Osteopontin Expression in Cardiomyocytes Is Increased in Pediatric Patients With Sepsis or Pneumonia

Sepsis and pneumonia are major causes of death in the United States, and their pathophysiology includes infection with inflammation and immune dysfunction. Both sepsis and pneumonia cause cardiovascular dysfunction. The expression of Osteopontin (OPN) in cardiomyocytes of patients with sepsis or pneumonia, and its role the induced cardiac dysfunction have not been thoroughly investigated. OPN is a matricellular protein synthesized by multiple diseased tissues and cells including cardiomyocytes. Here, we studied the expression of OPN protein using immunofluorescence in human myocardial autopsy tissues from pediatric and mid age or elderly patients with sepsis and/or pneumonia. Fourteen human myocardial tissues from six pediatric patients and eight mid-age or elderly patients were studied. Immunofluorescence was used to investigate the expression of OPN in paraffin-embedded heart sections co-stained with the myocyte markers Actin Alpha 1 (ACTA1) and Myosin Light Chain 2 (MLC2). A quantitative analysis was performed to determine the number of ACTA1 and MLC2 positive cardiomyocytes that express OPN. The results showed that OPN expression was significantly increased in cardiomyocytes in the hearts from pediatric patients with sepsis and/or pneumonia (N = 3) relative to pediatric patients without sepsis/pneumonia (N = 3), or adult to elderly patients with sepsis/pneumonia (N = 5). Among the older septic hearts, higher levels of cardiomyocyte OPN expression was seen only in conjunction with severe coronary arterial occlusion. This is the first study to document increased OPN expression in cardiomyocytes of pediatric subjects with sepsis or pneumonia. Our findings highlight a potentially important role for OPN in sepsis- or pneumonia-mediated cardiac dysfunction in pediatric patients

Avaliação de lesões intra-epiteliais escamosas e microbiologia em exames citológicos realizados no Laboratório da Disciplina de Análises Citológicas da Faculdade de Farmácia/UFRGS

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Osteopontin Expression in Cardiomyocytes Is Increased in Pediatric Patients With Sepsis or Pneumonia

Sepsis and pneumonia are major causes of death in the United States, and their pathophysiology includes infection with inflammation and immune dysfunction. Both sepsis and pneumonia cause cardiovascular dysfunction. The expression of Osteopontin (OPN) in cardiomyocytes of patients with sepsis or pneumonia, and its role the induced cardiac dysfunction have not been thoroughly investigated. OPN is a matricellular protein synthesized by multiple diseased tissues and cells including cardiomyocytes. Here, we studied the expression of OPN protein using immunofluorescence in human myocardial autopsy tissues from pediatric and mid age or elderly patients with sepsis and/or pneumonia. Fourteen human myocardial tissues from six pediatric patients and eight mid-age or elderly patients were studied. Immunofluorescence was used to investigate the expression of OPN in paraffin-embedded heart sections co-stained with the myocyte markers Actin Alpha 1 (ACTA1) and Myosin Light Chain 2 (MLC2). A quantitative analysis was performed to determine the number of ACTA1 and MLC2 positive cardiomyocytes that express OPN. The results showed that OPN expression was significantly increased in cardiomyocytes in the hearts from pediatric patients with sepsis and/or pneumonia ( N = 3) relative to pediatric patients without sepsis/pneumonia ( N = 3), or adult to elderly patients with sepsis/pneumonia ( N = 5). Among the older septic hearts, higher levels of cardiomyocyte OPN expression was seen only in conjunction with severe coronary arterial occlusion. This is the first study to document increased OPN expression in cardiomyocytes of pediatric subjects with sepsis or pneumonia. Our findings highlight a potentially important role for OPN in sepsis- or pneumonia-mediated cardiac dysfunction in pediatric patients

Abstract 12205: A New Mouse Model of Myocardial Lipids, Fibrosis, Arrhythmia and Diastolic Dysfunction Induced by Hyperlipidemia and Cardiac LDLR

