bluuebunny/crossref_metadata_2025 huggingface dataset:
5‐Hydroxytrptamine 7 receptors (5‐HT7R) in the nucleus tractus solitarii (nTS) modulate synaptic transmission.
Authors: Matott Michael P , Kline David D
Published: June 2021

5‐HT is a potent cardiorespiratory neuromodulator. 5‐HT7Rs are functionally located in the nTS, the central termination site of sensory afferents, where they modulate chemo and autonomic reflexes. Here we examined the mechanism of 5‐HT7Rs action in nTS by examining their influence on synaptic and membrane properties. Spontaneous (s) and solitary tract (TS)‐evoked excitatory post‐synaptic currents (EPSCs), and membrane potential, were examined in rat nTS slices following 5‐HT7R activation (LP‐44; 2–100 μM). Regardless of concentration, 5‐ HT7R activation decreased TS‐EPSC amplitude in the majority of cells (21/34); the remaining cells increased (11/34) or did not change TS‐EPSC amplitude. The magnitude of decrease was concentration‐related; with 100 μM producing complete synaptic failure in 40% of cells. 5‐HT7R‐induced synaptic depression was of presynaptic origin as indicated by an increased paired pulse ratio (PPR) of consecutive TS‐EPSCs. Conversely, 5‐HT7R‐induced synaptic augmentation did not change the PPR, suggesting a postsynaptic mechanism. 5‐HT7R did not alter sEPSCs or membrane potential, but did eliminate spiking to current‐depolarization in 24% of cells, with the remaining cells showing an increased spike half‐width. Altogether, these results suggest 5‐ HT7R's modulate cardiorespiratory reflexes by modulating nTS neurons via pre‐ and post‐synaptic mechanisms.
Acute hypoxia (AH) increases Fos‐IR in nNOS and AVP cells in the paraventricular nucleus of the hypothalamus (PVN)
Authors: Coldren Kevin Max , McCalmon Sean P. , King T. Luise , Kline David D. , Hasser Eileen M. , Heesch Cheryl M.
Published: June 2021

Acute hypoxia activates peripheral chemoreceptors. Previously we reported that PVN‐projecting second order nTS neurons were activated (Fos‐IR) by AH. We hypothesized that, within the PVN, AH increases Fos‐IR in RVLM projecting, nNOS, and vasopressin (AVP) neurons. Microinjection of fluorogold (FG) in the RVLM retrogradely labeled RVLM‐projecting PVN neurons. Rats (n = 6) underwent either 3 h normoxia (N, 21% O2) or 3 h AH (10% O2). Immunohistochemistry was performed on forebrain sections and cell phenotypes were quantified. AH increased the total number of Fos‐IR cells (N= 24±2; AH= 118±14) at Bregma ~ −1.8 in the PVN. Also at this level, AH increased the number and % of nNOS cells that were activated (nNOS‐Fos) (N = 3±.0.4, 2± 0.4%; AH =15± 2, 12±1%). Activation of AVP cells (AVP‐Fos) tended to increase (P = 0.06) with AH (N = 1±.0.3, 2± 0.5%; AH = 5± 1, 7±2%). Although AH increased the number of nNOS cells more caudally (Bregma ~ −2.1), activation of RVLM‐projecting cells at either level of the PVN was unchanged by AH. These preliminary data suggest that activation of nNOS and AVP containing cells in the PVN contribute to cardiorespiratory responses to AH. NIH HL 98602.
Exercise Training (ExT) Normalizes Subfornical Organ (SFO)‐ Mediated Sympathoexcitation in Chronic Heart Failure (HF)
Authors: Llewellyn Tamra L , Zheng Hong , Sharma Neeru M , Patel Kaushik P
Published: June 2021

ExT normalizes sympathoexcitation (SymEx) exhibited in HF, however the underlying mechanisms are not clear. The SFO is involved in dictating SymEx and is influenced by peptides such as angiotensin (Ang) II due to its weak blood‐brain barrier. We hypothesized that neuronal activity in the SFO would be enhanced in HF and contribute to increased Ang II‐mediated SymEx, and that ExT would reverse this process. HF induced by coronary artery ligation in Sprague‐Dawley rats was confirmed by echocardiography 4 weeks after surgery. HF rats displayed increased neuronal activity in the SFO, as assessed by FosB immunohistochemistry (101 ± 9 vs. 29 ± 2 cells). This increase was attenuated after 4 weeks of treadmill ExT (36 ± 7 cells). In anesthetized rats, microinjection of Ang II into the SFO increased renal sympathetic nerve activity (RSNA) to a greater extent in HF (23 ± 3 vs. 9 ± 1%; 100 pmol), and was normalized after ExT (8 ± 2%). ExT also abolished the decrease in RSNA in HF rats after losartan microinjection into the SFO (−21 ± 4 vs. −1 ± 3%; 200 pmol), implying higher endogenous Ang II tone in HF. ExT reversed the elevated mRNA and protein expression of AT1R in the SFO of rats with HF. The enhanced activation of the SFO by elevated tonic Ang II contributes to the enhanced SymEx exhibited in HF. The reduction in AT1 receptors in the SFO by ExT may be responsible for restoring the neuronal activation in the SFO and SFO‐mediated SymEx. Supp. by NIH HL62222.
Sympathetic perivascular nerves contribute to hypoxic transformation of smooth muscle phenotype in ovine cerebral arteries
Authors: Adeoye Olayemi O , Silpanisong Jinjutha , Williams James M , Pearce William J
Published: June 2021

