This article was downloaded by: [The University of British Columbia] On: 11 December 2014, At: 05:02 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Veterinary Quarterly Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tveq20
Short-term effect of granulocyte colony-stimulating factor in dogs with severe myxomatous mitral valve disease a
Min-Hee Kang & Hee-Myung Park
a
a
Department of Veterinary Internal Medicine, College of Veterinary Medicine, Konkuk University, Seoul, South Korea Published online: 24 Sep 2014.
To cite this article: Min-Hee Kang & Hee-Myung Park (2014) Short-term effect of granulocyte colony-stimulating factor in dogs with severe myxomatous mitral valve disease, Veterinary Quarterly, 34:2, 60-66, DOI: 10.1080/01652176.2014.954063 To link to this article: http://dx.doi.org/10.1080/01652176.2014.954063
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Veterinary Quarterly, 2014 Vol. 34, No. 2, 60 66, http://dx.doi.org/10.1080/01652176.2014.954063
ORIGINAL ARTICLE Short-term effect of granulocyte colony-stimulating factor in dogs with severe myxomatous mitral valve disease Min-Hee Kang and Hee-Myung Park* Department of Veterinary Internal Medicine, College of Veterinary Medicine, Konkuk University, Seoul, South Korea
Downloaded by [The University of British Columbia] at 05:02 11 December 2014
(Received 21 April 2014; accepted 8 August 2014) Background: Use of granulocyte colony-stimulating factor (G-CSF) to treat damaged myocardium is a relatively new concept. Clinical beneficial and safety outcomes are still controversial. Objective: The aim of this study was to evaluate recruitment of hematopoietic stem cells and therapeutic efficacy of G-CSF in the treatment of myxomatous mitral valve disease (MMVD) of dogs. Animals and methods: Thirty client-owned MMVD dogs with clinical signs of heart failure were enrolled in a prospective double-blind, randomized, placebo-controlled study to compare the short-term effect of G-CSF (n D 17) with control group (n D 13) for identical periods. Clinical, hematological, and cardiovascular assessments were performed on days 0, 1, 3, and 7. Follow-up examination was conducted four weeks after the study. Results: Dogs treated with G-CSF had a significantly elevated white blood cell (WBC) (£103/mL) count at day 3 compared with baseline (from 10.23 § 4.42 to 42.84 § 11.84; P D .000). The WBC population was also changed (elevated neutrophils and decreased lymphocytes) and the numbers of CD34C cells in the peripheral blood were also increased at day 3. However, the results of clinical, laboratory, and echocardiographic assessments did not differ significantly between the G-CSF treatment and control groups after four weeks. Conclusions: G-CSF administration elevated the peripheral WBC count, especially neutrophils, and recruited hematopoietic stem cells. However, positive effects of G-CSF on cardiac function were not detected during short-term monitoring. Keywords: granulocyte colony-stimulating factor (G-CSF); myxomatous mitral valve disease; canine; hematopoietic stem cells
1. Introduction Despite advances in diagnosis and treatment of congestive heart failure over recent decades, morbidity and mortality remain high both in dogs (Borgarelli et al. 2008) and humans (Haeck et al. 2012). The most frequent cause of heart failure is acute myocardial infarction (AMI) in humans (Jessup & Brozena 2003) and mitral valve degeneration in dogs (Aupperle & Disatian 2012). Myxomatous mitral valve disease (MMVD) is an acquired primary valvular disease which can result in progressive cardiac enlargement and congestive heart failure in dogs and cats due to valvular degeneration (Abbott 2008; Borgarelli & Haggstrom 2010). Even though the basic etiology of heart failure differs between dogs and humans, AMI also induces left ventricular (LV) remodeling, which could progress to other cardiovascular disorders including valvular heart disease (Takano et al. 2003). Like ischemic disease in humans, the myocardial changes, such as intramural arteriosclerosis and myocardial fibrosis in chronic MMVD dogs, are significant (Falk et al. 2006) and cardiac fibrosis appears to be important in pathology and dysfunction in valvular disease (Khan & Sheppard 2006). There is no conventional treatment for terminal heart failure in both species. Therefore, the discovery of novel treatment strategies is required to further improve clinical outcomes. Recently, gene- and cell-based therapies have been established. Use of hematopoietic cytokines, such as granulocyte
*Corresponding author. Email: [email protected] Ó 2014 Taylor & Francis
colony-stimulating factor (G-CSF), to treat damaged myocardium has been investigated in the last decade (Takano et al. 2006). Paracrine effects (growth factors, cytokines, and other signaling molecules) of transplanted cells appear to be the main mechanism of beneficial effects (Murry et al. 2005) and several studies demonstrated that stem cell therapy improved myocardial function through myocardial regeneration or neovascularization (Kocher et al. 2001; Orlic et al. 2001). More recent studies have demonstrated the beneficial effects of G-CSF such as improvement of the cardiac function and the regression of myocardial fibrosis on non-ischemic cardiomyopathy (H€uttmann et al. 2006; Szardien et al. 2012). To the authors’ knowledge, G-CSF efficacy in naturally occurring MMVD is not established. We hypothesized that, as it is in humans, cytokine therapy using G-CSF would be beneficial in treating MMVD dogs by improving cardiac function through preventing apoptosis and regression of cardiac fibrosis. Therefore, the purposes of this study were to evaluate hematopoietic stem cell recruitment and therapeutic efficacy of G-CSF in dogs with MMVD. 2. Material and methods 2.1.
Animals
This was a four-week, prospective double-blinded, randomized, placebo-controlled study. Dogs with naturally
Downloaded by [The University of British Columbia] at 05:02 11 December 2014
Veterinary Quarterly occurring MMVD were eligible for enrollment after client consent for participation was obtained. The study protocol was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the College of Veterinary Medicine, Konkuk University, South Korea. To be eligible for inclusion, the dog had previously been diagnosed with MMVD based on the following criteria: characteristic left-sided heart murmur of moderate to high intensity, echocardiographic evidence of MMVD (characteristic valvular lesions of the mitral valve apparatus such as leaflet thickening and/or valve prolapse, evidence of moderate to severe left atrial (LA) and/or left ventricular enlargement), and demonstrated mitral regurgitation on color Doppler echocardiography (Goncalves et al. 2002; Chetboul & Tissier 2012). The MMVD dogs were classified according to the International Small Animal Cardiac Health Council (ISACHC) scheme (Fox et al. 1999). The clinical status and medications received by each dog were stable for 1 month before enrollment in the study. Dogs were excluded from the study if they had a congenital cardiac disease, or had another clinically significant systemic disease, or had evidence of concurrent organ dysfunction such as respiratory tract disease. Asymptomatic dogs (ISACHC class I) were also excluded from the study. Dogs were randomly allocated into two groups (treatment and control groups) by a third party (a person that was independent of the research team). Owners were blinded to treatment allocation until the end of the study period (four weeks), and the clinician and technician responsible for recording the outcome measures were also blinded to treatment allocation until completion of data analysis. 2.2.
Concurrent medications
Patients were stabilized on current medications used to treat heart disease for at least one month prior to enrolment on the study and were continued at the same dose throughout the study. All the dogs were receiving diuretics (furosemide, 100% (range, 1 3 mg/kg q 12 hr); spironolactone, 13% (range, 1 2 mg/kg q 24 hr); hydrochlorothiazide, 3% (2 mg/kg q 12 hr)) and 83% of the dogs were receiving angiotensin-converting enzyme inhibitors (enalapril (range, 0.5 mg/kg q 12 hr) or ramipril (range 0.125 0.25 mg/kg q 24 hr); treatment-group 82%, control-group 85%). Positive inotropic agents were being administered to 63% of the dogs (pimobendan (range, 0.25 mg/kg q 12 hr), treatmentgroup 35%, control-group 46%; digoxin (range, 0.0050.01 mg/kg q 12 hr), treatment-group 29%, control-group 15%). Forty percent of the dogs were receiving hydralazine (range, 0.5 1 mg/kg q 12 hr) (treatment-group 41%, control-group 38%). One of the dogs in the treatment group was receiving sildenafil (1 mg/kg q 12 hr) and one of the dogs from the control group was receiving carvedilol (0.3 mg/kg q 12 hr). 2.3.
Granulocyte colony-stimulating factor treatment protocol
Treatment-group animals received 30 mg of G-CSF/kg (LeukokineÒ , CJ, Seoul, South Korea) by subcutaneous
61
injection once daily for three consecutive days. The treatment periods were determined based on our previous controlled experimental results, which showed that mobilization of peripheral blood progenitor cells were significantly increased on day 3 and gradually decreased, when given five successive days (data not shown). Control-group dogs were treated with the same volume of normal saline (placebo) for an identical period. In addition, all dogs continued preexisting treatment, consisting of diuretics, angiotensin converting enzyme inhibitors, and/ or inotropic agents, and the medication was not changed during the study period. 2.4.