Background: Hyperlipidemia is a major risk factor for CVD. Patients with HF with preserved ejection fraction (HFpEF) have more myocardial lipid accumulation than patients with reduced EF (HFrEF). RNASeq data from cardiac biopsies showed downregulation of the gene for lipoprotein lipase (LPL) that degrades triglycerides, in HFpEF patients compared to healthy and HFrEF controls. Poloxamer-407 (p407) induces hyperlipidemia by blocking LPL and subsequent increase in plasma triglycerides and low-density lipoprotein (LDL) cholesterol. We hypothesized that mice treated with p407 and cardiac LDL-Receptor (LDLR) over-expression (OE) develop hyperlipidemia, myocardial lipid accumulation, and diastolic dysfunction resulting in HFpEF and arrhythmias. Methods: Baseline cardiac function was assessed by echo for male and female C57Bl6 mice (n=9) for 2 groups: 4wk biweekly i.p. p407-injections with (n=4) or without (n=3) single i.v. injection with AAV9-cTnT-LDLR. Cardiac function was assessed by echocardiography at 3 and 4 wks. Blood Pressure (BP) and Whole Body Plethysmography (WBP) were assessed during wk4. Ttest was used for statistics. PR and ORO staining and telemetry were performed at wk4. Results: At wk3, P407 and LDLR OE led to alterations in diastolic function (increased IVCT, IVRT, MV E/E’, MPI, and NFT) and increased LV wall thickness, p<0.05. At wk4, there was pulmonary hypertension (increased mean pulmonary arterial pressure, decreased pulmonary acceleration time p <.05).Histology showed excessive myocardial lipids and fibrosis, and telemetry showed incidents of second-degree and higher-degree AV block. The group injected solely with p407 show e d alterations in diastolic function (increased IVCT, IVRT, NFT, LVMPI, LVMPI NFT p<.05 ) and decreased EDV, ESV, EDLVM, ESLVM, p<.05 at wk4. All groups had preserved %EF and no abnormalities in BP or WBP. Conclusions: P407 and cardiac LDLR OE induce a drastic decline in cardiac diastolic function over a shorter period of time compared to p407 alone. Diastolic dysfunction was observed in wk3 followed by pulmonary hypertension, arrhythmia, myocardial lipid accumulation and fibrosis in wk4. This new model may allow for more rapid investigations of cardiac abnormalities seen in HFpEF patients

Abstract P428: A New Mouse Model Of Myocardial Lipids, Fibrosis, Arrhythmia And Diastolic Dysfunction Induced By Hyperlipidemia And Cardiac LDLR

Background: Hyperlipidemia is a major risk factor for CVD. Patients with HF with preserved ejection fraction (HFpEF) have more myocardial lipid accumulation than patients with reduced EF (HFrEF). RNASeq data from cardiac biopsies showed downregulation of the gene for lipoprotein lipase (LPL) that degrades triglycerides, in HFpEF patients compared to healthy and HFrEF controls. Poloxamer-407 (p407) induces hyperlipidemia by blocking LPL and subsequent increase in plasma triglycerides and low-density lipoprotein (LDL) cholesterol. We hypothesized that mice treated with p407 and cardiac LDL-Receptor (LDLR) over-expression (OE) develop hyperlipidemia, myocardial lipid accumulation, and diastolic dysfunction resulting in HFpEF and arrhythmias. Methods: Baseline cardiac function was assessed by echo for male and female C57Bl6 mice (n=9) for 2 groups: 4wk biweekly i.p. p407-injections with (n=4) or without (n=3) single i.v. injection with AAV9-cTnT-LDLR. Cardiac function was assessed by echocardiography at 3 and 4 wks. Blood Pressure (BP) and Whole Body Plethysmography (WBP) were assessed during wk4. Ttest was used for statistics. PR and ORO staining and telemetry were performed at wk4. Results: At wk3, P407 and LDLR OE led to alterations in diastolic function (increased IVCT, IVRT, MV E/E’, MPI, and NFT) and increased LV wall thickness, p<0.05. At wk4, there was pulmonary hypertension (increased mean pulmonary arterial pressure, decreased pulmonary acceleration time p <.05).Histology showed excessive myocardial lipids and fibrosis, and telemetry showed incidents of second-degree and higher-degree AV block. The group injected solely with p407 show e d alterations in diastolic function (increased IVCT, IVRT, NFT, LVMPI, LVMPI NFT p<.05 ) and decreased EDV, ESV, EDLVM, ESLVM, p<.05 at wk4. All groups had preserved %EF and no abnormalities in BP or WBP. Conclusions: P407 and cardiac LDLR OE induce a drastic decline in cardiac diastolic function over a shorter period of time compared to p407 alone. Diastolic dysfunction was observed in wk3 followed by pulmonary hypertension, arrhythmia, myocardial lipid accumulation and fibrosis in wk4. This new model may allow for more rapid investigations of cardiac abnormalities seen in HFpEF patients

Tracking Acoustic and Electrophysiological Changes Associated with CKD-induced Cardiac and Valvular Remodeling in a Mouse Model

