bluuebunny/crossref_metadata_2025 huggingface dataset:
Dynamic turnover of L‐type calcium channels in rat mesenteric arteries in vivo
Authors: Srivastava Anup Kumar , Kharade Sujay V , Fletcher Terry W , Rhee Sung , Rusch Nancy J
Published: June 2021

Increased Ca2+‐dependent vascular tone is a hallmark feature of hypertension. The upregulation of the pore‐forming α1C subunit of the voltage‐gated L‐type Ca2+ (CaL) channel contributes to this increased Ca2+‐dependent tone. However, the mechanism by which high blood pressure drives CaL channel expression has been difficult to identify and may require monitoring CaL channel turnover in arteries in vivo. To enable these studies, we anesthetized Sprague‐Dawley rats with isoflurane and exposed intestinal loops containing mesenteric arteries (MA) through a minimal abdominal opening. Distal MA branches were removed immediately (0 hr). Then the MA bed was pinned flat in a silicone chamber and exposed either to physiological salt solution (PSS, control) or PSS + 20 μg/ml puromycin to inhibit de novo protein synthesis. After 6 hr, the exposed branches of the MA bed were removed. Western blots compared the expression of the CaL channel α1C pore protein between 0 and 6 hr. The immunoreactive band corresponding to α1C was maintained in MA exposed to PSS alone but reduced by 75% in MA exposed to puromycin. Thus, vascular CaL channel turnover is rapid at normal blood pressure levels, raising the possibility that reduced turnover during hypertension could increase CaL channel abundance. Funded by NIH R01 HL064806 (NJR)
Sildenafil stimulates heme oxygenase‐1 gene expression in vascular smooth muscle cells via a soluble guanylate cyclase‐independent pathway
Authors: Liu Xiao-ming , Peyton Kelly J. , Durante William
Published: June 2021

Sildenafil is a potent type 5 nucleotide‐dependent phosphodiesterase inhibitor that is used to treat erectile dysfunction and pulmonary arterial hypertension. Since heme oxygenase‐1 (HO‐1) exerts numerous beneficial effects in the circulation, the present study investigated whether sildenafil regulates the expression of HO‐1. Treatment of cultured rat aortic smooth muscle cells (SMCs) with sildenafil stimulated a concentration‐and time‐dependent increase in HO‐1 protein. A significant increase in HO‐1 protein was detected after 4 hours and levels progressively increased over 24 hours of sildenafil exposure. Sildenafil‐mediated increases in HO‐1 protein were paralleled by significant elevations in HO‐1 mRNA. The induction of HO‐1 by sildenafil was dependent on de novo RNA synthesis since it was blocked by the transcriptional inhibitor, actinomycin D. Sildenafil also stimulated the phosphorylation of vasodilator‐stimulated phosphoprotein and this was prevented by the soluble guanylate cyclase inhibitor ODQ. In contrast, ODQ failed to block the induction of HO‐1 by sildenafil. In conclusion, this study demonstrates that sildenafil stimulates HO‐1 gene expression in vascular SMCs via a transcriptional mechanism that is soluble guanylate cyclase‐independent. The ability of sildenafil to induce HO‐1 expression may contribute to the beneficial actions of this agent. Supported by NIH grant HL59976.
Depletion of platelet serotonin with a selective serotonin reuptake inhibitor does not improve survival in a mouse model of thromboembolism
Authors: Liu Henry F , Campbell Christina , Hegde Sharath , McNamara Alexander
Published: June 2021

Platelet serotonin (5‐hydroxytryptamine, 5‐HT) is released upon platelet activation and serves as a proaggregatory factor. Clinical and preclinical evidence suggest that selective serotonin reuptake inhibitors (SSRIs) deplete platelet 5‐HT and reduce platelet aggregation. The goal of this study was to determine if inhibition of the 5‐HT transporter (SERT) reduces platelet 5‐HT and improves survival in a mouse thromboembolism (MTE) model.

C57/BL6 mice were treated with vehicle (veh), fluoxetine (FX, 30 mg/kg, PO x 1 & 7d), paroxetine (PX, 30 mg/kg, PO x 7d), escitalopram (EC, 2 mg/kg, SC x 7d) or norfluoxetine (NFX, 5 mg/kg, IV x 1d). Mice were anesthetized and the right jugular vein cannulated. Thromboembolisms were induced with an infusion of collagen 200 μg/epinephrine 10 μg (Col/EPI), survival was monitored over the next 15 min and whole blood 5‐HT was measured.

Col/EPI produced mortality in vehicle‐treated mice (~66 %). FX (7d) and NFX significantly enhanced survival (69 & 70 %), whereas FX (1d), PX and ES offered no survival benefit (≤ veh). SSRI treatment reduced whole blood 5‐HT by 75 – 99%; however, the decreased concentration of 5‐HT did not correlate with improved survival. Thus, depletion of platelet 5‐HT through SERT inhibition does not prevent Col/EPI induced‐thromboembolism in the MTE model. The efficacy of FX and NFX in this model appears to be independent of their SSRI activity.

Source of Funding: None
Propofol protects the cardiomyocytes against hypoxia injury through inhibition of ER‐dependent apoptotic pathways
Authors: Lai Hui Chin , Liu Tsun-Jui , Lee Hsiao-Wei , Wang Li-Chuan , Yeh Yueh-Chiao
Published: June 2021

Recent observations indicate that in the heart, the UPR is activated during acute stresses that lead to cardiac hypertrophy and heart failure. Hypoxia was found to mediate UPR activation in cardiac myocytes in vitro. This study was designed to examine whether propofol, an anesthetic with pluripotent cytoprotective properties, could ameliorate reduce the endoplasmic reticulum (ER) stress and hypoxia‐induced cardiomycytes apoptosis. Propofol pretreatment ameliorates apoptosis of hypoxia on cardiomyocytes. By using of immunofluorescence, western blotting analysis and RealTime‐PCR, propofol pretreatment were reduced mRNA expression of the crucial ER stress sensors ATF6, reduced pro‐apoptotic transcriptional factor C/EBP homologous protein (CHOP) and glucose‐regulated protein 78 (GRP78) and leads to apoptotic cell death. Our data has shown that propofol can ameliorate ER stress‐induced cardiac UPR‐reproducted and cardiomyocytes apoptosis. The effect of propofol may provide a new therapy to prevent the cardiovascular damage in stroke patients.
ET‐1 induces COX‐2/PGE2 expression via an ETA/B/PKCδ/c‐Src/PI3K/Akt‐dependent pathway in murine osteoblast‐like MC3T3‐E1 cells
Authors: Li Ying-yi , Yang Chuen-Mao
Published: June 2021

