bluuebunny/crossref_metadata_2025 huggingface dataset:
Combinational treatment of colon cancer cells with bioactive food components (1147.2)
Authors: Hwang Sunil , Lee Jin‐Gyun , McKinney Kimberly , Bonkovsky Herbert
Published: June 2021

Using comparative proteomic analysis, we investigated the synergistic suppressive activity of natural components on the metastasis of colorectal cancer (CRC) cells. The isogenic SW480 and SW620 colon adenocarcinoma cell lines were compared to identify proteins overexpressed in metastatic SW620, which demonstrated altered expression levels following treatment with bioactive food components from natural sources and/or chemotherapeutic agents. SW620 cells were treated with three natural components, namely, curcumin, luteolin, and ginsenoside 20(S)‐Rg3 and three chemotherapeutic agents, 5‐fluorouracil, sorafenib, and oxaliplatin, which were selected by their cytotoxicity to SW620 cells. Comparative proteomic datasets were generated using linear ion‐trap mass spectrometry combined with nano‐flow ultra performance liquid chromatography. MS data files were searched using the SEQUEST algorithm. Significant overexpression of structural proteins, such as neurofilaments and histone proteins, was observed in untreated SW620 compared to untreated SW480 cells. Interestingly, treatment with curcumin or ginsenoside 20(S)‐Rg3 suppressed the overexpression more effectively than either 5‐FU or oxaliplatin, which are drugs currently used as primary therapy in the treatment of colorectal cancer. In addition, the overexpression of histone proteins in SW620 cells was down‐regulated by the co‐treatment of curcumin and oxaliplatin, which demonstrated synergistic cytotoxicity via combinational treatment. It has been known that chemical modification (e.g. acetylation) of histones is one of the major features of cancer metastasis. However, based on the current proteomic analysis, increased expression of histone proteins is also associated with the development of CRC metastasis. Furthermore, our data suggests that curcumin co‐treatment represents a promising adjuvant to standard oxaliplatin therapy for the suppression of metastasis in colorectal adenocarcinoma.
S‐phase kinase‐associated protein 2 gene aberration in ovarian cancer Saudi patients (1148.10)
Authors: Al‐Shabanah Othman , Hafez Mohamed , Sayed‐Ahmed Mohamed
Published: June 2021

Background: SKP2/Erb B/RAS signaling pathways play an important role in normal cell growth and malignant transformation in various malignancies. This pathway is misregulated and hyper activated in several cancers including ovarian cancer. Overexpression of this pathway is associated with the advanced stages of ovarian carcinoma. Therefore, this study investigated the genetic alteration of SKP2, P21, P27, Erb B2, Grb2, k‐Ras, c‐myc and Cox‐2 genes in the Erb B/RAS pathway in relation to the clinicopathological characteristics in 200 ovarian cancer tissues. Methods: DNA was isolated from 200 ovarian cancer FFPE sections. Clinico‐pathological data was obtained from the medical records. Relative quantitative reverse transcription‐polymerase chain reaction (PCR) and western blotting were performed to determine the genes and their protein expression levels. Results: The gene and protein expression levels of SKP2 were significantly up regulated in ovarian cancer tissue and were associated with the down regulation of both p21 and p27. The SKP2 expression is associated with the advanced FIGO stage III and IV and high grade of the tumor. There was genetic alteration in Erb B/Ras signaling pathway observed in the ovarian cancer samples compared to the normal adjacent tissues. This alteration was associated with the tumor stage and grade. Conclusion: The Skp2 expression in advanced stages might play an important role in the development and progression of ovarian cancer. Most cases of ovarian cancer with advanced stages are associated with EGFR over‐expression, mutation of KRAS. These findings suggest that blockade of EGFR signaling with EGFR‐targeted therapies might be an approach for the treatment of advanced stages ovarian cancer Saudi patients.
RNA‐guided transcriptional activation of TIMP2 utilizing CRISPR:Cas9 (1148.13)
Authors: Duellman Tyler , Warren Chris , Yang Jay
Published: June 2021

The clustered, regularly interspaced palindromic repeat (CRISPR) system is capable of genome‐specific targeting utilizing the CRISPR associated 9 (Cas9) endoribonuclease and effector targeting by a single guide RNA (sgRNA). We show that fusion of the transcriptional activating viral protein, VP64, to an enzymatically dead form of Cas9 (dCas9) is capable of enhancing gene‐targeted transcriptional activity. Using a luciferase reporter assay we show that a N‐terminal fusion of VP64 to dCas9 (dCas9.nVP64) results in higher transcriptional activity compared to the C‐terminal or flanked VP64 fusion to dCas9. Additionally, we find that there are optimal targets within the murine Tissue Inhibitor of Metalloproteinase 2 (TIMP2) promoter that results in maximal transcriptional activation and that multiple sgRNAs expressed simultaneously results in a synergistic enhancement of promoter activity. Finally, using a genome‐wide bioinformatics approach we identified a number of potential off target sites for a given sgRNA‐target within the promoter of other genes. Selective activation of TIMP2 with the CRISPR‐Cas9 system opens up a strategy for treating diseases associated with over activity of the Matrix Metalloproteinases.

Grant Funding Source: Supported by the Training Program in Translational Cardiovascular Science, 5 T32 HL 7936‐12
Temporal transcriptomic changes in canine liver slices compared to native liver (1148.3)
Authors: Liguori Michael , Klapczynski Marcin , Kowalkowski Kenneth , Ditewig Amy , Blomme Eric , Buck Wayne , Yang Yi
Published: June 2021

Precision cut tissue slices retain the multicellular, structural, and functional features of the original organ and therefore offer a potentially more relevant approach to interrogate toxicity and pharmacology. However, limited data are available regarding transcriptomic changes occurring after slicing and in culture. Here, global mRNA profiles of canine liver slices immediately after preparation (0 h) and up to 48 h in culture were evaluated relative to whole liver. Liver slices from a male beagle dog were prepared and cultured at 37°C in an O2 rich atmosphere for 0, 24 and 48 h. Integrity of the slices was monitored using routine viability markers. Total RNA was isolated for analysis on Affymetrix canine genome microarrays. At 0 h, only minor perturbation of the transcriptome was apparent, indicating similarity to liver at the transcriptional level. After 24 to 48 h, ~8300 expression changes were observed. At 0 h, mRNA levels of phase I/II drug metabolizing enzymes were unchanged, but thereafter declined. Apoptotic signaling and proteasome mRNAs increased after 24 h, but not at 0 h, suggesting minor early stress signals. After 24 h, fibrosis pathway activation was evident with substantial increases in IL‐6 and IL‐8 mRNA. Unlike primary hepatocytes, a test substance can be incubated in slices at 0 h, allowing for the retention of an organ‐like molecular environment and thus offering advantages compared to monolayers.

