bluuebunny/crossref_metadata_2025_split huggingface dataset:
Phenotypic abnormalities in microvascular endothelial cells isolated from the Sugen/Hypoxia rat
Authors: Suresh Karthik , Servinsky Laura , Huetsch John , Shimoda Larissa 
Published: November 2023

The mechanisms behind microvascular endothelial dysfunction in PAH are unclear, although mitochondrial dysfunction, increases in reactive oxygen species (ROS) and increased intracellular calcium concentration ([Ca2+]i) have all been implicated in endothelial cell samples from humans with PAH. To better understand the mechanisms behind endothelial dysfunction in PAH, we recently isolated lung microvascular endothelial cells (LMVEC) from normoxic rats (N‐LMVEC) and rats undergoing Sugen/Hypoxia (SuHx‐LMVEC), an experimental model of PAH.To induce PH, rats were injected with SU5416 (Sugen), a VEGFR inhibitor, and then placed in hypoxia for 3 weeks, followed by return to normoxia for 2 weeks. Following hemodynamic measurements, LMVEC were isolated from rats at 5 weeks using a magnetic bead approach, with anti‐CD31 and Griffonia simplicifolia (GS‐II) lectin conjugated beads. Calcium measurements were made in Fura 2‐AM loaded cells placed in a heated flow chamber perfused with Krebs buffer. Immunofluorescence was performed using primary and secondary antibodies and DAPI nuclear stain on rat LMVEC. To measure ROS, LMVEC were infected with a baculovirus vector containing a plasmid encoding roGFP, a ratiometric ROS dye. Cells were imaged 48 h after infection. Mitochondrial respiration was measured using the Seahorse metabolic analyzer system.N‐LMVEC rats appeared phenotypically similar to mouse and human LMVEC, while SuHx‐LMVEC appeared more spindle‐shaped. N‐ and SuHx‐LMVEC stained positive for GS‐II and CD31. Unlike N‐LMVEC, SuHx‐LMVEC additionally stained positive for smooth muscle actin and myosin heavy chain. Mitochondrial respiration was decreased in SuHx‐LMVEC compared to N‐LMVEC, whereas ROS, basal [Ca2+]I, migration and proliferation were all increased. However, basal [Ca2+]i, migration and proliferation in SuHx‐LMVEC were attenuated with treatment with TEMPOL, a ROS scavenger.We conclude that LMVEC isolated from SuHx rats exhibit metabolic and phenotypic differences from control cells that result from elevations in ROS, possibly due to mitochondrial dysfunction.Support or Funding InformationF32HL124930
Chronic Inflammation Deactivated the MAPK pathway and Decreased the TRPV1 Protein Expression in the 50B11 Dorsal Root Ganglion Cell Line
Authors: WANG HANJUN , Rozanski George J. , Zucker Irving H. 
Published: November 2023

It has been shown that skeletal muscle metabolic afferents are desensitized in the chronic heart failure (CHF) state, which is associated with a decreased transient receptor potential vanilloid 1 (TRPV1) receptor expression in the lumbar dorsal root ganglia (DRG). However, the molecular mechanisms underlying the downregulated TRPV1 protein expression in lumbar DRG neurons in CHF remain unclear. Because CHF is associated with chronic inflammation, we decided to investigate the potential effects of acute and chronic inflammation on TRPV1 protein expression in the 50B11 DRG Cell line. We used tumor necrosis factor (TNF) alpha (a pro‐inflammatory cytokine) to create an inflammatory environment. Since evidence suggests that the mitogen‐activated protein kinases (MAPK) pathway is involved in the regulation of TRPV1 channel function, we first investigated the time and dose‐dependent effects of TNF alpha on the phosphorylation of ERK (one of the MAPK components). Our time‐course experiments showed that TNF alpha (100 ng/ml) caused peak phosphorylation of ERK at 15 min and lasted up to 3 hours. TNF alpha also dose‐dependently (0.01, 0.1, 1, 10 and 100ng/ml) increased the phosphorylation of ERK with a peak effect at 10 ng/ml (1.6±0.26 at 10 ng/ml and 1.7±0.26 at 100ng/ml vs. Vehicle (1.0±0), n=6, p<0.01) at 15 min. However, either 20‐hour or 5‐day of continuous treatment with TNF alpha decreased the phosphorylation of ERK by 30% compared to vehicle treatment. Furthermore, a 20‐hour treatment with TNF alpha (100 ng/ml) decreased protein expression of TRPV1 by 30% whereas a 5‐day continuous TNF alpha treatment (10 ng/ml) further decreased TRPV1 protein expression by ~90% (0.10±0.04 vs. Vehicle (1.0±0.0), n=8, p<0.01). These data suggest that chronic inflammation causes a significant reduction in TRPV1 protein expression in DRG neurons which is associated with decreased phosphorylation of ERK. Ongoing experiments will determine if the decreased phosphorylation of ERK by chronic treatment with TNF alpha contributes to the loss of TRPV1 receptors in DRG neurons.Support or Funding InformationThis work was supported by by grants from the National Heart, Lung, and Blood Institute (1R01HL121012‐01A1 and R01 HL116608‐01A1).
