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The influence of physical activity in the progression of experimental lung cancer in mice
- PMID: 22683274
- DOI: 10.1016/j.prp.2012.04.006
GRUPO_AF1 – GROUP AFA1 – Aerobic Physical Activity – Atividade Física Aeróbia – ´´My´´ Dissertation – Faculty of Medicine of Sao Jose do Rio Preto
GRUPO AFAN 1 – GROUP AFAN1 – Anaerobic Physical Activity – Atividade Física Anaeróbia – ´´My´´ Dissertation – Faculty of Medicine of Sao Jose do Rio Preto
GRUPO_AF2 – GROUP AFA2 – Aerobic Physical Activity – Atividade Física Aeróbia – ´´My´´ Dissertation – Faculty of Medicine of Sao Jose do Rio Preto
GRUPO AFAN 2 – GROUP AFAN 2 – Anaerobic Physical Activity – Atividade Física Anaeróbia – ´´My´´ Dissertation – Faculty of Medicine of Sao Jose do Rio Preto
Slides – mestrado – ´´My´´ Dissertation – Faculty of Medicine of Sao Jose do Rio Preto
DMBA CARCINOGEN IN EXPERIMENTAL MODELS
Avaliação da influência da atividade física aeróbia e anaeróbia na progressão do câncer de pulmão experimental – Summary – Resumo – ´´My´´ Dissertation – Faculty of Medicine of Sao Jose do Rio Preto
Lung cancer is one of the most incident neoplasms in the world, representing the main cause of mortality for cancer. Many epidemiologic studies have suggested that physical activity may reduce the risk of lung cancer, other works evaluate the effectiveness of the use of the physical activity in the suppression, remission and reduction of the recurrence of tumors. The aim of this study was to evaluate the effects of aerobic and anaerobic physical activity in the development and the progression of lung cancer. Lung tumors were induced with a dose of 3mg of urethane/kg, in 67 male Balb – C type mice, divided in three groups: group 1_24 mice treated with urethane and without physical activity; group 2_25 mice with urethane and subjected to aerobic swimming free exercise; group 3_18 mice with urethane, subjected to anaerobic swimming exercise with gradual loading 5-20% of body weight. All the animals were sacrificed after 20 weeks, and lung lesions were analyzed. The median number of lesions (nodules and hyperplasia) was 3.0 for group 1, 2.0 for group 2 and 1.5-3 (p=0.052). When comparing only the presence or absence of lesion, there was a decrease in the number of lesions in group 3 as compared with group 1 (p=0.03) but not in relation to group 2. There were no metastases or other changes in other organs. The anaerobic physical activity, but not aerobic, diminishes the incidence of experimental lung tumors.
Copyright © 2012 Elsevier GmbH. All rights reserved.
@ The world needs more efficient and detailed researches to people live longer more and more urgently. & The difusion of very good ideias are fundamental to human health
- 1. Introduction
- 2. Materials and methods
- 3. Results
- 4. Discussion
- 5. Conclusion
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Volume 96, March–April 2019, Pages 78-86
Research articleA mouse model of heart failure exhibiting pulmonary edema and pleural effusion: Useful for testing new drugs
Author links open overlay panelXiuyingMaShahidTannuJohnAlloccoJiePanJanetDipieroPancrasWongShow morehttps://doi.org/10.1016/j.vascn.2019.02.001Get rights and contentUnder a Creative Commons licenseopen access
Introduction: Mouse models of chronic heart failure (HF) have been widely used in HF research. However, the current HF models most often use the C57BL/6 mouse strain and do not show the clinically relevant characteristics of pulmonary congestion. In this study, we developed a robust mouse model of HF in the BALB/c mouse strain, exhibiting pulmonary edema and pleural effusion, and we validated the model using the standard pharmacological therapies in patients with chronic HF and reduced ejection fraction (HFrEF) or acute decompensated HF.
Methods: After induction of myocardial infarction (MI) by permanent ligation of the left coronary artery in BALB/c mice, the cardiac function, pulmonary congestion, disease biomarkers, and survival were evaluated using the angiotensin converting enzyme inhibitor enalapril or the loop diuretic furosemide. Enalapril was administered 4 weeks post-MI for 6 weeks or furosemide was given 10 weeks post-MI for 4 days, when pulmonary congestion was evident.
Results: Compared to sham controls, MI mice developed systolic dysfunction, exhibited lung weight increase at 4 weeks, and progressively developed pleural effusion (60% of the animals) at 10 weeks. Compared to the vehicle, enalapril significantly reduced the lung weight and pleural effusion, preserved systolic function, and improved survival. Furthermore, furosemide completely abolished the pleural effusion. Enalapril or furosemide also reduced the plasma brain natriuretic peptide concentration.
Discussion: The post-MI HF in BALB/c mice shows reproducible and robust pulmonary congestion and may be a clinically relevant model for novel drug testing for treatment in patients with HFrEF or acute decompensated HF.