Analysis of peripheral blood
Blood samples were obtained prior to the G-CSF/placebo treatment (baseline) and on days 1, 3, 7, and 28 after commencing the G-CSF/placebo treatment. Complete blood count was determined by automatic analyzer (VetScanR HM2, ABAXIS, Union City, CA, USA) and a manual differential count was performed. 2.5.
Flow cytometric enumeration of CD34C hematopoietic stem cells
The number of CD34C hematopoietic stem cells in the peripheral blood was determined using a flow cytometer prior and on day 3 after commencing the G-CSF/placebo treatment. Hematopoietic stem cell counting was performed according to the dual-platform method of the International Society of Hematotherapy and Graft Engineering (ISHAGE) protocol (Ormerod 2000). Double staining with mouse anti-canine CD34:RPE (AbD Serotec) and CD 45: FITC (AbD Serotec) was used for fluorescence-activated cell sorting analysis (FACScalibur flow cytometer, Beckton Dickinson, CA, USA) with the CellQuest program (Becton Dickinson Immunocytometry Systems, San Jose, CA, USA) as reported previously (Jung et al. 2008). 2.6.
Clinical examination and hemodynamic assessment
All dogs underwent a thorough physical examination and blood biochemical analysis. Hemodynamic changes were monitored using indirect arterial blood pressure (Cardell Model 9401, Sharn Veterinary Inc., FL, USA), 6-channel electrocardiogram (ECG; Cardiofax GEM ECG-9020K, Nihon Kohden, Tokyo, Japan), two-view thoracic radiographs, and complete echocardiographic examination (Logiq400, GE healthcare Medical Systems, Milwaukee, WI, USA), which included transthoracic two-dimensional (2D), M-mode, spectral, and color-flow Doppler. The dogs were not sedated during the examinations. All cardiac valves were examined for evidence of insufficiency using color-flow Doppler. Systolic and diastolic dimensions of the left ventricular internal diameter (LVIDs and LVIDd, respectively) were measured on the right parasternal M-mode view, and LV fractional shortening (FS) was calculated. The end-diastolic volume (EDV) and the endsystolic volume (ESV) were calculated using the Teicholz
62
M.-H. Kang and H.-M. Park
method (Borgarelli et al. 2008), and LV ejection fraction (EF) was calculated according to the following formula: EF D [(EDV ¡ ESV)/EDV £ 100]. Stroke volume (SV) was calculated by subtracting ESV from EDV. Measurements of the aortic (AO) and LA diameters were obtained on the 2D right parasternal short-axis view, and the LA/ AO ratio was calculated. All echocardiographic measurements followed previously described methods (Goncalves et al. 2002; Chetboul & Tissier 2012). All assessments were obtained prior to the G-CSF/placebo treatment (baseline) and at days 1, 3, and 7, and 28 after commencing the G-CSF/placebo treatment.
Downloaded by [The University of British Columbia] at 05:02 11 December 2014
2.7.
Statistical analysis
The distribution of all baseline parameters was assessed using the Shapiro Wilk’s test and data that were not normal were analyzed using the Scheirer Ray Hare extension of the Kruskal Wallis test. Based on data distribution, baseline variables were compared between two groups using the paired t-test and Mann Whitney Utest. The paired t-test was used to investigate changes in the evaluated variables in the control and treated groups. The changes from baseline to follow-up between the control and treatment groups were compared with repeated measures analysis of variance (ANOVA). A separate analysis was performed to evaluate changes in hematologic values from baseline to follow-up between the control and treatment groups (repeated ANOVA) and the results over time as a within variable (oneway ANOVA), followed by post hoc Bonferroni tests. Optimal multilevel model (linear, quadratic, or cubic model) was applied for the comparison of repeated measures time effect. Post hoc power analyses (requesting 80% power) were performed to detect differences in diagnostic values. Statistical significance was defined as observed power (1 ¡ b) > 0.80 and P(a) < 0.05. SPSS version 19.0 (SPSS, Inc., Chicago, IL, USA) and GPower version 3 (Germany) were used to perform statistical analyses. 3. Results Thirty client-owned dogs with MMVD (ISACHC Class II (47%), Class IIIa (47%), and Class IIIb (7%)) were included in this study. The dogs were randomly divided into two groups (17 dogs in the treatment group and 13 dogs in the control group). The mean age of all dogs included in the study was 10.6 years (range, 7 16) and mean weight was 4 kg (range, 2 11.5 kg). The study population consisted of 16 Maltese terriers, 5 Shih-Tzus, 3 Yorkshire terriers, 2 Pomeranians, 2 mixed breeds, 1 Miniature Poodle, and 1 Miniature Pinscher. There were no significant differences between groups with regard to gender, age, or clinical heart failure classification (Table 1). Dogs treated with G-CSF had a significantly elevated white blood cell (WBC) (£103/mL) count three days after the treatment (baseline, 10.23 § 4.42; three days after treatment, 42.84 § 11.84; F D 66.822, P D .000).
Table 1. Baseline characteristics of 30 dogs with MMVD treated either with G-CSF or placebo. Parameters
Control group Treatment group
Number 13 17 Male/female 7/6 8/9 Age (years, mean § SD) 10.21 § 2.64 10.91 § 3.18 ISACHC class II/III 5/8 9/8
P .713 .324 .431
Breeds
n
n
Total n
Maltese terrier Shih-Tzu Yorkshire terrier Mixed breed Pomeranian Miniature poodle Miniature pinscher
5 4 3 1 0 0 0
11 1 0 1 2 1 1
16 5 3 2 2 1 1
Note: ISACHC, International Small Animal Cardiac Health Council.
Multilevel modeling with cubic model revealed significant difference of WBC count over time (F D 24.449, P D .000, partial h2 D 0.746, observed power D 1.000). The WBC population (%) was also changed at day 3, and elevated neutrophils (baseline, 67.67 § 10.82; three days after treatment, 85.08 § 4.85; F D 8.854, P D .000) and decreased lymphocytes (baseline, 20.50 § 7.98; three days after treatment, 9.58 § 2.84; F D 6.186, P D .001) were noted. Both neutrophils (F D 16.669, P D .001, partial h2 D 0.510, observed power D 0.969) and lymphocytes (F D 9.763, P D .007, partial h2 D 0.379, observed power D 0.835) population was significantly changed over time. The WBC count stabilized to the baseline at day 7. The changes of red blood cells, packed cell volume, and platelets by time course and the time by group interaction were not significantly different (Table 2). Increased numbers of CD34C cells in the peripheral circulation three days after the G-CSF treatment (baseline, 11.24 § 4.61 cells/mL; three days after treatment, 38.83 § 18.98 cells/mL, P D .002) compared to the control group (baseline, 11.88 § 7.99 cells/mL; three days after treatment, 12.28 § 6.97 cells/mL, P D 0.87) were noted. Results of physical and hemodynamic parameters before and four weeks after treatment in both groups are presented in Table 3. In both groups, body weight, heart rate, body temperature, and blood pressure did not change significantly during the study period. Changes in ECG and echocardiographic parameters were not significantly different before and after the G-CSF treatment. In repeated ANOVA test, FS results were elevated significantly at four weeks (F D 4.507, P D .043, partial h2 D 0.139, observed power D 0.536), but not between groups (F D 1.444, P D .240, partial h2 D 0.049, observed power D 0.213). Also, the time by group interaction did not differ significantly. We also observed significant effects of time, groups, and their interactions (Table 4). While not an objective of this study, no adverse effect regarding safety was noted during the study period.
Veterinary Quarterly
63
Table 2. Changes in hematologic parameters in G-CSF or placebo-treated dogs with MMVD.
Downloaded by [The University of British Columbia] at 05:02 11 December 2014
Parameters
Group
Baseline
3 days
7 days
28 days
F
P
WBC (£103/ul)
Control group Treatment group
10.68 § 2.39 10.23 § 4.42a
10.07 § 1.46 42.84 § 11.84b
10.51 § 2.99 11.06 § 3.76a
10.24 § 2.67 10.07 § 3.83a
.074 66.822
.973 .000
RBC (£106/ul)
Control group Treatment group
6.61 § 0.53 6.52 § 1.11
6.30 § 0.65 6.40 § 1.31
6.50 § 0.45 6.28 § 1.30
6.50 § 0.41 6.62 § 1.26
.351 .160
.789 .922
Hb (g/dl)
Control group Treatment group
14.25 § 0.78 15.72 § 2.90
14.47 § 0.87 15.28 § 2.69
14.22 § 0.48 15.08 § 3.13
14.68 § 0.84 16.02 § 3.16
.486 .243
.696 .866
PCV (%)
Control group Treatment group
41.81 § 2.69 44.69 § 7.36
42.37 § 2.90 42.94 § 7.96
42.02 § 1.87 43.14 § 8.35
42.72 § 2.51 45.70 § 8.27
.151 .324
.928 .808
PLT (£103/ul)
Control group Treatment group
442.33 § 101.76 424.21 § 174.88
444.50 § 118.51 453.50 § 174.12
456.67 § 130.64 450.25 § 134.02
476.00 § 100.39 499.33 § 181.69
.111 .418
.953 .741
Control roup Treatment group
7.00 § 2.68 8.00 § 3.05
6.17 § 1.83 4.50 § 3.87
5.33 § 2.16 5.50 § 4.10
7.00 § 2.19 7.58 § 2.39
.763 2.864
.528 .047
Lymphocyte
Control group Treatment group
20.00 § 4.60 20.50 § 7.98c
20.67 § 3.83 9.58 § 2.84a
19.33 § 3.56 17.50 § 6.04b
18.67 § 4.27 17.08 § 7.69b
.266 6.186
.849 .001
SegNe
Control group Treatment group
69.17 § 6.27 67.67 § 10.82a
71.17 § 3.82 85.08 § 4.85c
73.50 § 2.43 73.92 § 7.35b
73.33 § 4.37 71.00 § 10.72b
1.275 8.854
.310 .000
Differential count Monocyte
Note: WBC, white blood cells; RBC, red blood cells; Hb, hemoglobin; PCV, packed cell volume; PLT, platelet; and SegNE, segmented neutrophils. a c At each time point, values with different superscript letters differ significantly (P < .05).