Acute Electrical Vagal Nerve Stimulation Exerts Powerful Anti‐inflammatory Effects Through its Nicotinic Action in Rats with Pulmonary Arterial Hypertension
Authors: Yoshida Keimei , Saku Keita , Tanaka Mariko , Abe Kohtaro , Kishi Takuya , Tsutsui Hiroyuki , Sunagawa Kenji 
Published: November 2023

BackgroundsThe inflammatory mechanism plays a crucial role in the pathogenesis of pulmonary arterial hypertension (PAH). Since autonomic dysregulation, that has been reported in PAH, aggravates the inflammatory responses, we hypothesized that electrical vagal nerve stimulation (VNS) may help restore autonomic balance, thereby prevent the worsening of PAH. To clarify the beneficial mechanism of VNS, we investigated how acute VNS impacts on the inflammatory mechanism involved in PAH.MethodsWe used Fischer344 rats weighing 180–200g. As a model of PAH, Sugen5416 was injected (VEGF receptor inhibitor, 20mg/kg, subcutaneously), followed by hypoxia (O2 10%) for 3 weeks and normoxia for 2 weeks. We attached the electrode to the right cervical vagal nerve, applied VNS (20Hz, 0.1–0.15mA, n=5) or sham stimulation (n=4) for 90 minutes, measured hemodynamics and sampled tissues of lung at the end of experiment. We performed western blotting for eNOS and real time PCR for pro‐inflammatory cytokines, anti‐inflammatory cytokine, endothelin‐1 (ET‐1), and α7 nicotinic acetylcholine receptor (α7nAchR).ResultsVNS significantly reduced heart rate by about 10 % from baseline, while did not change mean pulmonary arterial pressure (before VNS: 39.3±8.8, after VNS: 35.9±8.7 mmHg, n.s.) or pulmonary vascular resistance (Fig. 1). Compared to sham, VNS did not alter the eNOS level or mRNA level of ET‐1, whereas attenuated the expressions of pro‐inflammatory cytokines, especially in Tnfa (p<0.05), and that of Il10, an anti‐inflammatory cytokine (p<0.01). In addition, VNS upregulated α7nAchR, the upstream regulatory factor of pro‐inflammatory cytokines (p<0.05) (Fig. 2).ConclusionsVNS exerted the powerful anti‐inflammatory effect through the nicotinic action in PAH model rats. Because the excessive inflammatory responses accelerate pulmonary vascular remodeling in PAH, VNS may serve as a potential therapeutic strategy for PAH. However, how those acute anti‐inflammatory effect of VNS translates into the improvement of PAH remains to be seen.Support or Funding InformationNothing to declare.
Disruption of Mitochondrial Redox and Metabolic States in Norepinephrine‐Exposed T‐lymphocytes
Authors: Case Adam J. , Collins Christopher W. , Roessner Colton T. , Tian Jun , Zimmerman Matthew C. 