Objective: Patients with chronic kidney disease (CKD) are at a higher risk of developing heart and valvular diseases than the general population. Non-invasive screening methods are needed to identify cardiovascular abnormalities in this population. In this study, we use a diet-induced CKD mouse to study AV and cardiac remodeling. We hypothesize that structural differences in AV and cardiac remodeling occur in CKD mice, and can be identified through phonocardiogram (PCG) and electrocardiogram (ECG) signals. Methods: Eight-week old C57BL/6J mice were assigned to the following groups: 1) control group, fed a normal chow diet for 12 weeks, and 2) CKD group, fed a chow containing 0.2% adenine, with normal (0.6%) phosphorus for 6 weeks to induce CKD, and 3) CKD+high phosphate (HP) group, fed CKD inducing diet for 6 weeks, and then fed for 6 weeks with a 0.2% adenine/1.8% phosphorus diet to induce cardiovascular calcification. PCG and ECG signals were recorded weekly and echocardiogram parameters were recorded at the experimental end-point. Results: The HP diet induced aortic valve calcification in the CKD+HP group. CKD and CKD+HP group had increased end-diastolic volume, which is indicative of left ventricular hypertrophy. Changes in ECG parameters indicate that CKD groups have delayed cardiac repolarization time, an early ECG marker associated with arrhythmias. PCG signals indicate the presence of a systolic and diastolic murmur in the CKD+HP group, a characteristic of both valve disease and cardiac dysfunction. Conclusions: We demonstrate that the diet-induced CKD mouse model can be used to study valve and cardiac remodeling, and ECG and PCG signals can noninvasively track valve and cardiac changes. Future studies will determine the adequacy of using these signals to predict cardiovascular diseases

Abstract 12042: A Mouse Model of Heart Failure With Preserved Ejection Fraction Driven by Hyperlipidemia and Enhanced Cardiac LDLR Expression

Byline: Monique M Williams, Univ of Miami, Miller Sch, Miami, FL; Jose Manuel Condor Capcha, Miami, FL; Camila Iansen Irion, Univ of Miami, Miami, FL; Grace Seo, Univ of Miami Miller SOM, Miami, FL; Guerline Lambert, Univ of Miami, Miami, FL; Ali Kamiar, Miami, FL; Rosemeire M Kanashiro-Takeuchi, UNIVERSITY OF MIAMI, Miami, FL; Lauro Takeuchi, Univ of Miami, Miami, FL; Ali Saad, Univ of Miami, Miller Sch of Medicine, FL; Armando J Mendez, UNIVERSITY MIAMI, Miami, FL; Keith A Webster, UNIVERSITY MIAMI, Miami, FL; Jeffrey J Goldberger, Univ of Miami, Miami, FL; Joshua M Hare, UNIVERSITY OF MIAMI, Miami, FL; Lina A Shehadeh, UNIVERSITY OF MIAMI MILLER SCH MED, Miami, FL Background: HFpEF prevalence has surpassed that of HFrEF. Elevated accumulation of myocardial lipids is a prominent feature of HFpEF patients and the gene for lipoprotein lipase (LPL) in cardiac biopsies is decreased compared with HFrEF or healthy controls. Poloxamer-407 (p407) induces hyperlipidemia by blocking LPL and increasing plasma triglycerides (TG) and LDL cholesterol. We hypothesized that hyperlipidemia driven by p407 and overexpression (OE) of cardiac LDLR in mice mimics a subset of human HFpEF. Methods: WT-129 mice were subject to 4 wks of biweekly i.p. p407-injections with or without a single i.v. injection of AAV9-cTnT-LDLR (n=31); AAV9-cTnT-Luciferase control (n=11), or single i.v. injection with AAV9-cTnT-LDLR alone (n=10). Treatment groups were compared to an untreated group (n=26). Echo, blood pressure (BP), whole-body plethysmography (WBP), ECG telemetry, activity wheel monitoring (AWM), biochemical tests, and histological changes were assessed at 4-8 wks. Results: At 4 wks, p407 and LDLR OE led to diastolic dysfunction (DD) (prolonged IVRT, decreased E/A (p<0.05 and p<0.001 respectively)), preserved EF and increased LV wall thickness (LVID and LVAW, p<0.0001). BP and WBP were normal at 4 wks but respiration (breaths per minute) decreased at 8 wks (p<0.01). ECG and AWM respectively indicated heart block and decreased exercise activity (p<0.001). With the double treatment, total cholesterol increased in heart, skeletal muscle and liver (p<0.05), while TG was decreased only in hearts (p<0.001) and liver (p<0.05). Double treatment also promoted elevated myocardial lipids, fibrosis, increased Wet/Dry lung (p<0.001) and HW/BW (p<0.05). Xanthelasma, ascites, and cardiac ischemia were noted in the double and single, p407 + luciferase groups. Sudden death (SD) occurred between 6-12 wks in double and p407+Luciferase treatment groups. Conclusion: We present a unique model of early onset, rapidly evolving HFpEF where double treatment mice acquire DD, arrhythmia, cardiac hypertrophy, fibrosis, pulmonary congestion, exercise intolerance, and preserved EF in the absence of obesity, HTN, CKD or DM. The model may represent a subset of HFpEF wherein hyperlipidemia drives a severe phenotype with SD as a prominent endpoint.Professiona