Induction of COX‐2 expression and PGE2 synthesis by endothelin‐1 (ET‐1) may play an important role in inflammation and directly stimulate osteoblastic activity. However, the molecular mechanisms underlying ET‐1‐induced COX‐2 expression in murine osteoblast‐like cell line (MC3T3‐E1) were largely unknown. Here, we found that ET‐1 time‐dependently induced COX‐2 protein and mRNA expression, and PGE2 generation in MC3T3‐E1 cells. Pretreatment with an inhibitor of ETA receptor (BQ123), ETB receptor (BQ788), Gi or Gs (GPAnt2), Gq (GPAnt2A), PKCδ (rottlerin), c‐Src (PP1), PI3K (LY294002), Akt (SH‐5), p38 MAPK (SB202190), or MEK1/2 (U0126) and transfection with siRNA of c‐Src, Akt, p42, or p38 significantly attenuated ET‐1‐induced COX‐2 protein expression and PGE2 generation. We also found that ET‐1‐induced the phosphorylation of PKCδ, c‐Src, Akt, p42/p44 MAPK, and p38 MAPK was attenuated by the inhibitors of PKCδ, c‐Src, PI3K, Akt, MEK1/2, and p38 MAPK, respectively. Moreover, ET‐1 stimulated ATF2 phosphorylation and translocation from the cytosol to the nucleus in MC3T3‐E1 cells, which were attenuated by preincubation with rottlerin, PP1, LY294002, SH‐5, U0126, or SB202190. These results suggest that ET‐1‐induced COX‐2 expression was mediated through an ETA/B/PKCδ/c‐Src/PI3K/Akt/p42/p44 MAPK and p38 MAPK pathway and ATF2 in MC3T3‐E1 cells.
Lipopolysaccharide induces ICAM‐1 expression in human pulmonary alveolar epithelial cells through a TLR4/MyD88/TRAF6/NADPH oxidase‐dependent pathway
Authors: Cho Rou-Ling , Yang Chuen-Mao
Published: June 2021

Lipopolysaccharide (LPS), a key component of the outer membranes of Gram‐negative bacteria, plays an important role in the induction of adhesion molecules expression and reactive oxygen species (ROS) generation in airway inflammatory diseases. Here, we investigated the mechanisms of LPS‐induced intercellular adhesion molecule‐1 (ICAM‐1) expression in human pulmonary alveolar epithelial cells (HPAEpiCs). We found that LPS induced ICAM‐1 protein and mRNA expression, promoter activity, and the adhesion of THP‐1 cells, as well as the phosphorylation of c‐Src, PDGFR, EGFR, Akt, and p65, which were attenuated by pretreatment with an anti‐TLR4 Ab or the inhibitor of c‐Src (PP1), PDGFR (AG1296), EGFR (AG1478), PI3K (LY294002), NADPH oxidase [diphenylene iodonium chloride (DPI) and apocynin (APO), ROS (edaravone), or NF‐κB (Bay11–7082) and transfection with siRNA of TLR4, MyD88, CD14, or TNFR‐associated factor (TRAF)6. LPS‐induced TLR4, MyD88, TRAF6, c‐Src, p47phox, and Rac‐1 complex formation was revealed by immunoprecipitation using an anti‐TLR4 Ab, followed by Western blot against an anti‐TLR4, anti‐MyD88, anti‐TRAF6, anti‐c‐Src, anti‐p47phox, or anti‐Rac‐1 Ab. These results demonstrated that LPS‐induced ICAM‐1 expression was mediated through TLR4/MyD88/TRAF6/p47phox/Rac‐1/c‐Src/EGFR, PDGFR/PI3K, in turn initiates NF‐κB activation, and ultimately induces ICAM‐1 expression and the adhesion of THP‐1 cells in HPAEpiCs.
Delayed Onset of Proteinuria by Selected Renal Cells in a Canine Model of Early Stage CKD
Authors: Guthrie Kelly Isley , Brands Michael , Rivera Elias , Genheimer Christopher , Sangha Namrata , Bravo Mike , Jayo Manuel , Jain Deepak , Bertram Timothy A. , Ludlow John W. , Presnell Sharon , Kelley Rusty
Published: June 2021

Surgical reduction of kidney mass (RKM) models have been used to study cardiovascular ramifications of early CKD, however long‐term features of the model have not been well defined. This study characterized a canine RKM model and its use in evaluating efficacy of autologous selected renal cells (SRC) through one‐year post‐nephrectomy by monitoring renal specific, whole animal, and terminal histological parameters. SRC preserve tubular and glomerular functions in rodent nephrectomy models. Dogs underwent 2‐step nephrectomy (RKM) and hemi‐nephrectomized dogs served as controls. Using International Renal Interest Society guidelines, 15 weeks post‐nephrectomy RKM dogs were in late Stage I/early Stage II CKD as defined by sCre (1.6), systolic BP (144–157) and mild proteinuria (UPC 0.2). SRC were then delivered to 4 RKM dogs. All animals demonstrated improving sCre values indicating sufficient nephron mass remained for increased singlular nephron GFR to compensate for RKM and maintain eGFR, thus limiting the sensitivity in detecting filtration related treatment effects. SRC therapy significantly reduced proteinuria and evidence of interstitial tubular disease and juxtamedullary glomerular degeneration. Analysis of urine proteins supports the presence of specific treatment benefit to the tubulointerstitial renal compartment. Since proteinuria is considered to be an early indicator of and a contributor to progression of human and canine CKD, this model may provide a useful approach to evaluate potential therapeutics for CKD and supports continued effort toward developing regenerative medicine products containing SRC.
Structure‐based design of inhibitory peptide for End Binding proteins
Authors: Saqib Uzma , Geyer Melissa , Minshall Richard , Malik Asrar , Komarova Yulia
Published: June 2021