Published: November 2023

Increased sympathoexcitation and norepinephrine (NE) outflow are hallmarks of chronic diseases such as heart failure, hypertension, diabetes, and even psychological stress. NE has been demonstrated to modulate the immune system, but mechanisms controlling this cross‐talk between the autonomic nervous and immune systems are complex and poorly defined. Recently, using a mouse model of elevated NE we elucidated that NE utilizes superoxide (O2•−)‐dependent redox signaling to modulate T‐lymphocyte function. However, the source of this O2•− and how it mechanistically contributes to T‐lymphocyte function remain elusive. Due to growing evidence that metabolism is a key regulator of T‐lymphocyte activation and differentiation, we hypothesized that NE mediates its effects on T‐lymphocytes via disruption of mitochondrial bioenergetics and redox state. To address this hypothesis, we utilized purified mouse splenic CD4+ and CD8+ T‐lymphocytes stimulated with NE and assessed subcellular O2•− levels, mitochondrial metabolism, cellular proliferation/differentiation, and cytokine profiles. First, we demonstrated the source of O2•− was indeed the mitochondria as evidenced by a 50% increase (p<0.01 vs vehicle) in MitoSOX Red oxidation as well as the attenuation of this signal by the addition of the mitochondrial‐targeted O2•− scavenging antioxidant MitoTempol. NE‐stimulated T‐lymphocytes also demonstrated a 25% (p<0.05 vs vehicle) decrease in mitochondrial respiratory capacity, which suggests disruption of mitochondrial metabolism and the potential source of increased mitochondrial O2•−. Next, NE inhibited proliferation of T‐lymphocytes by approximately 50% (p<0.01 vs vehicle), which was accompanied by a concomitant increase in the percentage of cells remaining in their naïve state (49.5% with NE vs 26.4% with vehicle, p<0.01). Interestingly, NE lead to significant increases (ranging from 1.8–4.1 fold, p<0.05) in O2•− levels in all T‐lymphocytes independent of activation state (i.e. naïve, memory, and effector). Last, mitochondrial O2•− was shown to be mechanistic to the NE‐mediated T‐lymphocyte phenotype as supplementation of MitoTempol could significantly reverse the approximate 50% decreases in IL‐2 and interferon gamma (p<0.01) and 2‐fold increases in IL‐6 and IL‐17 (p<0.01) levels observed with NE treatment. However, the decreases in proliferation could not be reversed by increased mitochondrial O2•− scavenging. Overall, our data suggest the potential for a new paradigm involving mitochondrial redox and metabolism as mechanistic elements in the regulation of T‐lymphocyte function by NE.Support or Funding InformationThis work is supported by the National Institutes of Health (NIH) National Heart, Lung, and Blood Institute (NHLBI) R00HL123471 (to AJC) and R01HL103942 (to MCZ).
Acute kidney injury induces oxidative stress and distant organ dysfunction due to glutathione depletion
Authors: Shang Yue , Siow Yaw L. , Isaak Cara K. , Karmin O 
Published: November 2023

Ischemia‐reperfusion is a common cause for acute kidney injury and often complicated by distant organ dysfunction. Oxidative stress plays an important role in ischemia‐reperfusion induced local and distant organ injury. However, the mechanisms of AKI induced distant organ injury are not well understood. Glutathione is a major endogenous antioxidant and its depletion directly correlates to ischemia‐reperfusion injury. The liver has a high capacity for producing glutathione and is a key organ that regulates local and systemic redox balance. Liver function is often compromised in patients with acute kidney injury. In the present study, we investigated the mechanism by which kidney ischemia‐reperfusion induced oxidative stress and downregulated glutathione biosynthesis in the liver. The left kidney of Sprague‐Dawley rats was subjected to 45min ischemia followed by 6h reperfusion. Renal ischemia‐reperfusion impaired kidney and liver function as indicated by increased plasma creatinine, aspartate aminotransferase and alanine aminotransferase levels. This was accompanied by a significant reduction in glutathione levels in the liver and plasma. There was a marked elevation of hepatic lipid peroxidation and plasma homocysteine levels. Renal ischemia‐reperfusion caused a significant decrease in mRNA and protein levels of glutamate‐cysteine ligase (catalytic and modifier subunits) which regulated the rate‐limiting reaction in glutathione biosynthesis. A decrease in gene expression of glutamate‐cysteine ligase subunits was mediated through inhibition of a transcription factor Nrf‐2. Renal ischemia‐reperfusion also inhibited hepatic expression of cystathionine‐gamma‐lyase (CSE), an enzyme responsible for producing cysteine (an essential precursor for glutathione synthesis) through the transsulfuration pathway. In conclusion, our study has identified that decreased expression of glutamate‐cysteine ligase, a key enzyme for glutathione biosynthesis as well as reduced CSE‐mediated cysteine production through the transsulfuration pathway may be responsible for hepatic glutathione depletion upon renal IR. This, in turn, dampens the antioxidant defense mechanism and contributes to renal ischemia‐reperfusion induced oxidative stress.Support or Funding InformationThis study was supported, in part, by NSERC and St. Boniface Hospital Research Centre.
