Subcutaneous Osmotic Pumps Research Articles

(Pro)renin Receptor Antagonism Attenuates High Fat Diet Induced Non‐alcoholic Fatty Liver Disease

Non‐alcoholic fatty liver disease (NAFLD) represents a diverse array of liver damage linked to obesity, and metabolic syndrome. The mechanisms and the treatment for NAFLD remains lacking; however, recent evidence suggests a critical importance of the (pro)renin receptor (PRR) and the renin‐angiotensin system (RAS) in liver lipid metabolism. Our laboratory recently developed a novel PRR antagonistic peptide PRO20 that blocks the fibrotic signaling of the PRR and the activation of the RAS. We hypothesize that PRR antagonism reverses NAFLD development by preventing the activation of liver RAS and fibrotic signaling. To test this hypothesis, wild‐type male C57Bl/6J mice (n=9–10/group) were fed with either HFD (60% calories from fat) or normal fat chow (NFD, 10% calories from fat) for 6 weeks. Two weeks following diet modification, mice were implanted with a subcutaneous osmotic pump containing either PRO20 or scramble peptide (700 μg/kg/d, 4 weeks). At the end of the 6‐week protocol, liver tissues were harvested and processed for Oil Red O (ORO) and Masson’s trichrome staining for assessment of lipid and collagen accumulation respectively. In scramble peptide treated groups, we found that 6 weeks of HFD induced a significant elevation on liver ORO staining (15.90 ± 1.49 %;) compared with the liver from mice fed with NFD (3.60 ± 1.61 %, p < 0.0001). More importantly, PRO20 (9.91 ± 1.46 %; p = 0.001) significantly reduced the ORO staining compared with scramble peptide in mice treated with HFD. Portal vein collagen deposition was significantly lower in mice treated with PRO20 (1.93 ± 0.60%) compared to the control peptide (4.05 ± 0.60%, p < 0.0211) following 6 weeks of HFD. Mechanistically, we found that PRO20 (1.99 ± 0.62) normalized the elevation of liver mRNA level (fold change) of peroxisome proliferator‐activated receptor gamma (PPARγ) induced by HFD (HFD Scramble peptide: 2.61 ± 0.47 vs. NFD Scramble peptide: 1.13 ± 0.47, p = 0.0037), a key transcription factor regulating lipogenic genes in the liver. In addition, PRO20 prevented the activation of key liver RAS components induced by HFD including the angiotensinogen (1.00 ± 0.62 vs. 6.153 ± 0.62, p < 0.0001), angiotensin converting enzyme (1.03 ± 0.63 vs. 2.91 ± 0.63, p = 0.0344), and angiotensin II type 1a receptor (0.35 ± 0.46 vs. 2.20 ± 0.46, p = 0.0091 value). In summary, our data showed that subcutaneous infusion of PRO20 attenuated the development of HFD‐induced NAFLD and portal fibrosis by regulating liver PPARγ and preventing liver local RAS activation. We conclude that PRR antagonism maybe a new therapeutic target for the treatment of NALFD.Support or Funding InformationNIH/NHLBI (R01HL122770), American Heart Association (17IRG33370128), NIH/NIGMS (1P20GM130459‐5451)

Cardiac AKAP12 Signalosome Overexpression Exacerbates Isoproterenol Induced Heart Failure

BackgroundHeart failure (HF), defined as an impaired ability of the heart to efficiently pump blood, is responsible for 1 out of 8 deaths per year in the U.S.A. and is the major cause of death globally. Physiologically, stimulation of β‐adrenergic receptors (β‐AR) by catecholamines, epinephrine (EP) and norepinephrine (NE), increases cardiac contractility. However, in HF, chronic β‐AR stimulation leads to reduced cardiac function due in part to the desensitization of these receptors. Previous in vivo studies reported by our group showed enhanced cardiac function after Isoproterenol (ISO) induced HF in male mice lacking functional cardiac AKAP12 compared to wild‐type (WT) mice. In this study, we aim to investigate the impact of cardiac AKAP12 overexpression (oxAKAP12‐Tg) on HF progression in both male and female mice and understand the underlying mechanisms involved. Our central hypothesis is that cardiac AKAP12 overexpression is involved in heart failure progression by influencing signaling downstream β‐AR.MethodsWT and oxAKAP12‐Tg mice (8–10) weeks old males and females were categorized into three groups; subcutaneous ISO osmotic pump group (ISO; 60 mg/kg/day X 14‐days), vehicle group (ascorbic acid 0.0002% X 14‐days), and sham group (no subcutaneous pump). Echocardiography and ECG were obtained from all groups 2‐days before subcutaneous insertion of the osmotic pumps as well as 7‐days and 14‐days after the osmotic pump insertion, followed by tissue harvest on the 14‐day. Adult mouse ventricular homogenates were used for protein expression levels analysis. Furthermore, CRISPR‐cas9 technology was used to overexpress AKAP12 into AC16 cells, a human cardiomyocyte cell line, to assess gene expression levels upon overnight treatment with 100 nmol of ISO.ResultsCardiac overexpression of AKAP12 in both males and females reduced left ventricular ejection fraction (EF) and fractional shortening (FS). Both males and females treated with ISO and overexpressing AKAP12 had a 22.7±2% reduction in EF and 14.5±2.5% reduction in FS after 14‐days of chronic ISO treatment when compared to control groups. However, the pattern of this decline in cardiac function was different between male mice and female mice; where male mice showed a slow decline in cardiac function over the two week period of chronic ISO treatment while female mice showed a steep decline within the first week of chronic ISO treatment that plateaued during the second week of treatment. Protein expression levels of β‐arrestin1 and β‐arrestin2 were significantly lower in cardiac homogenates of ISO treated oxAKAP12‐Tg when compared to ISO treated WT male and female mice, respectively. No significant differences were observed between control groups. This observation was further supported by QPCR gene expression studies; where we observed a 40% reduction of β‐arrestin‐1 gene expression in ISO treated AC16 overexpressing AKAP12 when compared to the ISO treated WT AC16 cells.ConclusionsCardiac overexpression of AKAP12 negatively influences systolic function in both male and female mice, potentially through affecting the β‐arrestin signaling pathway. Thus, AKAP12 might be involved in biased signaling downstream β‐AR which could serve as a potential target for designing drugs to ameliorate HF.

