bluuebunny/crossref_metadata_2025 huggingface dataset:
Anti‐tumor Effect of a Novel Soluble Recombinant Human Endostatin: Administered as a Single Agent or in Combination with Chemotherapy Agents in Mouse Tumor Models
Authors: Ren Zhihua , Jiang Wenhong , Dai Wei , Jiang Yongping
Published: June 2021

Endostatin is one of the potent anti‐angiogenesis agents, which suppress tumor formation through the inhibition of blood vessel growth. It is anticipated that combined therapy using endostatin and cytotoxic compounds may exert a synergistic effect. In the present study, we expressed and purified recombinat human endostatin (rhEndostatin) that contained 3 additional amimno acid residues (arginine, glycine, and serine) at the amino‐terminus and 6 histidine residues in its carboxyl terminus. Briefly, rhEndostatin cDNA was cloned into PQE vector and expressed in E. Coli. The protein was refolded through an optimized dialysis procedure for a large scale yield. To establish tumor models, nude mice were subcutaneously injected with human cancer cells (lung carcinoma A549, hepatocellular carcinoma QGY‐7703, or breast cancer Bcap37). For the tumor metastasis model, mice were injected intravenously with mouse melanoma B16 cells. A single dose of rhEndostatin, or in combination with CTX or DDP, was administered intravenously or at a site close to the tumor. Our results demonstrate that rhEndostatin reduced the growth of A549, QGY‐7703, and Bcap37 xenograft tumors in a dose dependent manner. When it was administered peritumorally, rhEndostatin exhibited a more potent inhibitory activity. Furthermore, rhEndostatin displayed a synergistic effect with CTX or DDP on the inhibition of metastasis of B16 tumors or growth of A549 tumors. In conclusion,soluble rhEndostatin exibibits a potent anti‐tumor activity in mouse xenograft models and it also has a synergistic effect with CTX and DDP, implying possible applications in clinical settings
Preclinical Efficacy of an Antibody‐Drug Conjugate Targeting TIM‐1 in Ovarian, Renal and Lung Tumor Models
Authors: Gergel Lauren , Forsberg Eric , Pilsmaker Catherine , Boyer James , Round Sarah , Borrelli Kathleen , Sandland April , Vargas Nohely , Marsh Henry , Keler Tibor , Thomas Lawrence
Published: June 2021

T cell immunoglobulin and mucin domain 1 (TIM‐1) is a type I transmembrane glycoprotein, also described as kidney injury molecule 1 (KIM‐1) and as hepatitis A virus cellular receptor 1 (HAVCR1). TIM‐1 has been shown to have upregulated expression in several human cancers, but has restricted expression in healthy tissues, thus it represents a promising target for antibody mediated therapy. To this end we have developed a fully human monoclonal antibody specific for the extracellular domain of TIM‐1, covalently linked to the potent cytotoxin monomethyl auristatin E (MMAE). We have designated this antibody‐drug conjugate as CDX‐014. Three TIM‐1 expressing xenograft models (Caki‐1, a renal clear cell carcinoma; IGROV‐1, an ovarian adenocarcinoma, and A549, a lung carcinoma) have been established to demonstrate the efficacy of CDX‐014 in vivo against human tumors. In all models, cells were grown in culture and then implanted subcutaneously into immunocompromised mice. When tumors reached an average of approximately 0.3cm3, mice were split into equivalently sized control and treatment groups. Dose dependent efficacy of CDX‐014 was shown in all models by a decreased rate of tumor growth and extended group survival. These studies support the further development of CDX‐014, including manufacturing activities and IND‐enabling studies, in order to advance towards clinical studies in clear cell carcinomas and potentially other TIM‐1 expressing tumors.
Inhibition of Geranylgeranyl Diphosphate Synthase Induces Apoptosis of Lymphocytic Leukemia Cells
Authors: Su Sherry , Hsiao Chia‐Hung , Jensen Christopher , Wiemer Andrew
Published: June 2021

Bisphosphonates are metabolically stable diphosphate analogs that inhibit downstream enzymes in the mevalonate pathway and induce cell death. Here, we compared the effects of a farnesyl diphosphate synthase inhibitor, zoledronate, and a geranylgeranyl diphosphate synthase inhibitor, digeranyl bisphosphonate (DGBP), on lymphocytic leukemia cell proliferation and apoptosis. Both zoledronate and DGBP prevented Molt‐4 cell proliferation with DGBP doing so more potently. Surprisingly, DGBP was markedly less toxic than zoledronate towards the viability of healthy human peripheral blood mononuclear cells. Furthermore, addition of geranylgeranyl diphosphate rescued the inhibitory effects of DGBP and partially rescued the effects of zoledronate. In order to further assess the growth inhibitory effects of the bisphosphonates, we analyzed annexin V and PI staining via flow cytometry and found that DGBP was more potent than zoledronate in inducing apoptosis. We demonstrate via Western blot analysis that incubation with DGBP causes cleavage of caspase 3/7, increases ERK phosphorylation, and elevates RhoA protein levels. Taken together, our findings indicate that DGBP is a more potent and selective compound than zoledronate in inducing apoptosis mediated through RhoA, ERK and the caspase pathway. These findings support the further development of geranylgeranyl diphosphate synthase inhibitors as anti‐cancer therapeutics.
Novel Strategy of Using Flaxseed Lignans as Adjuvant Therapy Against Prostate Cancer
Authors: Di Yunyun , Krol Ed , Alcorn Jane
Published: June 2021

