×10−7 M) decreased the contraction induced by PE (1×10−7 ‐ 3×10−5 M), which was partly blocked by eplerenone. These results indicate that aldosterone has biphasic effect on contractile response to PE of normal rat artery, mediated by aldosterone receptor. The effect of aldosterone on heart failure rat artery is monophasic, reducing the sensitivity to PE, which is partly mediated by aldosterone receptor.
ASIC2 protein is required for pressure‐induced constriction in mouse renal interlobar artery
Authors: Gannon Kim Parker , Chiposi Rumbidzayi , McKey Elizabeth , Drummond Heather
Published: June 2021

Pressure‐induced, or myogenic, constriction is a mechanically mediated event. Increased luminal pressure stretches vascular walls leading to the opening of presumptive mechanosensitive channels, and initiating the cascade for vascular contraction. Epithelial Na+ Channel and Acid Sensing Ion Channel 2 (ASIC2) proteins may form mechanosensitive channels. Myogenic constriction may protect against pressure‐induced renal injury. ASIC2 heterozygous (+/‐) mice have no cerebral myogenic response; however renal myogenic response and susceptibility to renal injury remains unknown. The current goal was to investigate the role of ASIC2 in renal interlobar arteries and renal injury. We examined agonist‐ and pressure‐induced constriction in isolated renal interlobar arteries from ASIC2 wildtype (+/+) and +/‐ mice. Constriction to KCl (20 ‐ 80 mM) and phenylephrine (10−7 ‐ 10−4 M) was not different. Constrictor responses to intraluminal pressure (25 ‐ 150 mmHg) were significantly impaired in ASIC2 +/‐ mice (31.6 + 3.7 and 2.4 + 1.2% myogenic tone at 150 mmHg in +/+ and +/‐ ASIC2 mice). Histological analysis demonstrated tubular casts and a 5‐fold increase in renal macrophage infiltration suggestive of renal injury in ASIC2 +/‐ mice. These results indicate normal ASIC2 is required for myogenic constriction in the renal interlobar artery and may protect against pressure‐induced injury. Supported by NIH‐NHLBI & NINDS.
Hypochlorous acid‐induced heme oxygenase‐1 gene expression promotes human endothelial cell survival
Authors: Liu Xiao‐ming , Wei Yong , Peyton Kelly J. , Wang Hong , Johnson Fruzsina K. , Johnson Robert A. , Durante William
Published: June 2021

Hypochlorous acid (HOCl) is a unique oxidant generated by the enzyme myeloperoxidase that contributes to endothelial cell (EC) dysfunction and death in atherosclerosis. Since myeloperoxidase localizes with heme oxygenase‐1 (HO‐1) in and around ECs of atherosclerotic lesions, the present study investigated whether there was an interaction between these two enzymes in vascular endothelium. Treatment of human ECs with the myeloperoxidase product HOCl stimulated a concentration‐ and time‐dependent increase in HO‐1 mRNA, protein, and promoter activity, and carbon monoxide synthesis that was preceded by a marked rise in Nrf2 protein. The HOCl‐mediated increase in HO‐1 promoter activity was blocked by mutating the antioxidant response element in the promoter or by overexpressing a dominant‐negative mutant of Nrf2. The induction of Nrf2 or HO‐1 by HOCl was prevented by N‐acetyl‐L‐cysteine. Finally, treatment of ECs with HOCl stimulated cell death that was potentiated by the HO inhibitor tin protoporphyrin‐IX or by the selective knockdown of HO‐1. These results demonstrate that HOCl induces HO‐1 gene transcription via the redox‐dependent activation of Nrf2 to counteract HOCl‐mediated toxicity. The ability of HOCl to activate Nrf2 and stimulate HO‐1 gene expression may represent a critical adaptive response to maintain EC viability at sites of vascular inflammation and atherosclerosis. Supported by NIH and AHA.
Rat thoracic aorta and mesenteric artery show a multiphasic contraction‐relaxation‐contraction response to sodium sulfide and sodium hydrosulfide
Authors: Predmore Benjamin Lee , Whidden Melissa A. , Erdos Benedek , Tumer Nihal , Julian David
Published: June 2021

