bluuebunny/crossref_metadata_2025 huggingface dataset:
Membrane mobility of aquaporin‐2 (AQP2) tagged with photoconvertible fluorescence protein in mpkCCD cells
Authors: Yip kay‐Pong , Amin Jahanshah , Tse Chung‐Ming , Cha Byeong
Published: June 2021

mEos is a photoconvertible fluorescence protein. Its emission can be switched from green to red by an UV pulse. Tagging AQP2 with mEos allows tracking of a subset of AQP2 after photoconversion. Here, we examined whether, 1) AQP2 tagged with mEos to amino‐terminus (Eos‐AQP2) or carboxyl‐terminus (AQP2‐Eos) has similar membrane mobility, and 2) photoconversion alters their membrane mobility. Cells grown on glass‐bottom dishes were transfected with either Eos‐AQP2 or AQP2‐Eos and their membrane mobility were measured by fluorescence recovery after photobleaching (FRAP). A 488 nm laser pulse was used to photobleach a 4 μm spot for FRAP. Fluorescence recovery half‐time (t1/2) was calculated with ImageJ. Eos‐AQP2 was found in cytosolic vesicles and plasma membrane. Apical accumulation of Eos‐AQP2 was stimulated by forskolin. AQP2‐Eos was localized in apical membrane. t1/2 of Eos‐AQP2 and AQP2‐Eos were in the same range (42–50 s). Local photoconversion of green‐Eos to red‐Eos was performed with a 405 nm laser pulse on the apical surface. Photoconversion did not alter the membrane mobility of EOS‐AQP2 or AQP2‐EOS as measured by the diffusion time course of red‐Eos from the photoconversion site. However, forskolin stimulation increased t1/2 of Eos‐AQP2 by 3 folds, and decreased the t1/2 of AQP2‐Eos by 25%. These results indicated that photoconversion did not alter AQP2 membrane mobility.
Peroxisome proliferator activated receptor – alpha regulation of sodium transport mechanisms in human primary renal proximal tubule epithelial cells during acute Angiotensin II treatment
Authors: Doh Kwame , Hudson Tamaro , Lee Dexter L
Published: June 2021

Angiotensin II (Ang II) is important for Na+ regulation in the kidney. Proteins in the proximal tubule (PT) epithelial cells such as the sodium hydrogen exchanger (NHE3), sodium hydrogen exchanger regulatory factor‐1 (NHERF‐1), Na+/K+ ATPase, NaPi2 serve as Na+ transporters. Studies have demonstrated that NHERF‐1 interacts with NaPi2 and NHE3 to regulate Na+ transport. Peroxisome proliferator activated receptor‐alpha (PPAR‐α) activation reduces Na+/K+ ATPase activity but is important for its expression. This study tests the hypothesis that PPAR‐α activation stimulates NHERF‐1 to interact with NHE3, NaPi2 and Na+/K+ ATPase to reduce Na+ reabsorption in PT cells during Ang II stimulation. Human primary renal proximal tubule epithelial cells (PREC) were used during passage 4 and plated at a density of 2 × 106/dish. PRECs were treated for 20 hours as follows: control plates, Ang II (10−9 M), fenofibrate (a PPAR‐α agonist)( 10−7 M) and Ang II + fenofibrate. Western blots were performed to determine the expression (optical density/area) of NHERF‐1, NHE3, Na+/K+ ATPase and NaPi2 in PRECs. Fenofibrate, Ang II and Ang II + Fenofibrate significantly increased NHERF‐1 expression in PRECs above control. We did not observe differences in NaPi2 or Na+/K+ ATPase expression between the groups. Our results suggest fenofibrate regulates Na+ reabsorption in PRECs by increasing NHERF‐1 expression. Our results also indicate that a low dose of Ang II directly stimulates NHERF‐1 expression in PRECs. Future studies are needed to determine the dose dependent effect of Ang II and fenofibrate on NaPi2, Na+/K+ ATPase, NHE3 expression and activity. Support: 5K01HL092593–05
Rh proteins mediated ammonia excretion in the skin of zebrafish larvae: evidence of active ammonia transport by ionocytes
Authors: Shih TinHan , Horng JiunLin , Lai YiTing , Lin LiYih
Published: June 2021

Rhesus (Rh) glycoproteins, Rhbg and Rhcg play a critical role in ammonia transport in mammalian renal collecting ducts. In the model animal, zebrafish, Rhcg1was found in the apical membrane of skin ionocytes (H+‐ATPase‐rich cells; HR cells) which is similar to α‐type intercalated cells in mammalian collecting ducts. However, the cellular distribution and role of Rhbg in zebrafish larvae has not been well investigated. In this study, Rhbg was localized to both apical and basolateral membrane of skin keratinocytes by using in situ hybridization and immunohistochemistry. A scanning ion‐selective electrode technique (SIET) was applied to measure NH4+ flux at the apical surface of keratinocytes and HR cells in intact larvae. Knockdown of Rhbg with morpholino oligonucleotides suppressed ammonia excretion by keratinocytes, and induced compensatory ammonia excretion by HR cells. To test if HR cells can excrete ammonia against higher external ammonia level comparing to keratinocyte, “reversal concentration (RC)” of ammonia excretion was determined by measuring zero NH4+ fluxes in a serial concentration of external NH4+. Results showed that the RC of HR cells was higher than that of keratinocytes demonstrating that HR cells can excrete ammonia against higher external ammonia levels. Either knockdown the expression of Rhcg1 or H+‐ATPase in HR cells suppressed the RC of HR cells to the level of keratinocytes, suggesting that Rhcg1 and H+‐ATPase are involved in the active ammonia excretion of HR cells.
Hypertension in a rat model of HELLP Syndrome is associated with Increased TNF‐alpha, IL‐6 and CD4+ T cell activation
Authors: Wallace Kedra , Darby Marie , Kyle Patrick , Morris Rachael , Moseley Janae , Frazier Krystal , LaMarca Babbette
Published: June 2021

HELLP syndrome (hemolysis, elevated liver enzymes, low platelets) is a hypertensive disorder that can occur during pregnancy with severe symptoms and consequences, however, the physiological mechanisms involved are unknown. Our previous studies have shown that women with HELLP syndrome have elevated sFlt‐1, sEndoglin, TNF‐α and IL‐6. In this study we utilized a HELLP syndrome rodent model to determine if immune activation occurs similarly to that seen in HELLP patients.