Myocardial infarctionHeart failureLung congestionPulmonary edemaPleural effusionBALB/c mouse
Heart failure (HF) is a leading cause of morbidity and mortality in the United States, affecting many people around the world (Heidenreich et al., 2013; Houser et al., 2012; Mozaffarian, 2016; Ponikowski et al., 2014). The incidence and prevalence of chronic HF continue to increase because of the aging of the general population (Heidenreich et al., 2013; Savarese & Lund, 2017) and the effective treatment of acute cardiac diseases, such as myocardial infarction (MI) (Bonneux, Barendregt, Meeter, Bonsel, & van der Maas, 1994; Dalen, Alpert, Goldberg, & Weinstein, 2014). In patients with chronic HF and reduced ejection fraction (HFrEF), angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers (ARB), and β-blockers have been the standard of care (SOC) pharmacological therapies. More recently, two new promising drugs, ivabradine and sacubitril/valsartan, have been approved and recommended in the United States and European guidelines for treatment in patients with HFrEF (McMurray et al., 2014; Swedberg et al., 2010; Yancy et al., 2017). Ivabradine is the selective sinus-node inhibitor that decreases heart rate on top of the maximally tolerated β-blocker therapy in patients with sinus rhythm and reduces HF hospitalization (Swedberg et al., 2010). Sacubitril/valsartan is the angiotensin receptor-neprilysin dual inhibitor, demonstrating a greater reduction in HF hospitalization and mortality in patients with HFrEF compared with the ACE inhibitor enalapril treatment (McMurray et al., 2014). Despite new therapies being developed and approved, the mortality and hospitalization rates are still quite high, and many HF patients remain at high risk for acute decompensation and are in need of additional treatments.
For patients with acute decompensated HF, new therapeutic targets and novel classes of drugs have been evaluated in randomized Phase II-III clinical trials (TRUE-HF, BLAST-AHF, RELAX-AHF2) (Packer et al., 2017; Pang et al., 2017; Teerlink et al., 2017). However, those trials failed to demonstrate beneficial clinical outcomes compared to placebo or SOC. In addition, the CUPID-2 clinical trial, employing the new gene therapy treatment modality, unfortunately, showed neutral results following AAV1/SERCA2a administration in advanced HF (Greenberg et al., 2016), and its companion AGENT-HF trial failed to demonstrate beneficial effects on ventricular remodeling (Hulot et al., 2017). To help better understand what is behind the unsuccessful development of innovative medicines, Tamargo, Rosano, Delpón, Ruilope, and López-Sendón (2017) thoroughly reviewed the key failed clinical trials and provided possible pharmacological reasons that may explain the lack of success. A repeated finding is that the encouraging results of the preclinical and early clinical trials do not translate to the clinical outcomes in the randomized Phase III trials. In addition to the complex pathophysiology of acute HF and an incomplete understanding of the pharmacology of the novel drugs, a key problem is the lack of adequate preclinical models. Therefore, it is critical and urgent to reestablish clinically relevant animal models of HF for preclinical testing, which may be more directly translatable to humans. Houser et al. (2012), on behalf of the American Heart Association, made a scientific statement recommending that the distinctive pathological features of HF in humans should be present in an animal model being used to test novel therapies that could improve cardiac structure and function.
Large animal models of HF in dogs, pigs, sheep and non-human primates are the closest models of human HF, whose cardiac physiology, pathophysiology and disease progression can be studied efficiently and reliably with translational applicability to humans (Dixon & Spinale, 2009). However, there are some disadvantages working with large species in terms of economics, logistics, and throughput (Camacho, Fan, Liu, & He, 2016). Undoubtedly, preclinical animal models of HF in small species have become one of the most important tools to support HF research and drug discovery. However, small animal models of post-MI HF, most often using the C57BL/6 mouse strain, do not have the clinically relevant features of lung congestion (van den Borne et al., 2009; Villalba-Orero et al., 2017). van den Borne et al. (2009) reported that the wound healing process after MI in the mouse model is strain-dependent, suggesting that C57BL/6 is good for infarct healing study and BALB/c is for HF. Therefore, in the present study, we fully characterized a post-MI model in the BALB/c mouse strain, including the measurements of the clinically relevant endpoints of survival, cardiac function, pulmonary congestion, and disease biomarkers, and we validated the model using the SOC therapies, enalapril and furosemide.
2. Materials and methods
The experiments were performed on BALB/c male mice that were 10–11 weeks of age and were obtained from Jackson Laboratories (Bar Harbor, ME, USA). All experimental procedures were approved by the Animal Care and Use Committee of Bristol-Myers Squibb Company and were in accordance with the NIH Guide for the Care and Use of Laboratory Animals.
2.1. Induction of MI
Myocardial infarction was induced by permanent ligation of the left anterior descending coronary artery (LAD) according to a novel method described previously (Gao et al., 2010). This method was highly efficient, without the need of artificial ventilation during surgery. Briefly, the chest region was shaved and treated with Nair prior to surgery. Under anesthesia with 2–2.5% isoflurane inhalation, a small incision over the left chest was made after scrubbing with alcohol. The chest cavity was exposed at the fourth intercostal space with a mosquito clamp. While keeping the clamp open, the heart was smoothly popped out through the hole with a gentle press on the right chest. The LAD was ligated using a 6–0 silk suture at a site approximately 3 mm below its origin. Successful LAD ligation was verified by the observed blanching of the left ventricular (LV) anterior wall. The heart was immediately repositioned in the chest cavity followed by manual evacuation of air and closure of the incision with surgical clips. The sham-operated animals underwent the same surgical procedures without the LAD ligation. Animals, post-surgery, were allowed to recover in a warm box and were returned to their home cages after they were fully awake.