The Microtubule (MT) cytoskeleton plays an important role in regulating paracellular permeability of the endothelium in the context of inflammation; however, the molecular mechanism involved in MT‐dependent increase in permeability is not yet fully understood. We have shown that End Binding (EB) protein 3, which binds to and regulates MT dynamics in the endothelium, interacts with ER associated inositol 1,4,5‐trisphosphate (IP3) receptor type 3 and facilitates thrombin‐induced release of calcium from ER stores. Based on the crystal structure of the EB interacting domain and using the computational alanine scan approach, we designed a 7‐amino acid synthetic inhibitory peptide that mimics interaction between EB3 and IP3R3. We have shown that treatment of human pulmonary artery endothelial cells with this peptide blocked the interaction between IP3R3 and EB3 and negatively regulated thrombin‐ and histamine‐induced increase in intracellular calcium. Consistent with this finding, EB inhibitory peptide attenuated histamine‐induced activation of endothelial nitric oxide synthase (eNOS), as determined by NO production but had no effect on basal level of NO. In an animal model of inflammation, EB peptide prevented histamine‐induces vasodilation. Our data suggests that EB proteins can be used as a potential target for development of novel therapies to prevent leaky vessel syndrome during inflammation.

Funding source: RO1 HL103922 to Yulia Komarova
Pulsed electrical fields cause activation of tyrosin kinase related cellular signalling in endothelial cells leading to transcription processes and angiogenesis
Authors: Dissing Steen , Tritsaris Katerina , Hansen Anker Jon
Published: June 2021

We investigated if pulsed electrical fields cause enhanced endothelial cellular signalling and angiogenesis. Pulsed electrical fields were imposed on human and porcine endothelial cell cultures (HDMEC, PAE and HUVEC) by use of coils that create pulsed electromagnetic fields (PEMF) that create pulsed electrical fields of a magnitude of 1–10 mV/cm. PEMF activates cellular signalling related to tyrosin kinase receptor function since it evokes increased phosphorylation of MAPK signalling and Akt signalling pathways. The tyrosine kinase Src becomes phosphorylated at Tyr416 in the activation loop of the kinase domain, and dephosphorylated at Tyr527 located in the negative regulatory tail of the protein. Both mechanisms imply activation of the kinase. We propose that the Src enzyme is the sensor or is located close to the site that senses the changes in the electrochemical potential of protein charges. RT‐PCR analysis of brain derived neurotrophic factor (BDNF) mRNA revealed an upregulation after 3 h of PEMF exposure in HDMEC. In animal angiogenesis models we found an enhanced angiogenesis after 24 hours. Thus, PEMF induce endothelial cell activation and angiogenesis. We hypothesize that activation of brain endothelial cells (blood brain barrier) contributes to the beneficial effects by PEMF observed in a study of patients with depression (Martiny et al., Biol. Psychiatry, 68: 163–9, 2010).
S‐Nitrosylation of Caveolin‐1 Cys156 Stimulates Src‐dependent Caveolin‐1 Tyr14 Phosphorylation Required for Caveolae‐mediated Endocytosis in Endothelial Cells
Authors: Bakhshi Farnaz Rose , Chen Zhenlong , Shajahan Ayesha , Hussain Yawer , Bonini Marcelo , Minshall Richard D.
Published: June 2021

Membrane‐associated Caveolin‐1 (Cav‐1) oligomers are required for caveolae formation. We previously showed that during an inflammatory response, Src‐dependent Cav‐1 phosphorylation increases transendothelial transport via caveolae and breakdown of the endothelial barrier, both of which were abolished in Cav‐1−/− lung microvessels. Thus, in this study we tested the hypothesis that TNFα‐induced NO production induces Cav‐1 S‐nitrosylation (SNO) resulting in Src displacement and activation which increases caveolae trafficking. Endothelial cells (ECs) stimulated with TNFα or NO donor showed reduced Cav‐1 oligomerization (MW > 250 kDa) as measured by immunoblot analysis of non‐reduced SDS PAGE gels and also increased Cav‐1 Tyr14 phosphorylation. Of the 3 Cys residues in the C‐terminus (Cys 133, 143, and 156), we observed S‐nitrosylation of Cys156by biotin switch assay. eNOS dependent increase in Cav‐1 Tyr14 phosphorylation and Cav‐1‐GFP labeled caveolae trafficking were blocked by L‐NAME and PP2. Cav1−/− ECs expressing C156S Cav‐1 mutants did not form stable oligomers and showed increased Src activity but were unable to form caveolae. When expressed in normal endothelial cells, C156S‐Cav‐1‐GFP and Y14D‐Cav‐1‐GFP had a dominant positive effect whereas Y14F‐Cav‐1‐GFP reduced caveolae‐mediated uptake and trafficking. These data suggest that Cav‐1 Cys156 SNO leads to Src activation and phosphorylation of Cav‐1 Tyr14 which increases caveolae trafficking dynamics in endothelial cells.
sGC in SMC acts as Nitrite Reductase leading to NO formation
Authors: Madrasi Kumpal Jagdish , Tsoukias Nikolaos , Joshi Mahesh
Published: June 2021

Several heme proteins including hemoglobin and nitric oxide synthase (NOS) have been documented to serve as nitrite reductase under anoxia. Since sGC is also a heme protein and an important signaling biomolecule, we hypothesized that it may function as nitrite reductase in cultured smooth muscle cells. Millimolar levels of nitrite were required to detect cellular NO as measured by NO specific fluorescent dye (CuFL2E). However, in chemiluminescence analysis of cell lysates, addition of 400–600 μM NO2− was sufficient to detect measurable NO formation under anoxic conditions. This NO formation was inhibited with ODQ showing sGC in the cell lysate as the source of nitrite reduction. CO is a known activator of sGC and thus CO donor (50 μM CORM‐2) significantly activated nitrite reduction to NO. The cellular uptake of NO2− was studied using the AE‐1 inhibitor, DIDS and intracellular NO2− was measured by tri‐iodide assay. DIDS (300 μM) significantly inhibited NO2− uptake and showing that NO2− uses AE‐1 transport mechanism. These results demonstrate that sGC mediates NO2− reduction to form NO and that the AE‐1 channel is one of the pathways for NO2− entry into SMC. This work was supported by NIH grant SC1HL095101.
TRIACSIN C, A FATTY ACYL COA SYNTHETASE (FACS) INHIBITOR, IMPROVES CARDIAC PERFORMANCE FOLLOWING GLOBAL ISCHEMIA
Authors: Weis Margaret Teresa , Tolson Jasmine , Chen Qian , Young Lindon
Published: June 2021

Extensive evidence shows that eNOS is protective in ischemia/reperfusion (I/R) injury. We have shown that triacsin C, a (FACS) inhibitor, increases eNOS activity in the post‐ischemic rat hind limb, suggesting that triacsin C might be useful in treatment of cardiac I/R. Isolated rat hearts, perfused at a constant pressure by the method of Langendorf, were subjected to a 30 minute period of global ischemia, followed by 45 minutes of reperfusion in the presence or absence of 5μM triacsin C.