On gestational day (GD) 12, sEndoglin (7ug/kg/day) and sFlt‐1 (4.7ug/kg/day) infusion began via miniosmotic pump into normal pregnant (NP) rats. On GD18 carotid catheters were inserted, GD19 mean arterial pressure (MAP), plasma and tissue collected.

MAP increased from NP (n=21) 101.1±1.7 to 113.4±3.2mmHg in HELLP rats (n=14; P<.05). Liver enzymes increased from 46.3±6.9 in NP to 63.8±4.6 IU/L in HELLP rats (P<.05) and platelets decreased in HELLP rats (7112±657.9uL) vs. NP rats (28714+5236uL, P<.5). TNFα and IL‐6 were increased in HELLP rats (54.7±12.6 vs. 25.6±3.5pg/mL; P<.5) and (103.6±26.5 vs. 40.2±8.2pg/mL; P<.05) compared to NP rats. HELLP rats had increased CD4+ T lymphocytes compared to NP rats, circulating 20.7 to 8.9% (P<.5); spleen 7.1 to 4.9% and liver 14.7 to 3.8% gated.

In this study we demonstrate that hypertension in a rat model of HELLP syndrome, is associated with a T helper 1 immune activation similar to that seen in HELLP patients.
Afferent Vagal Nerve Stimulation Resets the Baroreflex Neural Arc and Inhibits Sympathetic Nerve Activity
Authors: Saku Keita , Sakamoto Kazuo , Hosokawa Kazuya , Kakino Takamori , Ikeda Masataka , Oga Yasuhiro , Nishizaki Akiko , Ide Tomomi , Sunagawa Kenji
Published: June 2021

Background

Vagal nerve stimulation (VNS) benefits patients with heart failure. The afferent vagal nerve mediates cardiopulmonary reflex. We hypothesized that vagal afferent stimulation (AVNS) suppresses sympathetic nerve activity (SNA) and improves heart failure.

Methods and Results

In 5 ventilated rats, we controlled the isolated, bilateral carotid sinuses pressure (CSP) and measured celiac SNA and AP. In the presence of constant CSP, increasing the voltage of AVNS captured the vagal afferent nerves at 2.7±0.7 volts and dose dependently decreased SNA and AP with a maximal inhibition of SNA at 5.5±1.2 volts. We changed CSP stepwise and measured SNA and AP. AVNS shifted the CSP‐SNA relation (neural arc) downward, whereas unaffected the SNA‐AP relation (peripheral arc). To evaluate the dynamic impacts of AVNS, we randomly stimulated AVNS with binary white noise sequence, and identified the transfer functions from AVNS to SNA (HAVNS‐SNA) and from the SNA to AP (HSNA‐AP). HAVNS‐SNA resembles that of the neural arc, while HSNA‐AP resembles that of the peripheral arc indicating AVNS induces sympathoinhibition just like baroreflex.

Conclusion

AVNS resets the baroreflex neural arc and induces sympathoinhibition. It may in part attribute to the beneficial impacts of VNS on heart failure.
Rheumatoid Arthritis and Arterial Baroreflex Function
Authors: Adlan Ahmed M , Kitas George , Paton Julian F.R. , Lip Gregory Y.H. , Fisher James P
Published: June 2021

Rheumatoid arthritis (RA) is an inflammatory disorder associated with elevated cardiovascular mortality. Central administration of pro‐inflammatory cytokines decreases arterial baroreflex function (ABR) in rats. The optimal strategy for assessing ABR in RA patients remains unclear. To begin to investigate this, we monitored beat‐to‐beat blood pressure (BP) (Portapres) and heart rate (HR, ECG) in 9 RA patients (3 men, age 63 ± 3 yr, mean ± SEM) while resting supine. ABR gain was assessed using sequential infusion of sodium nitroprusside (100 μg) and phenylephrine (150 μg) (modified Oxford technique [GMOT]), and compared with ‘spontaneous’ indices of ABR gain (sequence technique [GSEQ], low frequency transfer function gain [GTF]) using Pearson correlation analyses. Resting HR was 65±2 bpm and mean BP 106±3 mmHg. GMOT, GSEQ and GTF were 4.4±1.0, 8.0±1.5 and 4.9±0.8 ms/mmHg, respectively. Although, GSEQ and GTF were significantly associated (P<0.05, R2=0.80), neither were significantly related to GMOT. These findings suggest that the ‘spontaneous’ and modified Oxford techniques evaluate different facets of ABR function in RA patients. Supported by Arthritis Research UK.
Cardiovascular responses during static skeletal muscle contraction following neuronal NOS blockade within the ventrolateral medulla
Authors: Ally Ahmmed , Maher Timothy J
Published: June 2021

We have previously shown that blockade of neuronal nitric oxide synthase (nNOS) within the rostral (RVLM) and caudal ventrolateral medulla (CVLM) differentially modulates cardiovascular responses via changes in glutamate and GABA concentrations during static skeletal muscle contraction (Brain Res. 977: 80–89, 2003). In this study, we determined if microdialyzing a specific nNOS antagonist into the left RVLM or the left CVLM and into both the left RVLM and CVLM would affect cardiovascular responses expression during the exercise pressor reflex in left‐sided stroked rats. Rats were stroked by employing a 90 minute left‐sided middle cerebral artery occlusion (MCAO) followed by 24 hours of reperfusion. In protocol 1, microdialysis of a selective nNOS antagonist, 1‐(2‐trifluoromethylphenyl)‐ imidazole (TFMP, 1.0 microM), for 120 min into the left RVLM significantly potentiated cardiovascular responses during a static muscle contraction compared to intact rats. In Protocol 2, application of TFMP into the left CVLM significantly attenuated cardiovascular responses during muscle contractions. Finally, administration of TFMP into both the left RVLM and left CVLM produced results similar to those observed with Protocol 1. These results demonstrate that blockade of nNOS within the ventrolateral medulla differentially modulates cardiovascular responses during static exercise in stroked rats.
Vitamin D Insufficiency is Associated with Greater Obesity‐Related Insulin Resistance
Authors: Sulistyoningrum Dian , Gasevic Danijela , Green Timothy J , Lear Scott A , Devlin Angela M
Published: June 2021