Atractylodes lancea Up‐Regulates ERK1/2 and p‐ERK1/2 Expression on Hypoxia/Reoxygenation‐Injured H9c2 Cells
Authors: Nusuetrong Punnee , Gerdprasert Orapin 
Published: November 2023

Atractylodes lancea (Thung) DC. has been used for treatment of fever, flu, sore throat, and common cold. Since, A. lancea contains antioxidant phenolics and flavonoids, A. lancea should be able to protect cardiomyoblasts against hypoxia/reoxygenation (H/R) injury. In the present study, the cardioprotective effect of A. lancea extract (AL) on H/R‐damaged H9c2 cells was elucidated. Incubation of AL (0.01–1 mg/mL) for 24 h showed no toxic effect on H9c2 cells, measured by MTT assay. Cardioprotective effect of AL was determined on the cells exposed to hypoxic buffer for 6 h followed by reoxygenation in complete medium for 24 h. Treated cells were measured by MTT assay as well as under fluorescent microscopy using Hoechst33342. AL at a concentration of 0.1 mg/mL showed an inhibition against H/R‐damaged H9c2 cells, using 6‐hydroxy‐2,5,7,8‐tetramethylchroman‐2‐carboxylic acid as a positive control. Nuclear morphology of H/R‐exposed cells showed shrinking in size, meanwhile normal cells and AL (0.1 mg/mL)‐treated H/R‐exposed cells were found healthy. AL at the same concentration was able to restore the protein expression of ERK1/2 and pERK1/2 when compared to the H/R control. In conclusion, AL inhibited H/R‐injured cardiomyoblasts, which was partly through up‐regulation of ERK1/2 and pERK1/2.Support or Funding InformationThis work was supported by research grant from the National Research Council of Thailand (NRCT 2009‐113) and grant from HRH Princess Maha Sirindhorn Medical Center, Faculty of Medicine, Srinakharinwirot University, Thailand (N0.359/2556). We specially thank Dr. Sanya Hokputsa for giving us the extracts. The authors gratefully acknowledge help and assistance Mr. Pirapas Yingdamnoona for laboratory manipulation of cell culture.