Increasing evidence from preclinical and clinical studies demonstrate that dietary flaxseed lignans may have a role in prevention and treatment of prostate cancer. The lignans enterolactone (ENL) and secoisolariciresinol (SECO) may contribute to the anticancer effect. Our in vitro cytotoxicity studies found that ENL and SECO reduced the IC50 values of chemotherapeutic agents in prostate specific membrane antigen (PSMA) positive and negative prostate cancer cell lines. However, following oral consumption the flaxseed lignans exist mainly as inactive glucuronic acid conjugates with very low systemic levels of ENL and SECO. Based upon their unusual pharmacokinetic profile, we propose the use of antibody directed enzyme prodrug therapy (ADEPT) as adjuvant therapy. ADEPT is a two‐step process by which a prodrug activating enzyme is delivered specifically to the tumor site by a tumor‐specific antibody following administration of a nontoxic prodrug. Activation of the prodrug by the localized enzyme will trigger cancer cell death directly or via bystander effect. An anti‐PSMA antibody conjugated with beta‐glucuronidase (fusion protein) was successfully generated. The fusion protein binds well against PSMA positive prostate cancer cell line, LNCap, as analyzed by flow cytometry. The enzymatic activity of the fusion protein was also tested using 4‐methylumbeliferone (positive control) and enterolactone glucuronide and demonstrated ability to cleave the glucuronide. The fusion protein will be tested in prostate cancer xenograft mouse model to determine the tumor specific distribution, enzymatic activity at the tumor site, and ability to reduce tumor size and prolong survival when used in combination with chemotherapeutic agents.
Exchange Protein Directly Activated by cAMP (EPAC1) Promotes Pancreatic Cancer Metastasis
Authors: Mei Fang , Almahariq Muayad , Chao Celia , Hellmich Mark , Cheng Xiaodong
Published: June 2021

Cyclic adenosine monophosphate (cAMP) plays a critical role in regulating migration of various cancers. This role is context dependent and is determined by which of the two main cAMP sensors is at play: PKA or EPAC. Recently, we have shown that the cAMP sensor protein EPAC1 promotes invasion/migration of pancreatic ductal adenocarcinoma (PDA) in vitro. Here we investigated the role of EPAC1 in invasion and metastasis of PDA in vivo, and evaluated the therapeutic potential of EPAC inhibitors as anti‐metastasis agents for this neoplasm. We employed an orthotopic metastatic mouse model in which the PDA cells MIA PaCa‐2 were injected into the pancreas of athymic nude mice, and their local and distant spread was monitored by in vivo imaging and histological evaluation of the number of metastatic foci in the liver. Either genetic suppression of EPAC1 or its pharmacologic inhibition with ESI‐09, an EPAC‐specific antagonist recently identified in our lab, decreased invasion and metastasis of the PDA cells. Mechanistically, EPAC1 promotes activation and trafficking of integrin β1, which plays an essential role in PDA migration and metastasis. Our data shows that EPAC1 facilitates metastasis of PDA cells and EPAC1 might be a potential novel therapeutic target for developing anti‐metastasis agents for PDA.
Contractile Properties of Adult Ventricular Myocyte in Long‐term Culture
Authors: Liu Shi
Published: June 2021

We have reported that the primary culture of adult ventricular myocyte (AVM) isolated from hearts of adult rat can be maintained in culture for several months (defined as LTC‐VM) and retains functional capacity. This study is to characterize contractile properties [including spontaneous contraction, frequency response and responsiveness to 1 μM isoproterenol (ISO) and 0.1‐1 mM ouabin (ouab)] of the LTC‐VM. Cells were maintained in antibiotic‐ and antimycotic‐free M199 culture medium with 2% fetal bovine serum in an incubator with 5% CO2 and 95% air at 37°C. Spontaneous contractile activities or paced cell contractions (measured as cell shortening, CS) were monitored using video microscopy or a video edge‐motion detector system, respectively. The spontaneous activity of LTC‐VM was decreased with time in culture; e.g., averaged frequency of 1 and 0.25 Hz for cells in one month and 3 months of culture, respectively. LTC‐VM of spontaneous activity and paced cells with a rate higher than the spontaneous contraction displayed post‐rest potentiation and a frequency response; i.e., increases in contraction amplitude and baseline until at 5 Hz. ISO induced positive inotropy and lusitropy, while ouab elicited only positive inotropy in cells at all ages of culture. Moreover, high concentrations of ISO and ouab induced arrhythmias. Both detection methods provide comparable results of contraction measurements, while video microscopy offers multi‐site measurements from a large area of LTC‐VM. In summary, these results are qualitatively consistent with those observed in freshly‐isolated AVM. In conclusion, LTC‐VM retains physiological contractile property of AVM and can be valuable to pathophysiological studies, cardiomyocyte senescence and drug screening. (Supported by UAMS departmental funding)
Progression of Cancer Cachexia Alters Muscle Lactate Metabolism In ApcMin/+ Mice
Authors: Brown Lemuel , Brown Jacob , Rosa Megan , Lee David , Perry Richard , Hardee Justin , Carson James , Greene Nicholas , Washington Tyrone
Published: June 2021

Roughly 100,000 individuals are diagnosed with colorectal cancer a year, 80% of these cases arise from adenomatous polyps. A mutation of the tumor suppressor gene, Adenomatous polyposis coli (Apc) results in multiple intestinal neoplasia (Min), thus the Apc (Min/+) mouse is a common colorectal cancer model. Cancer cells favor a highly glycolytic environment. As such, there is an induction of processes regulating lactate metabolism and lactate shuttling. How colorectal cancer influences skeletal muscle lactate metabolism is uncertain. Purpose: We investigated how muscle lactate metabolism is regulated during the progression of cancer cachexia in Apc (Min/+) mice. Methods: Male Min/+mice (n = 17) were classified by body mass loss: weight stable (WS, 蠄 5%, n = 5), and cachectic (CC, 6‐19%; n = 5). C57BL/6J (B6, n = 6) served as controls. Quadriceps muscle samples were extracted and real‐time PCR was conducted to quantify lactate‐related gene expression. Results: LDH‐A was reduced 47% in the CC mice compared to the WS mice (p < 0.05), while LDH‐B was not changed with cancer cachexia progression (p = 0.09). Monocarboxylate transporter 1 (MCT1) was induced 1.9‐fold in CC mice (p < 0.05) compared to the B6 mice. MCT4 was induced 1.7‐fold and 2.3‐fold in the CC mice compared to B6 and WS mice, respectively. Muscle CD147 was not changed with cancer cachexia progression (p = 0.31).