Grant Funding Source: Supported by AbbVie
Influence of beta‐sitosterol on ER and gene PS2 expression in breast cancer cell line T47D cells (1148.7)
Authors: Tao Shiying , Niu Jianzhao , Zhao Piwen , Hao Qinxiu , Yang Meijuan , Sun Yanling , Zhang Lijuan , Wang Jifeng
Published: June 2021

Objective: To observe the effect of beta‐sitosterol on ER and downstream gene PS2 expression in breast cancer cell line T47D cells, and explore its phytoestrogenic mechanism. Methods: mRNA expressions of ER‐alpha,ER‐beta and its downstream gene PS2 in T47D were assessed by RT ‐PCR, protein expressions of ER‐alpha,ER‐beta in T47D were assessed by western‐blot. ICI182 780 was employed to observe the influence on the gene expressions of PS2 and protein expressions of ER‐alpha,ER‐beta. Results: beta‐sitosterol up‐regulated ER‐alpha,ER‐beta mRNA expression.PS2 gene are induced in response to beta‐sitosterol treatment. The effect on PS2 gene expression was inhibited by pure estrogen receptor antagonist ICI182780. Beta‐sitosterol increased the level of T47D cells ER‐alpha, ER‐beta proteins expression, respectively. The effect was completely antagonized by ICI182 780. Conclusion: beta‐sitosterol up‐regulated gene PS2 expression through ER. And beta‐sitosterol possessed phytoestrogenic effect by activating ER. Supporttrd by 111 project (B07007)and NSFC(30772849,81273887).

Grant Funding Source: Suooorted by 111 projict(b07007) and NSFC（30772849,81273887）
Historical note: what's old is new again; exercise is medicine (1149.1)
Authors: Tipton Charles
Published: June 2021

In 2007, ACSM, AMA and the Surgeon General Office launched a national and international initiative to mobilize physicians, health care providers and professionals plus educators to promote and include exercise into their practices or activities to prevent, reduce, treat or manage diseases that impact health and the quality of life. Recently, ACSM issued a Position Stand recommending that healthy adults perform 150 min of moderate exercise per week. Although timely, admirable and needed, the initiative is not original as the basic concept has roots that began in antiquity (a time period that ended with the death of Galen in 210 AD). Specifically, the physician Susruta of India (600 B.C.E.) prescribed daily moderate exercise to his followers and patients for the purposes of promoting health, preventing corpulence, reducing diabetes, and improving appearances. He was followed by Hippocrates of Greece (480‐370 B.C.E) who was the first physician to provide a written exercise prescription for a patient suffering from consumption. He also promoted exercise to “ purge humors “ from body. Galen (129 ‐210 AD) of Rome should be remembered because he advocated exercise for diseases and conditions of consumption, depression, epilepsy, tuberculosis and vertigo. Furthermore, his views on the practice of medicine and on the role of exercise in the practice of medicine impacted Europe and the Arabic countries until the 16th century.
Myocardial force and strain comparisons in control and MLP deficient mice (1150.1)
Authors: Michaelides Marcos , Georgiadou Soteria , Constantinides Christakis
Published: June 2021

No prior study has directly characterized or quantified in‐vivo strain and force/stress responses from the left ventricular (LV) epicardium. This study quantifies and compares in‐vivo epicardial force and strain in control and transgenic hearts using micro sensors.

Male mice (N=39) [C57BL/6 (n=26), 129/Sv (n=5), wild‐type (WT) C57×129Sv (n=5), and MLP (LIM‐protein knockout) (n=3)] were studied under 1.5% isoflurane. Piezoelectric crystals were placed at longitudinal (L) epicardial loci at basal (B), middle (M) and apical (A) LV regions, with the independent and/or concurrent placement of a force sensor. Longitudinal contractile and relaxation strains ranged between 4.8‐9.3 % in (B/M/A) regions of C57BL/6 mice and in mid‐LV regions of 129/Sv, WT, and MLP mice. Measured forces ranged between 3.1‐8.9 mN. Measurements were also conducted in normal mice following afterload‐challenges. Concurrent B‐L force‐displacement recordings in C57BL/6 mice yielded stress‐strain responses exhibiting bi‐modal active force contractile characteristics. Middle‐LV forces were significantly lower in MLP mice compared to WT controls (5.9±0.4 vs. 8.9±0.2 mN, p<0.0001), evidence of a defective excitation‐contraction coupling in the dilated, fibrotic, and stiffer myocardium.

In‐vivo force and strain quantification is demonstrated in normal and trangenic murine hearts with potential usefulness to cardiac disease.