Abstract 10313: Gender and Strain Background Affect Cardiopulmonary Function in Col4a3 -/- Alport Mice

Background: Alport syndrome (AS) is a hereditary form of chronic kidney inflammation that results in renal failure (RF). We previously reported that Col4a3 -/- 129J mice, a model of AS and RF, exemplified multiple features of heart failure with preserved ejection fraction (HFpEF). This study investigates the effect of 3 different genetic backgrounds on cardiopulmonary function in Col4a3 -/- mice. Methods: Male and female Col4a3 -/- (Alport) and WT mice from 129x1/SvJ, C57Bl/6 and BALBC strain were examined using echocardiography, whole body plethysmography (WBP) and pressure-volume (PV) loop analysis. Ttest was used for statistical analysis. Four groups per strain were used (n= 4-15/group). Results: Compared with their respective age-matched WT controls, male and female 8-week-old Col4a3 -/- 129x1/SvJ mice demonstrated impaired systolic function (EF of 56 to 43.7%, p=.002 for females and 50.9 to 42% for males, p=.044; SV, CO, p<.05) and diastolic dysfunction (increased IVRT from 15.3 to 21.7ms, p=.0009 for females; 16.6 to 26.2ms, p<.0001 for males; increased MPI, P<.05). Additionally, PV loop analysis showed an increased EDPVR and end diastolic pressure, prolonged time constant of LV relaxation, and decreased CO and SV (p<0.05). For Col4a3 -/- BalbC and Col4a3 -/- C57Bl/6 strains, only females exhibited systolic (BalbC: EF% 51 to 45.66%, p=0.106 for females and 43.5 to 40.8% for males, p=.51; B6: EF% 51.3 to 40.8% p=.02 for females and 48.4 to 40.9%, p=.31 for males; SV, CO, p<.05) and diastolic dysfunction (IVRT from 16 to 24.1ms for BalbC and 18.20 to 25.63ms for B6, p<0.05). Male Col4a3 -/- BalbC had an increase in minimum and end-systolic pressures, and an increased pressure at dV/dt-max. Male Col4a3 -/- C57Bl/6 mice had an increased ESPVR slope (p<.05). All strains except BalbC males demonstrated alterations (p<.05) in pulmonary function including decreased ventilation rate (MV) and respiratory frequency, decreased peak expiratory flow, EF50, increased expiratory time, relaxation time, inspiratory time, and time of pause at end-of-expiration. Conclusion: Both male and female Col4a3 -/- 129x1/SvJ mice demonstrated impaired cardiopulmonary viability. However, Col4a3 -/- mice on BalbC or C57Bl/6 strains had cardiopulmonary deficits only in females

Abstract P390: Gender And Strain Background Affect Cardiopulmonary Function In Col4a3 -/- Alport Mice

