bluuebunny/crossref_metadata_2025_split huggingface dataset:
Novel Negative Gating Modulators of KCa2/3 as Pharmacological Tools for Novel Treatments
Authors: Oliván‐Viguera Aida , Valero Marta , Badorrey Ramón , Wulff Heike , Köhler Ralf 
Published: June 2021

IntroductionKCa2/3 channels are present in many organs and tissues such as vascular endothelia, intestinal and bronchial epithelia, neurons or blood cell lineages. Therefore, they play important roles in cardiovascular, immunological and neurological functions and emerge as therapeutic targets.Existing KCa2/3 pore blockers, due to its mechanism of action, could exert unspecific blocking effects at high dosages. Negative‐gating modulators, which modulates the regulatory mechanism of the active channel, seem to be better pharmacological tools.MethodsWe synthesized new small molecules and performed patch‐clamp experiments to evaluate their potency. We also evaluated their functional activity by isometric tension myography on porcine coronary arteries. We determined cardiovascular safety and efficacy by telemetric cardiovascular monitoring on mice.ResultsOur novel entities met Lipinski's rule of five and we identified RA‐2 as KCa2/3 pan‐negative gating modulator, with potencies in the nM range and with no considerable effects on distantly related human K channels. RA‐2 blocks EDH‐type relaxation and it is apparently safe in‐vivo, with no effects in blood pressure, although it moderately reduces heart rate.DiscussionWe identified new molecules that could serve as pharmacophores for structurally new chemical entities for specific negative‐gating modulation of KCa2/3. RA‐2 has nanomolar potency, and ex‐vivo and in‐vivo activity, and it could be a tool to study physiological and pathophysiological roles of the KCa2/3 channels and may be of therapeutic utility.
Targeted Delivery of TNF‐Alpha and IFN‐Gamma by Tumor Targeting Peptide Inhibited Orthotopic Colorectal Cancer Growth
Authors: Shen Jing , Lu Lan , Li Long Fei , Wu William Ka Kei , Li Zhi Jie , Cho Chi Hin 
Published: June 2021

ObjectiveTo determine the in vivo anti‐tumor activities of TNF‐alpha and IFN‐ gamma given alone or in combination and compare with the effects of their conjugates with tumor targeting peptide in a mouse colorectal tumor model.MethodsA mouse orthotopic colorectal tumor model was established. Treatment was given by a single i.v. injection. Mice were sacrificed and examined for their tumor sizes and other immuno and biological parameters in tumors 7 days after treatment. Apoptosis was determined by the TUNEL assay. Immunotherapeutic response was determined by immunofluorecence using antibodies against T lymphocytes, macrophages and NK cells.ResultsTNF‐alpha and IFN‐gamma showed marginal inhibitory effects on tumor growth, while their conjugates with tumor targeting peptide further decreased tumor sizes. Compared with the treatment with non‐targeted TNF‐alpha plus IFN‐gamma, targeted delivery by the peptide markedly alleviated the systemic toxicity as shown by a significant reduction in mortality after treatment. Moreover, targeted delivery of TNF‐alpha plus IFN‐gamma synergistically and significantly inhibited tumor growth.ConclusionsTargeted delivery of TNF‐alpha plus IFN‐gamma exhibited promising tumoricidal activity and at the same time alleviated the systematic toxicity induced by the immuno‐modulators in mice.Acknowledgements: The study was supported by the Innovation and Technology Support Programme and the General Research Fund from the Innovation and Technology Commission and the Research Grant Council from Hong Kong.
Immunohistological and Morphological Analysis from a Skeletal Muscle Microbiopsy
Authors: Townsend Jeremy , Fragala Maren , Oliveira Leonardo , Jajtner Adam , Gonzalez Adam , Wells Adam , Stout Jeffrey , Fukuda David , Hoffman Jay 
Published: June 2021