Grant Funding Source: Supported by the Research Promotion Foundation
Matrix metalloproteinase‐12 inhibition causes cardiac dysfunction post‐myocardial infarction in mice (1151.3)
Authors: Iyer Rugmani , Patterson Nicolle , Dive Vincent , Lindsey Merry
Published: June 2021

Matrix Metalloproteinases ‐12 (MMP‐12) deficiency diminishes the capacity to degrade extracellular matrix, but whether MMP‐12 inhibition post‐myocardial infarction (MI) is an effective therapeutic strategy has not been studied. Male C57BL/6J mice (3‐6 months old, n=60) were subjected to left coronary artery ligation. Saline or MMP‐12 inhibitor (MMP‐12i; 0.5 mg/kg/day) were delivered 3h post‐MI, and the mice were sacrificed at days 0 (no MI), 1 and 7 post‐MI. Infarct area was similar in the saline and MMP‐12i (p=0.70) groups, indicating similar initial injury. Survival rates were 33% for saline and 50% for the MMP‐12i (p=0.29). MMP‐12i group had 33±1% reduction in MMP‐12 activity at d1 post‐MI compared to the saline group (p<0.05). MMP‐12 inhibition worsened LV function at d7 post‐MI, as shown by larger LV volumes and decreased ejection fraction (all p<0.05). CD44, a cell adhesion molecule was 50±1% lower in RNA and protein levels in MMP12i group (both p<0.05). Hyaluronan (HA), a ligand of CD44, had reduced clearance at d1 and d7 post‐MI in MMP12i group. Accumulated HA increased levels of an apoptosis marker, caspase 3 at d7 post‐MI in the MMP12i group. Overexpression of caspase 3 leads to attenuated cardiac function post‐MI. In conclusion, MMP‐12 inhibition post‐MI reduced CD44‐HA interaction leading to increased apoptosis which resulted in LV dysfunction.

Grant Funding Source: Supported by HHSN268201000036C, HL051971, R01 HL‐075360, 5I01BX000505
Blockade of platelet‐derived growth factor on cardiac fibrogenic response (1152.10)
Authors: Liu Chang , Zhao Wenyuan , Zhao Tieqiang , Meng Weixin , Chen Yuanjian , Liu Hongyu , Sun Yao
Published: June 2021

Interstitial fibrosis appears in the viable myocardium of the infarcted heart (MI) and contributes to ventricular dysfunction. Platelet‐derived growth factor (PDGF) is a family of growth factors that stimulates cell growth, differentiation and migration. Our previous study has shown that PDGF‐D and PDGF receptors (PDGFR) were increased in the viable myocardium of the infarcted heart and cells expressing PDGF/PDGFR were predominantly fibroblasts. Herein, we sought to determine whether PDGF is involved in the development of interstitial fibrosis in the infarcted heart. MI was induced by the left coronary artery ligation in rats with or without receiving PDGF receptor antagonist (imatinib, 40mg/kg/d). Sham‐operated rats served as controls. We detected the effect of PDGF blockade on cardiac collagen synthesis and degradation at one week postMI. We found that compared to controls, TGF‐β1, type I collagen, TIMP‐1, TIMP‐2, MMP‐2 and MMP‐9 expressions were significantly increased in the viable myocardium of the left ventricle. PDGFR blockade significantly attenuated TGF‐β1, type I collagen, TIMP‐1/‐2 and MMP‐2 expressions compared to MI rats. These observations indicate that PDGF plays a role in the development of interstitial fibrosis in the viable myocardium of the infarcted heart. Our findings suggest the potential therapeutic effect of PDGFR blockade on interstitial fibrosis in the infarcted heart.

Grant Funding Source: NIH
Ethanol‐induced cardiac fibrosis is mediated by NADPH oxidase (1152.12)
Authors: Mouton Alan , Walker Morgan , El Hajj Milad , Molina Patricia , Gilpin Nicholas , Gardner Jason
Published: June 2021

Chronic ethanol (EtOH) abuse leads to cardiomyopathy that is characterized by extensive cardiac fibrosis and dilatation. We hypothesize that EtOH causes cardiac fibrosis by fibroblast activation via NADPH oxidase (NOX)‐induced oxidative stress. Male Sprague‐Dawley rats were intermittently exposed to EtOH vapor for 2 wks (14hr ON/10hr OFF; peak blood alcohol levels of ~200 mg/dL). Time‐matched controls were exposed to room air. Fixed mid‐LV sections stained with picrosirius red were assessed for collagen volume fraction (CVF). To examine the direct effects of EtOH on fibroblasts, isolated adult rat cardiac fibroblasts were treated with EtOH (50mM) for 24 hr, and cell‐conditioned media assayed for hydroxyproline (HPro). Protein expression was determined by Western blot; NOX activity was assessed by NADPH consumption assay; and cellular superoxide production was determined using DHE. Collagen gel contraction assay was used to assess fibroblast activity. In vivo EtOH‐exposure significantly increased LV CVF (1.6% vs 0.9% for control) and NOX‐2 and ‐4 expression (157.0% and 185.6% of control). EtOH significantly increased cardiac fibroblast secretion of collagen (HPro: 1.09±0.09 vs 0.75±0.07 µg/ml for control), and this was prevented by NOX inhibition (DPI; 10µM). EtOH increased fibroblast expression of α‐SMA (150.0% of control) and NOX‐2 (307.2%), but not NOX‐4. EtOH also significantly increased NOX activity (0.39±0.02 vs 0.27±0.00 nmol/mg/min) and superoxide production. EtOH increased gel contraction by fibroblasts (65.4% vs 42.4% control), which was also prevented by NOX inhibition. In conclusion, chronic intermittent EtOH vapor exposure produced cardiac fibrosis, which our data indicate is the result of a NOX‐dependent increase in fibroblast activity and collagen secretion.

Grant Funding Source: Supported by an LSUHSC ADACE Pilot Grant (jdg).
Scleraxis regulates myocardial extracellular matrix synthesis and homeostasis (1152.14)
Authors: Bagchi Rushita , Roche Patricia , Aroutiounova Nina , Schweitzer Ronen , Czubryt Michael
Published: June 2021

The cardiac extracellular matrix (ECM) functions as a source of mechanical and biochemical cues for cellular differentiation and growth in addition to its role as a physical scaffold for cellular constituents. The mechanisms governing basal ECM production are incompletely understood. Our laboratory has previously demonstrated the role of scleraxis (Scx) as a required regulator of human collagen Iα2 gene expression. Here we investigate the consequences of loss of Scx on matrix composition and turnover. We hypothesized that Scx functions to broadly regulate cardiac ECM production. DNA microarray analyses of Scx null (KO) murine hearts revealed marked changes in ECM components occurring due to loss of Scx. Histological assessment of Scx KO hearts revealed a marked reduction in abundance of collagen fibrils and overall fibrotic components as compared to wildtype (WT) hearts. Immunoblotting and qPCR analyses of these hearts confirmed a significant downregulation of fibrillar collagen types I, III and V. shRNA‐ mediated knockdown of Scx further refined the role of Scx as a direct regulator of these collagen genes. The expression of proteoglycan components including fibromodulin and lumican were reduced in Scx KO myocardium. Matrix metalloproteinases (MMPs), a prime regulator of matrix degradation and homeostasis, were also significantly altered in their expression particularly MMP2 and MMP9 as demonstrated by gelatin zymography and qPCR analyses. Thus Scx may be a direct modulator of the activity of these remodeling enzymes. The widespread effects of scleraxis deletion supports a central role for scleraxis in governing the normal expression and structure of the cardiac ECM.