Intranasal Leptin Prevents Opioid Induced Respiratory Depression in Obesity

IntroductionNorth America is currently facing obesity and opioid epidemics. Respiratory depression is the main cause of morbidity and mortality from opioids. Obese individuals with Obstructive Sleep Apnea have a higher risk of opioid induced respiratory depression (OIRD). Leptin acts as respiratory stimulant augmenting control of breathing and improving upper airway patency. We have previously shown that obesity‐induced leptin resistance can be circumvented by intranasal administration of leptin. In this study, we aimed to examine if intranasal administration of leptin prevents OIRD.MethodsMale diet‐induced obese C57BL/6J mice (n=8) were instrumented with headmount containing EEG and EMG leads under 1–2% isoflurane anesthesia. After recovery period polysomnograms were performed in a crossover manner with 1 week interval between studies. Mice received intranasal (IN) leptin or vehicle (BSA) at 0.4 mg/kg and 30 minutes later received a bolus of intraperitoneal (IP) morphine at 10 mg/kg followed by implantation of a subcutaneous osmotic pump delivering morphine at 2 mg/kg/h for the duration of the study (11am–5pm). The control group received IN vehicle, IP saline and a sham osmotic pump filled with saline. Minute ventilation (VE) was recorded through sleep/wake states and upper airway obstruction was defined by the presence of inspiratory flow limitation (IFL). Apneas were scored when flow was absent for two or more respiratory cycles. Excitatory post‐synaptic currents (EPSCs) were recorded in‐vitro from hypoglossal motor neurons (HGNs) and changes in EPSC amplitude and frequency were examined following application of the μ opioid receptor agonist (DAMGO) and leptin.ResultsMorphine induced severe inspiratory flow limitation and apneas during NREM sleep. Morphine increased IFL breath frequency (from 12% to 57% of all breaths, p<0.001) and apneas (from 13.5±3.5/h to 91.5±20.0/h, p<0.01) and decreased maximal inspiratory flow (VImax) during IFL breaths (from 3.4±0.4 to 2.3±0.4 mL/s, p<0.05) and VE (from 1.1±0.1 to 0.7±0.2 mL/min/g, p<0.05). Leptin attenuated effects of morphine. IN Leptin decreased IFL breath frequency (from 57% to 45%, p<0.005) and reduced the number of apneas (from 91.5±20.0/h to 34.3±7.0/h, p<0.01). Leptin reduced the severity of upper airway obstruction increasing VImax (from 2.3±0.4 to 2.8±0.4, p<0.05) and minute ventilation (from 0.7±0.2 to 1.0±0.2, p<0.05) during IFL breathing. Furthermore, in vitro studies showed that administration of DAMGO reduced EPSC frequency (but not EPSC amplitude) in HGNs that was reversed by application of leptin.ConclusionMorphine caused respiratory suppression and upper airway obstruction during sleep in DIO mice and decreased EPSC frequency in HGNs. Leptin relieved morphine‐induced hypoventilation and Obstructive Sleep Apnea and reversed the opioid induced depression of excitatory synaptic neurotransmission to HGNs. We conclude that intranasal leptin may prevent OIRD in obesity.Support or Funding InformationR01HL 128970, R01HL 138932, FAPESP 2018/08758‐3

Abstract P5-04-29: Osteoblasts regulate mobilization of the myeloid-derived suppressor cells from the bone marrow of breast cancer patients

Abstract MDSCs are T cell suppressive immature myeloid-lineage cells in the tumor microenvironent (TME). MDSCs originate in the bone marrow, circulate and accumulate in the spleen and tumor tissue. MDSCs consist of two morphologically and functionally distinct subpopulations, i.e. monocytic (M-) and polymorphonuclear (PMN-) MDSC. While PMN-MDSCs are more abundant and suppress T cells in an antigen-specific manner, M-MDSCs are earlier-stage MDSCs and suppress T cells in both antigen-specific and nonspecific manners. In contrast to clear functions in TME, how MDSCs are regulated in the bone marrow of tumor hosts remains unclear. We have previously reported that tumor-derived parathyroid hormone-related peptide (PTHrP) increased MDSCs in TME. In this study, we further investigated cellular and molecular mechanisms of PTHrP in mobilizing MDSCs from the bone marrow. Continuous infusion of PTHrP(1-34) in female Balb/C mice using subcutaneous osmotic pumps (80μg/kg, 3 weeks) increased M-MDSC, but not PMN-MDSC, in the circulation and bone marrow. Interestingly, a single administration of PTHrP(1-34) or PTH(1-34) in mice also rapidly increased M-MDSC in the circulation, suggesting that M-MDSC are mobilized from the bone marrow by PTH1R activation.RT-PCR analysis showed that PTH1R is expressed in osteoblasts, but not in human or mouse MDSC. In vitro cell binding assays measuring fluorescently labeled-human or mouse MDSC co-cultured with MC3T3E1 or hFOB osteoblasts were performed to determine the molecular mechanism of MDSC mobilization. We found that PTHrP(1-34) and PTH(1-34), but not PTHrP(7-34), released MDSC-osteoblast binding, and also that anti-VCAM 1 and/or -β1 integrin neutralizing antibodies blocked MDSC-osteoblast binding. Inhibitors of PKA, a downstream mediator of PTH1R, inhibited MDSC-osteoblast binding, while forskolin, a PKA activator, showed the opposite effects. Immunohistochemical staining of the bones of tumor-bearing mice showed that M-MDSCs localize adjacent to trabecular osteoblasts via VCAM-1 (expressed by osteoblasts) and β1 integrin (expressed by M-MDSC). Subsequent experiments demonstrated that PTH/PTHrP-stimulated osteoblasts release VEGF-A and IL-6 to stimulate M-MDSCs via phosphorylation of Src family kinase. Administration of dasatinib, a Src selective inhibitor, significantly suppressed PTHrP-mediated M-MDSC mobilization in vivo. Collectively, our data demonstrate that tumor-derived PTHrP circulate and activates osteoblasts, leading to release of M-MDSCs via disruption of VCAM-1 and β1 integrin retention axis by upregulation of VEGF-A and IL-6 in bone and by Src family kinase phosphorylation in MDCS. In conclusion, this study explains how MDSC, an essential bone marrow-derived cells in TME, are regulated in the bones of cancer patients. Further studies will provide a therapeutic approach to uncouple the partnership between tumor and bone. Citation Format: Serkin Park, Kyoung Jin Lee, Eun Jung Lee, Young Mi Whang, Sun Wook Cho. Osteoblasts regulate mobilization of the myeloid-derived suppressor cells from the bone marrow of breast cancer patients [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-04-29.