At 45 minutes of reperfusion, LVDP and dP/dt in treated hearts were significantly better than in vehicle controls. Coronary flow and heart rate were not changed by treatment. The infarct area, visualized with 1% 2,3,5‐triphenylterazolium, was significantly smaller in triacsin C treated hearts. The results support the hypothesis, and suggest that FACS inhibitors may represent a new category of drugs for I/R.
Sodium Nitrite Administered Immediately before Reperfusion Reduces in vivo Myocardial Infarction and Improves Postischemic Cardiac Function: Significance of Critical Dose Response and Safety Margin of Nitrite in Cardioprotection
Authors: Zweier Jay L. , Chae Jei-Keon , Talukder M.A. Hassan
Published: June 2021

Nitrite has recently been shown to confer protection against myocardial ischemia‐reperfusion (I/R) injury; however, there is a lack of information regarding the dose‐dependent effects of nitrite on myocardial infarction (MI) and cardiac function. Therefore, dose‐dependent effects of sodium nitrite were investigated in an in vivo rat model of I/R for MI and cardiac function. In anesthetized animals, I/R protocol was performed for 40‐min regional ischemia (left coronary occlusion) and 24‐hour reperfusion with intravenous nitrite (360 to 11520 nmoles) or saline 3‐min before reperfusion. The degree of protection was compared with that of saline group by measuring myocardial infarct size (Evan's blue and TTC staining) and postischemic cardiac function (echocardiography). Nitrite produced a bell‐shaped cardioprotection where lower doses significantly reduced myocardial infarct size and concurrently improved cardiac function. However, the beneficial effect of nitrite was lost at the higher doses and it was associated with significant myocardial dysfunction. There was also function (depressed contractility) ‐ morphology (reduced infarction) mismatching with nitrite at higher doses. Thus, these findings demonstrate a critical dose‐response effect and a narrow safety margin of nitrite, and it could be significant in its therapeutic efficacy. (NIH grants HL63744, HL65608, and HL38324 to JLZ)
Detrimental effects of antiretroviral treatment on contractile function of the rat heart
Authors: Reyskens Kathleen Maria Simone Elise , Essop M Faadiel
Published: June 2021

Although antiretroviral treatment decreases HIV‐AIDS morbidity/mortality, long‐term effects include the onset of insulin resistance and cardiovascular diseases. However, the underlying molecular mechanisms responsible for this are poorly understood. Here we hypothesized that protease inhibitor (PI) treatment elevates oxidative stress that attenuates cardiac contractile function at baseline and with ischemia. We established a model of chronic PI utilization, i.e. lopinavir/ritonavir dissolved in 1% ethanol (vehicle) was injected into mini‐osmotic pumps, surgically implanted into Wistar rats and exposed to PIs for 8 weeks vs. vehicle and sham controls. PI‐treated rats exhibited modest weight gain, with no indication of insulin resistance (homeostatic model of insulin resistance). Ex vivo Langendorff perfusions revealed that PI treatment significantly decreased contractile function at baseline, and in response to 30 min global ischemia and 60 min reperfusion vs. controls (p<0.01). Infarct sizes for PI‐treated rats increased vs. controls (p<0.01) after 30 min regional ischemia and 120 min reperfusion. In parallel, PI‐treated hearts displayed higher caspase 3 activity following ischemia‐reperfusion (p<0.001 vs. controls). Our data show that chronic PI treatment exerts direct effects on the rat heart that attenuates contractile function at baseline and in response to ischemia‐reperfusion.
The modulation of NF‐kappaB‐p65 with glutathionylation in 15‐Deoxy‐D12,14 ‐prostaglandin J2‐treated endothelial cells
Authors: Wung Being-Sun
Published: June 2021

Protein glutathionylation is a post‐translational modification of cysteine residues with glutathione in response to mild oxidative stress. Because 15d‐PGJ2 is an electrophilic prostaglandin that can increase glutathione (GSH) levels and augment ROS production, we hypothesized that it induces NF‐κB‐p65 glutathionylation and would exert anti‐inflammatory effects. Herein, we show that 15d‐PGJ2 suppresses the expression of ICAM‐1 and NF‐κB‐p65 nuclear translocation. 15d‐PGJ2 increases the GSH and ROS levels. Consistently, ECs treated with GSSG show increased thiol modifications of p65 and also a block in TNFα‐induced p65 nuclear translocation and ICAM‐1 expression, but not in IκBα degradation. However, the overexpression of glutaredoxin (GRX)‐1 was found to be accompanied by a modest increase in NF‐κB activity. Furthermore, we found that multiple cysteine residues in p65 are responsible for glutathionylation. 15d‐PGJ2 was observed to induce p65 glutathionylation and is suppressed by a GSH synthesis inhibitor, BSO, by catalase and by Nrf2 siRNA molecules. Our current results thus indicate that the GSH/ROS‐dependent glutathionylation of p65 is likely to be responsible for 15d‐PGJ2‐mediated NF‐κB inactivation and for the enhanced inhibitory effects of 15d‐PGJ2 upon TNFα‐treated ECs.
Opening of mitochondrial BK(Ca) channels at reoxygenation protects ventricular myocytes against injury via redox signaling
Authors: Kolar Frantisek , Borchert Gudrun H.
Published: June 2021