Conclusion

The progression of cachexia in mice with colon cancer alters markers of lactate metabolism that may play a role in muscle wasting associated with cachexia.
Effect of N‐2‐mercaptopropionyl glycine in postfatigue tension in avian skeletal muscle
Authors: Sánchez‐Duarte Elizabeth , Guzmán‐Ambriz Ana , Trujillo Xóchitl , Huerta Miguel , Saavedra‐Molina Alfredo , Cortés‐Rojo Christian , Montoya‐Pérez Rocío
Published: June 2021

Use of antioxidants can reduce the development of fatigue and reverse the decline of force induced by ROS. It have been reported that N‐2‐mercaptopropionyl glycine (MPG), a potent cell permeable ROS scavenger, prevents ischemic damage in heart during exercise; a process analogous in skeletal muscle fatigue, which also involves triggering of several signaling pathways, among those found the production of ROS. Thus, the purpose of this study was to determine the effect of MPG during fatigue. Anterior Latissimus Dorsi muscle of chicks was dissected and mounted on an experimental recording chamber by placing the distal end of the muscle to the hook of a mechanic‐electric transducer (Grass FT03), which through an amplifier and a analog‐digital interface (Digidata 1322A) allowed to acquire the muscle tension generated in a computer and a software of data acquisition (AXOTAPE, pCLAMP 9.2). We performed a fatigue protocol by twitches (100 V, 300 ms , 0.5 Hz). Once force decreased by 60 %, MPG was applied for 6 min (500 uM, 1 mM and 1.5 mM). The mayor effect observed by administration of MPG was at 1 mM on single twitch post‐fatigue tension. The post‐fatigue tension increased to 20.18 ± 5.92 % in peak tension (p=0.034), while in total tension was 39.95 ± 14.56 % (p= 0.046) compared to fatigued condition. Results suggest that the removal of certain amount of ROS can improve force, but it is also necessary a quantity of ROS for its normal function.
Cerebral Blood Flow Velocity Responses to an Acute Cognitive Challenge in Healthy Adults
Authors: Barnes Jill , Harvey Ronee , Malterer Katherine , Johnson Maja , Joyner Michael
Published: June 2021

Cerebral blood flow velocity responses to hypercapnia are blunted by the cyclooxygenase inhibitor indomethacin (INDO). We hypothesized that INDO would also attenuate middle cerebral artery blood flow velocity (MCAv) responses to an acute cognitive challenge. We evaluated the MCAv responses to an acute cognitive challenge in 20 healthy young (20‐35 yrs) and older (56‐76 yrs) healthy adults, including 10 men and 10 women before and 90 minutes after INDO administration. The cognitive challenge consisted of 2 blocks of the Stroop color word test: easy (3 colors) and difficult (8 colors). Mean arterial pressure (MAP), end‐tidal CO2, and MCAv were monitored throughout the protocol. There were no differences in MAP or end tidal CO2 between trials. Indeed, MCAv was lower during INDO than baseline for both the easy (50±4 vs. 68±4 cm/s; p<0.01) and difficult (49±3 vs. 66±3 cm/s; p<0.001) cognitive challenges. Additionally, there was an Age x Sex interaction for the increase in MCAv during the easy cognitive task, with older men having a greater increase than older women (15±5 vs. ‐1±5%, respectively; p<0.05). This interaction was not statistically significant for the difficult cognitive task or during the INDO trial. In conclusion, similar to hypercapnia, the cyclooxygenase inhibitor INDO blunts cerebral blood flow velocity responses to an acute cognitive challenge.

Funded by NIH grant HL 118154
Protective Effect of Transfusion of Carbon Monoxide‐Bound PEGylated Hemoglobin (PEG‐COHb) after Transient Focal Cerebral Ischemia
Authors: Koehler Raymond , Lui Xiaoguang
Published: June 2021

Transfusion of acellular PEG‐COHb at 20 min of middle cerebral artery occlusion (MCAO) maintains pial arteries in a dilated state and reduces infarct volume. We investigated whether the decrease in infarct volume could be attributed to improved oxygenation and to known anti‐inflammatory effects of moderate levels of CO. Anesthetized rats underwent MCAO for 2 h by the intraluminal filament technique. Transfusion of 10 ml/kg of a 4% solution of PEG‐COHb at 20 min of MCAO decreased Hif‐1a protein expression in the ischemic hemisphere by 83 (11)% (SE) at 2 h after onset of MCAO, consistent with improved intraischemic oxygenation. Next, we examined the effect of delaying transfusion until reperfusion. Infarct volume in cerebral cortex was significantly decreased from 36 (5)% in controls to 17 (6)%, 15 (5)%, and 17 (5)% in groups transfused with PEG‐COHb at 20 min, 2 h, and 4 h after MCAO, respectively. PEG‐COHb transfusion at reperfusion decreased TNFa and IL‐1b gene expression by 69% and 74%, respectively, in cortex 24 h after MCAO. To test for the direct effect of CO, another group received an injection of the CO‐releasing molecule‐3 (6.63 mg/kg, iv) at reperfusion; gene expression was significantly decreased by 84% for TNFa and by 83% for IL‐1b. These data indicate a significant therapeutic time window for transfusion of small amounts of PEG‐COHb after ischemic stroke. The protective effect of PEG‐COHb transfused at reperfusion appears to be related to the CO donor effect of PEG‐COHb and an associated suppression of neuroinflammation. In addition, the benefit of transfusion during the period of vascular occlusion may be related to its ability to improve tissue oxygenation via collateral vessels. (Supported by NIH NS038684.)
Hypoxic Remodeling of Fetal Cerebral Arteries Involves The NPY/Y1 Pathway
Authors: Adeoye Olayemi , Silpanisong Jinjutha , Kim Dahlim , Williams James , Pearce William
Published: June 2021