ABSTRACTThe study of skeletal muscle often requires invasive muscle biopsies, traditionally with the Bergstrom technique. Microbiopsies, or fine‐needle biopsies are used with many biochemistry applications but are scarcely used for histology due to inconsistencies with sample weight and integrity. Thus, the aim of the current study was to develop procedures for muscle fiber morphology and immunohistological analysis from a microbiopsy technique.METHODS1,366 muscle fibers were analyzed from 8 muscle biopsy samples obtained from four healthy men (26.8 ± 5.4y, 175.9 ± 4.3 cm, 91.9 ± 5.3kg). Percutaneous microbiopsies were taken of the vastus lateralis with a 14‐gauge microbiopsy needle on two separate occasions. Samples were removed from the needle and promptly weighed. The tissue was oriented in a cryomold, embedded in Tissue‐Tek® then frozen in liquid nitrogen cooled isopentane. The muscle sections were stained with haematoxylin and eosin or anti‐laminin and counterstained with AlexaFlour 488® and viewed under 4x magnification. Fiber number and mean fiber area was determined by Image J.RESULTSThe mean ± SD (range) microbiopsy sample weight was 18.3 ± 2.9 mg (14‐22mg). Fiber number within the microbiopsy specimens was 150.4 ± 120.6 (64–366). All viable fibers were measured in each sample and the mean fiber area was 4385.1 ± 1265.8 µm2 (977.0 – 10,132.93 µm2). The intra‐subject coefficient of variation for mean fiber area was 7.2%.CONCLUSIONResults show the potential of a minimally invasive muscle biopsy technique for use in immunohistological and morphological analysis. This could allow future advances in the study of skeletal muscle by allowing for smaller muscle groups to be examined.
Characterization of a Kbtbd13‐Deficient Mouse Model That Recapitulates Nemaline Myopathy With Mutations in The KBTBD13 Gene
Authors: de Winter Josine , Joureau Barbara , Stienen Ger , Granzier Henk , Ottenheijm Coen 
Published: June 2021

BackgroundNemaline myopathy (NM) is the most common non‐dystrophic congenital myopathy and is characterized by muscle weakness. Recently a novel implicated gene was discovered ‐ KBTBD13 – of which the function of its protein product is unknown. NM patients with KBTBD13 mutations have muscle weakness and show a typical slowness of movement. Here, we generated a mouse model that lacks the kbtbd13 gene to obtain insight into the role of this new gene and its protein product and to elucidate the pathophysiology of this new form of NM.MethodsKbtbd13‐KO mice are viable and live into adulthood. Muscle weights of cardiac and skeletal muscle were collected. Intact soleus muscles of 3 month‐old male kbtbd13‐KO and wild‐type (WT) mice were used for in vitro experiments. Muscle tendons were attached to a force transducer and a length motor. Muscles were electrically activated by field stimulation at 30°C.ResultsMaximal tetanic force at 150Hz was reduced in kbtbd13‐KO (n=10). The relative force‐frequency relationship in kbtbd13‐KO mice was shifted to the left compared to WT mice. Both activation and relaxation kinetics were slower in kbtbd13‐KO mice. Muscle weight data reveal atrophy of skeletal muscles, whereas the cardiac muscle shows hypertrophy of the left ventricle.ConclusionKbtbd13‐KO mice phenocopy the normal life span and typical muscle weakness observed in NM patients with mutations in KBTBD13. Hence, we present a novel NM mouse model that is a useful tool to elucidate the role of this new gene and its protein product and to study the mechanism underlying muscle weakness observed in patients.
Reduced Common Carotid Artery Blood Flow in Spontaneously Hypertensive Rats is Unaffected by Aging and/or Chronic In Vivo Resveratrol Treatment
Authors: Denniss Steven , Smith Christopher , Mariani Rachelle , Wood Laura , Rush James 
Published: June 2021