2.2. Experimental design and protocols
2.2.1. Mouse model of post-MI HF
In a preliminary study, we observed a significant increase in lung weight 4 weeks after MI. To further characterize the disease progression, a cohort of 38 mice (30 LAD ligation and 8 sham-operation) that survived through the subsequent 24 h post-surgery were monitored for the Kaplan-Meier survival rate analysis up to 7 weeks. All surviving animals, at the conclusion of the experiment, were comprehensively assessed for the cardiac function, pulmonary congestion, infarct size and interstitial fibrosis. The survival rate was used to predict the number of animals enrolled in the following studies.
2.2.2. Enalapril study
Enalapril was administered 4 weeks after MI, when animals already developed lung congestion. A cohort of 83 mice (68 LAD ligation and 15 sham-operation) underwent echocardiographic assessment 4 weeks after surgery, and the 68 mice with LAD ligation were randomly divided into 2 experimental groups, including the vehicle control (50%DMSO:50%Saline, n = 34) and the enalapril treatment (20 mg/kg/day, n = 34). The drugs were administered for 6 weeks via a subcutaneous infusion using osmotic pumps (Alzet, Model 2006). Animals that survived through the echocardiography and minipump implantation procedures were monitored throughout the study for the Kaplan-Meier survival rate analysis. The sham-operated controls (n = 15) underwent the same procedure without the minipump implantation.
Following completion of the echocardiography at the end of the 6-weeks drug treatment, animals were anesthetized under 2.5% isoflurane and were bled via the carotid artery. The chest cavity was then exposed and was carefully examined for determination of pleural effusion, which was defined by the amount of fluid accumulation >30 μl. The pleural effusion was ranked as moderate (30–200 μl) or severe (>200 μl). The hearts and lungs were harvested, grossly examined, and weighed immediately. The hearts were then fixed in 10% neutral buffered formalin and were prepared for the analysis of the infarct size and interstitial fibrosis. The right tibia was dissected, and its length (TL) was measured for the normalization of the heart weight (HW). The lung weight (LW) was measured again after air drying for approximately 60 h. The endpoints include cardiac function/dimension, pulmonary edema, pleural effusion, atrial size, survival rate, and the disease biomarker plasma brain natriuretic peptide (BNP) concentration as well as organ weights, infarct size, and fibrosis. The pulmonary edema was defined by a significant increase in the amount of fluid retention in the lungs, derived from the LW measured before and after air drying.
2.2.3. Furosemide study
Furosemide was administered 10 weeks after MI, when most of the animals developed severe decompensation. A cohort of 20 mice with LAD ligation was confirmed having a significant dilatation of the LV chamber assessed by echocardiography 10 weeks after surgery, and they were randomly divided into 2 experimental groups: the vehicle control (saline, n = 10) and the furosemide treatment (20 mg/kg, n = 10). The drugs were administered intraperitoneally twice daily for 4 days. Two hours after the last dose, animals were anesthetized under 2.5% isoflurane and bled via the carotid artery. The chest cavity was then exposed and carefully examined for determination of pleural effusion. The hearts and lungs were harvested, grossly examined, and weighed immediately. The LW was measured again after air drying for approximately 60 h. The endpoints include body weight (BW), pulmonary edema, pleural effusion, and plasma BNP concentration as well as the target engagement and renal function markers.
All studies were performed on anesthetized mice. Briefly, mice were anesthetized under isoflurane, 2–3% induction and 1% maintenance, and the echocardiographic assessment was conducted using the VisualSonics Vevo 2100 echocardiographic system equipped with a 30-MHz transducer (FUJIFILM VisualSonics Inc., Toronto, Canada). A 2-dimensional (2-D) parasternal short-axis view of the LV was obtained at the level of the papillary muscle. The cardiac function and dimension, including the ejection fraction (EF), fractional shortening (FS), LV mass, and LV volume (Vol), were all derived from the M-mode image. A 2-D parasternal long-axis view of the left atrium (LA) was obtained, and the M-Mode cursor was positioned to transect the anterior aortic root, aortic valve, the posterior aortic root, LA chamber and the posterior LA wall. From the M-mode image, the LA diameter was measured at the time of the aortic valve fully closing. The body temperature was maintained between 36 and 37.0 °C to avoid the confounding effects of hypothermia. All the echocardiographic measurements were taken in a blinded manner and in compliance with American Society of Echocardiography guidelines (Lang et al., 2015). Three independent measurements were obtained from each animal. The results are reported as the average of three cardiac cycles.
2.2.5. Osmotic minipump implantation
The osmotic minipumps (Alzet, Model 2006, Cupertino, CA) were loaded with dosing solution and were then placed in 0.9% saline for approximately 60 h at 37 °C before implantation. Animals were anesthetized under 2% isoflurane, shaved at the back of the neck and were scrubbed with betadine and alcohol. The pump was implanted subcutaneously along the left side of the back via an incision at the middle dorsal surface of the neck. The incision was closed with surgical staples.