P6279Mitochondria targeted catalase overexpression protects against beta-adrenergic receptor mediated cardiac remodeling in mice

Abstract Background Beta-adrenergic receptor signaling is widely recognized as a fundamental component in the pathogenesis of chronic heart failure. However, the mechanisms behind beta-adrenergic receptor-mediated remodeling in cardiomyocytes and the myocardium are not fully understood. Oxidative stress has been proposed as a central pathophysiological mediator in cardiovascular disease and heart failure. The triggers and sources of oxidative stress in heart failure remain unclear. In this study we use mice with mitochondria-targeted overexpression of the antioxidant enzyme catalase (mCAT) to link beta-adrenergic receptor-mediated stress to mitochondrial reactive oxidative species (ROS) and the progression of heart failure. Hypothesis Mitochondrial ROS, induced by beta-adrenergic receptor-activation, is a mediator in the progression of the heart failure phenotype. Methods mCAT and wild type mice (n=10) were administered the non-selective beta-adrenergic agonist Isoprotenerol (Iso; 50mg/kg/day) through subcutaneous osmotic pumps for 3 weeks. Hearts were taken for biochemistry (western blotting, qPCR). Cardiomyocytes were isolated and loaded with Fluo-3 AM to study intracellular Ca2+ transients and fractional shortening using confocal line scan microscopy. All experiments were performed in accordance with the Stockholm ethical committee for animal research. Results and conclusions The WT mice displayed an increased heart/body weight ratio following chronic Iso administration. In contrast, mCAT mice displayed resistance to Iso-induced cardiac hypertrophy (p<0.05). Furthermore, chronic Iso exposure in WT mice induced increased ROS-dependent post-translational protein modifications, impaired cardiomyocyte Ca2+ handling and reduced contractility in isolated cardiomyocytes (p<0.05). Cardiomyocytes from mCAT mice did not display the deleterious effects of chronic Iso exposure on cardiomyocyte Ca2+ and contractility. Our study demonstrates that beta-adrenergic receptor stimulation-induced remodeling of the heart, which is similar to what is seen in heart failure, can be prevented by overexpressing catalase in the mitochondria. This indicates an important role of mitochondrial ROS in the link between adrenergic signaling and the development of cardiomyopathy and heart failure. Acknowledgement/Funding Hjärtlungfonden

Effects of the mGluR2/3 agonist LY379268, alone and in combination with monoamineenhancing drugs, on cocaine self‐administration in rhesus monkeys

Cocaine addiction persists as a major public health problem but still lacks any FDA‐approved pharmacotherapies. Wake Forest's Center for the Neurobiology of Drug Abuse Treatment (CNAT) has demonstrated the effectiveness of ‘agonist medications’ such as the monoamine releaser phenmetrazine (PM) and its pro‐drug phendimetrazine (PDM) to decrease cocaine use in animal models. CNAT investigators have hypothesized that combination of a dopamine‐enhancing drug like PM/PDM with a drug with a different pharmacological and/or behavioral mechanism may display enhanced effectiveness and represent a promising pharmacotherapeutic approach to treating cocaine use disorder. The present studies characterized the effects of the mGluR2/3 receptor agonist LY379268, alone and in combination with daily PDM or the dopamine transporter inhibitor methylphenidate (MPD) on cocaine reinforcement in rhesus monkeys. In one experiment (n=4), a multiple schedule was used in which food reinforcers were available in the first component under a fixed‐ratio schedule of reinforcement and cocaine self‐administration was studied under a progressive‐ratio schedule. A second study (n=4) used a food‐cocaine choice procedure in which a complete cocaine dose‐effect curve was generated each day. Effects of acute and chronic LY379268 were studied. Under the multiple schedule, acute LY379268 decreased food‐but not cocaine‐maintained responding; when administered chronically (as an i.v. injection) during chronic oral PDM treatment, a drug combination that persistently decreased cocaine self‐administration was found in only one of four subjects. Under the choice procedure, LY379268 increased the reinforcing strength of cocaine relative to food. After 5 days of treatment, tolerance developed to this effect in a dose‐related manner but at no point were decreases in cocaine choice observed. Finally, when chronic LY379268 was administered in combination with chronic oral PDM or chronic MPD (via subcutaneous osmotic pump), dose combinations were observed that modestly decreased cocaine choice in two of four monkeys. Although some results were positive, taken together these studies, which used highly translational nonhuman primate models of cocaine use disorder, do not support the effectiveness of LY 279638 alone or in combination with agonist medications.Support or Funding InformationP50 DA06634This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