This study examined common carotid artery (CCA) blood flow (using a perivascular flowprobe) and pressure (using a catheter‐tip transducer) in anesthetized Adult (20‐24 wk old) and Aging (60‐70 wk old) Wistar Kyoto rats (WKY) and Spontaneously Hypertensive rats (SHR) following 28‐days of in vivo treatment (in drinking water, ab libitum) with either no trans resveratrol (RSV; No‐RSV), a moderate RSV dose (Mod‐RSV; ~0.075 mg/kg/day; to mimic daily red wine consumption), or a high RSV dose (High‐RSV; ~7.5 mg/kg/day). CCA mean, maximal systolic, minimum diastolic, and pulse pressure, and heart rate, were all significantly elevated in SHR vs. WKY (p<0.01). Aging was associated with a significant increase in pulse pressure in WKY (p<0.01). CCA mean, maximal systolic, and minimum diastolic blood flow, and vascular conductance, were all significantly reduced in SHR vs. WKY (p<0.05). Aging did not have a significant effect on the flow parameters in either SHR or WKY. Neither Mod‐RSV nor High‐RSV treatment had a significant effect on the CCA blood pressure and blood flow variables measured. These data demonstrate that the reduction in CCA blood flow found in SHR is unaffected by aging or chronic in vivo RSV treatment. Further investigation into what can account for and alter the reduced CCA blood flow found in SHR is warranted.Funded by NSERC.
The Role of ATP and P2X7 in Upregulation of Vasocontractile ET‐B Receptors in Basilar Arteries
Authors: Haanes Kristian , Edvinsson Lars 
Published: June 2021

Cerebrovascular disease is one of the main causes of death in the developed countries. Several G‐protein coupled receptors, such as the Endothelin‐B receptor (ET‐B), are upregulated after a stroke. These receptors cause constriction in the blood vessels and reduce blood flow, causing further damage and reduced chances of recovery. Our group has previously shown that MEK/ERK1/2 inhibition greatly reduced the receptor upregulation and the cerebral damage. Extracellular ATP is a short‐range signal important in the regulation of both the blood flow and also in long term cellular modifications. One of the receptors for ATP, the P2X7 receptor, is of high interest, as it can be involved in apoptosis, proliferation, and ERK1/2 signaling. We hypothesized that ATP could be an important activator of MEK/ERK1/2 and contribute to the upregulation of contractile receptors. Ex vivo ET‐B receptor upregulation in basilar arteries (assessed by the specific agonist sarafotoxin 6c (S6c) in a myograph) can be induced by incubation of artery segments in DMEM media for 48 hours (organ culture). The changes are similar to those observed in vivo in experimental stroke. Surprisingly, adding apyrase (an ATP degrading enzyme) further increased the contraction induced by S6c and was nearly mimicked by the specific P2X7 antagonist Az10606120. The addition of a P2X7 agonist (10µM BzATP) during organ culture had little effect, indicating that the lower ATP concentrations are the important stimuli. In conclusion, ATP and P2X7 appear to be important in the upregulation of ET‐B receptors during organ culture and play a beneficial role because inhibiting them causes an increased upregulation of contractile ET‐B receptors.Funding: Lundbeck foundation (R167‐2013‐15379) and Lundbeck Grant of excellence
Circulating MicroRNAs Characterize Insufficient Coronary Collateral Artery Development in Patients with Chronic Total Occlusion
Authors: Hakimzadeh Nazanin , Nossent A Yaël , van der Laan Anja , Schirmer Stephan , van Royen Niels , Quax Paul , Hoefer Imo , Piek Jan 
Published: June 2021

Coronary collateral artery growth, arteriogenesis, significantly impacts mortality after an acute coronary event. We aimed to identify microRNAs (miRNAs) that differentiate between insufficient or sufficient coronary collateral artery development in patients with chronic total occlusion (CTO).CTO patients (n=41) were dichotomized based on their pressure‐derived collateral flow index (CFI = (wedge pressure – central venous pressure)/(aortic pressure – central venous pressure)) into low (CFI< 0.39) and high arteriogenic responders (CFI > 0.39). MiRNA profiling on aortic plasma and verification by qPCR, revealed higher levels of miR423‐5p (p < 0.05), miR10b (p < 0.05), miR30d (p < 0.05) and miR126 (p < 0.001) were present in low arteriogenic responders. Receiver operator characteristic curves showed that only miR126 can distinguish patients with low arteriogenic response (area under curve 0.812, p < 0.01, cut‐off 0.037).In conclusion, we have identified miRNAs linked with poor collateralization and found miR126 as the most suitable circulating biomarker to distinguish patients with poor or adequate coronary artery collateral capacity.Supported by the Center for Translational Molecular Medicine (EMINENCE).
Release of microparticles from native coronary arteries and saphenous vein grafts in patients with elective stent implantation under protection with an aspiration device
Authors: Kleinbongard Petra , Baars Theodor , Horn Patrick , Kahlert Philipp , Heiss Christian , Westenfeld Ralf , Kelm Malte , Erbel Raimund , Heusch Gerd 
Published: June 2021