2.2.6. Infarct size and cardiac fibrosis
The hearts were harvested and cut longitudinally in half and were then fixed in neutral buffered 10% formalin for at least 24 h at room temperature. The tissues were processed overnight (LEICA ASP300S, Leica Biosystems Inc. Buffalo Grove, IL), embedded in paraffin blocks, and cut into 5 μm sections. The slides were stained with picro-sirius red, and bright field images were taken with a 20× objective. The infarct size was determined by a midline length measurement as described previously (Takagawa et al., 2007). The LV myocardial midline was drawn at the center between the epicardial and endocardial surfaces, and the length of the midline was measured as the midline circumference. The midline infarct length was taken as the midline of the length of infarct. The percent infarct was calculated by taking the length of the infarct divided by the midline circumference of the entire LV. Interstitial fibrosis was determined in the border zone of infarction by quantifying the total tissue stain and picro-sirius red collagen stain utilizing the Leica Ariol imager (Leica Biosystems, Inc., Buffalo Grove, IL) and reporting the percentage of collagen specific staining in a given field. A minimum of 10 fields per section was read randomly and averaged to give a final reading. All the analyses were performed in a blinded manner.
2.2.7. Plasma and serum biomarkers
All the blood samples were collected into EDTA containing blood collection tubes treated with protease inhibitor cocktail (Cat#11836145001, Sigma, St. Louis, MO) on ice and in serum separator blood collection tubes at room temperature, which were spun at 4 °C and immediately frozen in aliquots at −80 °C until analyzed. The plasma BNP concentration was measured by ELISA using a capture antibody (#sc-67455, Santa Cruz Biotech, Dallas, TX) and detection antibodies (#G-011-23, Phoenix Pharmaceuticals, Inc., Burlingame, CA and #R32AB-1, Meso Scale Discovery, Gaithersburg, MD). The plasma aldosterone concentration was determined using the Aldosterone ELISA kit (ab136933, Abcam, Cambridge, MA). The serum electrolytes and creatinine levels were measured by an automated chemistry analyzer (ADVIA 1800®, Siemens Healthcare Diagnostics, Inc., Tarrytown, NY) using the Siemens Advia® Chemistry Creatinine and electrolytes human reagent kit, which was previously validated for multispecies at the Bristol-Myers Squibb Company, Discovery Toxicology Clinical Pathology Laboratory in Lawrenceville, New Jersey where the testing was performed. All analyses were performed in a blinded manner.
2.3. Statistical analysis
The data are presented as the mean ± SEM. The statistical analysis was performed using the GraphPad Prism 7 statistical package (GraphPad Software, La Jolla, CA). The Kaplan-Meier survival curves were compared by the log-rank test, and the P value was adjusted by multiple comparisons. An adjusted P value that was <0.017 was considered significant. The echocardiographic and morphometric parameters were compared by a t-test or a one-way ANOVA with a Tukey’s multiple comparison test. Pleural effusion was compared by a Fisher’s exact test. P < 0.05 was considered significant. Unless specified, significance <0.05 is indicated by P < 0.05.
3.1. Post-MI model demonstrates distinctive features of congestive HF in BALB/c mice
Animals that survived through the LAD ligation procedure and the subsequent 24 h were continuously monitored up to 7 weeks. In the sham-operation group (n = 8), all animals survived. In the MI group (n = 30), there were a total of 8 deaths, and the mortality rate was approximately 7%, 14%, and 29% at weeks 1, 4 and 7, respectively. Fig. 1A shows the Kaplan-Meier survival curves. The LAD ligation caused an LV infarction in 20 out of 22 of the surviving mice, and the quantitative analysis of the picro-sirius red – stained sections revealed that the infarct size encompassed 39.2 ± 2.8% of the circumference of the LV. Compared to the sham controls, the LV infarction resulted in a significant LV dilation, systolic dysfunction, cardiac hypertrophy, and pulmonary edema at 7 weeks post-MI. Fig. 1B-D graphically display the average data of the EF, LV mass, and the amount of fluid in the lungs from the sham controls and MI mice. Table 1 summarizes all the echocardiographic and morphometric data. The 2 mice without the LV infarction in the MI group were excluded from the data analysis.
Table 1. Characterization of a mouse model of heart failure in BALB/c mice. Animals with either sham-operation or myocardial infarction (MI) were monitored up to 7 weeks post the surgery. Echocardiographic and morphometric data were collected at the end of the 7-week study.
|Echocardiographic data||Morphometric data|
|Sham (n = 8)||MI (n = 20)||Sham (n = 8)||MI (n = 20)|
|HR (bpm)||453 ± 23||485 ± 10||BW (g)||25.9 ± 0.4||26.3 ± 0.4|
|EF (%)||50.1 ± 1.9||25.4 ± 3.1⁎||HW/TL (mg/mm)||6.7 ± 0.1||9.8 ± 0.5⁎|
|FS (%)||25.1 ± 1.2||12.1 ± 1.6⁎||Wet LW (mg)||152.5 ± 4.5||241.2 ± 19.5⁎|
|LV mass (mg)||88.2 ± 5.4||122.9 ± 5.4⁎||Dry LW (mg)||33.6 ± 1.4||51.1 ± 3.9⁎|
|LV Vol, d (μl)||71.4 ± 3.0||139.0 ± 8.7⁎||Fibrosis (%)||4.2 ± 0.5||15.5 ± 1.4⁎|
|LV Vol, s (μl)||35.6 ± 1.6||107.8 ± 10.1⁎||Infarct size (%)||39.2 ± 2.8|
The results are expressed as the mean ± SEM. HR, heart rate; EF, ejection fraction; FS, fractional shortening; LV Vol, d, left ventricular volume at diastole; LV Vol, s, left ventricular volume at systole; BW, body weight; HW/TL, heart weight normalized by tibia length; LW, lung weight.⁎
P < 0.05 vs. sham by an unpaired t-test.