Table 3. Changes in physical and cardiac parameters in G-CSF or placebo-treated dogs with MMVD. Control group Parameters
Treatment group
Baseline
28 days
P
Baseline
28 days
P
4.74 § 2.69 153.38 § 21.34 38.72 § 0.55 138.71 § 15.72
4.65 § 2.49 156.15 § 23.06 38.65 § 0.40 138.74 § 14.80
.1408 .7353 .6535 .9945
3.44 § 1.08 161.41 § 24.47 38.75 § 0.36 138.74 § 15.95
3.35 § 0.99 154.94 § 20.48 38.78 § 0.40 137.10 § 11.49
.4238 .4094 .6783 .7232
Echocardiography FS (%) LVIDd (mm) LVIDs (mm) EF (%) SV (ml) EDV (ml) ESV (ml) LA/AO
53.34 § 9.20 27.33 § 4.80 12.52 § 2.88 86.54 § 6.42 22.58 § 13.72 25.97 § 15.43 3.38 § 2.28 1.94 § 0.77
54.66 § 8.00 29.31 § 6.98 13.36 § 4.24 87.30 § 6.39 28.65 § 18.67 33.37 § 22.74 4.71 § 5.06 2.23 § 1.30
.6782 .3731 .6155 .7476 .3190 .3059 .7546 .4635
48.86 § 6.96 28.25 § 4.35 14.66 § 3.54 83.23 § 7.22 22.23 § 9.12 27.15 § 12.11 5.00 § 3.90 1.87 § 0.40
52.88 § 7.46 26.84 § 7.96 13.38 § 3.60 87.36 § 6.24 23.32 § 9.94 26.92 § 11.91 3.61 § 2.86 1.80 § 0.43
.1141 .5262 .3037 .0838 .7412 .9558 .2447 .6265
Radiography VHS
11.17 § 0.96
11.25 § 1.22
.6093
11.18 § 0.70
11.14 § 0.68
.5533
Weight (kg) Heart rate (bpm) Body temp ( C) BP (mmHg)
Electrocardiography P width P height PR interval QRS width R height QT interval
0.05 § 0.005 0.32 § 0.056 0.07 § 0.009 0.05 § 0.007 2.08 § 0.505 0.20 § 0.024
0.05 § 0.006 0.34 § 0.061 0.07 § 0.009 0.05 § 0.007 2.12 § 0.483 0.19 § 0.024
.3370 .1111 1.000 .3370 .2676 .1745
0.05 § 0.006 0.31 § 0.064 0.07 § 0.009 0.05 § 0.008 1.93 § 0.474 0.19 § 0.023
0.05 § 0.005 0.32 § 0.075 0.07 § 0.008 0.05 § 0.008 1.94 § 0.491 0.19 § 0.022
Note: BP blood pressure; FS fractional shortening; LVIDd/LVIDs left ventricular internal dimension at end-diastole/end-systole; EF fraction; SV stroke volume; EDV end-diastolic volume; ESV end-systolic volume; LA/AO left atrial to aortic root ratio; and VHS heart score.
.3322 .2156 1.000 .3322 .6684 .5434 ejection vertebral
64
M.-H. Kang and H.-M. Park
Table 4. Summary of time, groups and their interaction on variables evaluated in the study. Time effect (P)
Group effect (P)
Interaction effect (P)
Test
WBC (£103/ul) RBC (£106/ul)
.000 .110
.001 .856
.000 .284
AV AV
Hb (g/dl) PCV (%) PLT (£103/ul)
.029 .108 .377
.367 .567 .974
.117 .129 .688
AV AV AV
.027 .007 .001
.983 .069 .338
.293 .002 .000
AV AV AV
.043 .646 .650 .146 .414 .593 .602 .859
.240 .806 .390 .412 .982 .917 .676 .932
.292 .436 .033 .310 .682 .549 .328 .474
AV SRH AV SRH SRH SRH SRH SRH
Downloaded by [The University of British Columbia] at 05:02 11 December 2014
Parameters
Differential count Monocyte Lymphocyte SegNe Echocardiography FS (%) LVIDd (mm) LVIDs (mm) EF (%) SV (ml) EDV (ml) ESV (ml) LA/AO
Note: WBC white blood cells; RBC red blood cells; Hb hemoglobin; PCV packed cell volume; PLT platelet; SegNE segmented neutrophils; FS fractional shortening; LVIDd/LVIDs left ventricular internal dimension at end-diastole/end-systole; EF ejection fraction; SV stroke volume; EDV end-diastolic volume; ESV end-systolic volume; LA/AO left atrial to aortic root ratio; AV analysis of variance (ANOVA); and SRH Scheirer Ray Hare test.
4. Discussion G-CSF is an effective stimulus for the mobilization of bone marrow-derived stem cells into the peripheral circulation and acts on hematopoietic stem (CD34C) cells to regulate neutrophil progenitor proliferation and differentiation (Serres et al. 2008). The cardioprotective effects of G-CSF might be attributable to its direct action on the myocardium. The peak concentration of hematopoietic stem cells in peripheral blood was reported to occur 4 7 days after commencing G-CSF treatment (Ripa et al. 2006). In this study, all dogs treated with G-CSF had a significantly elevated WBC count (especially neutrophils) three days after commencing treatment, which returned to baseline by day 7. We also confirmed increased numbers of CD34C cells in the peripheral circulation. This cell population change is similar to that in previous reports (Mishu et al. 1992). During the short-term follow-up (four weeks), LV size and systolic function were monitored using conventional echocardiography. No significant difference in overall cardiac status was detected between dogs receiving GCSF and normal saline (placebo). Despite the recent advances in ultrasound technology, accurate evaluation of the complex hemodynamic changes associated with disease progress is challenging. However, conventional echocardiographic measurements, such as FS, EF, and LVIDs, are still considered the non-invasive diagnostic test of choice for assessing myocardial function (Serres
et al. 2008; Chetboul & Tissier 2012). In both placebo and G-CSF treatment groups, FS and EF were slightly increased but no significant differences were observed in the short-term follow-up period. However, these variables are besides being dependent on intrinsic contractility, also known to be influenced by hemodynamic load and sympathetic tone. This could potentially mask significant myocardial dysfunction in dogs with MMVD. Assessment of LVIDs has been suggested to better reflect systolic dysfunction in the presence of mitral regurgitation (Chetboul & Tissier 2012). LVIDs were slightly decreased in G-CSF treatment group, but there was still no significant difference. The cardioprotective effects of G-CSF are mainly focused on the cardiomyocytes. After AMI, cardiomyocyte death is accelerated, especially in the ischemic region, by necrosis or apoptosis. G-CSF activates a variety of intracellular signaling cascades such as Janus kinase (Jak)-signal transducer and activator of transcription (STAT), Ras-Raf-mitogen-activated protein (MAP) kinase, and Scr family kinase pathways (Harada et al. 2005; Takano et al. 2006). These mechanisms might protect against cardiac fibrosis and cardiac remodeling. Compared to the pathophysiology of AMI in humans, myocardial lesions of MMVD dogs contain focal areas of myocardial fibrosis and necrosis, especially in the subendocardium and papillary muscles of the left ventricle (Falk et al. 2006). Cardiac fibrosis appears to be important in pathology and dysfunction in several heart diseases and it has now been suggested to play a major role in valvular disease (Khan & Sheppard 2006). A recent study in nonischemic, diabetic cardiomyopathy patients investigating the therapeutic potential of G-CSF showed inhibition of transforming growth factor-b1 (TGF-b1) and progression of fibrosis (Lim et al. 2011). In general, TGF-b1 is a profibrotic cytokine and appears to be one of several factors that cause disease by inducing cardiac fibrosis. Another experimental study in mice with cardiac hypertrophy and fibrosis induced by AO stenosis showed that G-CSF treatment improved cardiac function and led to the regression of myocardial fibrosis at two weeks (Szardien et al. 2012). They also demonstrated that administration of G-CSF provides beneficial degradation of the extracellular matrix (ECM), by increasing activity of both matrix metalloproteinase 2 (MMP-2) and MMP-9. Changes in ECM composition and structure have been reported in dogs with mitral regurgitation, and myocardial MMP activity was accompanied by alterations in cardiac ECM structure and progressive cardiac dysfunction (Spinale et al. 1998; Aupperle et al. 2009). The association between MMP-9 activity and systolic dysfunction was investigated in canine MMVD (Ljungvall et al. 2011). They reported that plasma MMP-9 activity decreased with the myocardial remodeling process in dogs with MMVD. The most important limitation of this study is the lack of functional tests of the injected G-CSF. The effect of GCSF was evaluated according to the degree of clinical improvement and echocardiographic change during a relatively short period. Therefore, we cannot completely exclude the beneficial effect of G-CSF in MMVD
Veterinary Quarterly treatment and further studies on the cellular and subcellular mechanisms, and longer term clinical implications of G-CSF would be desirable. In addition, this study was not designed to allow the determination of optimal dose and scheduling of G-CSF administration.