In addition to its role in angiogenesis, VEGF may also contribute to arterial remodeling, possibly through activation of VEGF receptors on sympathetic nerves leading to altered trophic input to the arterial wall. To test this hypothesis normoxic and chronically hypoxic fetal sheep (110 days at 3820m) underwent unilateral superior cervical sympathectomy (SANX) at 124d gestation, and then were harvested at 138d (term). In middle cerebral arteries (MCA), hypoxia increased dopamine beta hydroxylase 360% and SANX ablated this increase. Hypoxia also decreased by 34% colocalization between Non‐Muscle Myosin Heavy Chain (MHC) and smooth muscle α‐actin (AA) indicating increased contractile differentiation; SANX increased this colocalization 106%. Conversely, hypoxia increased by 124% SM2‐MHC colocalization with AA, again indicating increased differentiation; SANX decreased this colocalization by 81%. Organ culture of MCA with NE decreased colocalization of SM2‐MHC with AA by 88% in intact arteries, but increased it 62% in SANX arteries; all effects of NE were blocked by prazosin. These results suggest that hypoxic increases in VEGF alter sympathetic release of NE and/or other factors that transform the contractile phenotype of cerebrovascular smooth muscle. This transformation appears to be a central feature of hypoxic cerebrovascular remodeling in the fetus. Supported by PHS# P01‐HD31226.
Remodeling and impaired dilation in middle cerebral arteries from Deoxycorticosterone acetate (DOCA)‐salt rats
Authors: Matin Nusrat , Pires Paulo Wagner , Garver Hannah , Dorrance Anne McLaren
Published: June 2021

Genetic hypertension causes inward remodeling and impaired dilation in the middle cerebral artery (MCA). DOCA‐salt rats are widely used model of hypertension, yet few studies have been performed on their cerebral arteries. We hypothesized that MCAs from DOCA‐salt hypertensive rats will have a reduced lumen diameter and impaired endothelium dependent dilation. Male Sprague‐Dawley rats had uninephrectomy, and received either DOCA (150mg/kg) and salt water (1% NaCl and 0.2% KCl) or tap water. MCAs were studied by pressure myography. Data are mean±SEM, SHAM vs DOCA. DOCA showed impaired dilation to 10 μM ADP (change from baseline: 41.2±5.2 vs 24.7±4.7%, p< 0.05, ANOVA). Lumen diameter was reduced (295±18 vs 343±13 μm at 80 mmHg, p <0.05, ANOVA) in the DOCA rats. Interestingly the wall/lumen ratio (0.06±0.02 vs 0.07±0.01 at 80 mmHg, p>;0.05, ANOVA) did not change. These data suggest the MCAs from DOCA rats may exhibit inward hypotrophic remodeling. These data are contrary to the inward hypertrophic remodeling observed in MCAs from genetically hypertensive rats and imply that the mechanism of remodeling in DOCA‐salt rats may be different.
Impact of physical activity on age‐related insulin resistance: The contribution of post‐transcriptional gene silencing.
Authors: Bunprajun Tipwadee , Chatsudthipong Varanuj , Pedersen Bente K. , Green Charlotte J.
Published: June 2021

Insulin sensitivity decreases during aging, reflected by impaired insulin‐stimulated glucose uptake into skeletal muscle. It has been suggested that lifestyle changes, especially physical activity, may enhance insulin action in aged muscle. To investigate how physical activity improves age‐related insulin resistance, we studied muscle satellite cells isolated from young, ageing sedentary and ageing active participants matched for BMI. Ageing sedentary myocytes had markedly reduced insulin‐stimulated glucose uptake compared to young myocytes whereas ageing active myocytes retained insulin sensitivity. However, PKB phosphorylation in response to insulin remained intact in all groups. Interestingly, there was an up‐regulation of microRNA (miR) 193b and 195 expressions in ageing sedentary but not ageing active myocytes when compared to young. Our findings indicate that physical activity may help to maintain insulin sensitivity in ageing muscle potentially via a microRNA dependent and PKB independent mechanism.

This work was supported by the Royal Golden Jubilee Program and FP7 EU consortium, myoage.
Carboxylesterase 1 gene duplication and mRNA expression in adipose tissue are linked to obesity and metabolic function
Authors: Friedrichsen Martin , Poulsen Pernille , Wojtaszewski Jørgen , Hansen Peter Riis , Vaag Allan , Rasmussen Henrik Berg
Published: June 2021

Context and aims

Carboxylesterase 1 (CES1) plays an important role in the control of the metabolism of triglycerides and cholesterol in various cell types including adipocytes and hepatocytes. In this study we investigated CES1 mRNA expression in adipose tissue as well as CES1 gene duplication and their association with measures of adiposity and metabolic function in a large twin population.

Methodology

295 monozygotic and dizygotic twins (62–83 years) were subjected to a standard OGTT and excision of abdominal subcutaneous fat biopsies. Levels of CES1 mRNA and DNA copy number of the CES1 gene were assessed by quantitative PCR.

Results

CES1 mRNA expression in adipose tissue was positively associated with BMI (P<0.001), insulin resistance ([HOMA], P=0.003) and fasting levels of glucose (P=0.002), insulin (P=0.006), and triglycerides (P=0.003). Gene duplication of CES1 was not linked to altered CES1 mRNA expression (P=0.63). Nonetheless, CES1 gene duplication was associated with increased glucose tolerance (P=0.03) and decreased insulin resistance (P=0.02).

Conclusion

CES1 mRNA expression in adipocytes as well as CES1 gene duplication were associated with measures of glucose metabolism, which raises the possibility of a potential role for this enzyme in the development of type 2 diabetes.
Chymase Protein Expressed in Principal Cells of Inner Medullary Collecting Ducts in Diabetic Kidney Disease
Authors: Wood Kirsten Ali , Ford Stephen M. , Berner Paul M. , Naqvi Nawazish , Husain Ahsan , Harrison‐Bernard Lisa M.
Published: June 2021

Chymase, a serine protease contained in mast cells granules, is increased in mesangial cells, podocytes, and vascular smooth muscle cells under hyperglycemic conditions. We tested the hypothesis that chymase protein expression is enhanced in type II diabetic kidneys. Kidneys and blood were harvested from 18‐ (AJP 298:F37‐F48, 2010) and 36‐wk‐old diabetic and control mice. Body weight (54 ± 1 vs 30 ± 0 g), blood glucose (534 ± 18 vs 154 ± 6 mg/dl), and plasma insulin (20 ± 6 vs 2 ± 1 μg/L) were significantly higher in 18‐wk‐old diabetic compared to control mice. The 36‐wk‐old diabetic mice (n=7) had significantly lower body weight (33 ± 3 vs 40 ± 2 g), normal insulin (0.8 ± 0.1 vs 0.4 ± 0.1 μg/L), but higher plasma glucose (552 ± 40 vs 220 ± 21 mg/dl) compared to control (n=8) indicating transitioning to type I diabetes. Diabetic kidneys exhibited 2 ± 0.4 fold subcapsular mast cells compared to control (p<0.05). Nominal intrarenal chymasepositive mast cells were identified. Diabetic cortex and papilla density of chymase protein expression was 0.5 ± 0.1 and 28 ± 13 fold compared to control (Image Pro; p<0.05). Chymase protein was expressed in principal cells of collecting ducts suggesting increased serine protease activation of the epithelial sodium channel in diabetes. In conclusion, we propose that papillary chymase expression may be positively correlated with the progression of diabetic kidney disease. APS UGSRF, NHLBI, AHA GIA
Inhibition of Hyperhomocysteinemia‐Induced Inflammasome Activation and Glomerular Sclerosis by NLRP3 Gene Deletion
Authors: Boini Krishna M. , Xia Min , Abais Justine M. , Zhang Yang , Gehr Todd W. , Li Pin‐Lan
Published: June 2021