Insulin resistance (IR) and vitamin D insufficiency [circulating 25‐hydroxyvitamin D (25OHD) ≤50nmol/L] are associated with obesity (BMI≥30kg/m2). We reported that visceral adipose tissue (VAT) is a negative determinant of plasma 25OHD. Given that vitamin D may play a role in insulin sensitivity the goal of this study is to explore the association between a vitamin D insufficiency/obesity phenotype with IR in healthy adults (N=364). Subjects were assessed for socio‐demographics; anthropometrics; body fat distribution; fasting plasma glucose, insulin, and 25OHD; and the homeostasis model assessment (HOMA) of IR. Subjects were grouped as: vitamin D adequate/no obesity; vitamin D adequate/obesity; vitamin D insufficiency/no obesity; and vitamin D insufficiency/obesity. Data were analyzed by linear regression adjusted for age, sex, ethnicity, smoking, physical activity, season, and VAT. Vitamin D insufficiency/obesity was observed in 20% of subjects and HOMA‐IR was 21% higher [B=0.207; p=0.033] in these subjects than those in the vitamin D adequate/no obesity group. No associations were observed between HOMA‐IR and adequate vitamin D/obesity or vitamin D insufficiency/no obesity groups. These findings suggest that vitamin D insufficiency in subjects with obesity places them at greater risk for IR, even after adjusting for body fat distribution. Vitamin D may play a role in obesity‐related IR.
Epicardial Force and Strain Measurements from the In Vivo Murine Heart
Authors: Constantinides Christakis , Michaelides Marcos , Georgiadou Sotiria
Published: June 2021

Critical to myocardial functional characterization is the assessment of regional stress and strain behavior. This study quantifies in‐vivo left ventricular (LV) epicardial force and strain measurements of the normal murine heart using micro sensors. Male C57BL/6 mice (n=11, mean age=10.5 wks, weight=24.6 g) were intubated and studied under 1.5% isoflurane in 100% O2. Microsurgery allowed the placement of two piezoelectric crystals (od=0.3–0.7 mm, 1–2 mm apart) at longitudinal, basal and mid‐myocardial regions, and a miniature force sensor, between the crystals. The crystals were sutured on the epicardial LV wall, whereas the cantilever force sensor was placed on a platform stand and was connected to the epicardium via a thin silver wire, attached to a silk thread suture inserted superficially on the tissue. Findings demonstrate lack of ischemia (H&E histology) and epicardial longitudinal crystal displacements in the range of 0.19–0.27 mm (n=7), indicative of maximum evoked contractile and relaxation strains of 5–6.8 and 4.6–6.2 % for basal/mid‐ventricular regions. Force results (n=5, uncorrected for baseline offsets) yield mean contractile and relaxation forces of 12.7±1.2 and 12.9±1.4 mN (p=NS), respectively. The potential usefulness of in‐vivo force and strain measurements relates to transgenic and diseased mouse studies, where regional cardiac mechanical properties may be significantly altered.
Differential response of cardiac NOX‐2 and ‐4 to mild and severe pressure overload
Authors: Bradley Jessica M , El Hajj Milad C , Mouton Alan J , El Hajj Elia C , Gardner Jason D
Published: June 2021

Heart failure results when compensatory mechanisms fail to overcome injury and hemodynamic overload often caused by pressure overload (PO), myocardial infarction, diabetes, or hypertension. Oxidative stress, via NADPH oxidases, has been shown to exacerbate PO‐induced left ventricular (LV) remodeling. However, individual NOX isoforms may have different roles in LV remodeling. Our goal was to examine the role of NOX‐2 and ‐4 during PO‐induced cardiac injury. We hypothesized that severe PO causes an imbalance of NOX‐2/NOX‐4, favoring the formation of oxidative stress and resulting in a loss cardioprotection. PO was surgically induced in male C57/BL6 mice via transverse aortic constriction (TAC: mild‐26g or severe‐27g needle). After 4 wks, analysis of LV homogenates indicated that mild TAC resulted in increased protein expression of NOX‐2 and ‐4 compared to Sham. Mild TAC also led to marked increases in lysyl oxidase (LOX), TGF‐β, HIF‐1α, and collagens I and III. Yet, severe TAC resulted in attenuation of NOX‐4, as well as TGF‐β. LOX expression was further increased by severe TAC compared to mild. These results suggest that NOX‐2 and ‐4 are differentially regulated during the progression of cardiac remodeling, with NOX‐2 promoting adverse remodeling and NOX‐4 promoting compensatory remodeling.
Epidermal growth factor receptor‐tyrosine kinase inhibition by erlotinib causes hypomagnesemia, oxidative stress and cardiac dysfunction
Authors: Weglicki William B , Mak Iu Tong , Chmielinska Joanna J , Spurney Christopher F , Kramer Jay H
Published: June 2021

We assessed cardiac dysfunction using endocardiography in rats treated with the anticancer drug erlotinib (Tarceva) at a dose of 10 mg/kg/day over a 9 week period. We observed significant progressive hypomagnesemia (P<0.05) at 5 and 9 weeks, when a 3‐fold elevation (P<0.05) of neutrophil superoxide production was seen. Moderately decreased (P<0.05) cardiac LV ejection fraction and % fractional shortening were measured at 7 weeks, and diastolic dysfunction (lower mitral valve E/A ratio) achieved significance (P<0.05) at 9 weeks of erlotinib treatment. Our prior studies with another EGFR‐TKI agent, tyrphostin AG1478 (21 mg/kg/day), also showed similar hypomagnesemia, oxidative stress, elevated plasma substance P and cardiac dysfunction after 5 weeks of treatment (Can. J. Physiol. Pharmacol. 90:1145–1149, 2012). Clinical studies have documented persistent hypomagnesemia in cancer patients treated with cetuximab (EGFR‐blocking antibody). We conclude that EGFR‐TK inhibiting agents also cause hypomagnesemia; in addition, oxidative stress and impaired cardiac contractility also occur in our rodent model. The clinical relevance of these findings with the increasing use of EGFR‐TK inhibiting drugs remains to be determined.
Osteonectin protects against adverse cardiac inflammation during viral myocarditis
Authors: Papageorgiou Anna , Rienks Marieke , Vanhoutte Davy , Verhesen Wouter , Carai Paolo , Vandendriessche Thierry , Chuah Marinee , Heymans Stephane
Published: June 2021

Viral myocarditis is inflammation of the heart muscle as a consequence of infection by common viruses and outcomes for patients range from complete recovery to fatal dilated cardiomyopathy. We investigated the role of the matricellular protein Osteonectin (SPARC) in mitigating the cardiac injury during viral myocarditis.