Grant Funding Source: Supported by the Canadian Institutes of Health Research and St. Boniface Research Foundation
Inhibition of Na/K‐ATPase signaling regulates cardiac remodeling induced by partial nephrectomy in mice (1152.3)
Authors: Drummond Christopher , Liu Jiang , Cooper Christopher , Xie Zijian , Shapiro Joseph , Tian Jiang
Published: June 2021

Previous studies found that induction of renal dysfunction by 5/6th partial nephrectomy (PNx) resulted in cardiac hypertrophy and increased systolic blood pressure in rats and mice. Using Na/K‐ATPase (NKA) α1 heterozygous mice (α1+/‐) and their wild‐type (WT) littermates, we further demonstrate that PNx surgery or MBG infusion leads to increased cardiac cell death and decreased cell survival signaling in cardiac tissues compared to WT animals. To study the role of NKA signaling, we employed a newly developed Src inhibiting peptide (pNaKtide) derived from NKA. Both α1+/‐ and WT animals were subjected to PNx or sham surgeries. A 25mg/kg dose of pNaKtide was administered biweekly at the 12th, 14th, and 16th week after PNx surgery. One week after the first injection of pNaKtide, systolic blood pressure decreases significantly in α1+/‐ mice and slightly in WT animals. Significant decreases in diastolic dimension are also observed in both WT and α1+/‐ animals given PNx. In addition, both WT and α1+/‐ mice saw significant reversals of PNx induced cardiac hypertrophy indicated by heart weight/body weight ratio. This reversal was more significant in α1+/‐ vs WT animals. These data highlight the importance of Na/K‐ATPase signaling in cardiovascular remodeling.

Grant Funding Source: Supported by NIH grants R01 HL‐105649 and HL‐109015
Cardioprotective effects of prior transient ACE inhibition ő role of cardiac fibroblast (1152.7)
Authors: Hale Taben , Biwer Lauren , Ramaiah Priya , Shahid Wafa , D'Souza Karen
Published: June 2021

Transient ACE inhibition induces persistent changes that protect against future L‐NAME‐induced cardiac fibrosis and inflammation. This study investigates whether these observations can be mechanistically linked to changes in cardiac fibroblasts.

We measured cardiac levels of chemokines, macrophages, and proliferating cells following 7 days of L‐NAME (L) in SHR that were previously treated with placebo (C+L) or enalapril (E+L) for 2wks followed by 2wk washout. Cardiac fibroblasts were isolated and cultured to passage 1.

L‐NAME induced microinfarcts and vascular injury in both groups. In C+L, but not E+L there was an increase in macrophage‐recruiting chemokines. By day 7 of L‐NAME there was a marked increase in cardiac macrophages and proliferating cells in C+L, but not E+L. In vitro, fibroblasts from C+L were hyperproliferative, demonstrated increased Collagen I gene expression, and increased MCP‐1 production.

These findings demonstrate that L‐NAME produces phenotypic changes in fibroblasts that persist in vitro that suggest that fibroblasts may participate in macrophage recruitment in addition to collagen production. However, fibroblasts isolated from SHR previously treated with enalapril display a different phenotype that more closely resembles cells from control hearts. ACE inhibitor‐induced cardioprotection may result, in part, from a modification of the cardiac fibroblast population.

Grant Funding Source: Supported by: Sarver Heart Foundation, Steven M Gootter Foundation
Hyperoxia exposure results in decreased expression of pro‐resolution receptor ALX/FPR2 (1153.12)
Authors: Cox Ruan , Phillips Oluwakemi , Fukumoto Jutaro , Fukumoto Itsuko , Arias Stephen , Cho Young , Lockey Richard , Kolliputi Narasaiah
Published: June 2021

Rationale: Endogenously produced polyunsaturated fatty acids (PUFAs) are key mediators of the anti‐inflammatory response following acute injury. PUFAs such as resolvins and lipoxins have demonstrated potent pro‐resolutionary effects in acute lung injury. These molecules have been shown to bind and exert their effects through G‐coupled protein receptor Lipoxin A4/formyl peptide receptor 2 (ALX/FPR2). Previous reports suggest that various septic or sterile insults lead to an increase in ALX/FPR2 expression; however, the response of ALX/FRP2 to oxidative stress has not been elucidated. In this study, we investigated the role of ALX/FPR2 in a murine mouse model of hyperoxic acute lung injury (HALI).

Methods: C57BL/6 mice were exposed to hyperoxic (50, 75, or 95% O2) or normoxic (≍ 21% O2) conditions for 24, 48, or 72hrs. Following atmospheric treatment, mice were euthanized; bronchoalveolar lavage (BAL) fluid and lung tissue were collected for analysis. BAL fluid was used to assess alveolar protein leak. Lung tissue samples were used for histopathological analysis and to assess ALX/FPR2 protein and RNA expressiton following hyperoxia treatment.

Results: Results reveal that hyperoxia exposure results in a significant decrease in ALX/FRP2 expression in comparison to normoxia treated controls. This decrease was dose dependent and reduction in ALX/FRP2 was evident in as little as 24hrs following hyperoxia exposure. For the first time, our results reveal a significant decrease in ALX/FRP2 expression which has not been shown in other forms of acute lung injury.

Conclusion: While the production of PUFAs in HALI may be normal, their pro‐resolutionary effects may be dampened due to the decrease in receptor expression.