ObjectiveStent implantation into coronary vessels impacts on downstream microvascular function and induces the release of particulate debris and soluble substances, which differs between native right coronary arteries (RCAs) and saphenous vein grafts on right coronary arteries (SVGRCAs). We have now quantified the release of microparticles (MPs) during stent implantation into RCAs and SVG‐RCAs.MethodsPatients with a stenosis in their RCA or SVG‐RCA, respectively (n = 14/14), were enrolled. Coronary aspirate was retrieved during stent implantation with a distal occlusion/aspiration device. Platelet‐derived MPs (PMPs) were distinguished by flow cytometry as CD41+, endothelium‐derived MPs (EMPs) as CD144+, CD62E+ and CD31+/CD41‐, leukocyte‐derived MPs as CD45+, and erythrocyte‐derived MPs as CD235+.ResultsIntracoronary PMPs and EMPs were increased after stent implantation into their RCAs and SVG‐RCAs (CD41+: 2729.6±645.6 vs. 4208.7±679.4 and 2355.9±503.9 vs. 3285.8±733.2 nr/μL; CD144+: 451.5±87.9 vs. 861.7±147.0 and 444.6±74.8 vs. 726.5±136.4 nr/μL; CD62E+: 1404.1±247.7 vs. 1844.3±378.6 and 1084.6±211.0 vs. 1783.8±384.3 nr/μL, P<0.05), but not different between RCAs and SVG‐RCAs.ConclusionStenting in RCAs and SVG‐RCAs is associated with a substantial release not only of PMPs, but also of EMPs.
The Activation of Matrix Metalloproteinease‐2 by Mitochondrially‐Generated Reactive Oxygen/Nitrogen Species
Authors: Hughes Bryan , Schulz Richard 
Published: June 2021

Matrix metalloproteinase‐2 (MMP‐2), along with other MMPs, has been implicated in heart injury and disease. These pathologies are characterized by both acute and chronic oxidative stress. In vitro, low concentrations of reactive oxygen/nitrogen species (RONS) can directly activate MMP‐2 via post‐translational modifications. However, this has yet to be demonstrated in a living system. We investigated whether endogenous RONS produced by pharmacological inhibition of mitochondrial ubiquinol‐cytochrome c reductase with antimycin could increase MMP‐2 activity in cardiomyocyte and non‐cardiomyocyte cell lines. We exposed neonatal rat cardiomyocytes (NRVMs), H9c2 cardiomyoblasts and HT1080 fibrosarcoma cells to antimycin (0.01‐1, 0.3‐3.2 and 1‐100 µM for NRVMs, H9c2 and HT1080 respectively) in serum‐free media for short (0.5 h) or long (6 h) intervals to measure changes in MMP‐2 activity in cell lysates and conditioned media by gelatin zymography. The antimycin concentrations used did not affect viability or cell growth, but increased oxidative stress in a dose‐ and time‐dependent fashion as shown by decreased aconitase activity. Antimycin decreased lysate MMP‐2 activity in NRVMs, but increased it in H9c2 cells, despite comparable decreases in aconitase activity. HT1080 cells also exhibited increased MMP‐2 activities, but only at the lowest concentrations of antimycin used. The activity of secreted MMP‐2 was not consistently affected by antimycin treatment in any cell line. We conclude that intracellular MMP‐2 activity can be affected by endogenous oxidative stress in cultured cells, dependent upon cell type. This provides evidence that RONS could modulate MMP‐2 activity in vivo, independent of gene expression. Support: CIHR
Assisted Reproductive Technologies‐Induced Premature Vascular Ageing Persists and Evolves into Arterial Hypertension in Adolescents
Authors: Rexhaj Emrush , von Arx Robert , Cerny David , Soria Rodrigo , Bouillet Elisa , Sartori Claudio , Scherrer Urs , Rimoldi Stefano 
Published: June 2021