3.2. Validation of enalapril effects in the mouse model of congestive HF
3.2.1. Kaplan-Meier survival rate analysis
Enalapril was administered after the manifestation of the disease state at 4 weeks post-MI, when there was about a 2-fold increase in the LW (338.1 ± 46.2 vs. 177.5 ± 3.9 mg, MI vs. sham, n = 8/group, p < 0.05) demonstrated in a preliminary study. A large cohort of MI mice were randomized to 2 treatment groups, including the vehicle (n = 34) and the enalapril treatment (n = 34). Animals that survived through the echocardiography and minipump implantation procedures were monitored throughout the study. During the 6 weeks treatment, all the sham controls survived, and there was only 1 death in the MI + enalapril group compared with 9 deaths in the MI + vehicle group. Two out of the 34 MI mice in the enalapril group had no LV infarction and were excluded from the data analysis. As shown in Fig. 2A, enalapril significantly improved the survival rate from 73% to 97% (P = 0.009).
3.2.2. Systolic function and pulmonary congestion
Four weeks post-MI, prior to treatment, animals developed significant systolic dysfunction and LV dilation, which were not different between the MI + vehicle and MI + enalapril groups. Compared to the vehicle control, 6 weeks enalapril treatment preserved the EF (Fig. 2B), reduced the HR (Fig. 2C), and showed a trend of reduction in the LV mass but without an effect on the LV Vol. Table 2 summarizes the echocardiographic measurements before and after drug treatment.
Table 2. Echocardiographic and morphometric data in the sham-operation and myocardial infarction (MI) mice before and after the 6 weeks drug treatment with vehicle and enalapril.
|Week 4||Week 10|
|Sham (n = 15)||MI + vehicle (n = 34)||MI + enalapril (n = 32)||Sham (n = 15)||MI + vehicle (n = 25)||MI + enalapril (n = 31)|
|BW (g)||26.9 ± 0.3||26.4 ± 0.4||26.0 ± 0.4||29.1 ± 0.7||29.1 ± 0.5||28.9 ± 0.4|
|HR (bpm)||454 ± 11||450 ± 8||462 ± 10#||418 ± 12||496 ± 12⁎||449 ± 8#|
|EF (%)||40.6 ± 2.2||20.7 ± 2.0⁎||20.0 ± 2.1⁎||46.1 ± 2.0||13.6 ± 1.8⁎||19.6 ± 2.0⁎|
|FS (%)||19.8 ± 1.3||9.7 ± 1.0⁎||9.4 ± 1.0⁎||22.9 ± 1.2||6.3 ± 0.9⁎||9.2 ± 1.0⁎|
|LV mass (mg)||99.2 ± 4.7||109.3 ± 6.7||116.3 ± 7.1||96.9 ± 4.6||126.9 ± 7.6⁎||110.5 ± 5.3|
|LV Vol, d (μl)||83.9 ± 3.0||148.5 ± 5.3⁎||142.7 ± 5.6⁎||75.9 ± 4.3||169.6 ± 8.3⁎||158.5 ± 7.8⁎|
|LV Vol, s (μl)||50.5 ± 3.1||120.3 ± 6.8⁎||116.7 ± 7.1⁎||41.9 ± 3.5||148.8 ± 9.3⁎||131.3 ± 9.0⁎|
|Wet LW (mg)||176.9 ± 2.6||289.4 ± 17.8⁎||216.4 ± 8.9#|
|Dry LW (mg)||43.1 ± 0.7||65.4 ± 3.5⁎||50.71 ± 1.8#|
The results are expressed as the mean ± SEM. BW, body weight; HR, heart rate; EF, ejection fraction; FS, fractional shortening; LV, left ventricular; LV Vol, d, left ventricular volume at diastole; LV Vol, s, left ventricular volume at systole; HW/TL, heart weight normalized by tibia length; LW, lung weight.⁎
P < 0.05 vs. sham.#
P < 0.05 vs. vehicle by a one-way ANOVA followed by Tukey’s test.
Pulmonary congestion was assessed by the LW and pleural effusion. As displayed in Fig. 3A, the amount of fluid in the lungs was significantly increased in the MI + vehicle group (223.9 ± 14.3 mg, P < 0.05 vs. sham) compared with the sham controls (133.8 ± 2.0 mg) and was reduced by the enalapril treatment (165.7 ± 7.1 mg, P < 0.05 vs. vehicle). Most dramatically, pleural effusion was present in 60% of surviving animals (15 out of 25) from the MI + vehicle group and in 42% in the MI + enalapril group (13 out of 31) but none in the sham controls. Since the amount of fluid in the chest cavity was variable, ranging from 30 μl to over 1 ml, pleural effusion was ranked as moderate (30–200 μl) or severe (>200 μl). Out of the 15 animals presented with pleural effusion in the MI + vehicle group, 13 (87%) were severe, but it was only in 5 out of the 13 in MI + enalapril group (34%, P < 0.05 vs. vehicle, Fig. 3B). The body weight was similar in the 3 experimental groups, including the sham controls, MI + vehicle, and MI + enalapril before and after the 6 weeks treatment (Table 2).