The sympathetic perivascular innervation has long been suggested to exert trophic influences on cerebral arteries, particularly during hypoxic adaptation. Although most of this effect has been attributed to the actions of norepinephrine, sympathetic nerves also co‐release NPY together with norepinephrine. This study tests the hypothesis that NPY released from sympathetic nerves also contributes to hypoxic remodeling of fetal lamb cerebral arteries. Hypoxic acclimatization for 110 days increased NPY levels by 230% in nerve intact arteries. Whereas sympathectomy (SANX) had no significant effect in normoxic arteries, it decreased NPY levels in hypoxic arteries (25%). Hypoxia also increased Y1 receptor levels (70%) but only in nerve intact arteries. SANX decreased Y1 levels (30%) in hypoxic arteries. Confocal microscopy revealed that organ culture with 100 nM NPY potently decreased the colocalization of Non‐Muscle Myosin with Smooth Muscle Alpha‐Actin, indicating increased contractile differentiation. In nerve intact arteries, NPY decreased myogenic tone whereas in SANX arteries NPY increased myogenic tone. Co‐culture with the Y1 antagonist BIBP‐3226 (5 µM) reversed these effects of NPY. Taken together, these results suggest that NPY released from sympathetic nerves contributes to remodeling of the fetal cerebral vasculature during hypoxic acclimatization.
Chronic Hypoxia Modulates Endothelial Influences on Smooth Muscle Phenotype in Fetal Cerebral Arteries
Authors: Hubbell Margaret , Kim Dahlim , Thorpe Richard , Silpanisong Jinjutha , Pearce William
Published: June 2021

This study explores the hypothesis that chronic hypoxia modulates endothelial regulation of smooth muscle phenotype and function in fetal cerebral arteries. Endothelium denuded and intact middle cerebral arteries harvested from term fetuses from ewes kept at sea level (Normoxic) or 3, 820 m (Hypoxic) altitudes for the last 110 days of gestation were assessed for contractile function and smooth muscle phenotype as quantified by changes in the colocalization of Non‐Muscle (NM) and Smooth Muscle (SM) Myosin Heavy Chain (MHC) with Smooth Muscle alpha‐actin (SM‐αA). Chronic hypoxia reduced maximum myogenic stress, but increased maximum K+‐induced stress. Organ culture of arteries from normoxic animals with VEGF reproduced this increase in K+‐induced stress but only in the presence of an intact endothelium. For NM‐MHC/AA colocalization, an index of synthetic smooth muscle, organ culture for normoxic arteries with VEGF had little effect in the absence of endothelium but in the presence of endothelium enhanced colocalization. This effect of the endothelium on NM‐MHC/AA colocalization was enhanced in hypoxic arteries. For SM‐MHC/AA colocalization, a marker for contractile smooth muscle, organ culture of normoxic arteries with VEGF had little effect in the presence or the absence of endothelium, but in hypoxic arteries endothelium dramatically amplified VEGF‐induced increases in colocalization. These results demonstrate that chronic hypoxia modulates VEGF‐dependent endothelial regulation of smooth muscle phenotype in fetal cerebral arteries.

This work was supported by National Institutes of Health Grants HL‐54120, HD‐31266, HL‐64867, and NS‐076945 and the Loma Linda University School of Medicine.
The Role of Endothelial Cell Mineralocorticoid Receptors in Blood Pressure Regulation and Vascular Function
Authors: Barrett Mueller Katelee , Bender Shawn , Shahid Mohd , Aronovitz Mark , Kershaw Tanya , Hill Michael , Jaffe Iris
Published: June 2021

Vascular endothelial cells (EC) express mineralocorticoid receptor (MR) and contribute to vasoreactivity. Studies suggest EC MR may play a role in blood pressure (BP) control and vascular function. To address this, we made a mouse with EC MR deletion (EC MR KO). We confirmed EC‐specific MR deficiency, intact leukocyte MR expression and normal renal MR function. Characterization of BP phenotype by telemetry revealed EC‐MR deletion did not alter systolic, diastolic, circadian, or salt‐sensitive BP. Hypertensive response to renin angiotensin aldosterone pathway modulators – aldosterone + salt and angiotensin II (ang II) – were equal in EC MR KO and MR‐intact littermates. The role of EC MR in vasoconstriction was explored by wire myography in mesenteric arterioles showing no constriction difference to phenylephrine, KCl, endothelin 1, thromboxane agonist or ang II. EC dependent relaxation to acetylcholine and EC independent relaxation to sodium nitroprusside were unaffected by presence of EC MR in mesenteric, coronary, or basilar vessels in healthy mice. Yet the MR ligand aldosterone is elevated in patients with obesity and resistant hypertension and MR inhibitors improve EC function in those populations. In dyslipidemic ApoE KO mice, loss of EC MR seems to protect from high fat diet‐induced EC dysfunction. We conclude that in healthy mice, EC MR does not affect vasoreactivity or BP control and hypothesize that EC MR mediates vascular dysfunction induced by cardiac risk factors.
Depressed Sarcoplasmic Reticulum Activity Underlies Ca2+ Dyshomeostasis in A Rat Model of Metabolic Syndrome
Authors: OKATAN ESMA , TUNCAY ERKAN , TURAN Belma
Published: June 2021

High‐carbohydrate diet‐induced obesity and/or metabolic syndrom (MetS) is recognized as an independent risk factor for dysfunctioning of the heart. Although several mechanisms have been postulated for its identification, no precise mechanism is available yet for the cardiac remodeling and contractile dysfunction under MetS. Therefore, we aimed to investigate the role of sarcoplasmic reticulum (SR) on altered intracellular Ca2+ regulation in cardiomyocytes from MetS male rats, induced with high‐sucrose drinking water during 16‐week period. Basically, electrically‐stimulated Ca2+ transients and L‐type Ca2+‐currents (LTCC) were recorded in ventricular myocytes from MetS rats relative to myocytes from control rats. We also investigated SR‐Ca2+‐ATPase (SERCA) activity as well as phosphorylation levels of cardiac ryanodine receptor (RyR2) family proteins by Western blot analysis. The Ca2+ transients exhibited significantly reduced amplitude (~30%), and prolonged time courses (2‐fold) with no change in LTCC as well as depressed Ca2+ loading of SR (~55%) due to caffeine responses in cardiomyoctes from MetS rats with increased basal Ca2+ compared to aged‐matched control rats. Our data with caffeine responses under 0Na0Ca also demonstrated a depressed SERCA activity in MetS cardiomyocytes which supported further the data on a slowed cytosolic Ca2+ removal associated with a significant decrease in SERCA2‐mediated Ca2+ reuptake. Additionally, the data with tetracaine application assay was supporting a leaky‐RyR2 in cardiomyocytes from MetS group. Moreover, high phosphorylation levels of both RyR2 and phospholamban are supporting a depressed SR activity which underlies Ca2+ dyshomeostasis in MetS rat model.