Downloaded by [The University of British Columbia] at 05:02 11 December 2014
5. Conclusion G-CSF was an effective stimulus for mobilization of bone marrow-derived hematopoietic stem cells into the peripheral circulation, but short-term cardioprotective effects were not demonstrated in this study. Evaluation of longterm clinical efficacy and safety in MMVD dogs treated with G-CSF is required in further study. Acknowledgement This research was supported by the Basic Science Research Program through the NRF of Korea funded by the Ministry of Education, Science and Technology (2012R1A6A3A01038961).
References Abbott JA. 2008. Acquired valvular disease. In: Tilley LP, Smith FWK Jr, Oyama MA, Sleeper MM, editors. Manual of canine and feline cardiology. 4th ed. London: Elsevier Ltd.; p. 110 138. Aupperle H, Disatian S. 2012. Pathology, protein expression and signaling in myxomatous mitral valve degeneration: comparison of dogs and humans. J Vet Cardiol. 14:59 71. Aupperle H, Thielebein J, Kiefer B, M€arz I, Dinges G, Schoon HA, Schubert A. 2009. Expression of genes encoding matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in normal and diseased canine mitral valves. J Comp Pathol. 140:271 277. Borgarelli M, Haggstrom J. 2010. Canine degenerative myxomatous mitral valve disease: natural history, clinical presentation and therapy. Vet Clin North Am Small Anim Pract. 40:651 663. Borgarelli M, Savarino P, Crosara S, Santilli RA, Chiavegato D, Poggi M, Bellino C, La Rosa G, Zanatta R, Haggstrom J, Tarducci A. 2008. Survival characteristics and prognostic variables of dogs with mitral regurgitation attributable to myxomatous valve disease. J Vet Intern Med. 22:120 128. Chetboul V, Tissier R. 2012. Echocardiographic assessement of canine degenerative mitral valve disease. J Vet Cardiol. 14:127 148. Falk T, J€onsson L, Olsen LH, Pedersen HD. 2006. Arteriosclerotic changes in the myocardium, lung, and kidney in dogs with chronic congestive heart failure and myxomatous mitral valve disease. Cardiovasc Pathol. 15:185 193. Fox PR, Sisson D, Moise NS. 1999. Recommendations for diagnosis of heart disease and treatment of heart failure in small animals. In: Fox PR, editor. Textbook of canine and feline cardiology. 2nd ed. Philadelphia (PA): W.B. Saunders Company; p. 883 901. Goncalves AC, Orton EC, Boon JA, Salman MD. 2002. Linear, logarithmic, and polynomial models of m-mode echocardiographic measurements in dogs. Am J Vet Res. 63:994 999. Haeck ML, Hoogslag GE, Rodrigo SF, Atsma DE, Klautz Rj, van der Wall EE, Schalij MJ, Verwey HF. 2012. Treatment options in end-stage heart failure: where to go from here? Neth Heart J. 20:167 175.
65
Harada M, Qin Y, Takano H, Minamino T, Zou Y, Toko H, Ohtsuka M, Matsuura K, Sano M, Nishi J, et al. 2005. GCSF prevents cardiac remodeling after myocardial infarction by activating the Jak-Stat pathway in cardiomyocytes. Nat Med. 11:305 311. H€ uttmann A, D€ uhrsen U, Stypmann J, Noppeney R, N€ uckel H, Neumann T, Gutersohn A, Nikol S, Erbel R. 2006. Granulocyte colony-stimulating factor-induced blood stem cell mobilisation in patients with chronic heart failure feasibility, safety and effects on exercise tolerance and cardiac function. Basic Res Cardiol. 101:78 86. Jessup M, Brozena S. 2003. Heart failure. N Engl J Med. 348:2007 2018. Jung DI, Kim HJ, Kim JW, Kang BT, Yoo JH, Park C, Lee JH, Park HM. 2008. Canine mesenchymal stem cells derived from bone marrow: isolation, characterization, multidifferentiation, and neurotrophic factor expressionin vitro. J Vet Clin. 25:458 465. Khan R, Sheppard R. 2006. Fibrosis in heart disease: understanding the role of transforming growth factor-beta in cardiomyopathy, valvular disease and arrhythmia. Immunology. 118:10 24. Kocher AA, Schuster MD, Szabolcs MJ, Takuma S, Burkhoff D, Wang J, Homma S, Edwards NM, Itescu S. 2001. Neovascularization of ischemic myocardium by human bone-marrowderived angioblasts prevents cardiomocyte apoptosis, reduces remodeling and improves cardiac function. Nat Med. 7:430 436. Lim YH, Joe JH, Jang KS, Song YS, So BI, Fang CH, Shin J, Kim JH, Lim HK, Kim KS. 2011. Effects of granulocytecolony stimulating factor (G-CSF) on diabetic cardiomyopathy in Otsuka Long-Evans Tokushima fatty rats. Cardiovasc Diabetol. 10:92. Ljungvall I, Rajam€aki MM, Crosara S, Olsen LH, Kvart C, Borgarelli M, H€ oglund K, H€aggstr€ om J. 2011. Evaluation of plasma activity of matrix metalloproteinase-2 and -9 in dogs with myxomatous mitral valve disease. Am J Vet Res. 72:1022 1028. Mishu L, Callahan G, Allebban Z, Maddux JM, Boone TC, Souza LM, Lothrop CD Jr. 1992. Effects of recombinant canine granulocyte colony-stimulating factor on white blood cell production in clinically normal and neutropenic dogs. J Am Vet Med Assoc. 200:1957 1964. Murry CE, Field LJ, Menasche P. 2005. Cell-based cardiac repair: reflections at the 10-year point. Circulation. 112: 174 183. Orlic D, Kajstura J, Chimenti S, Jakoniuk I, Anderson SM, Li B, Pickel J, McKay R, Nadal-Ginard B, Bodine DM, et al. 2001. Bone marrow cells regenerate infracted myocardium. Nature. 410:701 705. Ormerod MG. 2000. Further clinical applications. In: Ormerod MG, editor. Flow cytometry: a practical approach. New York (NY): Oxford; p. 101 103. Ripa RS, Jørgensen E, Wang Y, Thune JJ, Nilsson JC, Søndergaard L, Johnsen HE, Køber L, Grande P, Kastrup J. 2006. Stem cell mobilization induced by subcutaneous granulocyte-colony stimulating factor to improve cardiac regeneration after acute ST-elevation myocardial infarction: result of the double-blind, randomized, placebo-controlled stem cells in myocardial infarction (STEMMI) trial. Circulation. 113:1983 1992. Serres F, Chetboul V, Tissier R, Poujol L, Gouni V, Carlos Sampedrano C, Pouchelon JL. 2008. Comparison of 3 ultrasound methods for quantifying left ventricular systolic function: correlation with disease severity and prognostic value in dogs with mitral valve disease. J Vet Intern Med. 22:566 577. Spinale FG, Coker ML, Thomas CV, Walker JD, Mukherjee R, Hebbar L. 1998. Time-dependent changes in matrix metalloproteinase activity and expression during the progression of
66
M.-H. Kang and H.-M. Park
Downloaded by [The University of British Columbia] at 05:02 11 December 2014
congestive heart failure: relation to ventricular and myocyte function. Circ Res. 82:482 495. Szardien S, Nef HM, Voss S, Troidl C, Liebetrau C, Hoffmann J, Rauch M, Mayer K, Kimmich K, Rolf A, et al. 2012. Regression of cardiac hypertrophy by granulocyte colonystimulating factor-stimulated interleukin-1b synthesis. Eur Heart J. 33:595 605.
Takano H, Hasegawa H, Nagai T, Komuro I. 2003. Implication of cardiac remodeling in heart failure: mechanisms and therapeutic strategies. Intern Med. 42:465 469. Takano H, Qin Y, Hasegawa H, Ueda K, Niitsuma Y, Ohtsuka M, Komuro I. 2006. Effects of G-CSF on left ventricular remodeling and heart failure after acute myocardial infarction. J Mol Med. 84:185 193.