Assisted reproductive technologies (ART) induce vascular dysfunction and premature vascular ageing in young apparently healthy children and mice (Scherrer et al. Circulation 2012; Rexhaj et al. JCI 2013). In adult ART mice, premature vascular ageing translates into arterial hypertension. Given the young age of the human ART population, the evolution and long‐term consequences of ART induced vascular alterations are unknown. We speculated that vascular alterations persist in ART adolescents and that arterial hypertension represents the first clinically relevant end‐point of this problem in humans. We, therefore, 5 years after the initial assessment, reassessed vascular function (flow mediated dilation, FMD, pulse wave velocity, PWV, and carotid intima media thickness, IMT) in 54 ART young adults (16.4,y) and 43 controls (17.6,y) together with 24h ambulatory blood pressure measurements.The main new findings were 2‐fold: First, premature vascular ageing persisted in ART adolescents, as evidenced by decreased FMD (P<.01), and increased PWV (P=.039) and IMT (P<.01), compared to controls. Endothelial dysfunction and arterial stiffness were comparable to the one observed 5 years ago, whereas IMT had increased in ART subjects (P<.01). Second and most importantly, 24h blood pressure was markedly higher in ART than in control subjects (119.8±1.3 vs. 115.7±1.1, P=.02, vs. ctrl).These findings provide the first evidence that ART‐induced premature vascular ageing persists in adolescents and translates into arterial hypertension. These data further underscore the potential of ART to increase cardiovascular risk in this exponentially growing population.
Renoprotective Effects of N‐acetyl‐seryl‐aspartyl‐lysyl‐proline (Ac‐SDKP) in Dahl Salt‐Sensitive Rats
Authors: Worou Morel , D'Ambrosio M , Liao T‐D , Nakagawa P , Peterson E , Janic B , Rhaleb N‐E , Carretero Oscar 
Published: June 2021

Ac‐SDKP is a natural tetrapeptide, hydrolyzed by angiotensin‐converting enzyme, with anti‐inflammatory and anti‐fibrotic properties. The effect of Ac‐SDKP on Salt‐Sensitive (SS) hypertension is unknown; here, we hypothesized that Ac‐SDKP prevents renal inflammation, fibrosis, glomerulosclerosis and albuminuria in Dahl SS rats on high salt diet, without affecting the blood pressure. Dahl SS rats and their consomic controls, SS‐13BN were fed either 0.23% NaCl (low salt, LS) or 4% NaCl (high salt, HS) diet and infused with vehicle or Ac‐SDKP at either low or high dose (800 or 1600 µg/kg/day, respectively) subcutaneously via osmotic minipump for 6 weeks. Animals were divided into the following groups: LS + vehicle, HS + vehicle, HS + low AcSDKP, and HS + high AcSDKP.HS increased systolic blood pressure (SBP) in SS rats (HS + vehicle 186±5 vs. LS + vehicle 141±3 mmHg), but not in SS‐13BN rats (HS+vehicle 140±3 vs. LS+vehicle 140±2 mmHg). Ac‐SDKP did not affect SBP. In both strains, Ac‐SDKP prevented HS‐induced renal macrophage and T cell infiltration, interstitial fibrosis and glomeruloslerosis. In SS‐13BN rats, low dose of Ac‐SDKP were able to prevent albuminuria (marker of renal function) induced by HS (LS+vehicle 7±1 vs. HS + vehicle 44±8 vs. HS + low Ac‐SDKP 24±3.18 mg/24h), while in SS rats only high Ac‐SDKP dose had the same effect.In summary, at low and high doses, Ac‐SDKP prevented renal damages in SS rats on HS diet without affecting the blood pressure. But, only at high dose, Ac‐SDKP prevented HS‐induced albuminuria in this strain. On the other hand, in consomic controls, both doses of Ac‐SDKP prevented HS‐induced renal damages and albuminuria.Supported by NIH PPG grant P01HL028982
Calcineurin homologous protein 1 interferes with calcineurin‐dependent regulation of the renal Na‐K‐2Cl‐cotransporter
Authors: Boldt Christin , Paliege Alexander , Jankowski Vera , Wakabayashi Shigeo , Hisamitsu Takashi , Bachmann Sebastian , Mutig Kerim 
Published: June 2021