(Supported by TUBITAK‐SBAG‐111S042)
Palmitic Acid Regulation of Cardiac Inotropy and Calcium Handling in Healthy and Angiotensin II‐Induced Hypertensive Rat
Authors: Jin Chun Li , Zhang Yin Hua
Published: June 2021

Background and aim

Palmitic acid (PA) is a predominant metabolic substrate. However, the effect of PA on cardiac contraction and underlying mechanisms in healthy and diseased heart remain unclear. Here, we analyzed PA‐regulation of myocyte contraction in left ventricular (LV) myocytes from sham and angiotensin II (Ang II)‐induced hypertensive rats.

Results

Our results showed that PA (100 μM) increased the amplitude of sarcomere shortening in LV myocytes (field stimulation, 2Hz,IonOptix Corp) from shams but not in hypertension. PA increased intracellular ATP level in shams.In contrast, etomoxir, a selective carnitine palmitoyl transferase I inhibitor, blunted the positive inotropic effect of PA, suggesting that increased beta‐oxidation may be fundamentally importantin PA‐regulation of cardiac inotropy. Mechanistically, PA did not increase the amplitude of Ca2+ transients or the density of peak L‐type Ca2+ current (ICa) or Na+‐Ca2+ exchanger activity, INCX, in either group. Paradoxically, PA did not affect the amplitude of Ca2+ transient or myofilament Ca2+ sensitivity (Myo‐Ca‐Sen) in shams. In hypertension, PA did not affect myocyte contraction or Ca2+ transient amplitude. Reactive oxygen species (ROS) scavenger did not affect PA‐induced increase in LV myocyte contraction in sham or hypertension.PA reduced neuronal nitric oxide synthase (nNOS) production of NO in shams but increased nNOS production of NO in hypertension. Importantly, nNOS inhibition with S‐methyl‐l‐thiocitrulline (SMTC) increased the amplitude of Ca2+ transients and restored PA‐induced cardiac inotropy in hypertension.

Conclusion

PA increases myocardial ATP production and potentiates LV myocyte contraction in healthy heart. nNOS restricts PA‐dependent cardiac inotropy by modulating Ca2+ handling in hypertension.
Distinct roles of neuronal nitric oxide synthase in metabolic substrates‐induced abnormal Ca2+ handling and cardiac arrhythmias in normal and hypertensive rats
Authors: Zhao Zai Hao , Zhang Yin Hua
Published: June 2021

Background & Aim

Metabolic syndrome is the precursor for ventricular arrhythmias. Here, we test metabolic syndromes' regulation of cardiac excitation‐contraction coupling (fatty acids, pyruvate, carnitine and 5 mM glucose, termed NF) and the role of neuronal nitric oxide syndrome, nNOS in left ventricular (LV) myocytes from sham and angiotensin (Ang II)‐induced hypertensive rats.

Result: NF increased basal and beta‐adrenergic (ISO) stimulation of LV myocyte contraction in sham and hypertension. Notably, ISO induced delayed‐aftercontraction (DAC) in NF and the frequency of DAC was ~3 fold higher in hypertension. Insulin, which exerted anti‐adrenergic effect on myocyte contraction in normal tyrode (NT), did not affect basal and ISO‐stimulated myocyte contraction or DAC in NF in two groups. Furthermore, tyrosine phosphorylation of insulin receptor substrate (IRS‐Tyr612) not insulin receptor (IRβ) or eNOS (eNOS‐Ser1177) was significantly reduced by NF, suggesting impaired insulin signaling. Inhibition of nNOS with S‐methyl‐l‐thiocitrulline (SMTC) did not affect NF and ISO‐enhanced LV myocyte contraction in two groups. Unexpectedly, SMTC increased DAC in sham but reduced it in hypertension. Diastolic and peak [Ca2+]i were elevated by NF and induced spontaneous Ca2+ release. SMTC increased peak [Ca2+]i in sham but reduced it in hypertension. KB‐R7943, an inhibitor of Na+‐Ca2+ exchanger, abolished ISO‐induced arrhythmias in NF.

Conclusion

NF impairs insulin‐dependent signaling, potentiates beta‐adrenergic stimulation of arrhythmias. nNOS prevents arrhythmias in normal heart but assists arrhythmogenesis in hypertension via its contrasting roles in [Ca2+]i handling.
Aspirate from stented aortocoronary saphenous vein grafts reduces coronary flow and left ventricular pressure in an isolated perfused rat hearts
Authors: Kleinbongard Petra , Lieder Helmut , Kahlert Philipp , Heusch Gerd
Published: June 2021

Background

Stent implantation into aortocoronary saphenous vein grafts (SVG) releases not only particular debris but also soluble factors which compromise coronary blood flow: using rat mesenteric arteries as bioassay, we have identified serotonin as the main vasoconstrictor. We now addressed the effects of aspirate on the coronary microcirculation and left ventricular function.

Methods

In 16 patients with significant SVG stenoses, arterial plasma was withdrawn before stent implantation and aspirate was retrieved after stent implantation under protection with a distal occlusion/aspiration device. Isolated rat hearts were perfused at constant pressure with Krebs‐Henseleit buffer containing 4% bovine albumin. Plasma was infused (2ml/min) into rat hearts (± NOS‐inhibition: L‐NAME 100µM; ± serotonin receptor blockade: ketanserin (10‐6 M) and pindolol (10‐7 M)). Changes of coronary flow (CF) and left ventricular pressure (LVP) are expressed relative to baseline before plasma application.