(283) Adjuvant Treatment with Dopamine Receptor Modulators Prevents the Development of Morphine Tolerance in a Rodent Model

Opioids have a prominent role in managing acute pain. However, long-term management of pain with opioids is complicated, as tolerance for the drug develops, increasing the risk for misuse and abuse. The need exists for chronically effective treatments that minimize the rewarding component. Our data shows that, in animal models, combining low-dose morphine with a dopamine 3 receptor (DR3) agonist or dopamine 1 receptor (D1R) antagonist provides superior analgesia against acute painful stimuli compared to morphine alone. The current study examined if adjuvant treatment with D3R agonists or D1R antagonists can also prevent the development of tolerance to and reduce the addictive potential of morphine. Thermal and mechanical thresholds were tested in 16, female Long-Evans rats before and after a single subcutaneous dose of morphine (2mg/kg). Rats were then randomized to 1 of 4 chronic drug conditions: saline; morphine (5mg/kg/day); morphine + pramipexole (D3R agonist, 0.1mg/kg/day); morphine + SCH39166 (D1R antagonist, 0.1mg/kg/day). Drugs were delivered for 14 days via subcutaneous osmotic pump. Analgesia provided by an acute dose of morphine was re-assessed at 7 and 14 days after initiation of drug delivery. Addictive potential of each drug combination was determined using conditioned place preference (CPP). Prior to drug delivery, acute morphine injection increased thermal and mechanical thresholds in all animals. Fourteen days after chronic administration of morphine, sensory thresholds did not change in response to acute morphine (i.e. animals were tolerant). In contrast, sensory thresholds were increased in animals that had received morphine in combination with either dopamine receptor modulator. The addition of either dopamine receptor modulator to morphine also reduced the preference for the drug compared to morphine alone. Adjuvant therapy with clinically-available dopamine receptor agonists or antagonists can prevent the development of morphine tolerance and reduce the rewarding effect of morphine alone.

COA-Cl prevented TGF-β1-induced CTGF expression by Akt dephosphorylation in normal human dermal fibroblasts, and it attenuated skin fibrosis in mice models of systemic sclerosis

BackgroundSystemic sclerosis (SSc) is characterized by fibrosis of the skin and internal organs. Although transforming growth factor (TGF)-β1-induced connective tissue growth factor (CTGF/CCN2) expression has been presented in SSc fibrosis, the therapeutic potential of targeting CTGF in SSc has not been fully explored. COA-Cl is a novel nucleic acid analog, which is reported to have pleiotropic beneficial biologic effects. ObjectiveWe examine the effects of COA-Cl on TGF-β1-induced CTGF expression in normal human dermal fibroblast (NHDF). We also examined the effects of COA-Cl on CTGF expression in a mouse SSc model of angiotensin II (Ang II)-induced skin fibrosis. MethodsNHDF was cultured for in vitro experiments. For in vivo experiments, C57BL/6J mice were treated with Ang II for 14 days by subcutaneous osmotic pump. Quantitative real-time polymerase chain reaction, western blot analysis, immunohistochemical staining and immunofluorescence staining were performed to examine the expression levels of CTGF and phosphorylation levels of Smad2/3, ERK1/2 and Akt. ResultsCOA-Cl attenuated the TGF-β1-induced expression of both CTGF mRNA and protein in NHDF. Although COA-Cl did not alter the TGF-β1-induced phosphorylation of Smad2/3 or ERK1/2, it reduced the TGF-β1-induced phosphorylation levels of Akt in NHDF. Notably, COA-Cl dephosphorylated the Akt of lysates of TGF-β1-treated NHDF. COA-Cl reduced the levels of CTGF mRNA, CTGF protein, dermal thickness, collagen content and Akt phosphorylation in the skin of mice SSc model. ConclusionThese results imply that the inhibition of TGF-β1-induced CTGF expression by COA-Cl may be a therapeutic approach for SSc.

Abstract TMP35: Perivascular Astrocytes Alter the Basement Membrane Through TGF-Beta in an Aged Brain Post-Stroke

Individuals over 65 make up the majority of stroke patients in the United States. Post-stroke recovery within the elderly population maybe impeded through enhanced glial scarring and reduced glymphatic flow. Following stroke, reactive astrocytes remodel the basement membrane through transforming growth factor-beta (B) signaling (TGF-B), altering glymphatic flow. Impaired glymphatic flow leads to a buildup of waste products, such as amyloid-beta, further inhibiting post-stroke recovery. We hypothesize that post-stroke astrogliosis and basement membrane fibrosis are induced through TGF-B signaling, resulting in chronic glymphatic impairment in aged mice. To test this hypothesis, we used an in vivo model of ischemia to measure TGF-B signaling and reactive astrogliosis. In vivo studies used young (3 month, n=5-8) and aged (20-month, n=5-8) male C57/Bl6 mice that underwent distal middle cerebral artery occlusion (DMCAO). Outcome measurements included TGF-B signaling, reactive astrogliosis within the glymphatic system, and impaired glymphatic flow. As a potential therapy, an implantable subcutaneous osmotic pump was inserted post-stroke with a TGF-B antagonist (GW788388 Hydrate-10mg/kg/day) to determine the impact on astrogliosis, glymphatic flow, and functional outcome. Results for in vivo studies using immunohistochemistry demonstrated a significant increase (p<0.05) in TGF-B expression in aged sham/DMCAO compared to young sham/DMCAO. There was a significant (p<0.01) increase in astrogliosis in aged sham/DMCAO compared to young sham/DMCAO. Glymphatic flow was assessed using an injected dextran dye, results demonstrated a significant (p<0.05) reduction in glymphatic flow for aged DMCAO compared to naïve mice. Functional measurements demonstrated that aged DMCAO had a significant reduction (p<0.05) in Paw Area/Time (Digigait) compared to naïve. Following TGF-B antagonist treatment, there was no difference between aged DMCAO and naïve mice in astrogliosis, glymphatic flow, or Paw Area/Time. This demonstrates that TGF-B impairs post-stroke recovery through astrogliosis, and impaired glymphatic flow. While this study demonstrated positive effects post-stroke, future studies should target downstream pathways of TGF-B.