3.2.3. LA diameter and plasma BNP concentration
Fig. 4A-B show representative echocardiographic images of the LA and its diameter measurements at the time of the aortic valve fully closed in the M-mode. LA diameter was much larger in the MI + vehicle group (2.7 ± 0.2 mm) compared with the sham controls (1.8 ± 0.1 mm) and was significantly reduced by the enalapril treatment (2.3 ± 0.1 mm, P < 0.05, Fig. 4C). Enalapril also reduced the plasma BNP concentration (Fig. 4D), and the average data were 89.6 ± 7.8, 522.9 ± 42.7, and 273.8 ± 32.5 pg/ml in the sham-operated control, MI + vehicle (P < 0.05 vs. sham), and MI + enalapril (P <0 .05 vs. vehicle) groups, respectively.
3.2.4. Correlation of pulmonary congestion with the plasma BNP concentration, LA diameter, and EF
Fig. 5A-C show the strong associations of the amount of fluid in the lungs with the plasma BNP concentration (r2 = 0.87, P < 0.0001), LA diameter (r2 = 0.74, P < 0.0001), and EF (r2 = 0.63, P < 0.0001) in the MI + vehicle group, indicating that these parameters might be good markers for the prediction of pulmonary congestion and might allow for a longitudinal assessment in the preclinical animal model of congestive HF in future studies.
3.2.5. Heart weight, infarct size and interstitial fibrosis
The heart weight was significantly increased in the MI + vehicle group compared with sham controls and was reduced by the enalapril treatment. The average values of the HW/TL were 7.8 ± 0.1, 12.1 ± 0.5, and 8.8 ± 0.2 mg/mm in the sham control, MI + vehicle (P < 0.05 vs. sham), and MI + enalapril (P < 0.05 vs. vehicle) groups, respectively. The infarct size was not different between the MI + vehicle vs. the MI + enalapril groups (38.8 ± 3.5 vs. 45.0 ± 3.9%, P > 0.05). The picro-sirius red staining demonstrated significant collagen deposition in the border zone of the infarction, and no difference was found between the MI + vehicle vs. the MI + enalapril groups (21.2 ± 1.9 vs. 25.2 ± 2.1%, P > 0.05).
3.3. Effect of furosemide in the mouse model of congestive HF
3.3.1. Pulmonary congestion
Ten weeks post-MI, animals underwent echocardiographic assessment and were randomly assigned to 2 treatment groups, including the vehicle (n = 10) and the furosemide treatment (n = 10). Prior to the treatment, the body weight was not different between the MI + vehicle and the MI + furosemide groups (27.9 ± 0.8 vs. 29.8 ± 0.6 g, P > 0.05). After 4 days of treatment, furosemide reduced the BW to 26.2 ± 0.8 g (P < 0.05), whereas the vehicle showed no effect (28.4 ± 0.8 g, P > 0.05). Fig. 6A displays the change in BW from the baseline. In the MI + vehicle group, 1 out of 10 animals died, and 5 out of the 9 surviving MI mice (56%) presented with pleural effusion (Fig. 6B). In the MI + furosemide group, all 10 mice survived through the protocol, and none presented with pleural effusion (Fig. 6B). Compared to the vehicle, furosemide significantly reduced the amount of fluid in the lungs (243.3 ± 25.7 vs. 189.6 ± 14.8 mg, P < 0.05 by 1-tail t-test) and the plasma BNP concentration (617.2 ± 60.9 vs. 417.2 ± 74.6 pg/ml, P < 0.05).
3.3.2. Target engagement and renal function markers
As anticipated, the furosemide treatment significantly reduced the serum electrolytes, including Na+, K+, and Cl−, which was accompanied by an increase in the plasma aldosterone concentration, indicating a feedback-mediated activation of the renin-angiotensin-aldosterone-system (RAAS). The furosemide treatment also increased the serum creatinine levels, a biomarker of worsening renal function. Table 3 summarizes all the parameters.
|Creatinine (mg/dl)||Na+ (mmol/l)||K+ (mmol/l)||Cl− (mmol/l)||Aldosterone (pg/ml)|
|Vehicle (n = 9)||0.13 ± 0.01||152.5 ± 0.5||5.5 ± 0.2||115.0 ± 0.4||1275.3 ± 52.1|
|Furosemide (n = 10)||0.17 ± 0.00⁎||150.4 ± 0.6⁎||4.9 ± 0.2⁎||103.0 ± 0.5⁎||3596.3 ± 663.5⁎|
The results are expressed as the mean ± SEM.⁎
P < 0.05 vs. vehicle by a t-test.
In this study, we present three major novel findings in a post-MI HF model. The first unique finding is that, unlike the C57BL/6 mouse strain, the post-MI model in BALB/c mice exhibits clinically relevant pathological features of HF as evidenced by the deterioration of cardiac function, the pulmonary edema and pleural effusion, and the progressive mortality. The second major finding is the demonstration of the clinical-like benefits of the ACE inhibitor enalapril (CONSENSUS Trial Study Group, 1987; SOLVD Investigators et al., 1992, SOLVD Investigators et al., 1991) in this mouse model of congestive HF as evidenced by the improvement in survival and cardiac function and the relief of pulmonary congestion. The last finding is that the loop diuretic furosemide behaves the same as shown in the clinic (Bayliss, Norell, Canepa-Anson, Sutton, & Poole-Wilson, 1987; Metra et al., 2008), exhibiting a decongestive effect that is accompanied by the activation of the RAAS and the worsening of renal function. To the best of our knowledge, this is the first study reporting a comprehensive assessment of the disease progression in a mouse model of post-MI HF in the absence and presence of SOC therapies.