Hyperhomocysteinemia (hHcys) has been reported to initiate NLRP3 inflammasome formation and activation in podocytes, leading to glomerular dysfunction and sclerosis. However, it remains unknown whether Nlrp3 gene is critical for the activation of inflammasomes in glomeruli of hHcys mice. Uninephrectomized Nlrp3 knockout (Nlrp3−/−) and wild type (Nlrp3+/+) mice were fed a folate free (FF) diet or normal chow (ND) for 4 weeks to produce hHcys. HPLC analysis revealed that plasma Hcys levels were significantly elevated in both Nalp3−/− and Nalp3+/+ mice fed a FF diet compared to ND fed mice. The FF diet significantly increased the colocalization of Nlrp3 with ASC or caspase‐1 in glomeruli of Nlrp3+/+, but not in Nlrp3−/−mice, suggesting that hHcys‐enhanced the formation of Nlrp3 inflammasomes. Furthermore, colocalization of Nlrp3 with podocin indicates the enrichment of Nlrp3 in podocytes. In addition, biochemical analysis demonstrated that hHcys significantly increased the caspase‐1 activity and IL‐1β production in Nlrp3+/+mice, but not in Nlrp3−/− mice. Correspondingly, the glomerular damage index (GDI) and urinary protein excretion were significantly higher in Nalp3+/+ mice compared to ND fed mice. However, the hHcys‐induced increase in GDI and proteinuria were significantly lower in Nlrp3−/− than in Nlrp3+/+mice (GDI in Nlrp3+/+: 2.9 ± 0.1 vs. Nlrp3−/−: 1.3 ± 0.1). Immunocytochemical analysis showed that hHcys decreased expression of podocin and nephrin, but increased desmin, CD43 and F4/80 expression in glomeruli of Nlrp3+/+ mice compared to Nlrp3−/− mice. Based on these results, it is concluded that Nlrp3 is an essential component of Nlrp3 inflammasomes and that targeting Nlrp3 may be important therapeutic strategy to prevent inflammasome activation and thereby protect podocytes and glomeruli from hHcys‐induced injury (supported by NIH grants DK54927 and HL75316).
Cyclic stretch induces IL‐8 secretion in human proximal tubular epithelial cells through cPLA2, MAPK, and NF‐κB signaling pathways
Authors: Pham Khoi M , Hamzeh Mona T , Alexander Larry D
Published: June 2021

Cyclic mechanical stretch represent a unique model to mimic transient increase in intrarenal pressure resulting in tubular mechanical stretch accompanying obstructive nephropathy and may provide a mechanism to stimulate cytokine/chemokine protein expression. The purpose of this study was to investigate the effects of cyclic mechanical stretch on interleukin‐8 (IL‐8) secretion in human proximal tubular epithelial cells (HK‐2). The concentration of IL‐8 was analyzed by ELISA, phosphorylated p38 MAPK, c‐Jun NH2‐terminal kinase (JNK), and extracellular signal‐regulated kinase (ERK1/2) was assessed by Western blot analysis, while nuclear factor‐κB (NF‐κB) was analyzed by an ELISA‐based NF‐κB‐binding assay. Exposure to cyclic mechanical stretch (20%) resulted in a time‐dependent increase in IL‐8 secretion. In addition, we showed that cyclic stretch induced the phosphorylation of p38 MAPK, ERK1/2, and JNK in HK‐2 cells. Cyclic stretch also increased NF‐κB‐binding activities and inhibition of NF‐κB prevented stretch‐induced IL‐8 secretion. Moreover, we showed that selective inhibitors of PLA2 (Mepacrine), cPLA2 (MAFP and AACOCF3), ERK1/2 (PD98059), JNK (SP600125), and p38 MAPK, could suppress the effect of cyclic stretch on IL‐8 secretion, which strongly suggests that these pathways play important roles in the stretch‐induced IL‐8 secretion in renal proximal tubular epithelial cells.
The HDAC inhibitor, vorinastat, prevents TGF‐β1 induced EMT and apoptosis in human renal proximal tubular cells
Authors: Hussain Faryal , Brimble Elise , Dickhout Jeffrey G.
Published: June 2021

Renal interstitial fibrosis is characterized by renal fibroblast proliferation. This process involves renal tubular epithelial cells adopting a fibroblast morphology through epithelial‐to‐mesenchymal transition (EMT). EMT produces phenotypic changes such as epithelial cell adherins junction loss and de novo α‐smooth muscle actin (SMA) expression. TGF‐ß1 has been shown to stimulate EMT. Identifying agents that inhibit TGF‐ß1 induced EMT may prevent patient progression towards chronic renal failure (CRF). Previous work from our laboratory has shown that 4‐phenylbuterate (4‐PBA), a histone deacetylase inhibitor (HDACi), inhibited EMT. In this study, we hypothesized that a broad spectrum HDACi, vorinastat, would inhibit TGF‐ß1 induced EMT and apoptosis in human proximal tubular epithelium (hPTE). We tested vorinastat and 4‐PBA on TGF‐ß1 induced EMT in hPTE. TGF‐ß1 induced EMT was inhibited by both 1mM 4‐PBA and 5μM vorinastat as shown by cadherin junction preservation and reduced de novo α‐SMA expression. Vorinastat was also shown to prevent TGF‐ß1‐induced decrease of E‐cadherin and increase of type I collagen transcript levels. Further, 4‐PBA and vorinastat inhibited TGF‐ß1 induced apoptosis in hPTE. These findings suggest that HDACi prevent EMT and apoptosis induced by TGFß1 and may reduce the progression of patients with chronic kidney disease towards CRF. Funding, CIHR OSO‐115895.
Heparan Sulfate‐Bound Dimeric IL‐2: A Potential Mediator of Renal Ischemic Injury
Authors: Dixon Lauren A , Miller John D , Clabaugh Suzanne E , Liu Gino C , Stuever Mary F , Wrenshall Lucile E
Published: June 2021