The Na‐K‐2Cl‐cotransporter (NKCC2) of kidney thick ascending limb (TAL) is crucial for renal salt handling. Its activity tightly regulated by phosphorylation. Our previous data suggested that calcineurin is involved in dephosphorylation and thus, deactivation of NKCC2. Further results suggested calcineurin activity may be modulated by members of the calcineurin homologous protein family (CHP). Among these, CHP1 has been implicated in the regulation of several membrane proteins including ion transporters. The role of CHP1 in calcineurin‐dependent regulation of NKCC2 activity has therefore been studied.CHP1 was co‐localized with NKCC2 in rat TAL by immunofluorescence. Both products co‐immunoprecipitated, suggesting their interaction. GST pull down assays with recombinant N‐terminal NKCC2 mutants, mimicking its (de)phosphorylation at functionally relevant residues (T96, T101, T114, and S126), suggested competitive interactions of CHP1 and calcineurin with phosphorylated NKCC2. Overexpression of CHP1 in cultured TAL cells substantially increased the phosphorylation of endogenous NKCC2 at baseline and upon low‐chloride stimulation. Acute stimulation of NKCC2 by administration of desmopressin in rats facilitated NKCC2 interaction with CHP1 while attenuating its interaction with calcineurin.In sum, our data provide evidence that CHP1 facilitates NKCC2 phosphorylation, likely by interfering with calcineurin function in TAL.
Renal Specific Upregulation of Mitochondrial Genes in a Translational Primate Model of Spontaneous Hypertension
Authors: Goleva Slavina , Rhoads Megan , Osborn Jeffrey 
Published: June 2021

Mitochondrial gene expression in African Green Monkeys (Chlorocebus aethiops sabaeus common name, vervet) may importantly influence renal function and long‐term blood pressure control. Vervets share over 90% of their genome with humans and exhibit heritable, spontaneous hypertension.We hypothesized that altered renal mitochondrial gene expression in hypertensive vervets may be associated with arterial hypertension. Vervets were phenotyped as normotensive (NT, systolic blood pressure (SBP) <120 mmHg) or hypertensive (HT, SBP > 140 mmHg). RNA was extracted from kidney cortex and liver tissues from 18 HT (mean SBP 166±7 mmHg) and 18 NT animals (mean SBP 98±3 mmHg) and gene expression determined using semi‐quantitative RT‐PCR. In renal cortical tissue of HT vervets, COX 3 (Complex IV) and Cyt B (Complex III) were upregulated by 1.6‐fold (p=0.03) and 1.4‐fold (p=0.05) respectively, while NADH4 (Complex I) and ATP8 (Complex V) expression were unchanged. In contrast, liver COX 3 expression was decreased in HT monkeys (1.9‐fold; p=0.04), 1.6‐fold for ATP8 (p=0.03), and 2.0‐fold for CYTB (p=0.01). Gene expression of nuclear encoded subunits in the oxidative phosphorylation chain SDH (Complex II) and COX4 (Complex IV) were not different in both kidneys and liver of HT vs. NT vervets. Selected mitochondrial genes are upregulated in renal cortex of hypertensive vervets and may be associated with excess reactive oxygen species. Mitochondrial gene downregulation in the liver may result from the systemic hypertension.(Supported by APS, Biomedical Sciences Research Group, LLC, Telabio, Inc and Behavioral Sciences Foundation)