4.1. Mouse models of post-MI HF
Many studies have been using the C57BL/6 post-MI model for validation of the beneficial effects of SOC therapies, such as ACEi and ARBs, on cardiac hypertrophy and systolic dysfunction (Liu et al., 2005; Pattern et al., 2003; Voros et al., 2006; Wang et al., 2004; Xu et al., 2004 & 2009). However, some studies report a lung weight increase in animals ranging from 14 to 24 weeks post-MI (Liu et al., 2005; Wang et al., 2004; Xu et al., 2004), but this is not seen or reported in other studies from 4 to 8 weeks (Patten et al., 2003; Voros et al., 2006; Xu et al., 2009). To better understand whether the disease progression after MI may be time-dependent, in a preliminary study in C57BL/6 mice, we extended the observation period up to 26 weeks. Unfortunately, we were not able to demonstrate any evidence of lung congestion at 7 or 26 weeks post-MI (unpublished data). Consistent with our findings, Villalba-Orero et al. (2017) reported systolic dysfunction but with no evidence of lung congestion 10 months post-MI in C57BL/6 mice. Taken together, these results indicate that the widely used post-MI model in the C57BL/6 mouse strain does not present with lung congestion and may not be adequate for testing novel drugs for treatment in patients with HFrEF or acute decompensated HF.
It is intriguing to note here that the disease progression in a mouse model of post-MI HF may be strain-dependent. van den Borne et al. (2009) found that the wound healing process after MI is significantly different among different mouse strains. Among the five tested mouse strains (BALB/c, C57BL/6, FVB, 129S6, and Swiss), BALB/c and Swiss mice have a lower incidence of infarct rupture compared with the other strains, and only these 2 strains of mice develop a lung weight increase 4 weeks after MI. In line with the previous findings, we confirmed that BALB/c mice shows lung congestion 4 weeks post-MI. Most importantly, the MI mice progressively develop pleural effusion accompanied with progressive mortality and deterioration of cardiac function within 10 weeks, which has never been reported previously. Thus, the congestive HF model in the BALB/c mouse strain enables us for a further characterization of the translatability of the model.
4.2. Evaluation of the translatability of the mouse model of congestive HF
ACE inhibitors consistently show favorable outcomes in reducing the hospitalization and mortality of patients with either asymptomatic or severely symptomatic HF and ARBs in populations with mild-to-moderate HF who are unable to tolerate ACE inhibitors (Yancy et al., 2017). In this study, we used the ACE inhibitor enalapril to test the translatability of the mouse model of chronic HF in a preclinical setting. The dose of enalapril was carefully chosen based on a report showing 80% inhibition of the vasopressor effect of exogenous Ang I when administered in drinking water (Wang et al., 2004), which was further confirmed in our pilot study, with an 80% inhibition of the plasma ACE activity when it was administered in an osmotic minipump. Consistent with the well-documented cardiac protective and anti-cardiac hypertrophic effects of ACE inhibitors in post-MI models in C57BL/6 mice (Pattern et al., 2003; Voros et al., 2006; Wang et al., 2004; Xu et al., 2004), we report, in the present study, that enalapril significantly reduces the heart weight and improves the systolic function in the BALB/c HF model. Most importantly, enalapril demonstrates the clinical-like favorable outcomes in slowing disease progression and reducing mortality as well as pulmonary congestion, LA diameter, and plasma BNP levels.
On the other hand, pulmonary congestion is the hallmark of acute decompensated HF and is the most common reason for hospitalization for patients with chronic HF (Adams et al., 2005; Ambrosy et al., 2013), and the immediate action is to relieve symptoms of dyspnea, most often, by using loop diuretics (Felker, 2010; Pham & Grodin, 2017). Therefore, in the present study, we further characterized the clinical relevance of this preclinical congestive HF model by utilizing the commonly used loop diuretic furosemide. Furosemide treatment was initiated 10 weeks after MI when approximately 60% of the animals are in a severely decompensated state, presenting with pleural effusion. Furosemide was administered intraperitoneally for better absorption and twice daily for a prolonged coverage due to its poor pharmacokinetic profiles. The dosing, frequency, and duration was also chosen on the basis of a 3–4-fold increase in Na+ or Ca2+ excretion and polyuria reported in C57BL/6 or C57BL/6/129Sv mice (Lee, Chen, Lai, Yong, & Lien, 2007; Sandulache et al., 2006; van Angelen, van der Kemp, Hoenderop, & Bindels, 2012). As anticipated, furosemide significantly lowers the serum electrolytes Na+, K+, and Cl− and also causes increases in the plasma aldosterone and serum creatinine levels in the BALB/c HF model. With the properly selected dose, furosemide attenuates fluid retention in the lungs and in the chest cavity accompanied with reduced body weight and lower plasma BNP concentration. The results shown here are completely in line with the clinical use of loop diuretics in patients with acute decompensated HF, while producing an immediate correction of volume overload and a relief of symptoms, loop diuretics also activate the neurohormonal systems that contribute to the worsening of renal function (Bayliss et al., 1987; Goldsmith, 2016; Metra et al., 2008).