Grant Funding Source: Supported By: NIH RO1 HL105932
Aerosolized heparin mitigates chlorine‐induced lung injury (1153.15)
Authors: Basappa Susanna , Zarogiannis Sotirios , Yu Zhihong , Anjum Naseem , Rodriguez Cilina , Fernandez Solana , Honavar Jaideep , Clark Michael , Doran Stephen , Pittet Jean‐Francois , Matalon Sadis
Published: June 2021

Chlorine (Cl2) is a toxic oxidative gas that can cause acute lung injury (ALI) and death when inhaled at high concentrations for extended periods of time. Used in the production of pharmaceuticals, Cl2 exposure can occur in industrial or transportation accidents, which are common enough to pose a serious health risk. Cl2 insult results in coagulatory dysfunction, inflammatory damage and increased lung permeability. Thus we hypothesized that delivery of aerosolized heparin, which is an anticoagulant that decreases fibrin deposition and does not cause significant systemic bleeding when administered intranasally, may decrease lung injury. C57BL/6 mice were exposed to 400ppm Cl2 for 30min in environmental chambers and returned to room air. Immediately following Cl2 exposure, they were connected to a nebulizer and breathed heparin or vehicle for 20 min. At 1hr post aerosol, blood and bronchoalveolar lavage fluid (BALF) were taken for native rotational thromboelastometry (NATEM) and thrombin antithrombin (TAT) formation measurements (an index of clotting). At 6hrs post aerosol, BALF from a second group of mice was taken for measurements of total protein (an index of increased blood‐gas permeability) and white blood cell (WBC) numbers (an index of inflammation). Mice that received heparin had significantly faster coagulatory times and significantly lower levels of total protein and number of inflammatory cells in the BALF. These results suggest that heparin mitigates Cl2 induced lung damage, especially in the reduction of lung permeability, WBC inflammatory responses and post injury hypocoagulation.

Grant Funding Source: Supported by: NIEHS grants 5U54ES017218, 5U01ES015676 and a European Respiratory Society Fellowship
Neuron‐specific expression of CuZnSOD prevents the loss of muscle mass and function that occurs in homozygous CuZnSOD knockout mice (1153.3)
Authors: Sakellariou Giorgos , Davis Carol , Shi Yun , Ivannikov Maxim , Zhang Yiqiang , Giakoumaki Ifigeneia , Vasilaki Aphrodite , Macleod Gregory , Richardson Arlan , Van Remmen Holly , Jackson Malcolm , McArdle Anne , Brooks Susan
Published: June 2021

Deletion of CuZnSOD in Sod1‐/‐ mice leads to accelerated loss of muscle mass and force during aging. To determine the role of motor neurons in the muscle declines, we generated a transgenic Sod1‐/‐ mouse in which CuZnSOD was expressed under control of a synapsin 1 promoter (SynTgSod1‐/‐ mice). These “rescue” mice expressed CuZnSOD in brain, spinal cord, and peripheral nerves, but not other tissues. Sciatic nerve CuZnSOD content in SynTgSod1‐/‐ mice was ~20% that of control mice, but no reduction in muscle mass or maximum isometric force was observed for the rescue mice compared with controls at 15 months, whereas muscles of age‐matched Sod1‐/‐ mice displayed 30‐35% reductions in mass and force. Increased oxidative damage and adaptations in stress responses observed in muscles of Sod1‐/‐ mice were absent in SynTgSod1‐/‐ mice and accelerated degeneration of neuromuscular junction (NMJ) structure occurred in Sod1‐/‐ mice but not SynTgSod1‐/‐ mice. Our data demonstrate that CuZnSOD expression in neurons is sufficient to preserve NMJ and skeletal muscle structure and function in Sod1‐/‐ mice and suggest that redox homeostasis in motor neurons plays a key role in the initiation of sarcopenia during aging.

Grant Funding Source: Supported by the US National Institute on Aging (Grant No: AG‐020591)
Capsaicin‐induced cardioprotection is driven by its hypothermic effect (1154.1)
Authors: Dow Joan , Simkhovich Boris , Hale Sharon , Kay Gregory , Kloner Robert
Published: June 2021

Objective. We hypothesized that cardioprotective properties of topical capsaicin (CAP) application are related to its hypothermic effect.

Methods. Anesthetized rats received CAP cream or vehicle (VCL) applied 15‐30 minutes before a 30‐minute coronary artery occlusion followed by 2‐hour reperfusion. At the end of the protocol, hearts were excised and the area at risk (AR), area of necrosis (AN) and infarct size (AN/AR) were measured. In additional set of animals, hearts were excised at the end of CAP/VCL application, and used for Western Blot assay of p‐Akt and p‐Erk1/2.

Results. CAP cream produced a rapid decrease in pre‐occlusion core temperature, ranging from 0.22 to 1.78°C, with a median reduction of 0.97°C. In a CAP subgroup with a temperature decrease of > 0.97°C, AN/AR was decreased by 31% (from 56±4% in the VCL group to 38±4% in the CAP group, p<0.05). In the CAP subgroup with temperature decrease < 0.97°C no difference in the infarct size was observed. CAP induced an increase in p‐Akt and p‐Erk1/2. Phosphorylation of downstream p70S6 was unaffected. Levels of p‐Akt‐ and p‐Erk1/2 did not correlate with temperature at pre‐occlusion.