Results

Aspirate plasma reduced CF and LVP more than arterial plasma, however, only during NOS‐inhibition (arterial vs. aspirate plasma; CF [Δml/min]: ‐1.2±0.3 vs. ‐1.6±0.6, LVP [ΔmmHg]:‐11±2 vs.‐15±‐3). Effects of aspirate on CF and LVP were largely attenuated by serotonin receptor blockade.

Conclusion

In isolated rat hearts with impaired endothelial function, soluble factors within coronary aspirate, notably serotonin, compromise coronary microcirculation and left ventricular function.
Heart Rate Reduction and Longevity in Mice
Authors: Kleinbongard Petra , Gent Sabine , Dammann Philip , Neuhäuser Markus , Heusch Gerd
Published: June 2021

Background

Heart rate correlates inversely with life span across all species, including humans. In patients with cardiovascular disease, higher heart rate is associated with increased mortality, and such patients benefit from pharmacological heart rate reduction. However, cause‐and‐effect relationships between heart rate and longevity, notably in healthy individuals, are not established. We therefore prospectively studied the effects of a life‐long pharmacological heart rate reduction on longevity in mice. We hypothesized, that the total number of cardiac cycles is constant, and that a 15 % heart rate reduction might translate into a 15 % increase in life span.

Methods: C57BL6/J mice received either placebo or ivabradine at a dose of 50 mg/kg/day in drinking water from 12 weeks to death. Heart rate and body weight were monitored. Autopsy was performed on all non‐autolytic cadavers, and parenchymal organs were evaluated macroscopically.

Results: Ivabradine reduced heart rate by 14 (median, interquartile range 12 to 15) % throughout life, and median life span was increased by 6.2 % (p=0.01). An inverse relationship existed between individual heart rate and life span in all tested mice (r=0.86; p=0.046). Body weight and macroscopic findings were not different between placebo and ivabradine.

Conclusion

Life span was not increased by the same extent as heart rate was reduced, but nevertheless significantly prolonged by 6.2 %, which in humans translates to an additional 5 years for a person of 80 years.
Mitochondria are a target of cardioprotection by ivabradine
Authors: Kleinbongard Petra , Gedik Nilgün , Heusch Gerd
Published: June 2021

Background

Ivabradine is a bradycardic agent, which inhibits the If‐current in the sinus node. In pigs, ivabradine reduced infarct size even when given only at reperfusion and in the absence of heart rate reduction. Ivabradine´s protective action at reperfusion, when a burst of reactive oxygen species (ROS) is released, suggests it might attenuate ROS formation. Mitochondria are a major source of ROS after ischemia/reperfusion (I/R). Thus, we wanted to analyze a potential effect of ivabradine on mitochondrial function during simulated I/R.

Methods: Mitochondria were isolated from C57Bl/6J mouse hearts and subjected to simulated I/R (6 min hypoxia/3 min normoxia) in glutamate/malate‐ and ADP‐containing buffer ±3 µmol/l ivabradine at 30°C. After simulated I/R, mitochondrial complex I respiration (n=32,preparations), ROS concentration (n=9) and ATP production (n=15) were quantified.

Results: After simulated I/R ivabradine did not affect mitochondrial complex I respiration, but reduced the ROS concentration (144±12 vs. 122±12 nmol H2O2/100 µg protein; p=0.01), and increased the ATP concentration (148±20 vs. 181±21 µmol/l; p=0.01).

Conclusion

Mitochondria seem to be a target of protection induced by ivabradine by reducing mitochondrial ROS formation and increasing mitochondrial ATP production. Further studies are necessary to identify such target structure/s of ivabradine on mitochondria.
Metoprolol Prevents Cardiac Oxidative Stress Associated with Myocardial Ischemia/Reperfusion Injury
Authors: Bao Weike , Zacco Anna , Chendrimada Thimmaiah , Toomey John , Willette Robert , Schnackenberg Christine
Published: June 2021

Metoprolol, a selective β1 adrenergic receptor blocker is cardioprotective in a setting of ischemia /reperfusion injury. However, it remains controversial whether the cardioprotection is mediated by reducing oxidative stress. We tested the hypothesis that metoprolol prevents early cardiac oxidative stress during myocardial ischemia/reperfusion injury. Male Sprague‐Dawley rats were pretreated with metoprolol (1 mg/kg, i.v.) 10 min prior to a 30 min myocardial ischemia followed by 24 h reperfusion. Cardiac troponin I and T, and fatty acid binding protein 3 were measured at baseline (pre‐ischemia), 1, 2, 4, and 24 h after reperfusion using an MSD assay kit. To determine whether oxidative stress is involved in early myocardial ischemia/reperfusion injury, cardiac myosin binding protein C (MyBPC) carbonylation was determined by western blotting at 1 h after reperfusion. Infarct size was determined by TTC and Evans blue staining at 24 h after reperfusion. Ischemia/reperfusion injury significantly elevated plasma levels of cardiac injury markers for up to 24 h, reaching the peak at 1 h after reperfusion, increased MyBPC carbonylation 3.9‐fold, and caused a 56±6.2% infarct of the area at risk (n=6). Metoprolol attenuated peak and temporal plasma levels of cardiac injury markers, reduced MyBPC carbonylation by 52% (p<0.01), and reduced infarct size to 33±6.9% of the area at risk (n=6, P<0.01). Cardiac injury markers were positively correlated with infarct size, suggesting that metoprolol prevents cardiac oxidative stress associated with myocardial ischemia/reperfusion injury.
Mitochondria Modulates Calcium Pulsars In Native Endothelial Cells
Authors: Toussaint Fanny , Béziau Delphine , Charbel Chimène , Blanchette Alexandre , Mayer Gaétan , Ledoux Jonathan
Published: June 2021