Osteocytes, Muscle and Exercise: Role in Healthy Aging
Authors: Bonewald Lynda 
Published: November 2023

The osteocyte is a mechanosensory, multifunctional cell regulating mineral and skeletal metabolism and functions as an endocrine cell secreting factors that target distant organs such as kidney. These cells form a highly connected intricate network within the bone matrix that can communicate with cells on the bone surface. Osteocytes can live for decades in the bone matrix, however, with age, the osteocyte is subjected to increased reactive oxygen species making the cell more susceptible to apoptosis. Whereas some cells develop a hypermineralized perilacunar matrix and some die resulting in empty lacunae that fill in with mineral, called micropetrosis, the majority develop a senescence‐associated secretory phenotype, SASP. In proliferating cells, SASP halts proliferation of dysfunctional cells, however, osteocytes are not proliferating cells, so it is not clear why they develop this phenotype. One reason may be to avoid apoptosis which would lead to micropetrosis and reduced connectivity of the osteocyte lacunocanalicular network. Development of the senescense phenotype may serve to maintain the function of the network.We have found that muscle factors, and one in particular, called beta‐aminoisobutyric acid are more potent than estrogen or n‐acetyl cysteine in preventing or reducing osteocyte cell death due to reactive oxygen species. These muscle factors are elevated and secreted with muscle contraction. Exercise in the form of voluntary wheel running maintains the capacity of muscle to secrete these osteocyte protective factors. The mechanism of protection is through preservation of mitochondrial structure and function in osteocytes. Conversely, we have found that the young, healthy osteocyte secretes molecules such as Wnt3a and PGE2 that are elevated in response to shear stress to promote muscle myogenesis and function. In summary, exercise can significantly ameliorate or delay the effects of aging through the maintenance of muscle secretory function to maintain an intact osteocyte lacunocanalicular network.Support or Funding InformationNIH NIA 1PO1AG039355
Berlin Questionnaire is a Useful Tool to Identify Risk of Obstructive Sleep Apnea in Metabolic Syndrome Patients
Authors: Cepeda Felipe Xerez , Virmondes Leslie , Rodrigues Sara , Dutra‐Marques Akothirene C.B. , Carvalho Jefferson C. , Toschi‐Dias Edgar , Alves Maria Janieire N.N. , Rondon Maria Urbana P.B. , Lopes‐Vicente Wanda R.P. , Hussid Maria Fernanda , Lorenzi‐Filho Geraldo , Trombetta Ivani C. 
Published: November 2023

Obstructive sleep apnea (OSA) is a risk factor frequently present in patients with metabolic syndrome (MetS). Despite of MetS, moderate and severe OSA has been associated with cardiovascular diseases, increasing 80% the risk of cardiovascular event. The gold standard diagnostic method for OSA is the overnight polysomnography (PSG), that is in general not accessible. The aim of the present study was to evaluate whether Berlin Questionnaire (BQ) is a good tool for assessing the risk of OSA in patients with MetS. We submitted 97 patients, untreated and newly diagnosed with MetS (ATP‐III) to a PSG. OSA was characterized by apnea‐hypopnea index (AHI). BQ was administered before PSG and we evaluated the sensitivity, specificity, positive and negative predictive values and accuracy. Of the 97 patients with SMet, 81 patients had OSA, being 47 (48.5%) with moderate and severe OSA. For all SMet with OSA (AHI>5 events/hour), the BQ showed good sensitivity (0.65) and fair specificity (0.38) with a positive predictive value of 0.84, negative predictive value of 0.18 and accuracy of 84%. Similarly, for moderate‐severe OSA (AHI>15 events/hour) we found good sensitivity (0.73) and fair specificity (0.40). Interestingly, for severe OSA (AHI>30 events/hour), there was a very good sensitivity (0.91) and moderate specificity (0.42). In conclusion, BQ is a good predictor for the screening of OSA in MetS patients in general, but it is a very good tool for predicting severe OSA.Support or Funding InformationFundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP) #2011/175336, #2015/175336 and #2016/168317
Cardiorespiratory Responses to Severe Arterial Hypoxemia with Increasing Remifentanil Plasma Concentrations in the Rabbit
Authors: Quail Anthony W , Cottee David BF , Johnstone Janice M , O'Hara Kate , White Saxon W 
Published: November 2023