Hydrogen sulfide (H2S) is now a recognized gas signaling molecule in the cardiovascular system, joining nitric oxide and carbon monoxide. H2S has been shown to cause relaxation in rat thoracic aorta (TA) and mesenteric artery (MA), as well as multiphasic responses in pulmonary artery and other vertebrate vessels. While relaxation occurs shortly after H2S addition, multiphasic responses require more time to observe. Therefore in this study, we hypothesized that TA and MA would show a multiphasic response to H2S if time were allowed for completion. TA and MA were dissected from Fisher 344 x Brown Norway rats. TA was sectioned into rings and run on a wire myograph, and MA was run on a pressure arteriograph. H2S was added via the donors sodium sulfide (Na2S) or sodium hydrosulfide (NaHS) after preconstriction with phenylephrine. Both TA and MA showed a triphasic contraction‐relaxation‐contraction response using either H2S donor. The first contraction was quick and small in magnitude. A sustained relaxation followed, after which a second sustained contraction much larger in magnitude than the first occurred. Overall the triphasic response was complete in 15‐30 min. These data show that H2S signaling in mammalian vessels is more complex than a monophasic relaxation, regardless of the H2S donor, and therefore the entire mulitphasic response to H2S should be considered when investigating potential H2S signaling mechanisms.
Regulation of cardiac NO bioactivity in response to low salt diet: A time course study on the role of NADPH oxidase.
Authors: Skayian Yester , He Arthur Yu , Ojaimi Caroline , Hintze Thomas H
Published: June 2021

A time dependent study of low salt diet (LSD) was conducted on wild type (WT) male mice to assess its effects on ANG II, cardiac MVO2 and NO bioactivity. WT mice on a LSD for 2 weeks showed an increase in plasma ANG II levels (207 ± 46 to 502 ± 65 pg/ml (n=6), P<0.005) with no significant increase in blood pressure. Microarray analysis revealed a large number of significantly expressed genes in LSD mice, 153 genes up‐regulated and 399 genes down‐regulated (P<0.05). Genes related to NOS, ANG II, AT1 receptor, and NADPH oxidase did not show any significant change between LSD and normal diet (ND) fed mice. Accordingly, mice were put on LSD for 8 weeks. On ND, stimulation of NO production by bradykinin (BK) induced a concentration dependent reduction in MVO2 (‐18 ± 1%) in vitro. BK‐induced reduction in MVO2 was significantly attenuated in WT mice (‐12 ± 1%, P<0.0001) on LSD. The attenuated BK‐induced reduction in MVO2 was restored by tiron (‐22 ± 2%), apocynin (‐25 ± 3%), or by losartan (‐18 ± 2%), on LSD. Similar effects were seen in WT mice on LSD for 2 weeks. Blood pressure was also significantly increased after 8 weeks on LSD (127±1/73±2 to 146±4/90±5 (n=11), P<0.0001). These findings suggest that increased plasma ANG II levels with LSD reduce NO bioavailability and alter the control of cardiac O2 consumption by NO. The assembly and activation of NADPH oxidase by ANG II may be the basis of low salt induced cardiac dysfunction. Supported by HL 50142.
The Effects of IgG Anti D, IgG Anti D Coated Red Blood Cells and Lysed Red Blood Cells on Macrophage Viability, Morphology and Function
Authors: Ellis April , Hickman Tyrah , Hullitt JoAnna , Tucci Michelle , Benghuzzi Ham , Cameron Joseph A.
Published: June 2021

Immunoglobulin G (IgG) deposition at tissue sites leads to macrophage accumulation and organ injury, although the mechanism for injury is not known. The goal of this study was to investigate the interaction of IgG anti D alone, IgG anti D coated red blood cells (RBCs), or lysed RBCs with macrophage cells over time. Cell viability, cellular glutathione membrane damage, morphology, and nitric oxide (NO) were evaluated. Cell numbers were elevated in the lysed red blood cell treatment for the duration of the experiment. Cellular glutathione concentrations were similar to control values for all treated groups compared to the control for the first 48 hours. Cells exposed to IgG anti D coated RBCs showed the lowest glutathione values after 72 hours. Cellular damage was evident for the duration of the study in treated groups compared to control. NO was evident in IgG anti D and IgG anti D coated RBCs for the duration of the study, whereas lysed red blood cells did not increase NO. Overall, the biochemical and cellular staining results possibly indicate that NO is a receptor mediated event and phagocytosis may cause increased malondialdehyde (MDA) levels. Evaluating the interaction of macrophages with IgG may advance our understanding of macrophage involvement in organ injury due to the deposition of IgG. Supported in part by NIGMS‐NIH Grant R25 GM50117.
Identification and Functional Characterization of Phosphorylation Sites on GTP cyclohydrolase I
Authors: Du Jianhai , Wei Na , Xu Hao , Vasquez‐Vivar Jeannette , Pritchard Kirkwood A , Shi Yang
Published: June 2021