Veterinary Quarterly Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tveq20
Short-term effect of granulocyte colony-stimulating factor in dogs with severe myxomatous mitral valve disease a
Min-Hee Kang & Hee-Myung Park
a
a
Department of Veterinary Internal Medicine, College of Veterinary Medicine, Konkuk University, Seoul, South Korea Published online: 24 Sep 2014.
To cite this article: Min-Hee Kang & Hee-Myung Park (2014) Short-term effect of granulocyte colony-stimulating factor in dogs with severe myxomatous mitral valve disease, Veterinary Quarterly, 34:2, 60-66, DOI: 10.1080/01652176.2014.954063 To link to this article: http://dx.doi.org/10.1080/01652176.2014.954063
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Veterinary Quarterly, 2014 Vol. 34, No. 2, 60 66, http://dx.doi.org/10.1080/01652176.2014.954063
ORIGINAL ARTICLE Short-term effect of granulocyte colony-stimulating factor in dogs with severe myxomatous mitral valve disease Min-Hee Kang and Hee-Myung Park* Department of Veterinary Internal Medicine, College of Veterinary Medicine, Konkuk University, Seoul, South Korea
Downloaded by [The University of British Columbia] at 05:02 11 December 2014
(Received 21 April 2014; accepted 8 August 2014) Background: Use of granulocyte colony-stimulating factor (G-CSF) to treat damaged myocardium is a relatively new concept. Clinical beneficial and safety outcomes are still controversial. Objective: The aim of this study was to evaluate recruitment of hematopoietic stem cells and therapeutic efficacy of G-CSF in the treatment of myxomatous mitral valve disease (MMVD) of dogs. Animals and methods: Thirty client-owned MMVD dogs with clinical signs of heart failure were enrolled in a prospective double-blind, randomized, placebo-controlled study to compare the short-term effect of G-CSF (n D 17) with control group (n D 13) for identical periods. Clinical, hematological, and cardiovascular assessments were performed on days 0, 1, 3, and 7. Follow-up examination was conducted four weeks after the study. Results: Dogs treated with G-CSF had a significantly elevated white blood cell (WBC) (£103/mL) count at day 3 compared with baseline (from 10.23 § 4.42 to 42.84 § 11.84; P D .000). The WBC population was also changed (elevated neutrophils and decreased lymphocytes) and the numbers of CD34C cells in the peripheral blood were also increased at day 3. However, the results of clinical, laboratory, and echocardiographic assessments did not differ significantly between the G-CSF treatment and control groups after four weeks. Conclusions: G-CSF administration elevated the peripheral WBC count, especially neutrophils, and recruited hematopoietic stem cells. However, positive effects of G-CSF on cardiac function were not detected during short-term monitoring. Keywords: granulocyte colony-stimulating factor (G-CSF); myxomatous mitral valve disease; canine; hematopoietic stem cells
1. Introduction Despite advances in diagnosis and treatment of congestive heart failure over recent decades, morbidity and mortality remain high both in dogs (Borgarelli et al. 2008) and humans (Haeck et al. 2012). The most frequent cause of heart failure is acute myocardial infarction (AMI) in humans (Jessup & Brozena 2003) and mitral valve degeneration in dogs (Aupperle & Disatian 2012). Myxomatous mitral valve disease (MMVD) is an acquired primary valvular disease which can result in progressive cardiac enlargement and congestive heart failure in dogs and cats due to valvular degeneration (Abbott 2008; Borgarelli & Haggstrom 2010). Even though the basic etiology of heart failure differs between dogs and humans, AMI also induces left ventricular (LV) remodeling, which could progress to other cardiovascular disorders including valvular heart disease (Takano et al. 2003). Like ischemic disease in humans, the myocardial changes, such as intramural arteriosclerosis and myocardial fibrosis in chronic MMVD dogs, are significant (Falk et al. 2006) and cardiac fibrosis appears to be important in pathology and dysfunction in valvular disease (Khan & Sheppard 2006). There is no conventional treatment for terminal heart failure in both species. Therefore, the discovery of novel treatment strategies is required to further improve clinical outcomes. Recently, gene- and cell-based therapies have been established. Use of hematopoietic cytokines, such as granulocyte
*Corresponding author. Email: [email protected] Ó 2014 Taylor & Francis
colony-stimulating factor (G-CSF), to treat damaged myocardium has been investigated in the last decade (Takano et al. 2006). Paracrine effects (growth factors, cytokines, and other signaling molecules) of transplanted cells appear to be the main mechanism of beneficial effects (Murry et al. 2005) and several studies demonstrated that stem cell therapy improved myocardial function through myocardial regeneration or neovascularization (Kocher et al. 2001; Orlic et al. 2001). More recent studies have demonstrated the beneficial effects of G-CSF such as improvement of the cardiac function and the regression of myocardial fibrosis on non-ischemic cardiomyopathy (H€uttmann et al. 2006; Szardien et al. 2012). To the authors’ knowledge, G-CSF efficacy in naturally occurring MMVD is not established. We hypothesized that, as it is in humans, cytokine therapy using G-CSF would be beneficial in treating MMVD dogs by improving cardiac function through preventing apoptosis and regression of cardiac fibrosis. Therefore, the purposes of this study were to evaluate hematopoietic stem cell recruitment and therapeutic efficacy of G-CSF in dogs with MMVD. 2. Material and methods 2.1.
Animals
This was a four-week, prospective double-blinded, randomized, placebo-controlled study. Dogs with naturally
Downloaded by [The University of British Columbia] at 05:02 11 December 2014
Veterinary Quarterly occurring MMVD were eligible for enrollment after client consent for participation was obtained. The study protocol was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the College of Veterinary Medicine, Konkuk University, South Korea. To be eligible for inclusion, the dog had previously been diagnosed with MMVD based on the following criteria: characteristic left-sided heart murmur of moderate to high intensity, echocardiographic evidence of MMVD (characteristic valvular lesions of the mitral valve apparatus such as leaflet thickening and/or valve prolapse, evidence of moderate to severe left atrial (LA) and/or left ventricular enlargement), and demonstrated mitral regurgitation on color Doppler echocardiography (Goncalves et al. 2002; Chetboul & Tissier 2012). The MMVD dogs were classified according to the International Small Animal Cardiac Health Council (ISACHC) scheme (Fox et al. 1999). The clinical status and medications received by each dog were stable for 1 month before enrollment in the study. Dogs were excluded from the study if they had a congenital cardiac disease, or had another clinically significant systemic disease, or had evidence of concurrent organ dysfunction such as respiratory tract disease. Asymptomatic dogs (ISACHC class I) were also excluded from the study. Dogs were randomly allocated into two groups (treatment and control groups) by a third party (a person that was independent of the research team). Owners were blinded to treatment allocation until the end of the study period (four weeks), and the clinician and technician responsible for recording the outcome measures were also blinded to treatment allocation until completion of data analysis. 2.2.
Concurrent medications
Patients were stabilized on current medications used to treat heart disease for at least one month prior to enrolment on the study and were continued at the same dose throughout the study. All the dogs were receiving diuretics (furosemide, 100% (range, 1 3 mg/kg q 12 hr); spironolactone, 13% (range, 1 2 mg/kg q 24 hr); hydrochlorothiazide, 3% (2 mg/kg q 12 hr)) and 83% of the dogs were receiving angiotensin-converting enzyme inhibitors (enalapril (range, 0.5 mg/kg q 12 hr) or ramipril (range 0.125 0.25 mg/kg q 24 hr); treatment-group 82%, control-group 85%). Positive inotropic agents were being administered to 63% of the dogs (pimobendan (range, 0.25 mg/kg q 12 hr), treatmentgroup 35%, control-group 46%; digoxin (range, 0.0050.01 mg/kg q 12 hr), treatment-group 29%, control-group 15%). Forty percent of the dogs were receiving hydralazine (range, 0.5 1 mg/kg q 12 hr) (treatment-group 41%, control-group 38%). One of the dogs in the treatment group was receiving sildenafil (1 mg/kg q 12 hr) and one of the dogs from the control group was receiving carvedilol (0.3 mg/kg q 12 hr). 2.3.
Granulocyte colony-stimulating factor treatment protocol
Treatment-group animals received 30 mg of G-CSF/kg (LeukokineÒ , CJ, Seoul, South Korea) by subcutaneous
61
injection once daily for three consecutive days. The treatment periods were determined based on our previous controlled experimental results, which showed that mobilization of peripheral blood progenitor cells were significantly increased on day 3 and gradually decreased, when given five successive days (data not shown). Control-group dogs were treated with the same volume of normal saline (placebo) for an identical period. In addition, all dogs continued preexisting treatment, consisting of diuretics, angiotensin converting enzyme inhibitors, and/ or inotropic agents, and the medication was not changed during the study period. 2.4.
Analysis of peripheral blood
Blood samples were obtained prior to the G-CSF/placebo treatment (baseline) and on days 1, 3, 7, and 28 after commencing the G-CSF/placebo treatment. Complete blood count was determined by automatic analyzer (VetScanR HM2, ABAXIS, Union City, CA, USA) and a manual differential count was performed. 2.5.