Conclusion. In the current study we for the first time demonstrated that cardioprotective effect of capsaicin might be related to mild hypothermia, caused by its topical application. Salvage kinase pathway appears not to be critical for the cardioprotective effects of capsaicin.
Mdivi‐1 mitigates cardiac dysfunction by attenuating mitophagy in diabetes (1155.3)
Authors: Mishra Paras Kumar , Nandi Shyam , Chavali Vishalakshi
Published: June 2021

Although Mdivi‐1 ameliorates heart failure by inhibiting autophagy, the mechanism of Mdivi‐1 mediated mitigation of cardiac dysfunction in diabetes is unclear. We hypothesize that Mdivi‐1 improves LV function by mitigating mitophagy in diabetic hearts. The three major sequencial steps required for mitophagy are: smaller size of mitochondria (by excessive fission), decrease in ΔΨm (for initiation of mitophagy) and increase in autophagophore formation (induction of beclin‐1 that initiates autophagy pathway). To investigate cardiac mitophagy, we measured dynamin like receptor‐1 (drp1, fission protein), JC1 (for ΔΨm) and beclin1, Atg3, LC3B (for autophagosome) by RT‐qPCR, Immunoblotting(IB) and immunofluoresence (IF) in male C57BL/6J and Ins2+/‐ (spontaneous, chronic,T1D) mice treated with Mdivi‐1 (50mg/kg, ip./day for 7days). The cardiac dysfunction was evaluated by M‐mode echocardiography. For in vitro study, HL1 cardiomyocytes were treated with low (5mM, LG) and high (25mM, HG) dose of glucose, and Mdivi‐1 (50µmol/L for 40 min). In Ins2+/‐ mice, Mdivi‐1 treatment decreases drp1 (IF,3.8±0.6 folds), beclin1 (IB,2.2±0.3 fold), Atg3 (IB,2.05±0.1 fold) and LC3b (IB,3.4±0.2 fold) but increases both %FS (32.6 ± 2.8 vs 42.6 ± 2.9) and %EF (60.7±4.3 vs 79.6±2.9). Mdivi‐1 did not have effect on blood glucose levels. In HL1 cardiomyocytes, Mdivi‐1 significantly decreases hypopolarized (mitophagy prone) mitochondria (JC1 staining green, IF, 2.3±0.1 fold) that prevents initiation of mitophagy in diabetic cardiomyocytes. This is a novel mechanism of Mdivi‐1 mediated mitigation of mitophagy and amelioration of diabetic cardiomyopathy.

Grant Funding Source: NIH, HL113281 and HL116205
Peripheral vascular function in heart failure patients with preserved ejection fraction (1156.4)
Authors: Barrett‐O'Keefe Zachary , Lee Joshua , Ryan John , Nativi Jose , Richardson Russell , Wray D.
Published: June 2021

While peripheral vascular dysfunction is well‐described in heart failure patients with reduced ejection fraction, disease‐related changes in vascular function in heart failure patients with preserved ejection fraction (HFpEF) remains unclear. Thus, in six HFpEF patients (73 ± 4 yrs) and six healthy controls matched for age (74 ± 4 yrs), sex, and brachial artery (BA) diameter, ultrasound Doppler was utilized to determine BA flow‐mediated vasodilation (FMD) and reactive hyperemia (RH) following 5 minutes of suprasystolic cuff occlusion. BA FMD was lower in HFpEF patients (3.9 ± 0.9 %) compared to controls (6.0 ± 1.0 %). Likewise, the RH response, calculated as the cumulative BA blood flow (area under the curve), was reduced by ~50% in patients with HFpEF compared to controls (385 ± 56 vs. 698 ± 104 ml, HFpEF vs. controls). These results indicate a clear impairment in peripheral vascular function in patients with HFpEF beyond that observed with healthy aging, providing new insight into the pathophysiology of this understudied disease.

Grant Funding Source: Supported by NIH PO1 HL‐091830, VA RR&D E6910R, and AHA 0835209N.
Enhanced vasoconstrictor sensitivity to exercise pressor reflex engagement in peripheral arterial disease (1156.7)
Authors: Gao Zhaohui , Blaha Cheryl , Cauffman Aimee , Brandt Kristen , Reed Amy , Sinoway Lawrence , Leuenberger Urs
Published: June 2021

Exercise is associated with a rise in heart rate, blood pressure (BP) and blood flow to active muscle mediated by the exercise pressor reflex and regional release of vasodilators. In peripheral arterial disease (PAD) the exercise pressor reflex is enhanced. To assess the functional impact of the exercise pressor reflex, we determined BP and mean blood velocity (MBV, Doppler) in the popliteal artery and calculated vascular conductance (VC, MBV/mean BP) in the exercising and contralateral legs during graded rhythmic plantar flexion in 7 patients with PAD (age 67±2 yrs, ankle‐brachial‐index 0.6±0.1 in the ischemic leg) and in 5 controls. At peak exercise (2.0 kg, 1 Hz), the pressor effect was greatest in PAD when exercise was performed with the ischemic leg, and lowest in the controls. In controls, exercise did not affect VC in the contralateral inactive leg (P=NS). Similarly, in PAD, exercise in the ischemic leg did not affect VC in the opposite inactive leg (P=NS). In contrast, during exercise with the non‐ischemic leg, VC in the contralateral ischemic leg decreased by 24±10% (P<0.05). Thus, activation of the exercise pressor reflex resulted in vasoconstriction in the inactive but diseased leg. This suggests that despite upregulation of peripheral vasodilator factors, the vasculature of the ischemic leg is exquisitely sensitive to reflex vasoconstriction.

Grant Funding Source: Supported by P01 HL096570 and UL1 TR000127
Renal responses to severe haemorrhage in conscious lambs: role of kappa opioid receptors (1157.1)
Authors: Smith Francine , Samhan Mohamed , Qi Wei
Published: June 2021

In previous studies, we showed that activation of kappa opioid receptors (KORs) alters cardiorenal function in conscious newborn lambs. The endogenous ligand for KORs, Dynorphin, is increased in response to blood loss, although any role for KORs in the renal responses to haemorrhage in the newborn is not known. Therefore, in conscious newborn lambs (N=7, aged 7 to 9 days), various parameters of glomerular and tubular function were measured for 30 min before (Control) and 120 min after 30% haemorrhage after pretreatment with vehicle (experiment one) or the selective KOR antagonist, GNTI (experiment two). After vehicle, glomerular filtration rate decreased 30 min after haemorrhage from 0.24±0.08 to 0.14±0.05 mL/min/g and remained at 0.19±0.08 mL/min/g at 120 min. Renal plasma flow decreased by 30 min from 1.5±0.5 to 0.7±0.2 and then increased to 1.1±0.4 mL/min/g after 120 min. Urinary flow rates and electrolyte clearances also decreased after haemorrhage. These renal responses were not altered by pretreatment with GNTI. In contrast, after vehicle, free water clearance decreased from ‐0.6±1.8 to ‐1.8±1.2 μL/min/g 30 min after haemorrhage, but increased after pretreatment with GNTI from ‐1.1±1.0 to 1.3±0.6 μL/min/g. Therefore, in the newborn period, KORs appear to regulate tubular water reabsorption but are not involved in other tubular or glomerular responses to severe blood loss.