SPARC mRNA and protein expression was induced in the mouse model of viral myocarditis at 7 and 11 days respectively. Under these conditions, SPARC co‐localized with CD‐3‐lymphocytes and VCAM‐endothelial cells. SPARC‐null mice and their wild type (WT) littermates were subjected to human coxsackie‐virusB3 (CVB3)‐induced myocarditis and the absence of SPARC significantly increased cardiac inflammation and mortality as compared to WT mice. We found that SPARC null mice had increased levels of Ly6G+ and Ly6C+ myeloid cells and fewer CD4+ lymphocytes in the heart during viral infection. Importantly, adenoviral overexpression of SPARC in WT animals resulted in improved cardiac function after viral injury as compared to control vector (fractional shortening was 28,2%±0,6 vs 21,2%±0,8 respectively). Collectively these data indicate a role for SPARC in endothelial cell function, leukocyte function and cardiomyocyte contractility. This multifunctional glycoprotein represents an exciting novel target in the treatment of inflammatory heart disease.
MMP‐9 dependent proteins regulate left ventricular remodeling following myocardial infarction
Authors: de Castro Bras Lisandra Eunice , DeLeon Kristine Y. , Yabluchanskiy Andriy , Ma Yonggang , Halade Ganesh V. , Hakala Kevin , Weintraub Susan T. , Lindsey Merry L.
Published: June 2021

Adverse remodeling of the left ventricle (LV) following myocardial infarction (MI) is a leading cause of congestive heart failure. Extracellular matrix (ECM) turnover is a key component of LV remodeling that involves the degradation of ECM to remove dead myocytes as well as synthesis of new ECM to form the infarct scar. Post‐MI, matrix metalloproteinase (MMP)‐9 is a prognostic indicator of LV dysfunction. Identification and quantification of ECM proteins are hampered by the low solubility of ECM components in standard tissue preparation reagents. We developed a novel differential solubility‐based‐method for protein fractionation optimized for the analysis of cardiac ECM. We used this novel method coupled with a proteomic approach to evaluate the LV infarct regions of wild type (WT; n=8) and MMP‐9 null (null; n=8) mice at 5 days post‐MI. Our method generated 3 protein fractions per sample: soluble, cytoplasmic and insoluble. By HPLC‐ESI‐MS/MS analysis of the insoluble fraction, we identified 157 proteins, of which 22 (14%) were ECM proteins. Fibronectin increased post‐MI in both genotypes (number of assigned spectra: 2 to 55 in WT vs 4 to 87 in null). Periostin was only observed post‐MI (number of assigned spectra: 6 WT vs 20 null). In conclusion, fibronectin and periostin may mediate LV remodeling through MMP‐9 dependent mechanisms.
Reciprocal Endothelial NO Synthase (eNOS) Ser1177 Phosphorylation and Thr495 Dephosphorylation is Key for Robust in vivo Cardioprotection: Therapeutic Implication of a Novel Ischemic Preconditioning Stimuli
Authors: Talukder M A Hassan , Yang Fuchun , Yang Changjun , Nishijima Yoshinori , Chen Chun‐An , Varadharaj Saradhadevi , Zweier Jay L.
Published: June 2021

Ischemic preconditioning (IPC) induces powerful cardioprotection, but its clinical application has been limited. The cardioprotective effect of IPC may be limited by the design of the IPC protocol and downstream mechanisms. While IPC‐induced eNOS phosphorylation is reported for in vitro cardioprotection; the importance of eNOS phosphorylation at Ser1177 (stimulation) and Thr495 (inhibition) is unknown for in vivo cardioprotection. Therefore, we investigate whether a novel rapid cycles IPC (RIPC) protocol induces in vivo cardioprotection; and if so, what is its cardioprotective efficacy and mechanism(s) relative to classical IPC (C‐IPC). Rats were subjected to in vivo ischemia/reperfusion (I/R) preceded by either C‐IPC (3 × 5‐min I & 5‐min R) or R‐IPC (6 × 1‐min I & 1‐min R). After 24‐hrs R, cardiac function, infarction, reactive oxygen species (ROS) generation, prosurvival kinase and eNOS proteins were characterized. R‐IPC conferred robust cardioprotection and that was superior to C‐IPC. The R‐IPC effect was manifested by reduced MI, improved function, reduced ROS, activation of Akt and ERK1/2, and reciprocal phosphorylation of eNOS at Ser1177 and dephosphorylation at Thr495. Thus, reciprocal eNOS phosphorylation plays an important role in acute cardioprotection, and the novel R‐IPC is highly effective in activating cardioprotective signaling pathways. (NIH grants HL63744, HL65608 and HL38324).
Different physiological adaptations to aerobic interval training between heart failure patients with reduced and preserved ejection fractures
Authors: Fu Tieh‐Cheng , Wang Jong‐Shyan
Published: June 2021

This investigation elucidated different adaptation strategies taking during exercise training among different heart failure (HF) phenotypes. Thirty‐five HF patients with reduced ejection fraction (HFrEF) and 31 HF patients with preserved ejection fraction (HFpEF) performed an incremental exercise test before and after 36 aerobic interval exercise courses. Cardiac output (CO) was measured by a non‐invasive bio‐reactance device and peripheral hemodynamic changes in frontal cerebral lobe and vastus lateralis were assessed by a near‐infrared spectroscopy. The results showed that 1) the HFrEF patients revealed smaller COpeak before training and larger improvement after training, but still less than those of HFpEF group, 2) the HFpEF patients had steeper VE‐VO2 slopes in pre‐training status but greater decrease in post‐training status, even less than those of HFrEF group, and 3) less perfusions in frontal cerebral lobe and vastus lateralis of HFpEF patients during pre‐training exercise and greater improvement during post‐training exercise performance, even better than those of HFrEF group. Therefore we conclude that different HF phenotypes have different physiological adaptations to aerobic interval training, i.e., HFrEF patients have greater heart adaptation and HFpEF patients have greater peripheral adaptation to this exercise regimen.
A complex interplay between NO, EDHFs, and KIR channels in cutaneous active vasodilation
Authors: Brunt Vienna E , Fujii Naoto , Minson Christopher T
Published: June 2021