Interleukin‐2 (IL‐2) is a cytokine critical to normal immune function. Our laboratory has shown that IL‐2 is retained in tissues by heparan sulfate (HS). While known as a monomer, a dimeric form of IL‐2 was previously identified in fish optic neurons, and this form was toxic to oligodendrocytes. Given this observation, we asked whether dimeric IL‐2 is found in mammalian tissues. Murine and human aorta and kidney were assessed by Western blot for the presence of IL‐2. Dimeric (30 kD) IL‐2 was identified in each tissue. Heparinase digestion of tissues liberated dimeric IL‐2, suggesting that the dimer is bound, at least in part, by HS. To ascertain whether dimeric IL‐2 is cytotoxic, we treated cultures of renal epithelial cells with increasing concentrations of dimeric IL‐2, isolated by electroelution. Signs of cytotoxicity were evident within 15 minutes of dimer addition. Commercial IL‐2, isolated identically to dimeric IL‐2, was not cytotoxic. Systemic administration to mice of 10 μg of dimeric IL‐2 induced vacuolization of renal epithelium, a morphology typically seen with ischemic injury. Finally, murine kidneys subjected to 60 minutes of ischemia, compared to sham controls, expressed greatly increased amounts of dimeric IL‐2 in tissue homogenates. These results suggest that dimeric IL‐2 may contribute to acute tubular necrosis and, in turn, renal dysfunction. This work was supported by institutional funds.
Meprins cleave OS‐9 present in mouse kidneys subjected to ischemia reperfusion acute kidney injury
Authors: Conley Sabena , Martin Barry , Ongeri Elimelda Moige
Published: June 2021

Meprins, metalloproteinases that are highly expressed in the brush border membranes of proximal kidney tubules have been shown to intensify renal injury in ischemia reperfusion (IR). Osteosarcoma‐9 (OS‐9) has been shown to interact with meprin β and the hypoxia inducible factor, HIF‐1α, suggesting a role for OS‐9 in the hypoxia response. However, it's not known if OS‐9 is a meprin substrate. The objective of this study was to determine if OS‐9 is a meprin substrate, and whether this interaction is significant in the hypoxia response in vitro and in vivo. Hypoxia was induced in human embryonic kidney cells (HEK293) transfected with meprin α or meprin β cDNA by exposure to cobalt chloride. Western blot analysis was used to determine the levels of HIF‐1α and OS‐9 in cytosolic‐, nuclear‐ and membrane‐enriched fractions. To determine if OS‐9 is a meprin substrate, purified OS‐9 was incubated with activated forms of homomeric meprin A (α‐α) and meprin B (β‐β). Fragmentation of OS‐9 in the kidney proteins from wild‐type and meprin knockout mice subjected to IR was also evaluated by Western blot. OS‐9 was degraded by meprin B, but not homomeric meprin A. Stabilization of nuclear HIF‐1α was observed in cobalt chloride treated cells, but there was no clear correlation between nuclear levels of HIF‐1α and OS‐9. Fragmentation of OS‐9 was observed in WT, but not meprin KO kidneys subjected to IR, suggesting in vivo degradation.
A mouse model of accelerated aging due to a defect in DNA repair
Authors: Wack Kathryn E. , Gregg Siobhan Q. , Robinson Andria Rasile , Ross Mark A. , Rigatti Lora , Stolz Donna Beer , Niedernhofer Laura J
Published: June 2021

Aging results in the loss of kidney function, leading to increased risk of chronic kidney disease in the elderly. With this demographic rapidly rising, it is critical to understand the molecular basis for age‐associated kidney dysfunction. To address this, we utilize a murine model (Ercc1−/Δ mice) of human progeria, an accelerated aging disease, caused by reduced ERCC1‐XPF DNA repair endonuclease expression. These mice accumulate endogenous DNA damage faster than normal organisms and develop premature onset of aging‐related degenerative diseases over their 28‐wk lifespan. To determine if renal changes that occur with normal aging occur in Ercc1−/Δ mice, we compared kidneys from Ercc1−/Δ mice to old (24–36 month) wild‐type (WT) mice. 23 wk Ercc1−/Δ mice exhibited glomerulopathies similar to that observed in old WT kidneys including albuminuria, fibrosis, podocyte effacement and GBM thickening, indicating glomerular sensitivity to endogenous DNA damage. Endothelial activation, macrophage infiltration, increased smooth muscle actin expression, and decreased Klotho expression were comparable between old WT and the 23 wk Ercc 1−/Δ mice. These data demonstrate that endogenous DNA damage drives age‐related renal degeneration, and therefore the utility of the Ercc1−/Δ model for discovering the molecular mechanism by which damage drives aging and for testing therapeutic strategies to delay renal pathology.
Bisphenol A (BPA) exposure perturbs cardiac electrical conduction in excised rat hearts
Authors: Posnack Nikki Gillum , Jaimes Rafael , Asfour Huda , Swift Luther M , Wengrowski Anastasia M , Sarvazyan Narine , Kay Matthew W
Published: June 2021

Bisphenol A (BPA) is used to produce polycarbonate plastics and epoxy resins that are used in everyday products. Biomonitoring studies indicate that >;90% of the population is routinely exposed to BPA. Recent studies reported alarming correlations between increased BPA urinary concentrations and cardiovascular disease; yet the direct effects of BPA on the heart are unknown. Our goal was to measure BPA's effect on cardiac impulse propagation ex vivo, using excised rat hearts. BPA exposure (1–100 μM) resulted in delayed atrioventricular conduction, prolonged PR segment, lengthened QRS interval and alteration of QRS morphology, decreased epicardial conduction velocity and prolonged action potential duration. These effects were observed after acute exposure (<15min), underscoring the potential detrimental effects in humans with continuous exposure. Higher BPA exposure (100 μM) also resulted in dropped ventricular beats and eventually led to complete heart block. Our results show that acute BPA exposure is cardiotoxic and perturbs electrical conduction in excised rat hearts. These findings emphasize the importance of examining BPA's effect on heart electrophysiology and determining whether chronic in vivo exposure can exacerbate conduction abnormalities in patients with pre‐existing heart conditions and other high‐risk populations. Supported by the NIH (F32 ES019057).
Implantable micro‐Electrode Array for Long‐term ECG Recording in Zebrafish
Authors: Cobo Angelica Maria , Yu Fei , Zhao Yu , Gu Jie , Cao Hung , Tai Yu‐chong , Hsiai Tzung K.
Published: June 2021