The aim was to find out whether opening of mitochondrial large conductance calcium‐activated potassium channels [BK(Ca)] protects cardiomyocytes against injury associated with simulated reoxygenation. Cells isolated from the left ventricles of adult male Wistar rats were subjected to 25‐min metabolic inhibition with NaCN and 2‐deoxyglucose followed by 30‐min reenergization. NS11021 (0.1 μM), a novel BK(Ca) channel opener, or hydrogen peroxide (2 μM) added at reenergization increased cell survival by about 30% and reduced the release of lactate dehydrogenase (LDH). The cytoprotective effects of NS11021 were completely abolished by paxilline, a BK(Ca) blocker, or tempol, a superoxide dismutase mimetic, but not by wortmannin, an inhibitor of phosphatidylinositol 3 kinase (PI3K). NS11021 significantly increased fluorescence of cells loaded with dichlorofluorescein diacetate (DCFDA) indicating an increased formation of reactive oxygen species (ROS). The NS11021‐induced ROS signal was abolished by paxilline or tempol. NS13558, an inactive structural analogue of NS11021, affected neither cell survival nor DCFDA fluorescence. These results suggest that opening of mitochondrial BK(Ca) channels protects isolated cardiomyocytes against simulated reoxygenation injury; the protective mechanism requires ROS formation but not the activation of PI3K/Akt pathway. Supported by the grant IAA500110804.
Racial Difference in Central Stiffness and Arterial Structure following Aerobic Exercise Training
Authors: Ranadive Sushant , Yan Huimin , Lane Abbi D , Sun Peng , Kappus Rebecca , Cook Marc D , Fernhall Bo
Published: June 2021

African‐Americans (AA) have higher prevalence of hypertension than Caucasians (CA) in the United States. In addition, AA have stiffer central arteries (carotid and aortic) and higher peripheral vascular resistance. These factors are known to directly contribute to end‐organ damage. The purpose of this study was to examine the effects of aerobic exercise training on carotid and central stiffness in young white and African American individuals. 24 AA (8 men and 16 women) and 32 CA (17 men and 15 women) between the ages of 18–35 volunteered for the study, and completed a 8 week aerobic exercise training period (30–60 min of exercise at 60–90% of maximal heart rate, 3 times/week). Women were tested during menses. Carotid and aortic blood pressures were measured using applanation tonometry. Carotid maximum diameter increased significantly following training in CA (7.2 ± 0.1 to 7.4 ± 0.1) as compared to AA (7.5 ± 0.1 to 7.4 ± 0.2). In addition, carotid minimum diameter increased significantly following training in CA as compared to AA (6.5 ± 0.1 to 6.9 ± 0.1 vs. 6.9 ± 0.1 to 6.9 ± 0.2). Carotid SBP, DBP, MAP, Carotid/aortic stiffness or augmentation index showed no changes following training (data not shown, p<0.05). These data suggest that even though 8 weeks of aerobic training had no effect on carotid BP and carotid/aortic stiffness in normotensive CA and AA there is significant arterial remodeling in CA as opposed to AA.
Impact of cholinergically‐mediated vasodilation on blood pressure at the onset of exercise in humans
Authors: Vianna Lauro C , Fadel Paul J , Secher Niels H , Fisher James P
Published: June 2021

Handgrip exercise evokes an initial decrease in the vascular resistance of the non‐exercising forearm which is antagonized by atropine, indicative of cholinergic‐mediated vasodilation in human skeletal muscle. However, the role of cholinergic‐mediated vasodilation in modulating the blood pressure (BP) response to exercise in humans remains unknown. To examine this, beat‐to‐beat BP (brachial artery), cardiac output (CO; Modelflow) and total vascular resistance (TVR; mean BP/CO) were continuously monitored in 9 men (23±5 yrs) while performing static handgrip at 40% MVC under control (no drug), cholinergic (glycopyrrolate) and β‐adrenergic (metoprolol or propranalol) blockade conditions. Exercise under control conditions produced a biphasic BP response at exercise onset, consisting of an initial rise (0–3s, +6±2 mmHg, P<0.05) and a subsequent fall to a nadir at 7–10s (+2±2 mmHg). TVR dropped progressively between 3–10s of exercise (−24±2% at 10s; P<0.05). β‐adrenergic blockade did not affect the magnitude or temporal pattern of the BP and TVR responses. However, cholinergic blockade abolished the exercise‐induced fall in TVR, and augmented the pressor response to exercise (+13±3 mmHg at 10s; P<0.05 vs. control). Changes in CO were similar between trials. These findings highlight for the first time the important role of cholinergic vasodilation on BP responses at exercise onset in humans.
Middle cerebral artery blood velocity during running: interactions of heart and stride impact rates
Authors: Wasson Elisa , Harper Cody , Subudhi Andrew , Schneider Sue , Maestas Nico , Gutierrez Daniel , Roach Rob , Greene Ernest
Published: June 2021

Vertical movement during running or treadmill exercise generates complex aortic blood pressure waveforms that are developed by the interactions of the cardiac and foot impact pressure pulses (>120bpm). Heart rate (HR) and stride rate (SR) can be synchronized in and out of phase with beat frequencies (difference in the HR and SR). Large variations in phasic aortic pulse pressures (40–120 mmHg) occur during running. Transcranial Doppler (TCD) measures dynamic middle cerebral artery blood velocity (CBFv), an index of hemispheric cerebral blood flow. Due to technical limitations, CBFv during running has not been reported. We hypothesized that running would significantly modulate CBFv. We used a 2 MHz TCD and a form fitting headset to minimize motion artifact in 5 healthy subjects (26 ±8SD) during: upright rest; walking (4 mph, no vertical motion); and running (7 mph, 4–5 inch vertical motion). The resting and walking waveforms were similar to reported ergometer waveforms. Running CBFv waveforms were similar to reported pressure waveforms and exhibited beat frequencies with a range of maximal and minimal CBFv. During in phase HR and SR periods, CBFv was 78±8 cm/s. Out of phase CBFv was 56±6 cm/s (p<0.05). Preliminary data suggest that CBFv is significantly modulated by the convolution of HR and SR during running. Dynamic cerebral blood flow may depend on the mode of exercise, and it could be optimized by timing of stride rate.
Effect of Vitamin A Fortified Milk Intake on Total Body Vitamin A Stores in Mexican Preschoolers
Authors: Lopez-Teros Veronica , Quihui-Cota Luis , Mendez-Estrada Rosa Olivia , Grijalva-Haro Maria Isabel , Valencia Mauro , Esparza-Romero Julian , Rascon-Duran Lucila , Tortoledo-Ortiz Orlando , Pacheco-Moreno Bertha , Green Michael H. , Astiazaran-Garcia Humberto
Published: June 2021

In Mexico, vitamin A deficiency (VAD) is a public health problem for preschool children. Vitamin A (VA) fortified milk (Liconsa) is distributed nationally to fight VAD, nevertheless its effect has yet not been assessed. The aim of this study was to assess the effect of daily intake of VA fortified milk on the total body VA (TBVA) stores of preschoolers.