Flow cytometric enumeration of CD34C hematopoietic stem cells
The number of CD34C hematopoietic stem cells in the peripheral blood was determined using a flow cytometer prior and on day 3 after commencing the G-CSF/placebo treatment. Hematopoietic stem cell counting was performed according to the dual-platform method of the International Society of Hematotherapy and Graft Engineering (ISHAGE) protocol (Ormerod 2000). Double staining with mouse anti-canine CD34:RPE (AbD Serotec) and CD 45: FITC (AbD Serotec) was used for fluorescence-activated cell sorting analysis (FACScalibur flow cytometer, Beckton Dickinson, CA, USA) with the CellQuest program (Becton Dickinson Immunocytometry Systems, San Jose, CA, USA) as reported previously (Jung et al. 2008). 2.6.
Clinical examination and hemodynamic assessment
All dogs underwent a thorough physical examination and blood biochemical analysis. Hemodynamic changes were monitored using indirect arterial blood pressure (Cardell Model 9401, Sharn Veterinary Inc., FL, USA), 6-channel electrocardiogram (ECG; Cardiofax GEM ECG-9020K, Nihon Kohden, Tokyo, Japan), two-view thoracic radiographs, and complete echocardiographic examination (Logiq400, GE healthcare Medical Systems, Milwaukee, WI, USA), which included transthoracic two-dimensional (2D), M-mode, spectral, and color-flow Doppler. The dogs were not sedated during the examinations. All cardiac valves were examined for evidence of insufficiency using color-flow Doppler. Systolic and diastolic dimensions of the left ventricular internal diameter (LVIDs and LVIDd, respectively) were measured on the right parasternal M-mode view, and LV fractional shortening (FS) was calculated. The end-diastolic volume (EDV) and the endsystolic volume (ESV) were calculated using the Teicholz
62
M.-H. Kang and H.-M. Park
method (Borgarelli et al. 2008), and LV ejection fraction (EF) was calculated according to the following formula: EF D [(EDV ¡ ESV)/EDV £ 100]. Stroke volume (SV) was calculated by subtracting ESV from EDV. Measurements of the aortic (AO) and LA diameters were obtained on the 2D right parasternal short-axis view, and the LA/ AO ratio was calculated. All echocardiographic measurements followed previously described methods (Goncalves et al. 2002; Chetboul & Tissier 2012). All assessments were obtained prior to the G-CSF/placebo treatment (baseline) and at days 1, 3, and 7, and 28 after commencing the G-CSF/placebo treatment.
Downloaded by [The University of British Columbia] at 05:02 11 December 2014
2.7.
Statistical analysis
The distribution of all baseline parameters was assessed using the Shapiro Wilk’s test and data that were not normal were analyzed using the Scheirer Ray Hare extension of the Kruskal Wallis test. Based on data distribution, baseline variables were compared between two groups using the paired t-test and Mann Whitney Utest. The paired t-test was used to investigate changes in the evaluated variables in the control and treated groups. The changes from baseline to follow-up between the control and treatment groups were compared with repeated measures analysis of variance (ANOVA). A separate analysis was performed to evaluate changes in hematologic values from baseline to follow-up between the control and treatment groups (repeated ANOVA) and the results over time as a within variable (oneway ANOVA), followed by post hoc Bonferroni tests. Optimal multilevel model (linear, quadratic, or cubic model) was applied for the comparison of repeated measures time effect. Post hoc power analyses (requesting 80% power) were performed to detect differences in diagnostic values. Statistical significance was defined as observed power (1 ¡ b) > 0.80 and P(a) < 0.05. SPSS version 19.0 (SPSS, Inc., Chicago, IL, USA) and GPower version 3 (Germany) were used to perform statistical analyses. 3. Results Thirty client-owned dogs with MMVD (ISACHC Class II (47%), Class IIIa (47%), and Class IIIb (7%)) were included in this study. The dogs were randomly divided into two groups (17 dogs in the treatment group and 13 dogs in the control group). The mean age of all dogs included in the study was 10.6 years (range, 7 16) and mean weight was 4 kg (range, 2 11.5 kg). The study population consisted of 16 Maltese terriers, 5 Shih-Tzus, 3 Yorkshire terriers, 2 Pomeranians, 2 mixed breeds, 1 Miniature Poodle, and 1 Miniature Pinscher. There were no significant differences between groups with regard to gender, age, or clinical heart failure classification (Table 1). Dogs treated with G-CSF had a significantly elevated white blood cell (WBC) (£103/mL) count three days after the treatment (baseline, 10.23 § 4.42; three days after treatment, 42.84 § 11.84; F D 66.822, P D .000).
Table 1. Baseline characteristics of 30 dogs with MMVD treated either with G-CSF or placebo. Parameters
Control group Treatment group
Number 13 17 Male/female 7/6 8/9 Age (years, mean § SD) 10.21 § 2.64 10.91 § 3.18 ISACHC class II/III 5/8 9/8
P .713 .324 .431
Breeds
n
n
Total n
Maltese terrier Shih-Tzu Yorkshire terrier Mixed breed Pomeranian Miniature poodle Miniature pinscher
5 4 3 1 0 0 0
11 1 0 1 2 1 1
16 5 3 2 2 1 1
Note: ISACHC, International Small Animal Cardiac Health Council.
Multilevel modeling with cubic model revealed significant difference of WBC count over time (F D 24.449, P D .000, partial h2 D 0.746, observed power D 1.000). The WBC population (%) was also changed at day 3, and elevated neutrophils (baseline, 67.67 § 10.82; three days after treatment, 85.08 § 4.85; F D 8.854, P D .000) and decreased lymphocytes (baseline, 20.50 § 7.98; three days after treatment, 9.58 § 2.84; F D 6.186, P D .001) were noted. Both neutrophils (F D 16.669, P D .001, partial h2 D 0.510, observed power D 0.969) and lymphocytes (F D 9.763, P D .007, partial h2 D 0.379, observed power D 0.835) population was significantly changed over time. The WBC count stabilized to the baseline at day 7. The changes of red blood cells, packed cell volume, and platelets by time course and the time by group interaction were not significantly different (Table 2). Increased numbers of CD34C cells in the peripheral circulation three days after the G-CSF treatment (baseline, 11.24 § 4.61 cells/mL; three days after treatment, 38.83 § 18.98 cells/mL, P D .002) compared to the control group (baseline, 11.88 § 7.99 cells/mL; three days after treatment, 12.28 § 6.97 cells/mL, P D 0.87) were noted. Results of physical and hemodynamic parameters before and four weeks after treatment in both groups are presented in Table 3. In both groups, body weight, heart rate, body temperature, and blood pressure did not change significantly during the study period. Changes in ECG and echocardiographic parameters were not significantly different before and after the G-CSF treatment. In repeated ANOVA test, FS results were elevated significantly at four weeks (F D 4.507, P D .043, partial h2 D 0.139, observed power D 0.536), but not between groups (F D 1.444, P D .240, partial h2 D 0.049, observed power D 0.213). Also, the time by group interaction did not differ significantly. We also observed significant effects of time, groups, and their interactions (Table 4). While not an objective of this study, no adverse effect regarding safety was noted during the study period.
Veterinary Quarterly
63
Table 2. Changes in hematologic parameters in G-CSF or placebo-treated dogs with MMVD.
Downloaded by [The University of British Columbia] at 05:02 11 December 2014
Parameters
Group
Baseline
3 days
7 days
28 days
F
P
WBC (£103/ul)
Control group Treatment group
10.68 § 2.39 10.23 § 4.42a
10.07 § 1.46 42.84 § 11.84b
10.51 § 2.99 11.06 § 3.76a
10.24 § 2.67 10.07 § 3.83a
.074 66.822
.973 .000
RBC (£106/ul)
Control group Treatment group
6.61 § 0.53 6.52 § 1.11
6.30 § 0.65 6.40 § 1.31
6.50 § 0.45 6.28 § 1.30
6.50 § 0.41 6.62 § 1.26
.351 .160
.789 .922
Hb (g/dl)
Control group Treatment group
14.25 § 0.78 15.72 § 2.90
14.47 § 0.87 15.28 § 2.69
14.22 § 0.48 15.08 § 3.13
14.68 § 0.84 16.02 § 3.16
.486 .243
.696 .866
PCV (%)
Control group Treatment group
41.81 § 2.69 44.69 § 7.36
42.37 § 2.90 42.94 § 7.96
42.02 § 1.87 43.14 § 8.35
42.72 § 2.51 45.70 § 8.27
.151 .324
.928 .808
PLT (£103/ul)
Control group Treatment group
442.33 § 101.76 424.21 § 174.88
444.50 § 118.51 453.50 § 174.12
456.67 § 130.64 450.25 § 134.02
476.00 § 100.39 499.33 § 181.69
.111 .418
.953 .741
Control roup Treatment group
7.00 § 2.68 8.00 § 3.05
6.17 § 1.83 4.50 § 3.87
5.33 § 2.16 5.50 § 4.10
7.00 § 2.19 7.58 § 2.39
.763 2.864
.528 .047
Lymphocyte
Control group Treatment group
20.00 § 4.60 20.50 § 7.98c
20.67 § 3.83 9.58 § 2.84a
19.33 § 3.56 17.50 § 6.04b
18.67 § 4.27 17.08 § 7.69b
.266 6.186
.849 .001
SegNe
Control group Treatment group
69.17 § 6.27 67.67 § 10.82a
71.17 § 3.82 85.08 § 4.85c
73.50 § 2.43 73.92 § 7.35b
73.33 § 4.37 71.00 § 10.72b
1.275 8.854
.310 .000
Differential count Monocyte
Note: WBC, white blood cells; RBC, red blood cells; Hb, hemoglobin; PCV, packed cell volume; PLT, platelet; and SegNE, segmented neutrophils. a c At each time point, values with different superscript letters differ significantly (P < .05).