Zebrafish is an emerging model for cardiac electromechanical coupling owing to its regenerative capacity after 20% ventricular amputation. We hereby developed a micro‐electrode array (MEA) for both long‐term and real‐time monitoring of zebrafish electrocardiogram (ECG) in response to injury and regeneration. The gold sputtered 5‐lead MEA was formed on a parylene base by micro‐fabrication processes (Fig. 1A). To test the MEA, we sedated Zebrafish in 0.04% tricaine methanesulfonate, followed by a 2‐mm‐long horizontal incision at 0.5 mm caudally to the heart. The MEA was embedded into the incision site so that the four working electrodes (WE) were close to the heart and the reference electrode (RE) positioned on the fish body. The entire device was then secured by medical epoxy (Fig. 1B). The ECG signal recording was performed by connecting the MEA to a differential amplifier and a computer loaded with Labview. The signal was processed using wavelet transform and thresholding algorithm. The ECG obtained from the top working electrode is shown in Fig. 1C. The processed data were comparable with ECG recorded by conventional electrodes in terms of signal‐to‐noise ratio (SNR) and patterns. The implantable devices remained attached to zebrafish for several days, allowing for reliable long‐term and multi‐site recordings for heart regeneration research. This research was supported by NIH HD069305 and HL111437 (TKH).
Glutaredoxin 1 regulates endothelial barrier function via activation of AKT/eNOS pathway
Authors: Han Jingyan , Cohen Richard , Matsui Reiko
Published: June 2021

The integrity and barrier function of the endothelial cell (EC) monolayer lining the vasculature are critical for the maintenance of cardiovascular homeostasis. Vascular hyper‐permeability is an essential component of inflammation, atherosclerosis and tumor metastasis. Glutaredoxin1 (Glrx), a cytosolic enzyme that reduces S‐glutathionylation (R‐SSG), may activate signaling molecules that are inhibited by R‐SSG including Akt and eNOS. We investigated the role of Glrx in endothelial barrier function. We found that adenoviral Glrx overexpression in human umbilical vein endothelial cells (HUVEC) increased the EC permeability manifested by elevated FITC‐dextran influx across the HUVEC monolayer. This was accompanied by increased phosphorylation of Akt (S473) and eNOS (S1177). Inhibition of eNOS by L‐NAME decreased Glrx‐mediated EC permeability indicating the importance of nitric oxide. Glrx‐induced eNOS phosphorylation was diminished by siRNA‐mediated depletion of Akt1. The PI3K inhibitor, LY294002, completely abolished Grx1‐induced phosphorylation of Akt and eNOS. These results together suggest that Glrx, at least in part via activation of the PI3K/Akt1/eNOS signaling pathway, increases the basal endothelial permeability.

Funding: R37 HL104017
Focal adhesion kinase leads paxillin in focal adhesion assembly at lamellipodial protrusion of migrating endothelial cells
Authors: Hu Ying‐Li , Lu Shaoying , Szeto Kai W. , Wang Yingxiao , Lasheras Juan C. , Chien Shu
Published: June 2021

The dynamics of focal adhesions (FAs) can regulate cell migration. Focal adhesion kinase (FAK) and paxillin are involved in FA disassembly and formation. We used double‐color imaging to monitor concurrently the spatial‐temporal dynamics of FAK and paxillin in live endothelial cells (ECs) co‐transfected with GFP‐FAK and DsRed‐paxillin. The fluorescence intensities (FI) of paxillin‐FAs and FAK‐FAs were determined in cell front, center and rear. The mean FAK/Paxillin FI ratio (n=5) is highest at cell front (mean±SEM: 4.73±0.48), not significantly different from 1 at cell center (0.95±0.11), and lowest at cell rear (0.64±0.05). In ECs co‐transfected with RFP‐actin and GFP‐FAK, FAK is found to be associated with actin filaments at cell leading edge. Determination of the time difference between the assemblies of FAK and paxillin at nascent FA of lamellipodial protrusion (LP) by computational analysis indicates that FAK reaches its maximum intensity earlier than paxillin (mean±SEM: 3.63±0.68 min). Our results suggest that FAK is assembled in FAs at LP earlier than paxillin. While both FAK and paxillin play important roles in modulating FA dynamics, FAK promotes FA formation ahead of paxillin at the cell leading edge. This work was supported by NHLBI Research Grants HL‐104402 and HL‐106579 (S.C.).
Extracellular matrix associated protein CCN1 regulates shedding of endothelial ICAM1
Authors: Ringo Katherine , Norman Rosemary , Young Jennifer L
Published: June 2021

The extracellular matrix (ECM) provides essential contextual cues for appropriate cellular behavior. CCN1, an ECM associated protein, signals through integrins and heparan sulfate proteoglycans (HSPGs) to mediate multiple cellular responses including proliferation, differentiation, adhesion, migration, and apoptosis. Gene array analysis has identified CCN1 as one of the most highly upregulated genes in a number of TNFα‐mediated inflammatory disorders, including asthma, hyperoxia‐ and ventilator‐induced lung injury, and rheumatoid arthritis. Cytokines such as TNFα upregulate intercellular adhesion molecule 1 (ICAM1) expression in endothelial cells and stimulate shedding of soluble ICAM1. We demonstrate that in endothelial cells in culture, CCN1 inhibits TNFα‐stimulated increases in ICAM1 protein and this inhibition is transcription‐independent. Interestingly, we found that CCN1 promotes shedding of ICAM1 from TNFα‐stimulated endothelial cells via the action of a metalloproteinase. Further, we show that CCN1 acts through αV integrins but not HSPGs to regulate ICAM1 shedding. Together, these findings establish a novel role for CCN1 in regulating ICAM1, and identify CCN1 as a potential therapeutic target in inflammatory disorders. Supported by American Heart Association and NIEHS ES7026.
ERM proteins may regulate deactivation of PAF‐induced endothelial hyperpermeability
Authors: Mujica Patricio , Durán Walter N.
Published: June 2021