EDHFs contribute largely to skin local hyperemia. We sought to determine if they also contribute to active vasodilation (AVD) during whole‐body heating. Skin blood flow was measured at 4 sites perfused via microdialysis with 1) Lactated Ringer's, 2) tetraethylammonium (TEA, calcium‐sensitive potassium channel & EDHF inhibitor), 3) L‐NAME (NOS inhibitor), and 4) TEA + L‐NAME. Data is presented as % of max cutaneous vascular conductance. TEA had no effect on AVD (plateau at the end of heat stress: control=55.2±4.6% vs. TEA=63.2±5.2%, p=0.34), indicating EDHFs are not obligatory. L‐NAME attenuated plateau (33.7±5.4%); while TEA+L‐NAME augmented plateau compared to L‐NAME alone (49.7±5.3%, p=0.02 vs. L‐NAME), and did not differ from control (p=0.72). From these data, it appears that combined blockade of EDHFs and NOS forces dilation to occur through other means, possibly through inward‐rectifying potassium (KIR) channels. To test this second hypothesis, we measured AVD at the following sites: 1) Lactated Ringer's, 2) L‐NAME, 3) L‐NAME + TEA, and 4) L‐NAME + TEA + barium chloride (BaCl2, KIR channel inhibitor). The addition of BaCl2 to L‐NAME + TEA reduced plateau to 25.4±6.2% (p=0.01 vs. L‐NAME + TEA), which did not differ from the L‐NAME site (p=0.59). These data combined demonstrate a complex interplay between vasodilatory pathways, with cross talk between NO, EDHFs, and KIR channels.

Supported by NIH Grant HL081671
Investigating relationships between arterial stiffness and collagen turnover in humans
Authors: Cotie Lisa , Currie Katharine , de Zepetnek Julia Totosy , Josse Andrea , McGill Greg , Phillips Stuart , MacDonald Maureen
Published: June 2021

Understanding the factors regulating changes in vascular structure may assist in designing strategies for decreasing the development and progression of cardiovascular disease. Previous research has shown links between various markers of collagen turnover and arterial stiffness in clinical populations (McNulty, 2006 & Chatzikyriakou, 2008). The purpose of this study was to investigate the relationship between functional assessments and blood markers of arterial wall structure. Arterial stiffness and markers of collagen turnover were assessed in 20 overweight women, 20 elderly healthy men, 23 individuals with coronary artery disease, 16 individuals with spinal cord injury and 10 young healthy men. Carotid artery stiffness was determined through assessment of common carotid artery distensibility with simultaneous B‐mode ultrasound imaging and applanation tonometry. Fasting venous blood samples were analyzed for serum levels of pro‐collagen type I (PIP) and cross‐linked telopeptide of collagen type I (CTX); markers of type I collagen synthesis and degradation, respectively. There was a significant positive relationship observed between CTX and carotid artery distensibility (R = 0.330, p = 0.001) and a weak but significant inverse relationship (R = −0.224, p = 0.034) observed between carotid artery distensibility and type I collagen turnover, expressed as a ratio of PIP to CTX. These findings suggest a link exists between type I collagen turnover and central arterial distensibility, however this relationship appears to be opposite to that previous observed in the literature in older hypertensive individuals (McNulty, 2006). Funded by Canadian Institute of Health Research, Natural Sciences and Engineering Research Council, Innovation Center for US Dairy, Dairy Farmers of Canada and the Ontario Neurotrauma Foundation.
Increased respiratory rhythm and O2 and CO2 chemosensitivity in juvenile rats submitted to perinatal protein undernutrition
Authors: Costa‐Silva João Henrique , Alves José Luiz B. , Nogueira Viviane O. , Oliveira Gerliny B. , Da Silva Glauber S.F. , Wanderley Almir G. , Leandro Carol V.G.
Published: June 2021

We evaluated the effects of perinatal protein undernutrition on the respiratory pattern and responses to hypercapnia and hypoxia. Pregnant Wistar rats received normoproteic (NP, 17% of protein) or hypoproteic diet (HP, 8% of protein) during gestation and lactation. Male offspring (NP: n=10; HP: n=10) at the 30‐day‐old were evaluated for respiratory frequency (Rf), tidal volume (VT) and ventilation (VE), and ventilatory responses to hypercapnia (10% CO2) and hypoxia (7% O2). At rest, HP rats showed increased Rf (NP: 105.3±3.1 vs. HP: 135.1±2.2 cycles.min−1, P<0.0001) and Ve (NP: 987.4±36.1 vs. HP: 1384.9±57.1 mL.kg− 1.min−1, P<0.0001), but no change in VT. After exposure to hypercapnia, HP showed higher ventilatory responses than NP rats in Rf (NP: 170.5±2.8 vs. E: 181±3.5 cycles.min−1, P=0.02) and Ve (C: 2349.1±111.1 vs. E:2769.8±151.8 mL.kg−1.min−1, P=0.03). Likewise, hypoxia‐induced ventilatory responses were increased in HP rats in Rf (NP: 116.9 ±4.1 n= 5 vs. HP: 146.1±4.4 resp.min−1, p=0.0010), VT (NP: 12.0±0.7 vs HP: 16.5 ±1.2 mL.kg−1, p=0.0135) and Ve (NP: 1449.5 ±114.9 vs. HP: 2454.3 ± 175.0 mL.kg−1.min−1, p=0.0013). Our data suggest that juvenile rats submitted to perinatal protein undernutrition had increase in ventilation at rest and in ventilatory responses to hypercania and hypoxia, which could be a potential hidden basis for the development of arterial hypertension in adulthood.