4.3. New insights in the mouse model of congestive HF
In human HF, the LA volume is increased and the enlarged LA reflects an increased wall tension as a result of an elevated LV filling pressure, which is an important predictor of clinical outcomes (Suh et al., 2008; Triposkiadis et al., 2016). Therefore, in the present study, we assessed the LA diameter in this mouse model of HF by simply using the traditional echocardiographic method. It is interesting that we demonstrate a strong association of the pulmonary edema with the LA diameter as well as the plasma BNP levels, and animals with an LA diameter >3 mm consistently present with pleural effusion. The efficacy of enalapril on these parameters may be partly due to the reduction of the LV filling pressure, resulting from the lowered systemic blood pressure.
Furthermore, an elevated LV filling pressure in patients with HF, known as hemodynamic congestion, often presents in the days and weeks leading up to overt clinical congestion and is manifested as signs and symptoms of edema and dyspnea (Gheorghiade, 2006; Gheorghiade, Filippatos, De Luca, & Burnett, 2006). In some cases, hemodynamic congestion may persist following the resolution of signs and symptoms of edema and dyspnea (Klein, 2016). However, an early and accurate detection of pulmonary congestion has been very challenging, and no specific, non-invasive or quantitative gold standard protocol is applied to patients (Platz et al., 2012, Platz et al., 2016). The recent progress in the lung ultrasound technology seems promising to enable the speed and accuracy of diagnosis and to facilitate an early and thorough treatment in patients with HF (Platz et al., 2016). To keep up to date with the current technological advancements in the clinic, Villalba-Orero et al. (2017) described the feasibility of the lung ultrasound approach in detecting lung congestion in the genetically-engineered mice with HF. We anticipate that the noninvasive echocardiographic measurement of the LA diameter or the lung ultrasound method may be useful in evaluating longitudinally pulmonary congestion in the preclinical animal models of HF, and it may be helpful in defining the entry criteria for the optimization of animal selection and interpreting subsequent study outcomes.
The post-MI HF in the BALB/c mouse strain is a reproducible, inexpensive, and robust congestive HF model. The results reported here provide strong evidence of the translational and clinical relevance of the model. Therefore, it may be a better preclinical animal model to bridge the translational gap to the clinic and to improve the clinical success rate of testing novel drugs for treating patients with chronic HFrEF as well as those with acute decompensated HF.
The authors are employees of the Bristol-Myers Squibb Company, United States.
The authors thank Dr. Erhe Gao, Temple University, for providing the mouse LAD surgery training, Marisa Tonner and Cory Vernon for surgical assistance and animal dosing, and Earl Crain for echocardiographic assistance.
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BIOCHEMISTRY AFFILIATE WORKS TO DEVELOP NEW BIOENGINEERING STRATEGY
Posted on October 25, 2019
Andrew Buller, professor of chemistry at UW-Madison and affiliate of the Department of Biochemistry, is working to develop methods to create new protein building blocks in living systems. This chemical research, recently recognized with a $2.2 million grant from NIH, could make these processes more affordable for and accessible to scientists with expertise in other backgrounds.
Buller will receive a 2019 National Institutes of Health (NIH) Director’s New Innovator Award from the High-Risk, High-Reward program for his project, titled Engineering In Vivo Biomolecular Synthesis with Nonstandard Building Blocks.
“We want to bring new kinds of building blocks into biological systems, with the goal of interfacing with living systems,” Buller said, explaining that his group will repurpose how enzymes use vitamin B6, the cofactor that is preeminent in amino acid biosynthesis, to perform new types of reactions.
The biological building blocks of life include amino acids, vitamins and minerals, which are all accessible to living organisms through the foods they consume. Food goes through chemical processes to convert it to a set of proteins needed for life processes. There are 20 key amino acids that make up proteins, but Buller says scientists can create more.
“You could think of this as having a Lego kit with just 20 different pieces,” Buller said. “What could you build if you had more? We build these new Lego bricks and then teach biological systems how to make them too.”
This unique way of thinking and the possible long-term benefits of creating these systems won Buller the NIH award, which funds “highly innovative and unusually impactful biomedical or behavioral research, proposed by extraordinarily creative scientists.”
“We evolve enzymes to take relatively simple building blocks, that we can buy from a common chemical manufacturer, and evolve enzymes that can convert small building blocks into the side chain of an amino acid,” Buller said. “This lets us bring in new functional groups that weren’t present – new kinds of elements and new kinds of reactivity.”
Over the past two decades, scientists have been adding amino acids, produced by organic chemists in the lab, to proteins. Buller’s project will set up a system any molecular biologist can use, with enzymes and an off-the-shelf chemical, to create the amino acid they need in a cell.
“People have wanted to do this for a long time, but there are a lot of barriers,” he said. “We are addressing one of the biggest, which is that it’s really expensive and it takes a lot of training in two different areas. If we can make it cheaper and so that you need one skillset instead of two, then everybody else is going to be able to use this technology.”
Substantial awards, like this one, allow researchers time to work, discover, and create, because they remove some of the pressure to spend time searching for funding.
“Now we get to focus on the science,” Buller said, adding that this research could spawn methods of cheaper drug development. “and I think this is an important challenge for scientists to work on. We want to lower the barriers, both in terms of cost and difficulty, to make it easier to make new kinds of molecules. My hope is that they would have that kind of impact – that biochemists would have that expanded repertoire of new building blocks in their probing a structure-function relationship of molecules, or in the process of optimizing an antibody to bind to an antigen.”
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