Calcium (Ca2+) homeostasis is critical to endothelial function. Indeed, localized endothelial Ca2+ signals, such as Ca2+ pulsars ‐ restricted IP3R‐dependent Ca2+ releases within myoendothelial projection (MEP) ‐ emerge as major modulators of vascular tone. However, Ca2+ pulsars regulation remains yet unexplored. Conversely, mitochondria appear to be an important regulator of intracellular Ca2+ signals in several cell types. We thus hypothesized that mitochondria regulates endothelial Ca2+ pulsar. Electron microscopy first showed an asymmetrical mitochondrial distribution in endothelium of mice mesenteric arteries, with a higher mitochondrial density near MEP. High‐speed confocal microscopy was next used to study the impact of mitochondria on Ca2+ pulsars. Mitochondrial uncoupling by FCCP application unveiled pulsars active sites (+60%) but decreased discrete site frequency (‐30%). Moreover, pharmacological alteration of mitochondrial Ca2+ import/export mechanisms (Kaempferol, Ru360, CGP37157) had a significant impact on Ca2+ pulsar dynamics. In summary, near‐MEP mitochondria might be an important modulator of Ca2+ pulsars and hence of endothelial function. Support: FRQS, HSFC, CFI, SQHA and CIHR.
Deficiency of the T‐type Calcium Channel Cav 3.1 attenuates Plasma Aldosterone and Cardiac Hypertrophy despite similar Ang II‐induced Hypertension
Authors: Thuesen Anne , Jensen Boye , Hansen Pernille
Published: June 2021

Background

T‐type calcium channels affect adrenal aldosterone stimulus‐secretion coupling. It was hypothesized that Ang II infusion led to an attenuated blood pressure increase and aldosterone response in Cav 3.1‐/‐ mice.

Method

Mean arterial pressure was measured continuously with chronically indwelling catheters. Plasma aldosterone and renin were measured with RIA.

Results

Ang II increased significantly mean arterial pressure in Wt and Cav 3.1‐/‐ mice with no differences between genotypes. Heart rate in Ang II‐infused Cav 3.1‐/‐ mice was significantly lower compared to control. Plasma aldosterone was significantly lower in the Cav 3.1‐/‐ mice compared to the Wt mice after Ang II infusion. At baseline, there was a tendency towards a lower aldosterone level in the in the Cav 3.1‐/‐ mice compared to the Wt mice. The heart/body weight ratio was significantly higher after Ang II infusion in Wt mice compared to Cav 3.1‐/‐. The Ang II‐induced heart weight increase in Wt mice was inhibited by mineralocorticoid receptor blocker administration with no effect on MAP. Plasma renin was significantly lower after Ang II infusion in both groups. Summary; global deletion of T‐type Cav 3.1 subtype attenuates AngII‐induced aldosterone plasma increase and heart rate. The attenuated plasma aldosterone impairs cardiac hypertrophy in Ang II induced hypertension.

Conclusion

Cav 3.1 channels affect the plasma aldosterone levels but not Ang II induced hypertension. Aldosterone contributes significantly to cardiac hypertrophy in vivo independently of blood pressure and Ang II concentration.

Foundation

Danish Research Council and Danish Heart Foundation
Metformin Blunts Reductions in Serum Retinol Binding Protein 4 Following Exercise Training
Authors: Brandauer Josef , Malin Steven , Berardinelli Danielle , Viskochil Richard , Chipkin Stuart , Braun Barry
Published: June 2021

The adipokine serum retinol binding protein 4 (sRBP4) impairs skeletal muscle insulin sensitivity (IS). Metformin treatment improves IS but attenuates exercise training‐induced gains in cardiorespiratory fitness (i.e., VO2 peak) and IS. We assessed the interactive effects of training and metformin on sRBP4 in humans with impaired glucose tolerance (IGT).

20 female and 12 male subjects with IGT were randomized to placebo (P), metformin (M; 2000 mg/d), exercise (combined aerobic/resistance training)+placebo (EP), or exercise+metformin (EM) for 12 weeks. IS and non‐oxidative glucose disposal (NOGD) were assessed via euglycemic‐hyperinsulinemic clamps before and after intervention. VO2 peak was assessed via cycle ergometry. Serum RBP4 levels were determined via immunoblots.

After 12 weeks, sRBP4 decreased after EP and M but rose slightly in P and EM (time x treatment interaction; p=0.006). A blunted sRBP4 response was associated with smaller gains in VO2 peak (r=‐0.486, p=0.006), peripheral IS (r=‐0.431, p=0.017), and NOGD (r=‐0.408, p=0.025). Changes in sRBP4 were also linked to fasting plasma glucose (r=0.394, p=0.028).

In conclusion, the addition of metformin blunts exercise‐training induced reductions in sRBP4. This effect is correlated with attenuated adaptations of IS and VO2 peak. Our findings suggest that these alterations are caused partly by modulating inflammatory pathways in adipose tissue.
Change in microRNA profile predicts diabetes associated renal pathology non‐invasively
Authors: Mohan Aradhana , Singh Ravi , Garg Devika , Upadhyay Aditya , Ghildiyal Sneha , Tiwari Swasti
Published: June 2021

Over a third of diabetes patients develop diabetic nephropathy. To categorize such subset of diabetic patients there is a need for non‐invasive biomarker that regulate very early in the pathogenesis. We tested miRNAs in urinary exosomes (UE) as candidate molecule using serial urine sample from diabetic rats before and during the course of diabetes. Wistar rats were injected with streptozotocin (50 mg/kg. body weight). For exosome isolation, serial urine samples were collected before (ND) and after diabetes induction. Differentially expressed miRNAs were identified using small RNA sequencing, followed by Taqman based qRT‐PCR validation on UE RNA. Histomorphometric analysis of kidneys was performed at the end of the study. Heat maps revealed 114 miRNAs, common at all 4 stages, including miR 215 and 494, miRs reported to have predominant expression in the kidney relative to other tissues. Moreover, certain miRNAs, such as miR 451 and 10, although absent in the common heat maps, had significantly altered expression at 9th week of diabetes relative to ND or initial diabetic period i.e., 3rd week. qRT‐PCR validated significantly decreased levels of all 4 miRNAs in UE after diabetes induction. At 9th week, however, miR 494 restored to similar level as ND stage while miR 451 showed robust rise. Moreover, levels of 215 and 494 were significantly different between the diabetic rats with severe renal pathology, higher glomerulosclerosis index, relative to diabetic rats with moderate pathology. Overall, the findings suggest that change in miRNA expression in urinary exosomes during the course of diabetes can non‐invasively predict extent of renal pathology. DBT funding to ST
Acute Blockade of PDGF Receptors Decreases Arterial Blood Pressure and Renal Vascular Resistance in Cyp1a1‐Ren2 Transgenic Rats with Angiotensin II‐Dependent Malignant Hypertension
Authors: Haney Nora , Mitchell Kenneth
Published: June 2021