Endothelial hyperpermeability is a hallmark of inflammation. Endothelial barrier function is restored after a period of hyperpermeability, but the mechanisms that deactivate hyperpermeability are largely unknown. Exchange protein activated by cAMP (Epac) is emerging as a possible deactivating/restorative factor in the endothelium. Epac localization, which is modulated by Ezrin/Radixin/Moesin (ERM) proteins, may determine its function due to cellular compartmentalization of cAMP signaling. We report preliminary results testing the hypothesis that ERM proteins facilitate deactivation of hyperpermeability by localizing Epac to the plasma membrane. We demonstrated association between Epac and ERM proteins in human microvascular endothelial cells (HMVEC) by co‐immunoprecipitation. Depletion of Radixin and Moesin by siRNA increased baseline HMVEC monolayer permeability to macromolecules, as well as PAF (platelet‐activating factor)‐induced permeability. Inhibition of PKC abrogated PAF‐induced ERM phosphorylation. We propose that deactivation of PAF‐stimulated hyperpermeability is implemented in a time‐orchestrated manner, by PAF‐induced activation of ERM proteins, which locate Epac to the plasma membrane promoting its interactions with endothelial junctional proteins. (Supported by NIH grants 5RO1 HL070634 & 5RO1 HL088479).
C‐type natriuretic peptide treatment and cardiovascular function in spontaneously hypertensive rats.
Authors: Costa María Angeles , Caniffi Carolina , Cerniello Micaela , Romero Mariana , Sueiro Laura , Radionovas Vanesa , Arranz Cristina
Published: June 2021

The aim was to evaluate the effects of C‐type natriuretic peptide (CNP) on systolic blood pressure (SBP), cardiac and vascular function, and nitric oxide synthase (NOS) activity on cardiac left ventricle (LV) and aorta artery (AA), in spontaneously hypertensive rats (SHR).

Methods

12 weeks‐old male SHR were infused (14 days, osmotic pumps) with CNP (0,75 μg/hr.rat) or saline (S). SBP (mmHg) was recorded and echocardiography was performed. Animals were decapitated and NOS activity (pmol 14C L‐citrulline/g.tissue. min) was measured in AA and LV. Vascular reactivity in AA was studied, evaluating maximal response to phenylephrine (Rmax) and concentration of acetylcholine (expressed as negative log molar) producing 50% of maximum relaxation (pEC50). Statistics: t‐test.

S
CNP

SBP
175±3
159±5*

Heart rate (bpm)
435±10
434±15

End‐diastolic volume (ml)
0,16±0,02
0,23±0,03*

End‐systolic volume (ml)
0,05±0,02
0,07±0,03

Stroke volume (ml/min)
0,11±0,01
0,16±0,02*

Cardiac output (ml)
47,8±2,0
68,8±7,4*

Ejection fraction %
69±6
71±6

Fractional shortening %
34±5
35±4

NOS LV
255±5
371±8*

NOS AA
117±16
202±4*

Rmax
1,01±0,16
0,46±0.06*

pEC50
7,4±0,1
9,3±0.2*

Values are means ± SEM.

p<0.05 vs S; n=6 rats/group.

Conclusion

Chronic treatment with CNP decreased blood pressure, activating cardiovascular NOS, improving cardiac efficiency and vascular function, in this model of hypertension. Supported by: UBA‐Conicet.
Circulating fragmented mitochondria induce maternal hypertension, placental inflammation and apoptosis in pregnant rats
Authors: Goulopoulou Styliani , Matsumoto Takayuki , Wenceslau Camilla F , Spitler Kathryn , McCarthy Cameron G , Webb R. Clinton
Published: June 2021

Mitochondrial fragments released from necrotic cells (mitochondrial damage‐associated molecular patterns, mitDAMPs) have pro‐inflammatory and immunogenic properties. Placentas from pregnancies with preeclampsia (PE) show exaggerated rates of cell death. We tested the hypothesis that circulating fragmented mitochondria would increase maternal blood pressure and induce placental inflammation and apoptosis in pregnant rats. Mitochondria were isolated from rat liver and sonication was used to disrupt their integrity. Pregnant rats were injected with fragmented mitochondria (mit‐treated; 4 mg/tissue) or saline (control) on gestational day (gd) 15. On gd17, systolic blood pressure, as measured with the tail cuff method, was increased in mit‐treated compared to saline‐treated dams (mit‐treated vs. control: 140±3 vs. 114±6 mmHg, p<0.05). Protein levels of cyclooxygenase 1 (COX1) and 2 (COX2) and pro‐apoptotic protein BAX were increased in placentas from mit‐treated rats (mit‐treated vs. control: COX1, 6.3±1.8 vs. 1.9±1.3; COX2, 2.6±0.2 vs. 1.7±0.3; BAX: 9.1±1.3 vs. 4.4±0.02, p<0.05). Systemic treatment with fragmented mitochondria increases maternal blood pressure, placental inflammation and apoptosis in pregnant rats. MitDAMPs released from necrotic trophoblasts may be the source of maternal hypertension and placental dysfunction in pregnancies with PE.

Support: NIH, SWHR
Ablation of MMP‐9 gene ameliorates paracellular permeability and fibrinogen‐amyloid beta plaque formation during hyperhomocysteinemia
Authors: Muradashvili Nino , Tyagi Reeta , Tyagi Suresh C. , Lominadze David
Published: June 2021

Many inflammatory and cognitive disorders including Alzheimer's disease are accompanied by hyperhomocysteinemia (HHcy). We tasted the hypothesis that HHcy increases microvascular permeability affecting mainly paracellular transport via activation of matrix metalloproteinase‐9 (MMP‐9) leading to an increased formation of fibrinogen (Fg) and β‐amyloid (Aβ) complex.