Table 3. Changes in physical and cardiac parameters in G-CSF or placebo-treated dogs with MMVD. Control group Parameters
Treatment group
Baseline
28 days
P
Baseline
28 days
P
4.74 § 2.69 153.38 § 21.34 38.72 § 0.55 138.71 § 15.72
4.65 § 2.49 156.15 § 23.06 38.65 § 0.40 138.74 § 14.80
.1408 .7353 .6535 .9945
3.44 § 1.08 161.41 § 24.47 38.75 § 0.36 138.74 § 15.95
3.35 § 0.99 154.94 § 20.48 38.78 § 0.40 137.10 § 11.49
.4238 .4094 .6783 .7232
Echocardiography FS (%) LVIDd (mm) LVIDs (mm) EF (%) SV (ml) EDV (ml) ESV (ml) LA/AO
53.34 § 9.20 27.33 § 4.80 12.52 § 2.88 86.54 § 6.42 22.58 § 13.72 25.97 § 15.43 3.38 § 2.28 1.94 § 0.77
54.66 § 8.00 29.31 § 6.98 13.36 § 4.24 87.30 § 6.39 28.65 § 18.67 33.37 § 22.74 4.71 § 5.06 2.23 § 1.30
.6782 .3731 .6155 .7476 .3190 .3059 .7546 .4635
48.86 § 6.96 28.25 § 4.35 14.66 § 3.54 83.23 § 7.22 22.23 § 9.12 27.15 § 12.11 5.00 § 3.90 1.87 § 0.40
52.88 § 7.46 26.84 § 7.96 13.38 § 3.60 87.36 § 6.24 23.32 § 9.94 26.92 § 11.91 3.61 § 2.86 1.80 § 0.43
.1141 .5262 .3037 .0838 .7412 .9558 .2447 .6265
Radiography VHS
11.17 § 0.96
11.25 § 1.22
.6093
11.18 § 0.70
11.14 § 0.68
.5533
Weight (kg) Heart rate (bpm) Body temp ( C) BP (mmHg)
Electrocardiography P width P height PR interval QRS width R height QT interval
0.05 § 0.005 0.32 § 0.056 0.07 § 0.009 0.05 § 0.007 2.08 § 0.505 0.20 § 0.024
0.05 § 0.006 0.34 § 0.061 0.07 § 0.009 0.05 § 0.007 2.12 § 0.483 0.19 § 0.024
.3370 .1111 1.000 .3370 .2676 .1745
0.05 § 0.006 0.31 § 0.064 0.07 § 0.009 0.05 § 0.008 1.93 § 0.474 0.19 § 0.023
0.05 § 0.005 0.32 § 0.075 0.07 § 0.008 0.05 § 0.008 1.94 § 0.491 0.19 § 0.022
Note: BP blood pressure; FS fractional shortening; LVIDd/LVIDs left ventricular internal dimension at end-diastole/end-systole; EF fraction; SV stroke volume; EDV end-diastolic volume; ESV end-systolic volume; LA/AO left atrial to aortic root ratio; and VHS heart score.
.3322 .2156 1.000 .3322 .6684 .5434 ejection vertebral
64
M.-H. Kang and H.-M. Park
Table 4. Summary of time, groups and their interaction on variables evaluated in the study. Time effect (P)
Group effect (P)
Interaction effect (P)
Test
WBC (£103/ul) RBC (£106/ul)
.000 .110
.001 .856
.000 .284
AV AV
Hb (g/dl) PCV (%) PLT (£103/ul)
.029 .108 .377
.367 .567 .974
.117 .129 .688
AV AV AV
.027 .007 .001
.983 .069 .338
.293 .002 .000
AV AV AV
.043 .646 .650 .146 .414 .593 .602 .859
.240 .806 .390 .412 .982 .917 .676 .932
.292 .436 .033 .310 .682 .549 .328 .474
AV SRH AV SRH SRH SRH SRH SRH
Downloaded by [The University of British Columbia] at 05:02 11 December 2014
Parameters
Differential count Monocyte Lymphocyte SegNe Echocardiography FS (%) LVIDd (mm) LVIDs (mm) EF (%) SV (ml) EDV (ml) ESV (ml) LA/AO
Note: WBC white blood cells; RBC red blood cells; Hb hemoglobin; PCV packed cell volume; PLT platelet; SegNE segmented neutrophils; FS fractional shortening; LVIDd/LVIDs left ventricular internal dimension at end-diastole/end-systole; EF ejection fraction; SV stroke volume; EDV end-diastolic volume; ESV end-systolic volume; LA/AO left atrial to aortic root ratio; AV analysis of variance (ANOVA); and SRH Scheirer Ray Hare test.
4. Discussion G-CSF is an effective stimulus for the mobilization of bone marrow-derived stem cells into the peripheral circulation and acts on hematopoietic stem (CD34C) cells to regulate neutrophil progenitor proliferation and differentiation (Serres et al. 2008). The cardioprotective effects of G-CSF might be attributable to its direct action on the myocardium. The peak concentration of hematopoietic stem cells in peripheral blood was reported to occur 4 7 days after commencing G-CSF treatment (Ripa et al. 2006). In this study, all dogs treated with G-CSF had a significantly elevated WBC count (especially neutrophils) three days after commencing treatment, which returned to baseline by day 7. We also confirmed increased numbers of CD34C cells in the peripheral circulation. This cell population change is similar to that in previous reports (Mishu et al. 1992). During the short-term follow-up (four weeks), LV size and systolic function were monitored using conventional echocardiography. No significant difference in overall cardiac status was detected between dogs receiving GCSF and normal saline (placebo). Despite the recent advances in ultrasound technology, accurate evaluation of the complex hemodynamic changes associated with disease progress is challenging. However, conventional echocardiographic measurements, such as FS, EF, and LVIDs, are still considered the non-invasive diagnostic test of choice for assessing myocardial function (Serres
et al. 2008; Chetboul & Tissier 2012). In both placebo and G-CSF treatment groups, FS and EF were slightly increased but no significant differences were observed in the short-term follow-up period. However, these variables are besides being dependent on intrinsic contractility, also known to be influenced by hemodynamic load and sympathetic tone. This could potentially mask significant myocardial dysfunction in dogs with MMVD. Assessment of LVIDs has been suggested to better reflect systolic dysfunction in the presence of mitral regurgitation (Chetboul & Tissier 2012). LVIDs were slightly decreased in G-CSF treatment group, but there was still no significant difference. The cardioprotective effects of G-CSF are mainly focused on the cardiomyocytes. After AMI, cardiomyocyte death is accelerated, especially in the ischemic region, by necrosis or apoptosis. G-CSF activates a variety of intracellular signaling cascades such as Janus kinase (Jak)-signal transducer and activator of transcription (STAT), Ras-Raf-mitogen-activated protein (MAP) kinase, and Scr family kinase pathways (Harada et al. 2005; Takano et al. 2006). These mechanisms might protect against cardiac fibrosis and cardiac remodeling. Compared to the pathophysiology of AMI in humans, myocardial lesions of MMVD dogs contain focal areas of myocardial fibrosis and necrosis, especially in the subendocardium and papillary muscles of the left ventricle (Falk et al. 2006). Cardiac fibrosis appears to be important in pathology and dysfunction in several heart diseases and it has now been suggested to play a major role in valvular disease (Khan & Sheppard 2006). A recent study in nonischemic, diabetic cardiomyopathy patients investigating the therapeutic potential of G-CSF showed inhibition of transforming growth factor-b1 (TGF-b1) and progression of fibrosis (Lim et al. 2011). In general, TGF-b1 is a profibrotic cytokine and appears to be one of several factors that cause disease by inducing cardiac fibrosis. Another experimental study in mice with cardiac hypertrophy and fibrosis induced by AO stenosis showed that G-CSF treatment improved cardiac function and led to the regression of myocardial fibrosis at two weeks (Szardien et al. 2012). They also demonstrated that administration of G-CSF provides beneficial degradation of the extracellular matrix (ECM), by increasing activity of both matrix metalloproteinase 2 (MMP-2) and MMP-9. Changes in ECM composition and structure have been reported in dogs with mitral regurgitation, and myocardial MMP activity was accompanied by alterations in cardiac ECM structure and progressive cardiac dysfunction (Spinale et al. 1998; Aupperle et al. 2009). The association between MMP-9 activity and systolic dysfunction was investigated in canine MMVD (Ljungvall et al. 2011). They reported that plasma MMP-9 activity decreased with the myocardial remodeling process in dogs with MMVD. The most important limitation of this study is the lack of functional tests of the injected G-CSF. The effect of GCSF was evaluated according to the degree of clinical improvement and echocardiographic change during a relatively short period. Therefore, we cannot completely exclude the beneficial effect of G-CSF in MMVD
Veterinary Quarterly treatment and further studies on the cellular and subcellular mechanisms, and longer term clinical implications of G-CSF would be desirable. In addition, this study was not designed to allow the determination of optimal dose and scheduling of G-CSF administration.