Grant Funding Source: Supported by the CIHR.
Pulmonary permeability after hemorrhage and resuscitation in the obese Zucker rat (1157.2)
Authors: Clemmer John , Xiang Lusha , Lu Silu , Mittwede Peter , Hester Robert
Published: June 2021

Acute lung injury is a major cause of mortality and is the most frequent and earliest outcome seen in obese trauma patients. Moreover, obese patients are associated with increased morbidity and mortality after hemorrhage and resuscitation (H/R), however, the mechanisms are unclear. We hypothesized that, as compared to lean Zucker rats (LZ), obese Zucker rats (OZ) have increased lung vulnerability to H/R. Conscious animals were hemorrhaged 35% of their total blood volume and resuscitated with an equivalent volume of Ringer’s lactate. Three hours after H/R, OZ had an increased bronchoalvolar lavage to plasma protein ratio (0.12 ± 0.03) as compared to LZ (0.05 ± 0.01) suggesting increased permeability in OZ. This was associated with a greater increase in lung NADPH oxidase activity in OZ as compared to LZ. In order to consider superoxide as a factor that could play a role in lung permeability, isolated lung experiments were performed to measure basal lung capillary permeability (Kf) with or without the superoxide donor, PMS. Acute PMS treatment increased baseline lung Kf in LZ from 11 ± 2 to 53 ± 9 ml/mmHg/g/min and in OZ from 21 ± 3 to 98 ± 17 ml/mmHg/g/min, with a significantly greater increase in OZ. Basal lung Kf was significantly greater in OZ compared to LZ. These data suggest that obesity may increase vulnerability to H/R due to increased basal lung Kf and/or exacerbated production or sensitivity to lung superoxide.

Grant Funding Source: Supported by AHA‐12SDG12050525, NIH HL‐51971,HL‐89581, and T32 HL‐105324
Serum pregnancy‐associated plasma protein‐A levels in a cohort with cardiovascular disease (1158.2)
Authors: Gerthoffer William , Hellmich Alexandria , Aragon Ileana , Massey Clara
Published: June 2021

Patients with acute coronary syndrome (ACS) who have high serum levels of pregnancy‐associated plasma protein‐A (PAPP‐A) are at increased risk of cardiac events. PAPP‐A levels have been reported to indicate elevated risk in ACS patients who were troponin I‐negative. The long range goal is to assess changes in PAPP‐A levels in a longitudinal study of subjects diagnosed with cardiovascular disease to further test the validity of serum levels of PAPP‐A as a predictor of adverse cardiovascular events. Initial serum levels of PAPP‐A were measured in a cohort of 243 subjects to establish the reference range for the cohort. Serum PAPP‐A levels were measured using a commercial sandwich type immunoassay using dual wavelength colorimetric detection. Serum PAPP‐A levels ranged from 0.43 to 4.2 ng/ml with geometric mean and 95% CI of 0.95 (0.87 ‐ 0.97) ng/ml. A correction factor of 2.7 international units/ng PAPP‐A was used to calculate the reference range in mIU/L (0.4 ‐ 11). Median values of serum PAPP‐A did not vary significantly with gender or race, nor were there significant correlations with increasing age, mean arterial blood pressure or total cholesterol. Although PAPP‐A levels did not correlate with these known cardiovascular risk factors in this cross‐sectional analysis the data do provide a reference range of the cohort that is similar to published reports of PAPP‐A levels in subjects at other clinical centers.

Grant Funding Source: Supported by grant P20MD0002314‐06 from NIMHD
Increased Rho‐kinase activity and reactive oxygen species contribute to the enhanced vasoconstrictor effects of angiotensin II in aortic rings from sepsis‐surviving rats (1158.3)
Authors: de Souza Priscila , da Silva Luisa , Scheschowitsch Karin , Guarido Karla , Werner M. Fernanda , Assreuy Jamil , Marques M. Consuelo , da Silva‐Santos José Eduardo
Published: June 2021

We hypothesized that changes in vascular reactivity contribute to the high mortality rate among patients discharged after severe sepsis/septic shock. Male Wistar rats were subjected to cecal ligation and puncture to induce sepsis (mortality around 30%) and aortic rings from survived animals were tested at 30 or 60 days after (S30 and S60 groups). The effects of KCl, phenylephrine, angiotensin I, CaCl2, acetylcholine and sodium nitroprusside did not change, but angiotensin II (AII)‐induced constriction was increased by 80% in aortic rings from S60. The removal of endothelium, incubation with L‐NAME (inhibitor of nitric oxide synthase), or inhibition of Rho‐kinase by Y‐27632 did not change the effects of AII in these vessels, which showed increased protein levels of both RhoA and ROCK II. Incubation with apocynin (NADPH oxidase inhibitor), SOD, or tempol (O2‐ scavenger) did not change the effect of AII in control rings, but avoided the increased reactivity to AII in vessels from S60 rats. The endogenous activity of SOD was decreased by 45 ± 3%, while the generation of LOOH and GSH were increased by 41 ± 12% and 98 ± 3.9% in the aorta of S60 rats. A selective fluorescent probe revealed an increased ROS generation (up to 30%) in vessels from S60 rats after stimulation with AII. In conclusion, aortic rings from sepsis‐surviving rats display increased reactivity to AII, which involves augmented activity of ROCK and oxidative stress.