Previous studies have demonstrated that chronic blockade of PDGF receptors prevents the decreases in renal hemodynamic function and renal injury that occur in Cyp1a1‐Ren2 transgenic rats with angiotensin (ANG) II‐dependent malignant hypertension. The present study was performed to determine the effects of acute administration of the PDGF receptor blocker, imatinib mesylate, on mean arterial blood pressure (MAP) and renal hemodynamics during the development of ANG II‐dependent malignant hypertension. Male Cyp1a1‐Ren2 transgenic rats (n=5) were induced to develop malignant hypertension by dietary administration of indole‐3‐carbinol (0.3% wt/wt) for 7 days. Mean arterial pressure (MAP) and renal hemodynamics were measured in pentobarbital‐anesthetized male Cyp1a1‐Ren2 rats during control conditions and following a single intravenous bolus injection of imatinib mesylate (0.2 mg/kg). Imatinib administration decreased MAP (165±5 to 135±9 mmHg, P<0.05) but did not alter either renal plasma flow (2.51±0.11 vs. 2.95±0.21 ml/min.g, NS) or glomerular filtration rate (0.85±0.04 vs. 0.97±0.10 ml/min.g, NS). However, imatinib administration decreased renal vascular resistance in the hypertensive rats (32.3±1.01 to 24.6±1.7 mmHg/ml/min/g, P<0.05). The present findings demonstrate that acute blockade of PDGF receptors elicits decreases in MAP and renal vascular resistance in Cyp1a1‐Ren2 transgenic rats with ANG II‐dependent malignant hypertension. Thus, the data indicate that activation of PDGF receptors plays an important role in maintaining the elevated MAP and RVR that occurs in ANG II‐dependent malignant hypertension.
Intravital Imaging of the Kidney in Salt‐Sensitive Hypertension
Authors: Palygin Oleg , Endres Bradley , Sandoval Ruben , Rhodes George , Campos‐Bilderback Silvia , Kamocka Malgorzata , Molitoris Bruce , Staruschenko Alexander , Geurts Aron
Published: June 2021

Previous studies performed in rat kidneys shed light on the complexities of glomerular filtration of albumin and reabsorption at the proximal tubule (PT). It is now established that both PT reabsorption and transcytosis and glomerular filtration are responsible for determining the extent of albuminuria. Since hypertension is one of the most prevalent diseases worldwide, and is a major risk factor for developing albuminuria and renal failure, we investigated the mechanisms leading to albuminuria in the kidney of a rat model of salt‐sensitive hypertension, the Dahl SS rat. To determine the relative contributions of the glomerulus and PT to albuminuria in the SS rat, we applied the novel intravital two‐photon imaging to investigate the changes in renal function that occur during the development of salt‐sensitive hypertension (at 3, 7 and 14 days of high salt (HS) diet). Our results indicate that high amounts of albumin are filtered at the glomerulus in SS rats fed a low salt (LS) diet (GSCalb = 0.0155). Following HS diet consumption for 14 days, we observed significant elevations in blood pressure and albuminuria, increased prevalence of white blood cells (WBCs) within the kidneys (+Δ1.33 WBCs/image), and significant tubular damage as indicated by increased abundance of cast material (+Δ2.20 casts/image), compared to LS‐fed rats. We also observed a 57% increase in glomerular sieving of albumin (GSCalb = 0.0244) and a 58% reduction in PT reabsorption of albumin. Collectively, these results indicate that both the glomerulus and PT contribute to albuminuria in SS hypertensive rats.
Stem Cells Improved Renovascular Hypertension Independently of the Change of Renal Water and Sodium Transporters
Authors: Varela Vanessa , Oliveira‐Sales Elizabeth , Maquigussa Edgar , Borges Fernanda , Boim Mirian
Published: June 2021

Mesenchymal stem cells (MSC) improved renal morphology, inflammation, microvascular rarefaction, fibrosis and proteinuria in the stenotic kidney of 2Kidney‐1Clip (2K‐1C) model. Here, we investigated the effect of MSC on tubular function by evaluating the expression of the water and sodium transporters in the clipped kidney. Adult male Wistar rats were divided into groups: Control, Control+MSC, 2K‐1C, 2K‐1C+MSC. MSC was infused in the 3rd week after clipping into the tail vein (2x105 cells) with an interval of 2 weeks. Tail blood pressure (BP) was monitored weekly. Six weeks after clipping, 24 hours urine and blood were collected for biochemical analysis. Gene expression of the Na/Hexchanger (NHE3), sodium channel (ENaC), Na/KATPase, Na/K/2Cl cotransporter and aquaporins1 and 2 (AQP1 and AQP2).The 2K‐1C animals showed gradual increase of BPwhich was prevented by MSC treatment.There was a rise in the diuresis in the 2K‐1C rats, in parallel with a reduction in the AQP1 in the medullae but not in the cortex. Sodium excretion was reducedin spite of a decrease in ENaC expression with no changes in the other sodium transporters. This phenomenon may be explained by the increase in the free water excretion. Vascular rarefaction in the medulla (previously observed) together with the reduction in the expression of AQP1,mainly in the medulla suggests a deficit in the concentrating ability of the stenotic kidney. MSC did not corrected these tubular transport defects. Thus, MSC treatment was effective to minimize the renovascular hypertension and microvascular loss, however it was not sufficient to improve the concentrating ability of the stenotic kidney. Supported by FAPESP.