We conducted a randomized controlled clinical trial in Mexico to assess the effect of daily intake of 250 mL of VA fortified Liconsa milk for 12 weeks on VA status of 24 VA‐deficient preschoolers. Subjects were randomly allocated to treatment (n=11) or control (n=13) groups respectively. TBVA stores were calculated using the Olson equation at baseline and three months after daily intake of VA fortified “Liconsa” milk.

TBVA stores of the treatment group had a significant increase compared to their baseline value (P<0.05) and stores had a significant difference of 0.75 mmol in treatment vs control groups (P=0.01). Serum retinol concentration was significantly reduced compared to its baseline value in the control group (P<0.05) and serum retinol was 0.291μmol/L greater in the treatment vs control group (P=0.01).

Daily intake of vitamin A fortified “Liconsa” milk during 12 weeks, significantly increased TBVA stores and serum retinol levels of Mexican preschoolers.

Study supported by the IAEA. LTV received a fellowship from CONACyT.

Grant Funding Source : IAEA‐CONACyT
Arctium lappa improve Penile Erection in Penile Smooth Muscle
Authors: Lee Xiang , Kim Hye Yoom , Kho Joung Hyun , Kwon Oh Jeong , Kang Dae Gill , Lee Ho Sub
Published: June 2021

The aim of the present study was to investigate whether an ethanol extract of Arctium lappa (EAL) augments penile erection in vitro. Isolated rabbit endothelium‐intact corpus cavernosum was preconstricted with phenylephrine (PE). EAL relaxed penile smooth muscle in a dose‐dependent manner, which was significantly inhibited by pretreatment with NG‐nitro‐L‐arginine methyl ester (L‐NAME), a nitric oxide synthase inhibitor, and 1H‐[1,2,4]‐oxadiazole‐[4,3‐¥á]‐quinoxalin‐1‐one (ODQ), a soluble guanylyl cyclase (sGC) inhibitor. EAL‐induced relaxation was also significantly attenuated by pertreatment with verapamil and diltiazem, respectively. On the other hand, relaxation of penile smooth muscle induced by EAL was not blocked by several inhibitors including tetraethylammonium (TEA), glibenclamide, indomethacin, atropine and propranolol, respectively. In the perfusion model of penile tissue, EAL also relaxed PE‐precontracted penile tissue tension in a dose‐dependent manner. Moreover, perfusion of penile tissues with EAL significantly increased the levels of cGMP and cAMP. Taken together, these results suggest that EAL may be induced penile erection through the activation of penile tissue NO/cGMP system and/or Ca2+channels in corpus cavernosum.
Involvement of vascular Kv7 potassium channels in the pathogenesis of cerebral vasospasm
Authors: Mani Bharath K , Byron Kenneth L
Published: June 2021

Cerebral vasospasm is a grave complication after subarachnoid hemorrhage characterized by persistent intense constriction of large conducting arteries in brain. Spastic contraction of the arterial myocytes is associated with membrane depolarization implicating calcium entry through voltage‐gated calcium channels. Endothelin, serotonin and vasopressin are spasmogens involved in the initiation and maintenance of vasospasm. We recently reported that Kv7 potassium channels are expressed in rat basilar artery myocytes and potassium conductance through the channels determine resting membrane potential and contractile status of basilar artery. Here we use patch‐clamp electrophysiology and pressure myography to test the involvement of Kv7 channels in the pathogenesis of cerebral vasospasm. Spasmogens suppressed Kv7 currents and produced membrane depolarization of basilar artery myocytes. The effects were reversed by Kv7 channel activator retigabine. Additivity experiments using pressure myography reveal the involvement of Kv7 channel suppression in the constriction produced by spasmogens. Activated PKC, a signal transduction intermediate of Gq‐coupled spasmogen receptor stimulation, suppressed Kv7 currents in myocytes and constricted basilar artery. These results indicate suppression of Kv7 currents as a mechanism for membrane depolarization and constriction of arteries by spasmogens resulting in vasospasm.
Dicer‐deletion in the detrusor reduces contractility and expression of L‐type Ca2+ channels
Authors: Sadegh Mardjaneh Karbalaei , Ekman Mari , Rippe Catarina , Uvelius Bengt , Swärd Karl , Albinsson Sebastian
Published: June 2021

MicroRNAs (miRNAs) are small noncoding RNAs which modulate gene expression. Production of most mature miRNAs requires the endonuclease Dicer. Here we investigated the role of miRNAs in the urinary bladder using conditional smooth muscle‐specific Dicer knockout (KO) mice. miR‐145, miR‐22, miR‐125b‐ 5p, miR27a, and miR‐1 were the most highly expressed miRNAs in the detrusor. The knockdown level for these miRNAs ranged between 68 and 99%. Using strip preparations we found that the muscarinic component of the neurogenic contraction was decreased in Dicer‐deficient bladder. A substantially reduced stress in response to high K+ was seen. The expression of L‐type Ca2+ channels (LTCC) was reduced, and only modest reductions of other differentiation markers (desmin, calponin) were noted. Assessment of micturition patterns revealed changes in both frequency and spatial voiding distribution in KO mice. We conclude that the biomechanical consequences of Dicer deletion in the detrusor are dominated by reduced LTCC expression. Deletion of Dicer‐dependent and smooth muscle‐specific miRNAs moreover results in a reduced cholinergic component of neurogenic activation. The effects of Dicer‐deletion on contractility and neuro‐effector transmission act in concert to cause a disturbed micturition pattern in vivo. Supported by Swedish Research Council, Faculty of Medicine at Lund University.
Biochemical diversity of human skeletal muscle
Authors: Tirrell Timothy F , Cook Mark , Carr John A , Choi Anthony J , Lin Evie , Esparza Mary C , Ward Samuel R , Lieber Richard L
Published: June 2021

INTRODUCTION

The molecular components largely responsible for muscle attributes such as passive tension development (titin and collagen), active tension development (myosin heavy chain [MHC]), and mechanosensitive signaling (titin) are well studied in animals but less is known about their roles in humans.