Support: FACEPE and CNPq.
Thiosulfate: a Readily Accessible Source of Hydrogen Sulfide in Oxygen Sensing
Authors: Olson Kenneth R. , DeLeon Eric R. , Gao Yan , Hurley Kevin , Saduskas Victor , Batz Catherine , Stoy Gilbrian
Published: June 2021

We have shown that H2S produced from organic thiol metabolism and metabolized by mitochondrial oxidation is an oxygen sensor in the vasculature and in numerous other oxygen sensing tissues. Thiosulfate, an intermediate in oxidative H2S metabolism, can be disproportionated by reducing agents and regenerate H2S. We propose that this contributes to H2S signaling during hypoxia when the mitochondrial matrix becomes more reduced. H2S formation from thiosulfate in buffers and in a variety of mammalian tissues and in lamprey dorsal aorta was examined in real‐time using a polarographic H2S sensor. Inferences of intracellular H2S production was made by examining hypoxic pulmonary vasoconstriction (HPV) in bovine pulmonary arteries under conditions in which increased H2S production would be expected. In buffer, H2S was spontaneously generated from thiosulfate by the reducing agent dithiothreitol (DTT). Both the magnitude and rate of H2S production was greatly increased in the presence of tissue with the most notable effects occurring in the liver. H2S production was only observed when tissues were hypoxic; exposure to room air, or injecting oxygen inhibited H2S production and resulted in net H2S consumption. DTT also dose‐dependently augmented HPV. Substituting the endogenous reductant, dihydrolipoic acid for DTT also generated H2S and augmented HPV. These results indicate that thiosulfate can contribute to H2S signaling under hypoxic conditions and that this is not only a ready source of H2S production but also serves as a means of recycling sulfur and thereby conserving biologically relevant thiols. Support: NSF 1051627.
Arterial Stiffness in Diet‐induced Obese Mice is Reversed by Weight Loss
Authors: Shiang Tina , Al Sayah Leona , Fry Jessica , Weisbrod Robert , Bajpai Saumendra , Reinhart‐King Cynthia , Cohen Richard , Seta Francesca
Published: June 2021

BACKGROUND

We previously showed that C57Bl/6J male mice fed a diet rich in fat (35%) and sucrose (16.5%) (HFHS) develop obesity, glucose intolerance, insulin resistance, chronic inflammation, cardiac diastolic and kidney dysfunctions compared to normal diet (ND). We also showed that HFHS‐fed mice develop arterial stiffness (AS), measured in vivo by pulse wave velocity (PWV), within 2 mos and systolic hypertension, measured by radiotelemetry, after 6 mos compared to ND.

AIM

We sought to test the hypothesis that AS and hypertension would normalize in obese mice after reversal to ND.

METHODS AND RESULTS

Mice fed HFHS for 5 mos were fed ND for 2.5 mos with bi‐weekly assessments of metabolic and cardiovascular parameters. Diet reversal induced a severe weight loss in obese mice with a 2‐fold reduction in fat mass and hyperinsulinemia and no change in lean mass. PWV was significantly reduced from 5.3 ± 0.7 to 3.37 ± 0.4 m/sec after 1.5 mo reversal to ND. Similarly, systolic blood pressure was significantly decreased at this time point (from 135.7 ± 3.8 to 124.2 ± 2.7 mmHg). Atomic force on aortic rings, an in vitro indicator of structural stiffness of the intima, was increased after HFHS and significantly decreased after 4 mos reversal to ND.

CONCLUSIONS

Obesity‐induced arterial stiffness and hypertension are reversible by fat loss with normalization of metabolic state. NHLBI grant HL105287–01.
Inhibition of Vascular Smooth Muscle Growth by the Soluble Guanylyl Cyclase Activator BAY 60–2770
Authors: Martin Danielle Nicole , Adderley Shaquria P , Joshi Chintamani N , Durante William , Tulis David A
Published: June 2021

Abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) are key components of pathologic vessel growth and efforts aimed at their reduction are clinically essential. Cyclic nucleotide signaling has long been studied for its growth‐mitigating properties, and in the current study we hypothesized that the novel soluble guanylyl cyclase activator BAY 60–2770 (BAY) inhibits proliferation and migration of rat primary VSMCs through PKA and PKG. BAY significantly reduced cell numbers in concentration‐dependent fashion after 72 h (0.1–10 μM) and S‐phase cell numbers (~40%) after 16 h with no effects on cell viability. Interestingly, BAY caused nominal reduction (~20%) in cell migration after 16 h. Mechanistically, BAY significantly induced cGMP in concentration‐and time‐dependent fashion without apparent effects on cAMP. In‐Cell Western analysis showed that BAY significantly increased phosphorylation of vasodilator‐stimulated phosphoprotein (VASP) at Ser157 and Ser239, respective indicators of PKA and PKG signaling. Lastly, BAY doubled PKG activity without observable effects on PKA activity. These data suggest that BAY has capacity to inhibit VSMC proliferation through cGMP/PKG/VASP signaling. This work was supported by NHLBI grants HL59976 and HL81720 and by an ECU Undergraduate Research and Creative Activity Award.
Hydrogen peroxide modulates the contractile response induced by phenylephrine in renal hypertensive rat aorta
Authors: Silva Bruno Rodrigues , Pernomian Laena , Grando Marcella Daruge , Bendhack Lusiane Maria
Published: June 2021

Reactive oxygen species (ROS) can increase contractile responses in hypertensive rat vessels. This study aimed to evaluate the role of the endothelial cells (EC) and ROS to the contraction induced by phenylephrine (PE) in aorta from renal hypertensive (2K‐1C) and normotensive rat (2K). Concentration‐effect curves for PE were constructed in intact endothelium (E+) and denuded aorta (E−), with or without Catalase. Expression of phosphorylated Ser1177eNOS was evaluated by Western‐Blot. In isolated EC incubated with PE, it was measured the fluorescence intensity (FI) of the ROS sensitive dye (DHE) by flow citometry, with or without Catalase. The H2O2 production was measured through assay kit. The maximum contraction to PE was decreased only in 2K‐1C E+, which was partially reversed by Catalase. PE induces higher Ser1177phosphorylation of eNOS in 2K‐1C aorta than in 2K. PE increased the FI only in EC from 2K‐1C, and it was abolished by Catalase. PE stimulated higher H2O2 production in 2K‐1C (E+) than in 2K (E+). In conclusion, the reduced contraction to PE in E+ rat aorta from 2K‐1C is due to high eNOS‐Ser1177 phosphorylation and H2O2 production in the EC. Supported by FAPESP, CAPES and CNPq.
Nitric Oxide Bioavailability in Patients with Cystic Fibrosis
Authors: Harris Ryan A , Seigler Nichole , Fox Brandon , White Claire , Brantley Lucy , Eidson Dabney , McKie Kathleen T
Published: June 2021