GTP cyclohydrolase I (GCH‐1) is the rate‐limiting enzyme for the biosynthesis of tetrahydrobiopterin (BH4), an essential cofactor for nitric oxide synthases (NOS) and aromatic amino acid hydrolases. In this study, we aim to identify specific phosphorylation sites on GCH‐1 and characterize their function. Rat GCH‐1 with FLAG tag was subcloned into a Tet‐On plasmid which was stably expressed in Flp‐in 293 cells. Mass spectrometry studies revealed GCH‐1 was phosphorylated in vivo at serine(S) 167 and threonine (T) 231. Sequence analysis of GCH‐1 protein showed 8 potential phosphorylation sites [S51, S72, T85, T91, T103, S130, S167 and T231]. Functional studies demonstrated that GCH‐1 activity and BH4 levels were significantly decreased in cells expressing phospho‐defective alanine (A) mutants S72 A, T85A, T91A, T103A or S130A while they were increased in cells expressing the T231A mutant. Furthermore, GCH1 activity, and BH4 levels were increased in cells expressing the phospho‐mimic glutamic acid (E)/aspartic acid (D) mutants S72E, T85E, T91E, T103D or T130D, while they were decreased in cells expressing the T231D mutant. Interestingly, cells expressing the S51E, but not S51A had increased cellular BH4 production, while cells expressing S167A or S167E mutant did not affect BH4 synthesis. In summary, our study suggests that GCH‐1 is positively and negatively regulated by multiple phosphorylation sites.
Adaptation to hypoxia increases NO stores: A defense for cerebral blood vessels against NO overproduction in experimental Alzheimer's disease
Authors: Manukhina Eugenia B. , Goryacheva Anna , Mashina Svetlana , Alexandrin Valery , Downey H. Fred
Published: June 2021

Injury of cerebral blood vessels (CBV) and neurons in Alzheimer's disease (AD) partially results from nitric oxide (NO) overproduction in microglia and astrocytes. Formation of NO stores protects CBV against NO toxicity by binding excessive NO. We have shown that prior adaptation to intermittent hypoxia (AH; simulated altitude 4,000 m; 4 h daily, 14 days) reduced memory loss and neurodegeneration in rats with experimental AD (EAD), and we proposed that enhancing protective mechanisms against NO overproduction might contribute to the beneficial effect of AH. Here EAD was modeled in rats by a bilateral injection of beta‐amyloid peptide (Ab) fragment (25‐35) into n. basalis magnocellularis. NO production was assessed in brain tissue by measuring NO2+NO3. NO stores were detected by increases in Doppler‐measured cerebral blood flow (CBF) in response to N‐acetylcysteine (NAC) which releases NO from NO stores. Ab significantly increased NO production in rat brain (25±2.0 vs 40±3.3 μmol/g tissue, respectively). AH prevented NO overproduction in Ab‐treated rats (19±1.6 μmol/g tissue). In rats with EAD, NAC increased CBF by 18.4±6.2%, which reflected formation of NO stores; NO stores were absent in CBV from control rats. Size of NO stores in adapted rats with EAD was significantly larger (31±3.5%) than in nonadapted rats with EAD. Thus, AH protection against Ab‐induced NO overproduction involves two mechanisms: restricting excessive NO synthesis and expanding NO‐storing capacity of CBV. (Supported by RFBR grant 07‐04‐00650)
The use of MP4 file format for the delivery of educational resource material to students in the health sciences.
Authors: Gonzalez Ramon R
Published: June 2021

The availability of study reference material delivered by computer has gone through a significant metamorphosis in the 20 years of my experience with development. In the late eighties students did not have ready access to inexpensive, compact laptop machines. The sources were generally found in computer labs in the library, and sometimes in the academic departments. Moreover, the applications were very expensive, and had to be purchased by academic institution. Now, with the advent of compression software, students can download material directly from a variety of websites. These applications are free, or generally very inexpensive. The student has the options of running them from their personal desktop or laptop computer, porting them to a handheld device or viewing them from a television set. On line delivery is a major method of instruction in our school; therefore, I have developed a number of lessons in cardiovascular and pulmonary physiology lessons that the students can download to their MP4 devices. I will be offering a large sample of these lessons for your review, and some tips as to how one can easily convert most computer delivered presentations to portable MP4 files.
The role of Physiology in the development of a highly integrated clinically based curriculum at the Paul L. Foster School of Medicine
Authors: Osborne David Lee , Janssen Herbert Frederick , Busselberg Dietrich
Published: June 2021

The establishment of the new four year allopathic medical school in El Paso has provided the opportunity to develop an integrated organ based curriculum. This presentation describes the new curriculum and discusses the role of physiology. Here we focus on the first two years which are devoted to basic science delivery in the context of clinical presentations. Our El Paso curriculum is based on a model originally developed and proofed at The University of Calgary Medical School in Calgary Canada. There are 120 clinical presentations based on why a patient would seek medical attention. Each clinical presentation is illustrated by a scheme which represents the expert physicians approach to the patient's complaint. Following scheme presentation, faculty present the basic science needed to understand the decision tree presented in the scheme. The current faculty in El Paso consists of 15 PhD basic scientist representing the traditional disciplines and 4 MD clinical medical educators. Physiology represents 16 % of the contact time with a total of 185 hours of physiology including 60 hours of neurophysiology. According to the Chairs of Physiology report (2006), this emphasis on non‐neural physiology is 37% higher than the national average. While we teach 60 hours of neurophysiology compared to a national average of 9 hours. Physiology is playing a vital role in the overall development of this integrated curriculum.
Integrating physiology and cell biology in undergraduate inquiry‐based laboratory courses
Authors: Laverty Gary , Hong Seung M.
Published: June 2021