Grant Funding Source: Supported by CAPES, CNPq, FAPESC
Conjugated linoleic acid may decrease arterial calcification via induction of heat shock proteins (1158.7)
Authors: Hsieh Tsung‐Hsiu , Xu Jen , Hsieh Yu‐Chung , Kao E‐Jay , Wu Yong , Lim Kenneth , Kong Tianqing , Park Yeonhwa , Lu Tzongshi
Published: June 2021

Vascular calcification (VC) is a significant contributor to cardiovascular mortality in patients with chronic kidney disease and chronic inflammatory conditions. Despite this, there are currently no ideal therapies directed at the prevention or treatment of VC in clinical practice. We recently reported that heat shock protein 72 (HSP72) could be induced in human arteries and prevent human aortic smooth muscle cell (HA‐SMC) calcification. Conjugated linoleic acid (CLA) is a group of positional and geometric isomers of octadecadienoic acid (linoleic acid) with a conjugated double bond system, and cis9, trans11‐CLA form has been shown to have HSP72 inductive properties. In this study, we postulate that CLA may exert vasculo‐protective effects through induction of HSP72. Our results show that inducible HSP72 is significantly expressed following CLA treatment in HA‐SMCs, in vitro as well as in aorta from mice treated with CLA, in vivo. We next showed that JC‐1 complex, an indicator of mitochondrial membrane potential stability was preserved in HA‐SMCs treated with calcification medium following induction of HSP72 by CLA. In conclusion, induction of HSP72’s anti‐calcific effects by CLA involve stabilization of mitochondrial function. We suggest treatment strategies involving induction of HSP72 by CLA as a new approach to inhibit VC.
Effect of short‐term dietary curcumin supplementation on mitochondrial regulatory proteins in muscle and brown adipose tissue of aged mice (1159.4)
Authors: Wawrzyniak Nicholas , Duarte Andrew , Nguyen Linda , Joseph Anna‐Maria , Layne Andrew , Criswell David , Adhihetty Peter
Published: June 2021

Curcumin, a polyphenol found in the spice turmeric, is shown to have antioxidant and anti‐inflammatory properties in multiple tissues, but whether it alters mitochondrial biogenesis/apoptotic pathways is unknown. The aging process is associated with impaired mitochondrial function and elevated mitochondrial apoptotic susceptibility. Potential pharmacological and/or neutraceutical therapeutic interventions capable of improving mitochondrial function, like curcumin, have been postulated to delay this process. Thus, we investigated whether short‐term (21d) dietary curcumin supplementation (5% diet) altered mitochondrial biogenesis in muscle and brown adipose tissue (BAT) of aged mice (24 month; C57BL/6) compared to control diet mice (n=4‐6/group). While curcumin supplementation increased the mitochondrial content markers cytochrome c (14%) and COX Vb (26%) and enhanced the mitochondrial regulators Tfam (25%) and NRF‐1 (14%) in BAT, it suppressed and/or caused no change in these mitochondrial indices in muscle. In contrast, curcumin treatment evoked significant decreases (P<0.05) in the pro‐apoptotic BAX protein in both BAT (20%) and muscle (55%). Our data indicate short‐term curcumin treatment in aged mice causes tissue‐specific mitochondrial biogenesis adaptations in BAT and muscle while potentially suppressing mitochondrial apoptotic susceptibility in both tissues.
Potential role of AT2R on the cardiac mitochondrial metabolism in the hyperthyroidism condition (1159.8)
Authors: Silva Ivson , Lima Vanessa , Souza dos Santos Robson , Ferreira Julio Cesar , Barreto‐Chaves Maria Luiza
Published: June 2021

Thyroid hormone (TH) is known to influence the mitochondrial metabolism. Recently, it has been proposed that the intracellular renin angiotensin system (iRAS) seems to be activated by high levels of (TH). However, the contribution of AT2R in the regulation of cardiac mitochondrial metabolism or the possible effect of TH on this event was not elucidated yet. We investigated the potential role of AT2R on the cardiac mitochondrial metabolism and the possible influence of the hyperthyroidism on this process. Four groups of mice were used: wild‐type (WT), WT treated with TH (0.7 µg/Kg BW/day, i.p.) for 14 days, knockout (KO AT2R‐/y) mice and KO AT2R‐/y mice treated with TH. Cardiac mitochondria were isolated and the oxygen consumption was assessed by oxymetry. The H2O2 production was also analyzed. The determination of maximal oxygen uptake was determined by indirect calorimetry open circuit. Data were analyzed by two‐way ANOVA. The KO mice presented a reduction in all parameters analyzed: states 2, 3, 4 and respiratory control ratio in relation to control. The TH‐treated WT did not present changes on H2O2 production. However, H2O2 production was higher on KO mice treated with TH. Indeed, the calorimetry showed a decrease (17%) on basal metabolic rate of on KO mice. Taken together, our data show that AT2R may be involved in the control of cardiac mitochondrial metabolism and influence the effect of hyperthyroidism in this parameter.

Grant Funding Source: Supported by CNPq/CAPES and FAPESP
Brown adipose tissue thermal activity is influenced by season in healthy children (1160.1)
Authors: Astle Val , Zhang Taouri , Robinson Lindsay , Law James , Townsend Ellen , Symonds Michael , Pitchford Nicola , Budge Helen
Published: June 2021

Brown adipose tissue (BAT), present in the supraclavicular region of humans, can be activated by cold stimuli, and even small quantities could result in a large increase in energy expenditure. PET‐CT studies have identified more glucose uptake into BAT in winter months. This study was conducted to determine if seasonality had an effect on thermal activity of BAT. Infrared thermography, a technique previously established for this use in children, was used to detect the skin temperature overlying BAT depots in the supraclavicular region. Skin temperatures were recorded in the steady state and 5 minutes after a cool stimulus (hand placed into water at 20°C). The study was conducted at schools in age‐matched children aged 8‐11 years in the Summer (n=27) and in the Winter (n=25). The interoperator coefficient of variation (CV) (mean 4.11% (range 0.98‐9.04%) and intraobserver CV (mean 0.18% (range 0.01‐0.38%) were within acceptable limits. In line with our previous published studies, we found that BMI had a significant negative effect on baseline temperature (p<0.001). In addition, baseline skin temperature overlying BAT in the supraclavicular region was significantly greater in Winter (p=0.017, R2=0.31) than Summer independent of BMI, gender, age and room temperature. Future studies of brown adipose tissue activity during daily activities are required to determine possible factors which may be utilised to increase metabolic activity and, therefore, decrease white fat deposition.

Grant Funding Source: Action Research