METHODS

599 biopsies were obtained from 6 human cadaveric donors. Three assays were performed on each biopsy ‐ titin molecular weight determination, hydroxyproline content (a surrogate for collagen content), and MHC isoform distribution.

RESULTS

Discriminant function analysis (DFA) showed that titin MW was the strongest predictive factor of anatomic region and muscle functional group. On average, human muscles had more slow myosin than lower mammals. Average %MHC‐1 of muscles in this study was 65% (compare to 6% for mouse, 8% for rat, and 19% for rabbit). Overall, larger titins were associated with faster muscles.

DISCUSSION

The finding that titin was the strongest discriminating factor for DFA with anatomic region and muscle functional group as grouping variables is unexpected; titin is currently primarily considered to be a signal transduction cascade activator or determinant of passive tension. The titin‐MHC‐1 relationship we observed is opposite to that previously observed in rabbits, where large titins were associated with slower muscles.
Hypoxic Exercise Training Affects Thrombin Generation by Modulating Platelet‐derived Procoagulant Microparticles under Shear Flow
Authors: Chen Yi-Ching , Chen Yu-Wen , Wang Jong-Shyan
Published: June 2021

This study investigated how exercise training with/without hypoxia affects thrombin generation (TG) mediated by platelet‐derived micropraticles (PDMPs) under physio‐pathological shear flows. Seventy five sedentary males were randomly divided into five groups. The subjects in each group (n=15) were exposed to 21% (NC) or 15% (HC) O2 at rest or were trained at 50% of Wmax under 21% (NT) or 15%O2 (HAT), or 50% of heart rate reserve under 15%O2 (HRT) for 30 minutes daily, 5 days weekly for 4 weeks. Before the intervention, strenuous exercise increased the levels of coagulant factor V/Va (FV/FVa)‐/FVIII‐/tissue factor (TF)‐rich and phosphatidylserine (PS)‐exposed PDMPs and the rate of TG in PDMP‐rich plasma (MRP) under 100 dyne/cm2 of shear stress. The NT and HRT, but not NC and HC, for 4 weeks attenuated the enhancements of shear‐induced FV/FVa‐/FVIII‐/TF‐rich and PS‐exposed PDMP formation and TG rate in MRP by severe exercise. Conversely, the HAT regimen promoted shear‐induced formation and procoagulant activation of PDMPs, concurrence with increasing plasma FV and TF levels at rest or following exercise. We conclude that a hypoxic exercise regimen, that performs a relative‐intensive workload according to exercise heart rate, may depress shear‐mediated procoagulant activity of PDMPs during exercise.
Maternal Continuous vs. Intermittent Exercise and the Fetal heart
Authors: May Linda Elizabeth , Suminski Richard R , Gustafson Kathleen M
Published: June 2021

Objective

We tested the hypothesis that regular intermittent exercise (i.e. yoga) during pregnancy is associated with changes in fetal cardiac development.

Methods

We used magnetocardiograms from 32 and 36 week fetuses (N=60). We marked normal R‐peaks to measure heart rate (HR) and heart rate variability (HRV). Activity questionnaires were used to calculate time (minutes) and intensity (kcal/min) for continuous and intermittent exercise per trimester. Correlation and regression analysis was performed. Maternal pre‐pregnancy BMI, weight gain, and fetal activity state were also assessed.

Results

Maternal body weight and BMI was not correlated with fetal measurements. Only 3rd trimester maternal activity was significantly related to fetal measures. Total continuous time is significantly associated with fetal HRV (VLF; p=0.04). Continuous intensity is significantly associated with lower fetal HR (p=0.03). Total time spent in intermittent activity is significantly associated with increased HRV (p≤0.04). There is no significant effect on fetal sympathovagal balance.

Conclusion

Maternal time spent exercising appears to be consistently important in influencing fetal heart outcomes. These data suggest a combination of continuous and intermittent exercise provide fetal heart benefits. Further studies need to be done to determine if noncontinuous exercise alone provides fetal cardiac changes.

Grant Funding Source : none
Bad to Bone: Homocysteine
Authors: Tyagi Neetu , Narayanan Nithya , Givvimani Srikanth , Tyagi Suresh C
Published: June 2021

Children born with severe homocystinuria i.e. mutation of the cystathionine‐â‐synthase (CBS) gene resulting in an elevation in plasma homocysteine (Hcy) levels, known as hyperhomocysteinemia (HHcy), develop skeletal mal‐formation with weaker bones.. Therefore present study examined the mechanism of changes in bone structure/function during HHcy. There were four groups of mice: WT, CBS+/−, WT+ Folic Acid (FA), CBS+/− + FA (0.03gm/L in drinking water for 6 weeks). The tibial bone blood flow was measured by laser Doppler. The tibial bone density was assessed by dual energy X‐ray absorptiometry. The tibia bone homogenates were analyzed for total Hcy, SAHH, CBS and MTHFR levels. MMP‐9 activity was measured by zymography and RT‐PCR. The levels of TIMPs were measured by Western blots, reverse zymography, RT‐PCR and immunehistochemistry. The levels of osteoblast/osteoclast/osteocyte were detected by immunohistochemistry and TRAP staining. The results suggested that there was decrease in bone blood flow in CBS+/− mice. The bone density was also reduced in CBS+/− mice. There was an increase in SAHH, MMP‐9 protein as well as MMP‐9 activity in CBS+/− mice and decrease in MTHFR, TIMP‐1,‐2 protein as well as mRNA levels, in part by increasing total Hcy level in CBS+/− mice. Interestingly, these effects were ameliorated by FA and suggested therapeutic role of FA in genetically HHcy induced bone disease.
Mitochondrial glutathione depletion reveals a novel role for pyruvate dehydrogenase as a key H2O2 emitting source
Authors: Fisher-Wellman Kelsey H , Lin Chien-Te , Gilliam Laura A. A. , Cathey Brook L. , Neufer P. Darrell
Published: June 2021