Major advances in medical research and therapies have resulted in marked increases in longevity and lung function in patients with cystic fibrosis (CF); however, non‐pulmonary factors have been understudied. We recently published the presence of endothelial dysfunction in young patients with CF who exhibit normal lung function. This study tested the hypothesis that increasing NO bioavailability would improve endothelial function in patients with CF. Seventeen patients (7 boys and 10 girls) with CF (FEV1=89.5±19.4 % predicted) participated in this study. Flow‐mediated dilation (FMD) was performed at baseline and 3 hours following 5 mg/kg of Kuvan™ (BH4) dissolved in apple juice. No differences in FMD was observed following BH4; however, a significant relationship between the change in FMD following BH4 and baseline FMD was observed (r=−0.608; p=0.010). Absolute change in FMD was greater (p=0.047) in boys (0.021±0.007cm) compared to girls (0.012±0.009cm). Additionally, a statistical time x sex interaction (p=0.02) was observed, such that girls exhibited an increase in FMD (0.6±2.3%) following BH4, whereas a decrease in FMD (−2.1±2.0%) was observed in boys. These data indicate that the FMD response to BH4 in a fairly healthy cohort of patients with CF is in part dependent on baseline FMD. Supported in part by the American Heart Association, Child Health Discovery Institute and BioMarin Pharmaceutical Inc. (RAH).
The role of the type III transforming growth factor‐β receptor Tgfbr3 in the regulation of pulmonary vascular development
Authors: Niess Gero , Wygowski Jegor , Seeger Werner , Morty Rory Edward
Published: June 2021

Bronchopulmonary dysplasia (BPD) is associated with poor alveolar and vascular development. The molecular basis of this is not well understood. Expression studies in the lung of hyperoxia (85% O2) treated mice, a model of BPD, have revealed that the protein level of the type III TGF‐β receptor (Tgfbr3) is reduced by 4‐fold. Laser microdissection confirmed downregulated expression (mRNA level) of tgfbr3 in the pulmonary vasculature of the developing mouse lung. Immunohistochemical studies suggested that the changes in TGF‐β receptor expression were largely confined to the pulmonary vascular smooth muscle, suggesting a possible role for these receptors in the smooth muscle tissue in the development of BPD. Knockdown of TGFBR3 in primary pulmonary artery smooth muscle cells (PASMC) increased proliferation by 2‐fold, in a TGF‐β‐independent manner (observed in the presence of the TGF‐β pathway inhibitor SB431542). Migration of PASMC assessed by Boyden Assay was increased 4‐fold. However, apoptosis of PASMC was not affected by the knockdown of TGFBR3. Taken together, these data suggest a role for TGFBR3 in vascular smooth muscle cell function which could lead to a dysregulation of TGF‐β signalling in the pulmonary vasculature which in turn could contribute to the impaired pulmonary vascular growth and development associated with the lung hypoplasia observed in patients with BPD.

German research foundation.
GPCR activation of CFTR through two independent phosphorylation pathways
Authors: Billet Arnaud , Hanrahan John William
Published: June 2021

Cystic Fibrosis Transmembrane conductance Regulator (CFTR) activation by PKA and PKC has been extensively studied, however its regulation by receptors is less well understood. To study signaling upstream of the kinases, we measured whole‐cell Cl‐currents in BHK cells cotransfected with GPCRs and CFTR. In cells expressing the M3 muscarinic acetylcholine receptor, the agonist carbachol (Cch) caused strong activation of CFTR through two pathways; the canonical PKA‐dependent mechanism and a second mechanism mechanism that involves tyrosine phosphorylation. The role of PKA was suggested by partial activation of a mutant lacking tyrosine residues that mediate stimulation by the tyrosine kinase c‐Src. The role of tyrosine kinases was suggested by the ability of Cch to stimulate 15SA CFTR, a mutant which lacks 15 potential PKA sites and is unresponsive to PKA stimulation, and also by the sensitivity of the Cch response to inhibitors of the tyrosine kinase c‐Src. The results suggest that CFTR phosphorylation by PKA and tyrosine kinases both contribute to CFTR regulation by GPCRs that are expressed at the apical membrane of intestinal and airway epithelia.

Supported by GEPROM, GRASP, the McGill (CIHR) Chemical Biology program, and the Canadian Foundation for Innovation.
Sarcopenia and hypertrophy in aged skeletal muscle is independent of lifelong muscle stem cell depletion
Authors: Lee Jonah Dan , Mula Jyothi , Fry Christopher S , Kirby Tyler J , Jackson Janna R , Beggs Jake A , Campbell Marilyn S , Kmiec Tyler E , Dupont‐Versteegden Esther E , McCarthy John J , Peterson Charlotte A
Published: June 2021

Unresolved questions in aged muscle involve whether the loss of muscle stem cells, satellite cells, contributes to sarcopenia and impedes hypertrophy of aged muscle. To directly address these questions, we developed the Pax7‐DTA mouse that conditionally and specifically ablates Pax7+ satellite cells following tamoxifen administration (TM). Four month old mice were treated with TM or vehicle and at 5 (young) or 24 (old) months of age were subjected to a synergist ablation (SA; two weeks of overload) or sham surgery. With age, vehicle treated muscles demonstrated a 52% reduction in Pax7+ satellite cells. TM treated muscles showed >;90% ablation after 1 month and no recovery after 20 months, such that the aged mice lived the majority of their lives with a significantly reduced satellite cell pool. Despite Pax7 depletion with TM, muscle mass, fiber cross‐sectional area and function were all reduced with age independent of Pax7 number. After SA, TM mice increased muscle mass and fiber cross‐sectional area to the same extent as vehicle, with old mice demonstrating an attenuated hypertrophy compared to young. Large myofiber distribution decreased with age and increased with SA, independent of Pax7 number. However, the total number of small fibers (<600 um2), relatively more abundant in aged muscle, were reduced with TM. These data provide convincing evidence the satellite cell niche does not play a role in maintenance of skeletal muscle mass across the lifespan or in hypertrophy of young or old muscle.