Background: Alport syndrome (AS) is a hereditary form of chronic kidney inflammation that results in renal failure (RF). Our group previously reported that Col4a3 -/- mice, a model of AS and RF, on 129J background exemplified multiple features of heart failure with preserved ejection fraction (HFpEF). This study investigates the effect of 3 different genetic backgrounds on cardiopulmonary function in Col4a3 -/- mice. Methods: Male and female Col4a3 -/- (Alport) and WT mice on 3 different genetic backgrounds, 129x1/SvJ, C57Bl/6 and BALBC, were examined using echocardiography, whole body plethysmography (WBP) and pressure-volume (PV) loop analysis. Ttest was used for statistical analysis. Four groups per strain were used (n= 4 to 15 per group). Results: Compared with their respective age-matched WT controls, both male and female 8-week-old Col4a3 -/- 129x1/SvJ mice demonstrated impaired systolic function (EF of 56% to 43.7%, p=0.0018 for females and 50.9% to 42% for males, p=0.044; SV, CO, p<0.05) and diastolic dysfunction (increased IVRT from 15.32ms to 21.72ms, p=0.0009 for females; 16.57ms to 26.21ms, p<0.0001 for males; increased MPI, P<0.05). Additionally, PV loop analysis showed an increased EDPVR and end diastolic pressure, prolonged time constant of LV relaxation, and decreased CO and SV (p<0.05). However for Col4a3 -/- BalbC (8-week-old) and Col4a3 -/- C57Bl/6 (20-week-old) strains, only females exhibited systolic (BalbC: EF% 51% to 45.66%, p=0.106 for females and 43.5% to 40.8% for males, p=0.508; B6: EF% 51.30% to 40.80% p=0.024 for females and 48.43% to 40.91%, p=0.3098 for males; SV, CO, p<0.05) and diastolic dysfunction (IVRT from 15.96ms to 24.91ms for Balb/C and 18.20ms to 25.63ms for B6, p<0.05). Male Col4a3 -/- BalbC had an increase in minimum pressure and end-systolic pressure, and an increased pressure at dV/dt-max. Male Col4a3 -/- C57Bl/6 mice had an increased ESPVR slope (p<0.05). All strains except BalbC males demonstrated alterations (p<0.05) in pulmonary function including decreased ventilation rate (MV, mL/min) and respiratory frequency (BPM), decreased peak expiratory flow, EF50, increased expiratory time, relaxation time, inspiratory time, and time of pause at end-of-expiration. Conclusion: Both male and female Col4a3 -/- 129x1/SvJ mice demonstrated impaired cardiopulmonary viability. However, Col4a3 -/- mice on BalbC or C57Bl/6 strains had cardiopulmonary deficits only in females

Abstract P420: Nano-Triciribine Reduces SARS2-CoV-2 Infection By Sequestering ACE2 And The Novel Host Factor LDLR

Background: People with previous CVD hospitalized for COVID-19 have elevated death rate. We reported that patients with diabetes and HF higher protein levels of the low density lipoprotein receptor (LDLR). We hypothesized that LDLR is a novel host factor for the SARS-CoV-2-Spike (S2S) protein that may be regulated by the Akt inhibitor Triciribine (TCN), a drug being tested in Phase III studies for breast cancer. We also hypothesized that nano-formulation of Triciribine (NanoTriciribine; NTCN) would enhance its efficacy and allow for intranasal delivery. Methods: Interactions between the recombinant proteins Spike-RBD (receptor binding domain), ACE2, LDLR and its ectodomains (EGFA-EFFB, C2-C5 and C2) were analyzed by binding assays and co-IP in HepG2, HK2, and 293T cells. Viral entry assays were performed with 2 S2S pseudoviruses using 293T cells + hACE2 and TMPRSS2 or Furin protease. The effect of NTCN or the LXR agonist GW-3965 on viral uptake (pseudotyped VSVΔG-GFP*S2S or chimera VSV-S2S-eGFP virus) was assessed. Akt, pAkt, ACE2, and LDLR levels were determined in 293T+hACE2 by flow cytometry. Assays were done in triplicates and 1-way-ANOVA with Tukey’s correction was used for statistics. Results: RBD protein binds modestly to the human LDLR (EC 50 :10μM) and its C2-C5 ectodomain (EC 50 :13.8μM). Co-IP revealed a novel and strong LDLR-ACE2 interaction in several human cell lines. LDLR overexpression in human cells increased the uptake of VSVΔG-GFP*S2S (FC=2.32;p<0.001) and chimera virus (FC=.33; p<.0001). NTCN and TCN each reduced pAkt/Akt ratio. 1μM TCN or NTCN reduced LDLR (7.2%;p<.01 & 15.6%;p<0.0001) and ACE2 (32%;p<0.05 & 44.7%;p<.01) cell surface expression, respectively. 1μM NTCN or GW-3965 reduced S2S viral entry by 64.2% (p<.0001) and 40.7% (p<.01), respectively, confirming a role for LDLR in S2S infection. In hACE2tg mice, chimera VSV-S2S caused significant lung infection as measured by qPCR, GFP expression in proximal and distal lung airway epithelial cells, and electron microscopy. Intranasal delivery of NTCN was well tolerated. Conclusions: LDLR enhanced S2S viral entry supporting the elevated COVID-19 susceptibility seen in patients with heart disease. NTCN is a promising candidate for prophylactic treatment against COVID-19