Downloaded by [The University of British Columbia] at 05:02 11 December 2014
5. Conclusion G-CSF was an effective stimulus for mobilization of bone marrow-derived hematopoietic stem cells into the peripheral circulation, but short-term cardioprotective effects were not demonstrated in this study. Evaluation of longterm clinical efficacy and safety in MMVD dogs treated with G-CSF is required in further study. Acknowledgement This research was supported by the Basic Science Research Program through the NRF of Korea funded by the Ministry of Education, Science and Technology (2012R1A6A3A01038961).
References Abbott JA. 2008. Acquired valvular disease. In: Tilley LP, Smith FWK Jr, Oyama MA, Sleeper MM, editors. Manual of canine and feline cardiology. 4th ed. London: Elsevier Ltd.; p. 110 138. Aupperle H, Disatian S. 2012. Pathology, protein expression and signaling in myxomatous mitral valve degeneration: comparison of dogs and humans. J Vet Cardiol. 14:59 71. Aupperle H, Thielebein J, Kiefer B, M€arz I, Dinges G, Schoon HA, Schubert A. 2009. Expression of genes encoding matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in normal and diseased canine mitral valves. J Comp Pathol. 140:271 277. Borgarelli M, Haggstrom J. 2010. Canine degenerative myxomatous mitral valve disease: natural history, clinical presentation and therapy. Vet Clin North Am Small Anim Pract. 40:651 663. Borgarelli M, Savarino P, Crosara S, Santilli RA, Chiavegato D, Poggi M, Bellino C, La Rosa G, Zanatta R, Haggstrom J, Tarducci A. 2008. Survival characteristics and prognostic variables of dogs with mitral regurgitation attributable to myxomatous valve disease. J Vet Intern Med. 22:120 128. Chetboul V, Tissier R. 2012. Echocardiographic assessement of canine degenerative mitral valve disease. J Vet Cardiol. 14:127 148. Falk T, J€onsson L, Olsen LH, Pedersen HD. 2006. Arteriosclerotic changes in the myocardium, lung, and kidney in dogs with chronic congestive heart failure and myxomatous mitral valve disease. Cardiovasc Pathol. 15:185 193. Fox PR, Sisson D, Moise NS. 1999. Recommendations for diagnosis of heart disease and treatment of heart failure in small animals. In: Fox PR, editor. Textbook of canine and feline cardiology. 2nd ed. Philadelphia (PA): W.B. Saunders Company; p. 883 901. Goncalves AC, Orton EC, Boon JA, Salman MD. 2002. Linear, logarithmic, and polynomial models of m-mode echocardiographic measurements in dogs. Am J Vet Res. 63:994 999. Haeck ML, Hoogslag GE, Rodrigo SF, Atsma DE, Klautz Rj, van der Wall EE, Schalij MJ, Verwey HF. 2012. Treatment options in end-stage heart failure: where to go from here? Neth Heart J. 20:167 175.
65
Harada M, Qin Y, Takano H, Minamino T, Zou Y, Toko H, Ohtsuka M, Matsuura K, Sano M, Nishi J, et al. 2005. GCSF prevents cardiac remodeling after myocardial infarction by activating the Jak-Stat pathway in cardiomyocytes. Nat Med. 11:305 311. H€ uttmann A, D€ uhrsen U, Stypmann J, Noppeney R, N€ uckel H, Neumann T, Gutersohn A, Nikol S, Erbel R. 2006. Granulocyte colony-stimulating factor-induced blood stem cell mobilisation in patients with chronic heart failure feasibility, safety and effects on exercise tolerance and cardiac function. Basic Res Cardiol. 101:78 86. Jessup M, Brozena S. 2003. Heart failure. N Engl J Med. 348:2007 2018. Jung DI, Kim HJ, Kim JW, Kang BT, Yoo JH, Park C, Lee JH, Park HM. 2008. Canine mesenchymal stem cells derived from bone marrow: isolation, characterization, multidifferentiation, and neurotrophic factor expressionin vitro. J Vet Clin. 25:458 465. Khan R, Sheppard R. 2006. Fibrosis in heart disease: understanding the role of transforming growth factor-beta in cardiomyopathy, valvular disease and arrhythmia. Immunology. 118:10 24. Kocher AA, Schuster MD, Szabolcs MJ, Takuma S, Burkhoff D, Wang J, Homma S, Edwards NM, Itescu S. 2001. Neovascularization of ischemic myocardium by human bone-marrowderived angioblasts prevents cardiomocyte apoptosis, reduces remodeling and improves cardiac function. Nat Med. 7:430 436. Lim YH, Joe JH, Jang KS, Song YS, So BI, Fang CH, Shin J, Kim JH, Lim HK, Kim KS. 2011. Effects of granulocytecolony stimulating factor (G-CSF) on diabetic cardiomyopathy in Otsuka Long-Evans Tokushima fatty rats. Cardiovasc Diabetol. 10:92. Ljungvall I, Rajam€aki MM, Crosara S, Olsen LH, Kvart C, Borgarelli M, H€ oglund K, H€aggstr€ om J. 2011. Evaluation of plasma activity of matrix metalloproteinase-2 and -9 in dogs with myxomatous mitral valve disease. Am J Vet Res. 72:1022 1028. Mishu L, Callahan G, Allebban Z, Maddux JM, Boone TC, Souza LM, Lothrop CD Jr. 1992. Effects of recombinant canine granulocyte colony-stimulating factor on white blood cell production in clinically normal and neutropenic dogs. J Am Vet Med Assoc. 200:1957 1964. Murry CE, Field LJ, Menasche P. 2005. Cell-based cardiac repair: reflections at the 10-year point. Circulation. 112: 174 183. Orlic D, Kajstura J, Chimenti S, Jakoniuk I, Anderson SM, Li B, Pickel J, McKay R, Nadal-Ginard B, Bodine DM, et al. 2001. Bone marrow cells regenerate infracted myocardium. Nature. 410:701 705. Ormerod MG. 2000. Further clinical applications. In: Ormerod MG, editor. Flow cytometry: a practical approach. New York (NY): Oxford; p. 101 103. Ripa RS, Jørgensen E, Wang Y, Thune JJ, Nilsson JC, Søndergaard L, Johnsen HE, Køber L, Grande P, Kastrup J. 2006. Stem cell mobilization induced by subcutaneous granulocyte-colony stimulating factor to improve cardiac regeneration after acute ST-elevation myocardial infarction: result of the double-blind, randomized, placebo-controlled stem cells in myocardial infarction (STEMMI) trial. Circulation. 113:1983 1992. Serres F, Chetboul V, Tissier R, Poujol L, Gouni V, Carlos Sampedrano C, Pouchelon JL. 2008. Comparison of 3 ultrasound methods for quantifying left ventricular systolic function: correlation with disease severity and prognostic value in dogs with mitral valve disease. J Vet Intern Med. 22:566 577. Spinale FG, Coker ML, Thomas CV, Walker JD, Mukherjee R, Hebbar L. 1998. Time-dependent changes in matrix metalloproteinase activity and expression during the progression of
66
M.-H. Kang and H.-M. Park
Downloaded by [The University of British Columbia] at 05:02 11 December 2014
congestive heart failure: relation to ventricular and myocyte function. Circ Res. 82:482 495. Szardien S, Nef HM, Voss S, Troidl C, Liebetrau C, Hoffmann J, Rauch M, Mayer K, Kimmich K, Rolf A, et al. 2012. Regression of cardiac hypertrophy by granulocyte colonystimulating factor-stimulated interleukin-1b synthesis. Eur Heart J. 33:595 605.
Takano H, Hasegawa H, Nagai T, Komuro I. 2003. Implication of cardiac remodeling in heart failure: mechanisms and therapeutic strategies. Intern Med. 42:465 469. Takano H, Qin Y, Hasegawa H, Ueda K, Niitsuma Y, Ohtsuka M, Komuro I. 2006. Effects of G-CSF on left ventricular remodeling and heart failure after acute myocardial infarction. J Mol Med. 84:185 193.