Recent curriculum revisions for our Biological Sciences majors (BA degree) include a requirement of an inquiry‐based laboratory course in which students spend 5‐6 weeks on independent, hypothesis‐driven research projects. For students not involved in Independent Study or Senior Thesis, this requirement also fulfills a University "Discovery Learning Experience" requirement. Several lab courses are offered, including Experimental Cell Biology (BISC 315) and Experimental Physiology (BISC 316), which are complementary to, but independent of corresponding lecture courses. We have recently begun a collaboration to encourage students to "cross over" between these two disciplines. In particular, physiological color changes in killifish (Fundulus) and other teleost species provide an excellent model system for study of melanophore pigment cells, which are known as one of the fastest translocators of pigment vesicles. Whole animal experiments are used to measure differential kinetics of darkening vs lightening, to compare neuronal vs hormonal mechanisms of color change between different species, or to look at effects of environmental variables (background color, salinity, temperature, pH) on color change. At the same time, isolated scales or short‐term cultures of melanophores can be used to address questions about the cytoskeleton, motor proteins or cell signaling. Student presentations and discussions provide a forum for students to integrate physiological and cell biology aspects of this system. Supported by a grant for Undergraduate Education from the Howard Hughes Medical Institute.
Demonstration of hormonal regulation of metabolism: a teaching laboratory exercise
Authors: Cline Mark Andrew
Published: June 2021

Some of the most difficult topics to demonstrate within the Anatomy and Physiology laboratory are the endocrine and metabolic systems due to time and budget constraints. Thus, the activity presented here was developed to demonstrate these systems within one laboratory meeting. Juvenile chicks are injected with either thyrotropin‐releasing hormone (TRH), corticotrophin releasing factor (CRF) or a combination of both. The chicks are then placed in a respirometer for 15 min with soda lime. Students maintain a constant pressure in the system with a 60 mL syringe and manometer. Metabolic rate is calculated based on the chick's body weight, surface area and volume of air consumed during the observation period. This exercise demonstrates synergistic contributions of the TRH and CRF systems and gives students laboratory animal handling experience. Behavioral modifications causes by the hormones may also be observed during the exercise. Students prepare a formal laboratory report on the activity and include a detailed depiction of how these two systems interact to alter metabolism.
The use of technology and interactive case laboratory modules in the study of neurophysiologic concepts and the development of research skills
Authors: Griffin John David
Published: June 2021

Recent advances in technology have improved the ability to present complex physiologic concepts. However, if used only as lecture enhancements, their potential as educational tools is limited. In an upper division neurophysiology course, I have integrated the use of an interactive case method to improve student learning through hands on experimentation and peer evaluation. The course lab sessions are divided into several modules. The first session of each module begins with a case presentation from which student groups develop an original hypothesis and experimental methodology. For their experimental design, students may integrate the use of a physiologic recording system (iWorx Inc.) which allows for a wide range of experimental protocols. During the second session, students employ their experimental design in the actual testing of a "patient" (i.e., the instructor). They must then analyze their data, prepare a formal research paper, and present their project during the final modular session. Following each group's presentation, peer evaluations are done using the eInstruction classroom response system. Through the use of this technology enhanced case method, students gain a better understanding of neurophysiology, learn how to perform an original research project, present their work, and do peer evaluation. (Support: NIH, Mellon Foundation, and the HHMI W&M Science Education and Research Program.)
K‐12 Minority Outreach Fellowship: Bridging generations among minority scientists
Authors: Bruthers C. Brooke , Frank Martin , Matyas Marsha Lakes
Published: June 2021

The APS K‐12 Minority Outreach Fellowship cultivates partnerships between minority physiologists and K‐12 teachers and students. Activities foster communication between minority trainees (graduate and postdoctoral students) and middle/high school minority students. Physiology trainees visit K‐12 classrooms, help conduct teacher professional development workshops, and attend scientific meetings. Since the program's official inception in 2006, Outreach Fellows have reached almost 800 students in five states in schools and learning centers. Presentations range from large to small groups and included demonstrations on topics such as: blood flow and oxygen delivery changes from rest to exercise; the relationship of exercise to hypertension and heart disease; the importance of exercise to human health; the scientific method; careers in physiology, and diversity among scientists. Program impacts have been positive and are assessed through pre/post surveys and fellow reflections.