Reveromycin A Inhibits Bone Loss in Ovariectomized Mice

Background and objectives: Reveromycin A inhibits bone metastasis of lung cancer cells in natural killer cell-depleted immunodeficient mice with increasing apoptosis of osteoclasts. This knowledge suggests that reveromycin A is a potent inhibitor for osteoclasts in vivo. To determine the activity of bone loss in acute estrogen deficiency, we studied ovariectomized mice treated with reveromycin A. Materials and Methods: Eight-week-old female C57BL/6 mice were either ovariectomized or sham-operated. Reveromycin A was continuously administered for the first 2 weeks (Group RA) or the last 2 weeks (Group RP) using a subcutaneous mini osmotic pump. Mice were sacrificed 4 weeks after surgery. Inhibition was detected by images as well as serum carboxyterminal telopeptide of type I collagen (CTX). Results: BMD reduction in the RA group was significantly inhibited and was comparable with that in the sham group. In contrast, the RP group failed to inhibit BMD reduction. The plasma CTX level at the 28th day of the experiment was significantly increased in the RP and OVX groups compared with that in the sham group, while the increase was inhibited in the RA group. Morphometric analysis revealed that absorption of trabecular bone was strongly inhibited in the RA group. Conclusion: The inhibitory effects of reveromycin A administered immediately after ovariectomy was the most effective at the early phase of bone loss than at the later stage. A new reagent that inhibits bone loss in acute estrogen deficiency might be a new tool to experimentally study osteoporosis and may have clinical implications.

Aldosterone induces renal fibrosis by promoting HDAC1 expression, deacetylating H3K9 and inhibiting klotho transcription.

Aldosterone has an important role in the progression of renal fibrosis. In the present study, the concentration of aldosterone and klotho (KL) in the serum of patients with chronic kidney disease (CKD) were analyzed. A negative correlation was observed between aldosterone and KL, suggesting that KL may serve a protective role in CKD. Subsequently, an aldosterone‑induced CKD mouse model was established using a single nephrectomy and subcutaneous osmotic pump with aldosterone and 1% high‑salt drinking water. It was demonstrated that fibronectin 1 (Fn1) expression levels were higher in high aldosterone mice, whereas KL expression levels were low. In addition, the results demonstrated that histone deacetylase1 (HDAC1) protein expression levels were upregulated in the renal distal convoluted tubules of high aldosterone mice, whereas acetylated H3K9 (H3K9Ac) was significantly downregulated. To determine the transcriptional activation status, chromatin immunoprecipitation polymerase chain reaction (PCR) was used to validate binding of H3K9Ac to the KL gene promoter site. It was revealed that the binding product of the KL promoter could be PCR‑amplified at the H3K9Ac site from wild‑type and low aldosterone mice; however, amplification of the binding product was not observed in high aldosterone mice. In conclusion, aldosterone significantly inhibited H3K9 acetylation by upregulating HDAC1 protein expression levels in the renal distal convoluted tubule cells, resulting in its inability to bind to the KL promoter, loss of transcription of the KL gene and increased expression of the renal fibrosis gene, Fn1.

Nicotine impairs intra-substance tendon healing after full thickness injury in a rat model.

Nicotine is harmful to many bodily systems; however, the effects of nicotine on intra-substance tendon healing remain largely unexplored. The purpose of this study was to examine the functional, structural, and biomechanical effects of nicotine on the healing of Achilles tendons in rats after an acute full-thickness injury. Sixty Sprague-Dawley rats were enrolled in this study. Half were exposed to 0.9% saline and half to 61 ng/mL of nicotine for 3 months via subcutaneous osmotic pumps. At 3 months, all rats underwent blunt full thickness transection of the left Achilles tendon and were immobilized for one week in plantarflexion. In-vivo assays were conducted prior to injury, at 21 days, and at 42 days post-injury and included the following: Functional limb assessment, passive joint mechanics, and vascular evaluation. Rats were sacrificed at 21 and 42 days for biomechanical testing and histologic evaluation. Rats exposed to nicotine demonstrated decreased vascularity, greater alteration in gait mechanics, and increased passive ROM of the ankle joint. Biomechanically, the nicotine tendons failed at lower maximum loads, were less stiff, had smaller cross-sectional areas and had altered viscoelastic properties. Histologically, nicotine tendons demonstrated decreased vessel density at the injury site. This study demonstrates that nicotine leads to worse functional outcomes and biomechanical properties in tendons. The decreased vascularity in the nicotine group may suggest an underlying mechanism for inferior tendon healing. Patients should be counseled that using nicotine products increase their risk of poor tendon healing and may predispose them to tendon re-rupture. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

Fucoidans inhibit the formation of post-operative abdominal adhesions in a rat model.