Pyruvate dehydrogenase (PDH) is one of the seven potential sites for O2−• generation within mammalian mitochondria; however its role in contributing to H2O2 emission in vivo has historically been viewed as negligible. To determine if pyruvate‐stimulated H2O2 emission is sensitive to an oxidative shift in redox environment, saponin permeabilized fibers were prepared from red gastrocnemius muscle of rats in the presence of varying concentrations of the glutathione (GSH) depleting agent 1‐chloro‐2,4‐dinitrobenzene (CDNB). Compared to GSH‐replete fibers, an exponential increase in pyruvate‐stimulated H2O2 emission was observed following a 25% depletion in GSH (Ctrl 1.08 ± 0.11, CDNB 61.36 ± 12.34; pmoles/min/mg, mean ± SE), with peak rates of emission reaching 265.65 ± 19.68 in maximally (98%) depleted fibers. This striking sensitivity to GSH depletion was specific to PDH as GSH depletion had minimal effect on H2O2 emission in fibers energized with glutamate, α‐ketoglutarate, or succinate alone. Pyruvate‐stimulated H2O2 emission in GSH‐depleted fibers was significantly lower in the presence of the PDH kinase inhibitor dichloroacetic acid (− DCA 273.90 ± 22.14, + DCA 51.76 ± 4.55; P<0.001), implying that pyruvate‐stimulated O2−• is sensitive to PDH phosphorylation status. These data suggest PDH may be a significant source of H2O2 production when the mitochondrial redox environment is in a more oxidized state.
Effects of non‐steroid anti‐inflammatory drugs on the human hypoxic ventilatory response and acclimatization
Authors: Ellis Erika Marie , Zarndt Rachel , Ho Baran , Hopkins Susan , Powell Frank L.
Published: June 2021

Recent studies show that inflammation contributes to respiratory chemoreflex plasticity during chronic hypoxia. Ibuprofen (a non‐steroidal anti‐inflammatory drug, NSAID) prevents the normal time‐dependent increase in the hypoxic ventilatory response (HVR) in chronically hypoxic rats (Respir. Physiol. Neurobiol. 178: 381–386, 2011). We hypothesized that inflammatory pathways contribute to ventilatory acclimatization to hypoxia in humans also. To test this, we measured the isocapnic HVR in 5 subjects at sea level and at high altitude (3,800m) using a randomized, double‐blind, repeated measures protocol. Ibuprofen or placebo was administered (400mg TID) over 48 hours at sea level and over 48 hours at 3,800m. We measured HVR at 24 and 48 hours of treatment at sea level and 3,800m. Results showed no significant difference in the HVR between placebo and ibuprofen treatments at sea level. A significant increase in the HVR at altitude occurred with placebo, indicating normal ventilatory acclimatization. However, ibuprofen blocked the increase in HVR at altitude, showing significantly less HVR increase with ibuprofen versus placebo. The results may have implications for the treatment of acute mountain sickness at high altitude with NSAIDs. Supported by UC White Mt. Res. St., NIH RO1 HL‐ 081823 & 1P01 HL 098053.
1H/13C NMR metabolomics in a neonatal rat brain slice model of early and late mild hypothermia treatments of asphyxia
Authors: Liu Jia , Litt Lawrence , Pelton Jeffrey G. , Segal Mark , Kelly Mark J.S. , Kim Myungwon , James Thomas L.
Published: June 2021

Clinical trials with neurological outcomes have led to using mild therapeutic hypothermia (≈4°C decrease) in early treatments of newborns with hypoxic‐ischemic encephalopathy from birth asphyxia, which we simulated by oxygen‐glucose deprivation (OGD) in superfused brain slices. Temperature differently modulates metabolic fluxes and concentrations. Although 4°C changes are small, three hypothermia treatment groups (nothing, immediate, and delayed) were distinctly separated by PLS‐DA multivariate analyses in 14.1 T (Tesla) and 21.15 T NMR metabolic profiling of perchloric acid brain slice extracts. Changes in tricarboxylic acid cycle (TCA) fluxes and neuron‐glial metabolic differences were measured after simultaneous administration of [1‐13C]glucose and [1,2‐13C]acetate. High energy phosphates, quantified by 31P NMR at 14.1 T, were quantified by 1H NMR at 21.15 T (900 MHz). Mild hypothermia's influence on neuron/glial metabolic differences was measured via two ratios: acetate/glucose utilization, which had significant changes; and (pyruvate carboxylase)/(pyruvate dehydrogenase) activity, which had minimal changes. Combining 13C data with 1H data further sharpened metabolomic distinctions. Support: NIH GM34767
Significantly increased plasma levels of unbound‐VEGF in overweight/obese women
Authors: Makey Kristina L , Miele Lucio , Chinchar Edmund , Pei Ivy , Robinson James , Loftin Mark , Waddell Dwight , Huang Min , Gu Jian-Wei
Published: June 2021

Angiogenesis promotes obesity. However, it is not clear whether overweight/obese women have a pro‐angiogenic phenotype in the circulation. Present study determines plasma levels of unbound‐ VEGF, endostatin, sFlt‐1 in 63 lean and overweight/obese adult women volunteers aged from 18–44. Average BMI of this group was 29.1±0.82 kg/m2, in which lean BMI was 21.95±0.47 kg/m2 (n=22) and overweight/obese BMI was 32.83±0.79 kg/m2 (n=41). ELISA assay (R&D Systems) showed that that plasma levels of unbound‐VEGF in overweight/obese young adult women were 52.26±9.6 pg/ml, significantly higher than plasma levels of unbound‐VEGF in lean young adult women, 27.34±4.99 pg/ml (P<0.01). Plasma levels of sFlt‐1 in lean (48.4±7.1 pg/ml) and overweight/obese women (52.1±9.5 pg/ml) were similar (P=0.557). There was no significant difference (P=0.442) in plasma levels of endostatin in lean (120±25 ng/ml) and overweight/obese women (109±27 ng/ml). The results support the hypothesis that overweight/obese women have a pro‐angiogenic phenotype in the circulation characterized by increased plasma levels of unbound‐VEGF. A potential therapeutic strategy for obesity will be targeting the VEGF pathway. (NIH/AA013821 and NIH/HL051971)