Using a dual‐tracer probing method, changes in cerebrovascular permeability were assessed in male wild type (WT, C57BL/6J), cystathionine β‐synthase heterozygote (CBS+/−, a genetic model of HHcy), MMP‐9 gene knockout (MMP9−/−), and CBS and MMP‐9 double‐knockout (CBS+/−/MMP9−/−) mice. Expression of vascular endothelial cadherin (VE‐cadherin) and Fg‐Aβ plaque formation was assessed in mouse brain cryo‐sections.

We found that cerebrovascular permeability in CBS+/− mice was mainly via paracellular transport pathway and it was greater than that in other groups. Expression of VE‐cadherin was lowest and Fg‐Aβ plaque formation was greatest in CBS+/− mice. Absence of MMP‐9 activity in MMP9−/− mice ameliorated these effects.

Thus, HHcy increases cerebrovascular permeability by downregulating VE‐cadherin through MMP‐9 activation and enhances Fg‐Aβ plaque formation. These data may lead to identification of new targets for therapeutic intervention that can modulate inflammation‐induced cerebrovascular permeability.
Redefining control O2 conditions for rat medullary slices via quantification of oxidative and nitrosative stress: experiments in the caudal solitary complex (cSC)
Authors: Ciarlone Geoffrey , Matott Michael , Landon Carol , Dean Jay
Published: June 2021

We have postulated that the control oxygen level (95%) currently used in brain slices is too high, potentially modulating redox sensitive systems and inducing redox stress. This can lead to hyperexcitability, increased metabolism, and cell death. To test this hypothesis, we have decreased our control oxygen level from 95% to 40% while using electrophysiology, amperometry (WPI Apollo 4000), and real‐time fluorescence imaging to measure changes in the cell membrane and levels of reactive oxygen and nitrogen species (RO/NS) production. At 6mL/min, pO2 measurements in the aCSF superfusion bath were 283.9 ± 0.13mmHg (mean ± SEM) at 40% O2 and increased to 593.3 ± 0.38mmHg in 95% O2. pO2 measurements recorded at the surface of the tissue slice were 119.18 ± 0.5mmHg in 40% O2 and increased to 356.2 ± 1.2mmHg in 95% O2. The decrease in recorded pO2 when measurements were taken on the surface of the slice indicates active O2 consumption by the tissue. Nitric oxide levels also significantly increased from 56.43 ± 0.14nM in 40% O2 to 65.45 ± 0.16nM in 95% O2. Electrophysiology recordings indicate that 95% O2 stimulates firing rate in ~70% of cSC neurons tested. Overall, use of a 40% control level of oxygen has proven to be effective as slices are still viable, showing normal electrical activity in addition to active consumption of oxygen and subsequent increases in production of RO/NS. (ONR)
Serum 25‐hydroxyvitamin D predicts muscular strength after a muscle‐damaging event
Authors: Barker Tyler , Dixon Brian M. , Schneider Erik D. , Weaver Lindell K.
Published: June 2021

The primary purpose of this study was to identify if serum 25‐hydroxyvitamin D (25(OH)D) concentrations predict muscular weakness after a muscle‐damaging event. The secondary purpose was to determine if exercise‐induced muscle damage causes a fluctuation in serum 25(OH)D concentrations, and if serum 25(OH)D fluctuations are predicted by an inflammatory cytokine. Fourteen recreationally adults participated in this study. Each subject had one leg randomly assigned as a control. The other leg performed an intense exercise protocol to induce muscle damage. Single‐leg peak isometric force and blood 25(OH)D, aspartate (AST) and alanine (ALT) aminotransferases, albumin, and interferon (IFN‐γ) were measured prior to and following the exercise protocol. Days after the exercise protocol, plasma AST (p < 0.05) and ALT (p < 0.05) increased and muscular weakness persisted within the exercise leg (p < 0.05) and compared to the control leg (p < 0.05). A prior elevation in serum 25(OH)D predicted the attenuation in immediate (p < 0.05) and persistent (p < 0.05) muscular weakness after the muscle‐damaging event. Circulating IFN‐γ increases predicted (p < 0.05) serum 25(OH)D decreases, while albumin increases predicted (p < 0.05) serum 25(OH)D increases. We conclude that serum 25(OH)D predicts muscular strength after a muscle‐damaging event and that IFN‐γ and albumin changes predict serum 25(OH)D fluctuations.
Aging influences the expression of early response genes following acute resistance exercise in trained skeletal muscle
Authors: Liu Dongmei , Sartor Maureen , Pistilli Emidio , Nader Gustavo , Visich Paul , Hoffman Eric , Gordon Paul
Published: June 2021

To identify the influence of age on skeletal muscle transcriptional responses to resistance exercise (RE), we recruited 14 young (<40 y) and 8 older (>;60 y) participants. They underwent 12‐week unilateral arm RE training, and conducted one bout of acute RE one week later. Biceps brachii muscle biopsies were obtained 4h and 24h post exercise (PE). Gene expression was assessed using Affymetrix U133A microarrays, and analyzed using an intensity‐based Bayesian moderated paired t‐test. The immediate‐early response genes FOS, EGR1, JUNB, and NR4A3, and other key genes for muscle metabolism including IL6R, PRKAG2, EIF2AK3, HEY1, and ANKRD1, showed striking differences between young and old muscle. They were dramatically up‐regulated PE in the young, but highly down‐regulated in the old (Table 1). This age effect might implicate the diminished metabolic and hypertrophic benefits of RE with aging.

Funding for this study was provided by NIH RO1 NS40606–01
Effect of ageing and physical activity on cardiovascular function
Authors: Munch Gregers Druedal Wibe , Calbet José Antonio Lopez , Boushel Robert , Pedersen Bente Klarlund , Saltin Bengt , Mortensen Stefan Peter
Published: June 2021

Ageing is associated with vascular dysfunction and a reduced capacity of the central circulation to deliver O2 to the contracting muscles. The purpose of present study was to investigate whether a physical active lifestyle can maintain a sufficient exercise hyperemia and the influence of the active muscle mass. Central and limb hemodynamics were determined in 8 young (Y) (23±1 years), 8 lifelong sedentary elderly (LSE) (66±2 years) and lifelong active elderly (LAE) (62±2 years) men during incremental cycling with and without simultaneous arm cycling (50w). Peak workload was higher in the LAE compared to Y and LSE (294±15; 216±10; 213±8 W; P<0.05). Leg blood flow (LBF) increased similarly in LAE and LSE, but peak values were higher in LAE and Y compared to LSE (7.8±0.5; 8.6±0.4 vs. 6.4±0.4 l/min; P<0.05). Leg a‐vO2 difference was higher in LAE compared to the Y and LSE at all workloads (P<0.05), whereas leg VO2 was higher in LAE and Y compared to LSE at exhaustion (P<0.05). During combined arm and leg cycling LBF, leg a‐vO2 difference and VO2 was lower in Y and LSE compared to leg cycling alone (P<0.05). Mean arterial pressure increased in all groups but was lower in Y (P<0.05). These results demonstrate that lifelong physical activity can maintain aerobic power despite a lower exercise hyperemia by an increase in O2 extraction.