PurposeFibrin clot is essential for post-operative abdominal adhesion formation. Fucoidans, sulfated polysaccharides, inhibit fibrin clot formation. In addition, they inhibit inflammation and fibrosis, which also play important roles in adhesion formation. The purpose of this study was to evaluate fucoidans’ potential for inhibiting post-operative abdominal adhesions and measure their effects on systemic coagulation parameters when administered intraperitoneally (IP).Methods and materialsFemale Sprague Dawley rats were studied. A 2.5x2.5cm full thickness segment of abdominal wall was excised. The skin edges were approximated. This model induces extensive adhesions and allows objective quantitation. Three fucoidans were evaluated- Sigma Fucoidan Crude (SFC), Fucus vesiculosis 95% (Sigma) and, Peridan. One protocol involved continuous infusion into the abdomen from a subcutaneous osmotic pump. Alternatively, boluses of the solutions were injected IP at the end of the operation. Rats were sacrificed a week later. Adhesion extent was scored. Systemic coagulation effects of fucoidans were also evaluated. INR and aPTT were measured following IP injection of the fucoidan solutions and after 7 days of continuous infusion.ResultsAnimals given a continuous infusion of either SFC or Peridan yielded adhesion reduction of 80 to 90% from control. Bolus Peridan had no discernable influence on adhesion formation, but a single bolus of SFC caused significant adhesion reductions. Peridan resulted in prompt aPTT elevations which fell to nearly normal by 5 hours. The maximum peak value after SFC injection was seen in 15 hours. The maximal INR elevations were around 2. Measurement of INR and aPTT after a week of continuous infusion of either Peridan or SFC, were always in the normal control range. The third agent, Sigma, frequently yielded intraperitoneal infection found at autopsy.ConclusionsThese findings indicate that selected fucoidans infused intraperitoneally for a week after abdominal operations reduce adhesion extent by up to 90%.

Intrarenal ghrelin receptor inhibition ameliorates angiotensin II-dependent hypertension in rats.

The intrarenal ghrelin receptor (GR) is localized to collecting duct (CD) cells, where it increases epithelial Na+ channel (αENaC)-dependent sodium reabsorption in rodents. We hypothesized that chronic GR inhibition with intrarenal GR siRNA lowers blood pressure (BP) in angiotensin II-dependent hypertension via reductions in αENaC-dependent sodium reabsorption. Uninephrectomized Sprague-Dawley rats ( n = 121) received subcutaneous osmotic pumps for chronic systemic delivery of angiotensin II or vehicle (5% dextrose in water). Rats also received intrarenal infusion of vehicle, GR siRNA, or scrambled (SCR) siRNA. In rats receiving intrarenal vehicle or intrarenal SCR siRNA, systemic angiotensin II infusion increased sodium retention and BP on day 1, and BP remained elevated throughout the 5-day study. These rats also demonstrated increased CD GR expression after 5 days of infusion. However, intrarenal GR siRNA infusion prevented angiotensin II-mediated sodium retention on day 1, induced a continuously negative cumulative sodium balance compared with angiotensin II alone, and reduced BP chronically. Glomerular filtration rate and renal blood flow remained unchanged in GR siRNA-infused rats. Systemic angiotensin II infusion also increased serum aldosterone levels, CD αENaC, and phosphorylated serum and glucocorticoid-inducible kinase 1 expression in rats with intrarenal SCR siRNA; however, these effects were not observed in the presence of intrarenal GR siRNA, despite exposure to the same systemic angiotensin II. These data demonstrate that chronic inhibition of intrarenal GR activity significantly reduces αENaC-dependent sodium retention, resulting in a negative cumulative sodium balance, thereby ameliorating angiotensin II-induced hypertension in rats. Renal GRs represent a novel therapeutic target for the treatment of hypertension and other sodium-retaining states.

Inhibition of interleukin-1 suppresses angiotensin II-induced aortic inflammation and aneurysm formation

BackgroundAngiotensin II (Ang II) activates components of the inflammatory cascade, which promotes hypertension and development of abdominal aortic aneurysm (AAA). This study aimed to elucidate the effects of an IL-1 receptor antagonist (IL-1Ra) and an anti-IL-1β antibody (01BSUR) on Ang II-induced AAA. Methods and resultsMale wild-type (WT) and IL-1Ra-deficient (IL-1Ra−/−) mice were infused with Ang II (1000 ng/kg/min) using subcutaneous osmotic pumps for 28 days. Fourteen days post-infusion, both systolic blood pressure (SBP) (Ang II-treated IL-1Ra−/−:149 ± 2 vs. Ang II-treated WT:126 ± 3 mm Hg, p < 0.001) and abdominal aortic width (0.94 ± 0.09 vs. 0.49 ± 0.03 mm, p < 0.001) were significantly higher in IL-1Ra−/− mice than in WT mice. Because 28-day infusion with Ang II in IL-1Ra−/− mice significantly increased the occurrence of fatal aortic rupture (89% vs. 6%, p < 0.0001), both types of mice were infused with Ang II for only 14 days, and histological analyses were performed at 28 days. Interestingly, AAA increased more significantly in IL-1Ra−/− mice than in WT mice (p < 0.001), although SBP did not differ at 28 days in IL-1Ra−/− and WT mice (117 ± 4 vs. 115 ± 3 mm Hg, p = 0.71 (after cessation of Ang II infusion)). Histological analyses showed numerous inflammatory cells around the abdominal aorta in IL-1Ra−/− mice, but not in WT mice. Finally, compared with IgG2a treatment, treatment with 01BSUR decreased Ang II-induced AAA in IL-1Ra−/− mice. ConclusionsThe present study demonstrates that inhibition of IL-1β significantly suppresses AAA formation after Ang II infusion, suggesting that suppression of IL-1β may provide an additional strategy to protect against AAA in hypertensive patients.