Remifentanil is an ultra‐short acting synthetic opiate employed clinically as an infusion for total intravenous anesthesia, sedation and analgesia. We have previously reported that vagal control of heart rate (HR) with remifentanil during severe arterial hypoxia (PaO2 < 35 mm Hg) is maintained, while its effects on control of minute ventilation (VE), tidal volume (VT) and respiratory frequency (f) are selective (Quail et al., 2016). Although opiates can act at the carotid body (McQueen and Ribeiro 1980; Mayer, et al., 1989) and the NTS (Zhang et al. 2011), our data suggest the actions of remifentanil on cardiorespiratory control during hypoxia are mainly in the CNS, rather than at the carotid body. We now report additional data concerning the plasma concentrations of remifentanil corresponding to each dose of the drug producing these effects. An ear artery and vein of NZ White rabbits (n=6) were cannulated (lidocaine 1%). Measurements of VE, VT, f, HR, mean arterial pressure (MAP) and oxygen saturation (ear SpO2) were made during normoxia, followed by 5 minutes of hypoxia (FiO2 7–8%). After a one hour recovery, rabbits breathed air during an intravenous remifentanil infusion and the hypoxia was repeated. Arterial blood samples were taken for blood gas analysis and measurement of plasma concentrations of remifentanil (HPLC and mass spectrometry). Two doses of remifentanil were studied on each of two days, one week apart. Data were analysed using repeated measures ANOVA. In rabbits breathing air remifentanil mean plasma concentrations after 12 minutes of infusion (0.1, 0.2, 0.3, or 0.4 mcg/kg/min) were respectively 1.3, 3.1, 4.5 and 11.7 ng/ml. Remifentanil caused dose‐related falls in f, VE and HR (P<0.01), while VT and MAP were unchanged. Remifentanil concentrations during hypoxia were similar to normoxia and the reflex bradycardia observed in conscious rabbits was maintained at the lower concentrations (P<0.05). The VT response remained intact but the increase in VE was attenuated with f increasing from a lowered baseline. We conclude that at similar target plasma concentrations to those employed clinically, vagal control of heart rate with remifentanil during severe hypoxia is maintained, while the components of ventilation are selectively modified.Support or Funding InformationJohn Hunter Charitable Research Trust and Hunter Medical Research Institute
JGA‐Derived CCN1 is a Systemically Acting Angiogenic Hormone
Authors: Desposito Dorinne , Moon Ju‐Young , Riquier‐Brison Anne , Peti‐Peterdi Janos 
Published: November 2023

The kidneys are major regulatory organs of whole body homeostasis including body fluid and electrolyte balance, blood pressure maintenance, and blood cell production. Renal control mechanisms of these key systemic physiological functions include the local production and secretion of peptide hormones, and their actions at distant organ sites. Classic examples are erythropoietin, and renin, which is released from the highly perfused juxtaglomerular apparatus (JGA). The present studies aimed to test the hypothesis that the newly identified JGA‐derived cysteine‐rich protein CCN1 is released into the blood and serves as a novel systemic angiogenic hormone promoting general cardiovascular health. Blood was collected before and at different times after bilateral nephrectomy in both mice and rats. Plasma CCN1 levels measured by CCN1 ELISA and immunoblotting continuously decayed after nephrectomy, suggesting that the kidney/JGA is a significant source of systemic CCN1. Immunostaining of mouse and human kidneys showed a robust and constitutive expression of CCN1 in the JGA. The tube formation assay of glomerular endothelial cells (GEnCs) was performed to test the effect of exogenous CCN1 on in vitro angiogenesis. CCN1 appeared to be more effective on in vitro angiogenesis than the gold‐standard angiogenic factor VEGF. Other classic in vitro assays of GEnC cell proliferation and cell migration (scratch opening assay) showed similar robust effects of CCN1. In vivo, CD34 immunostaining of kidney tissues collected from C57BL/6 mice treated with subcapsular injection of CCN1 peptide showed a significant increase of angiogenesis compared to untreated mice. Kidney, aorta, heart, and brain tissues were collected from mice with JGA‐specific CCN1 KO before and three weeks after tamoxifen induction, and sections stained for TUNEL assay of apoptosis. Compared to nearly undetectable levels in normal tissues before, a high number of vascular endothelial cells in all tissues stained TUNEL+ after JGA CCN1 deletion, indicating the presence of widespread systemic endothelial injury when renal CCN1 synthesis is compromised. These findings provide strong support for our new concept that JGA‐derived CCN1 is released into the blood, acts as a new systemic angiogenic hormone, and is essential for maintaining vascular integrity throughout the body.Support or Funding InformationNIH R01 64324, AHA, ADA