Supported by the Lundbeck Foundation and Augustinus Foundation
The Effects of Tai Chi plus behaviorally‐based dietary weight loss on physical functioning in community‐dwelling obese older women
Authors: LETENDRE JONATHAN M , Bekke Jillian , Beebe Nowen , Lofgren Ingrid E , Xu Furong , Delmonico Matthew J
Published: June 2021

Older obese women are at risk of obesity‐related disability, but Tai Chi has not been explored as an exercise modality along with weight loss for their effects on physical function. A 16‐week community‐based intervention was conducted to assess the impact of Tai Chi plus behaviorally‐based weight loss (TCWL, n=29) on obese (BMI=35.4 ±0.8 kg/m2) older (68.2±1.5 yr) women compared to a control group (CON, n=9, BMI=38.0±1.5kg/m2, 65.6±2.7 yr). The major outcome was the short physical performance battery (SPPB), consisting of 4‐m gait speed (GS), standing balance, and chair stand (CS) tests. The TCWL group participated in two Tai Chi sessions and one dietary weight loss session per week with a goal of 5–10% weight loss. The CON group was asked to maintain their normal lifestyle. The TCWL group lost weight, (1.6±2.9 kg, P=.006) while the CON group showed no significant change (1.2 ± 1.9 kg P=.11). SPPB score improved in TCWL (0.59±1.28, P=.023) when compared to the CON group (−0.56±1.81, P=.38). TCWL group improved GS (−0.14±0.76 sec, P=.34) time better (P=.004) than CON (0.56±0.81 sec, P=.07) and also improved CS (−0.80±1.71 sec, P=.02) time better (P=.065) than CON (0.28±2.83 sec, P=.77). Tai Chi may represent an effective intervention strategy to maintain and improve physical function while losing weight..
Interval training program improves cardiac function and physical performance in heart failure mice
Authors: ABAD CESAR CAVINATO CAL , Mostarda Cristiano Teixeira , Nascimento Ademir Manuel , Santos Mirailton Alves dos , Souza Leandro Eziquiel , Figueroa Diego M. T. , Santos Fernando , de Souza Pâmella Ramona Moraes , De Angelis Kátia , Irigoyen Maria Claudia
Published: June 2021

This study was designed to verify the effect of an eight week interval training program (IT) in systolic and diastolic functions and physical performance in heart failure mice (C57B16, n=24). The animals were divided in three groups: 1) Intact sedentary (C), 2) infarcted sedentary (MI‐s); and 3) infarcted trained (MI‐i). At the begining (M1) and at the end of IT (M2), time of maximal running performance (Tvmáx) was quantified. The ejection fraction (EF), shortening fraction (SF) and isovolumetric relaxation time (IVRT) were accessed by echocardiography in M2. At 60 days after coronary artery occlusion the IT was started (4min at 80% Vmáx × 4min at 40% Vmáx during 60min). The results are presented in mean and SEM. Myocardial infarction area was not different between groups in both evaluation time (M1vsM2). The EF and SF in MI‐s were lower than C (44±3.7vs58±3.0% and 22±2vs31±2%, respectively; p≤0.05) but similar between MI‐i and C (56±5vs58±3% and 25±3vs31±2%, respectively). The IVRT was higher (p≤0.05) in MI‐s (25.6±4ms) than C (13±1ms) while similar in MI‐i (15±0.8ms) and C. At the M1 both MI‐s and MI‐i Tvmáx decreased in comparison with C (620±16 and 644±28vs840±17s, respectively). At the M2, Tvmáx delta percent was higher in MI‐i than MI‐s (64±8vs15±6%, respectively; p≤0.05) and C (0.53±3%). The results suggest that IT improves cardiac function and physical performance in heart failure mice.

Financial Suport: CnPq.
Endurance exercise does not protect the decrease in bone associated with Type 1 diabetes in adult rats
Authors: Hazell Tom J , Olver T Dylan , Kowalchuk Hana , McDonald Matthew W , Dey Adwitia , Grise Ken N , Lavery Paula , Weiler Hope A
Published: June 2021

Streptozotocin (STZ) induced type 1 diabetes mellitus (T1DM) decreases trabecular bone volume in mice. This study investigated the potential protective effects of endurance training (ET) on bone in STZ‐induced T1DM adult rats. Sixty four 8‐week old Sprague‐Dawley rats were randomly divided into 4 groups of 16: non‐T1DM sedentary (CS) and exercised (CX), T1DM sedentary (DS) and exercised (DX). Blood glucose was maintained at 9–15 mmol/L using subcutaneously implanted insulin pellets (Linplant, Linshin Canada, Inc.). ET was at ~75% VO2max for 1 h/d, 5 d/wk for 10 wk. Areal and volumetric bone mineral density (aBMD and vBMD; excised femur) was measured using dual‐energy x‐ray absorptiometry (DXA; QDR 4500A) and micro computed tomography (μCT; Aloka LCT200); bone strength was estimated using μCT. Two‐way ANOVA was used to test for diabetes and exercise differences; significance was set at P<0.05. T1DM had lower body weight (20.0%), aBMD (8.6%), cortical vBMD (1.6%), and bending (12.4%) and torsional strength (13.4%) vs control (P<0.001) with no differences in trabecular vBMD. Exercise also decreased body weight (4.7%) vs sedentary (P=0.019). These results suggest that 10 wk of ET does not have bone protective effects and T1DM rats have lower aBMD and vBMD than control, which may be secondary to effects on body weight. Funded by CIHR.