Relaxin-2 therapy reverses radiation-induced fibrosis and restores bladder function in mice.

To determine the efficacy of human relaxin-2 (hRLX2) in reversing radiation-induced bladder fibrosis and lower urinary tract dysfunction (LUTD). Radiation cystitis is a consequence of radiotherapy for pelvic malignancies. Acutely, irradiation leads to reactive oxygen/nitrogen species in urothelial cells, apoptosis, barrier disruption, and inflammation. Chronically, this results in collagen deposition, bladder fibrosis, and attenuated storage and voiding functions. In severe cases, cystectomies are performed as current therapies do not reverse fibrosis. We developed a mouse model for selective bladder irradiation (10 Gray; 1 Gy = 100 rads) resulting in chronic fibrosis within 6 weeks, with decreased bladder compliance, contractility, and overflow incontinence. Seven weeks post-irradiation, female C57Bl/6 mice were continuously infused with hRLX2 (400 μg/kg/day/14 days) or vehicle (saline) via subcutaneous osmotic pumps. Mice were evaluated in vivo using urine spot analysis, cystometrograms and external urethral sphincter electromyograms; and in vitro using length-tension measurements, Western blots, histology, and immunohistochemistry. hRLX2 reversed fibrosis, decreased collagen content, improved bladder wall architecture, and increased bladder compliance, detrusor smooth muscle Cav1.2 expression and detrusor contractility in mice with chronic radiation cystitis. hRLX2 treatment outcomes were likely caused by the activation of RXFP1/2 receptors which are expressed on the detrusor. hRLX2 may be a new therapeutic option for rescuing bladders with chronic radiation cystitis.

Growth Arrest Specific 6 in Regeneration after Skeletal Muscle Injury

Growth arrest‐specific 6 (Gas6) belongs to the family of vitamin K‐dependent proteins. Its functions in the cardiovascular, pulmonary and immune systems have been reported on in depth, but very little is known about its role in skeletal muscle. Because of its roles in the regulation of inflammation and cell proliferation in other tissues, our objective was to determine whether Gas6 plays an important role in skeletal muscle repair and/or regeneration after injury.Therefore, we unilaterally injured the tibialis anterior (TA) muscles of young adult (YA; 5 mo.) and old (O; 30 mo.) Fisher Brown‐Norway rats with barium chloride (n=5–8/group). At the time of injury we also implanted a subcutaneous osmotic pump that continuously infused Gas6 (4 mg/ml) or saline onto the TA muscle through silicone tubing. The contralateral muscles served as controls. One week after injury, blood and the TA muscles were collected and the rats were euthanized. Muscle sections were prepared and stained with hematoxylin and eosin. Muscle were homogenized and Gas6 concentration was measured by ELISA in both plasma and muscle homogenates.We found that muscle injury greatly increased Gas6 concentration in muscle, but not plasma. [Gas6] was greater in O vs. YA plasma, but age had no effect on Gas6 levels in skeletal muscle. Gas6 infusion increased regenerating muscle fiber size in YA rats, but not in O rats (n=3), despite a similar increase in Gas6 concentration with injury.These results suggest that Gas6 is likely an important player in the regeneration of skeletal muscle after injury, and that the impaired recovery of old skeletal muscle after injury may be due in part to Gas6 resistance.Support or Funding InformationResearch Support: BYU Gerontology Program GrantThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Trametinib prevents mesothelial-mesenchymal transition and ameliorates abdominal adhesion formation

BackgroundIntra-abdominal adhesions are a major cause of morbidity after abdominal or gynecologic surgery. However, knowledge about the pathogenic mechanism(s) is limited, and there are no effective treatments. Here, we investigated a mouse model of bowel adhesion formation and the effect(s) of an Federal Drug Administration-approved drug (trametinib) in preventing adhesion formation. Materials and methodsC57BL/6 mice were used to develop a consistent model of intra-abdominal adhesion formation by gentle cecal abrasion with mortality rates of <10%. Adhesion formation was analyzed histologically and immunochemically to characterize the expression of pro-fibrotic marker proteins seen in pathologic scaring and included alpha smooth muscle actin (αSMA) and fibronectin EDA (FNEDA) which arises from alternative splicing of the fibronectin messenger RNA resulting in different protein isoforms. Trichrome staining assessed collagen deposition. Quantitative polymerase chain reaction analysis of RNA isolated from adhesions by laser capture microscopy was carried out to assess pro-fibrotic gene expression. To block adhesion formation, trametinib was administered via a subcutaneous osmotic pump. ResultsAdhesions were seen as early as post-operative day 1 with extensive adhesions being formed and vascularized by day 5. The expression of the FNEDA isoform occurred first with subsequent expression of αSMA and collagen. The drug trametinib was chosen for in vivo studies because it effectively blocked the mesothelial to mesenchymal transition of rat mesothelium. Trametinib, at the highest dose used (3 mg/kg/d), prevented adhesion formation while at lower doses, adhesions were usually limited, as evidenced by the presence of FNEDA isoform but not αSMA. ConclusionsCecal abrasion in mice is a reliable model to study abdominal adhesions, which can be ameliorated using the MEK1/2 inhibitor trametinib. While blocking adhesion formation at the cell and molecular levels, trametinib, at the therapeutic doses utilized, did not impair the wound healing at the laparotomy site.