Funding: NIH R01AR060701, R21AG34453. Ellison Foundation/American Federation of Aging Research EPD12102.
Mitochondrial division inhibitor (Mdivi‐I) ameliorates post myocardial infarction via stimulating stem cell by elevating level of MiR‐499 in diabetes
Authors: Qipshidze Natia , Mishra Paras K , Tyagi Suresh C
Published: June 2021

Cardio vascular disease (CVD), particularly myocardial infarction (MI) is the major cause of morbidity and mortality in the world wide. The majority of CVD cases occur in patients with underlying acute or chronic heart disease. The incidence of MI increases in the presence of diabetes and increase further in the presence of structural heart disease. It has been recognized that diabetics experience a greater mortality during the acute phase MI and a higher morbidity in the post‐MI period. To improve regeneration of the injured myocardium, it is necessary to enhance the intrinsic capacity of the heart to regenerate itself and/or replace the damaged tissue by cell regeneration. Several studies have demonstrated that microRNAs (miRs) are important for stem cell maintenance and differentiation via translational repression. miR‐499 was produced almost exclusively in the heart. We hypothesize that miR‐499 improves post‐MI condition in diabetic mice via regenerating myocytes and promoting new vessels toward the damaged part, improving myocytes contractility and ejection function. Mdivi‐1 improves miR‐499 in heart post‐MI, which inhibits mitophagy in cardiomyocyte through decreasing Drp‐1 accumulation in mitochondria. Our data shows that miR‐499 regulates the mitochondrial fission, contractility was improved for 34±3% in treated mice. New vessles were formed from ligated part toward the MI part, and ejection fraction was increased for 18±2 % compared to untreated mice. Based on our data we suggest that modulation of miR‐499 levels may provide a therapeutic approach for treating Heart failure in the presence of structural heart defect during diabetes.
GLP‐2 receptor is required for glucose homeostasis and energy balance
Authors: Shi Xuemei , Wen Shixiang , Chang Benny , Chan Lawrence , Guan Xinfu
Published: June 2021

Glucagon‐like peptides (GLP‐1/2) are co‐secreted from enteroendocrine L cells and preproglucagon neurons, and proposed as key promoters of glucose homeostasis and insulin sensitivity after gastric bypass surgery. GLP‐2 receptor (GLP‐2R) activation stimulates intestinal growth and absorption, while decreasing gut motility to inhibit food intake. However, it is unknown if GLP‐2 plays a physiological role in the long‐term control of energy balance and glucose homeostasis. To determine the physiological relevance of GLP‐2R in glycemic control and body adiposity, we generated Glp2r knockout (KO) mice. First, we validated that Glp2r mRNA and protein were not expressed in the KO gut and brain, and GLP‐2‐stimulated cell proliferation was negated in the KO gut. In comparison with wide‐type (WT) littermates, Glp2r KO mice displayed higher (+38%) fat body mass and lower lean body mass. The KO mice had higher food intake during refeeding while basal energy expenditure was not altered. Moreover, the KO mice displayed higher levels of fasting glucose and slower removal of postprandial glucose. After ip glucose challenge, the KO mice showed glucose intolerance. Using 6,6‐2H2‐glucose and 2H2O tracers, we quantified that the KO mice had higher basal hepatic glucose production (5.44 ± 0.20 vs 7.02 ± 0.26 mmol/kg/h, respectively, for the WT and KO mice), while they had lower glucose infusion rate (GIR) during hyperinsulinemic euglycemic clamp. Furthermore, iv infusion of GLP‐2 (500 pmol/kg/h) increased the GIR by +33% in the WT mice, but not in the KO mice. GLP‐2‐mediated improvement in insulin sensitivity was largely attributed to enhancing glucose disappearance rate during insulin stimulation. Our data indicate that Glp2r deficiency displays fasting hyperglycemia, glucose intolerance, and insulin resistance, pointing to a novel physiological role of GLP‐2R in glucose homeostasis and insulin sensitivity. In addition, Glp2r deficiency displays hyperphagic obesity, revealing a long‐term effect of GLP‐2R in energy balance.
Roux‐en‐Y Gastric Bypass Surgery Prevents Diet‐Induced Hepatic Steatosis and Endoplasmic Reticulum Stress in the Liver of Obese Rats
Authors: Mosinski John David , Pagadala Mangesh , Huang Hazel , Dan Olivia , Shimizu Hideharu , Batayyah Esam , Schauer Philip R. , Brethauer Stacy A. , Kirwan John P.
Published: June 2021

Obesity is associated with hepatic lipid accumulation, which may increase endoplasmic reticulum stress and contribute to insulin resistance. We hypothesized that bariatric surgery would counter the effects of diet‐induced obesity and prevent steatosis and endoplasmic reticulum (ER) stress in the liver. Sixteen adult male Sprague‐Dawley rats were randomized to Sham (N=8) or Roux‐en‐Y gastric bypass (RYGB) surgery (N=8), and compared to a lean control (N=8) group. Rats were fed a high fat diet for 90 days and insulin resistance was assessed by oral glucose tolerance test and insulin tolerance test. Liver was subsequently assessed for hepatic steatosis, triglyceride accumulation, and ER stress related gene and protein expression. Body weight was decreased by 20.7±6% (P<0.01) and insulin sensitivity was improved (P<0.05) in RYGB versus Sham. ER stress gene and protein expression was normalized in the RYGB group compared to Sham, and was similar to lean controls. Specifically, glucose‐regulated protein 78 (P<0.001), X‐box binding protein‐1 (XBP‐1) and spliced XBP‐1 (P<0.01) was lower in RYGB compared to Sham. Spliced XBP‐1 protein expression was also lower in RYGB versus Sham (P<0.05). These data support the metabolic effectiveness of RYGB surgery and suggest that in rats fed a high fat diet, the prevention of hepatic steatosis and reversal of insulin resistance after RYGB surgery may be mediated through attenuation of hepatic ER stress responses.