Abstract P436: Intrarenal Ghrelin Receptor Inhibition Prevents Sodium Retention and Reduces Blood Pressure in Angiotensin II-induced Hypertension

The renal ghrelin receptor (GR) is localized to collecting duct (CD) cells where it increases E Na C-dependent sodium (Na + ) reabsorption. We have previously shown that in uninephrectomized Sprague-Dawley (SD) rats, systemic Ang II infusion induces hypertension after 1 day (129.2 ± 5 vs. 158.2 ± 2.6 mmHg; P&lt;0.01), and this increase is sustained over a 6 day period (191.2 ± 4.0 mmHg; P&lt;0.001). The increase in BP is attenuated with concomitant intrarenal (IR) GR siRNA infusion (F=28.9; P&lt;0.001), but the mechanism is unknown. In this study, we tested whether the reduction in Ang II-induced hypertension in the presence of IR GR siRNA is the result of the prevention of antinatriuresis. Uninephrectomized SD rats (N=33) received a subcutaneous osmotic pump for chronic systemic delivery of Ang II (200 ng/kg/min) or 5% dextrose in water (D 5 W). Rats received IR bolus infusion of D 5 W, GR siRNA (15 μg x 3 over 6 days), or scrambled siRNA (SCR siRNA, 15 μg x 3 over 6 days). Systemic Ang II + SCR siRNA reduced 24 h U Na V from 0.90 ± 0.04 to 0.34 ± 0.08 μmol/min (P&lt;0.001) and increased cumulative Na + balance from 0 to 0.54 mEq Na + (P&lt;0.01) on day 1, with no significant differences for either group for the remainder of the study. IR GR siRNA infusion prevented Ang II-mediated Na + retention (0.86 ± 0.06 vs. 0.87 ± 0.06 μmol/min) and induced a decrease in cumulative Na + balance from 0 to -0.37 ± 0.08 mEq on day 1 with no significant differences for either group for the remainder of the study. At 24 h of Ang II + SCR siRNA infusion, IR GR expression was increased in whole cell kidney (0.55 ± 0.03 vs. 0.91 ± 0.08 DU; P&lt;0.05) and major CD Na + transporter E Na C translocated from intracellular sites to apical plasma membranes to mediate Na + reabsorption (0.34 ± 0.05 vs. 0.51 ± 0.02 DU; P&lt;0.05). In rats receiving IR GR siRNA infusion, however, Ang II failed to recruit E Na C to the apical plasma membranes of CD cells. There was no change in total whole cell E Na C for all conditions. These data demonstrate that chronic inhibition of IR GR activity significantly reduces E Na C-dependent Na + retention, resulting in a negative cumulative Na + balance, thereby preventing Ang II-induced hypertension in rats. Thus, renal GRs represent a novel therapeutic target for the treatment of hypertension and other Na + -retaining states.

Abstract 5435: Beta-3 adrenergic antagonism can alleviate energy wasting associated with cancer-induced cachexia

Abstract Introduction: Cancer-associated cachexia (CAC) is a wasting syndrome characterized by atrophy of adipose tissue and skeletal muscle as a consequence of cancer. CAC has been shown to be responsible for approximately 20% of cancer deaths alone and reduces tolerance to treatment. The pathological mechanisms underlying this syndrome remains elusive and the treatment options are scarce. One proposed mechanism is the tumor-induced activation of brown adipose tissue (BAT); a thermogenic organ that wastes energy by producing heat via activation of uncoupling protein 1 (UCP1). β3 adrenergic signaling is a strong activator of BAT and the browning of white adipose tissue (WAT). We hypothesized that treatment with a specific β3 antagonist could alleviate CAC. Method: To study cachexia, the LuCAP 32 xenograft model of human prostate cancer was utilized in immunodeficient balb/ca nude mice. After two weeks of tumor growth, treatment with the specific β3 antagonist SR59230A (SR) was started using subcutaneous micro osmotic pumps. Vehicle substance and nontumor bearing (NTB) mice were used as controls. The mice were sacrificed after a treatment period of four weeks and tissues were harvested for subsequent analysis. The PCR analysis was normalized to NTB mice. Results: The mice with tumor started losing weight 20 days after tumor implantation while the nontumor bearing controls gained weight throughout the experiment. The group treated with SR also lost weight but to a significantly lesser degree than vehicle treated animals (-1.7 vs -3.2 g, p=0.032). This was also seen when looking at individual fat depots. Whole body composition, measured by echo MRI, showed that all groups equally lost fat mass over time. However, lean mass in the vehicle group decreased whereas the SR group gained lean mass (-0.41 vs 1.18 g, p=0.036). With respect to total body weight this gain in lean mass was counteracted by the loss of fat mass. PCR analysis of subcutaneous WAT showed a significant decrease in expression of ATGL (2.84 vs 1.98 FC, p=0,006), PPARγ (2.33 vs 1.45 FC, p=0.026) and CD36 (2.11 vs 1.3 FC, p=0.008) for the SR group compared to vehicle, indicating a decreased lipolysis and influx of fatty acids to WAT. The SR treated mice also had a significantly lower expression of UCP1 in WAT compared to controls (2.23 vs 0.58 FC, p=0.023), supporting the notion that the browning of WAT is a driving force for CAC. Conclusion: Our data show that treatment with a specific β3 adrenergic antagonist alleviates CAC through decreased lipolysis of WAT and decreases the expression of markers for WAT browning. This indicates a correlation between decreased browning and severity of CAC. We also provide evidence that β3 antagonism helps preserve muscle mass, which is of major importance for treatment of CAC and something that few other treatment options have been able to show. Citation Format: Fredrick Anderson, Stefan K. Nilsson, Jonas A. Nilsson. Beta-3 adrenergic antagonism can alleviate energy wasting associated with cancer-induced cachexia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5435. doi:10.1158/1538-7445.AM2017-5435