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Treatment of dilated cardiomyopathy in rabbits with mesenchymal stem cell transplantation and platelet-rich plasma
Accepted Manuscript Title: Treatment of dilated cardiomyopathy in rabbits with mesenchymal stem cell transplantation and platelet-rich plasma Author: P.D. Mörschbächer, T.N.A. Garcez, A.H. Paz, A.B. Magrisso, H.F. Mello, V.M. Rolim, E.B. Neuwald, D. Driemeier, E.A. Contesini, E. CirneLima PII: DOI: Reference:
S1090-0233(15)00480-3 http://dx.doi.org/doi: 10.1016/j.tvjl.2015.11.009 YTVJL 4707
To appear in:
The Veterinary Journal
Accepted date:
15-11-2015
Please cite this article as: P.D. Mörschbächer, T.N.A. Garcez, A.H. Paz, A.B. Magrisso, H.F. Mello, V.M. Rolim, E.B. Neuwald, D. Driemeier, E.A. Contesini, E. Cirne-Lima, Treatment of dilated cardiomyopathy in rabbits with mesenchymal stem cell transplantation and platelet-rich plasma, The Veterinary Journal (2015), http://dx.doi.org/doi: 10.1016/j.tvjl.2015.11.009. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Treatment of dilated cardiomyopathy in rabbits with mesenchymal stem cell transplantation and platelet-rich plasma
P.D. Mörschbächer a,b*, T.N.A. Garcez a, A.H. Paz b, A.B. Magrisso b, H.F. Mello b, V.M. Rolim a, E.B. Neuwald a, D. Driemeier a, E.A. Contesini a, E. Cirne-Lima a,b a
Graduate Program in Veterinary Science, Federal University of Rio Grande do Sul, Av. Bento Gonçalves 9090 CEP 91540-000, Porto Alegre, Brazil b Laboratory of Embryology, Porto Alegre Clinical Hospital, Porto Alegre, Brazil
*
Corresponding author. Tel: +55 51 8121630. E-mail address: [email protected] (P.D. Mörschbächer)
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Highlights
18 19
A rabbit dilated cardiomyopathy model was used.
20 21
Mesenchymal cell (MSC) transplantation may be promising for dilated cardiomyopathy.
22 23
Platelet-rich plasma (PRP) should be further studied before its use in dilated cardiomyopathy.
24 25
Further investigations are required to understand the mechanisms of MSC transplantation.
26
Abstract
27
Dilated cardiomyopathy (DCM) is a major cause of cardiovascular mortality and
28
morbidity, and there is evidence to suggest that stem cell transplantation may be a
29
viable treatment option for this condition. Therefore, the goal of the present study was
30
to assess myocardial regeneration in rabbits with doxorubicin-induced DCM treated
31
with adipose mesenchymal stem cells (MSC) alone or in combination with platelet-rich
32
plasma (PRP). Twenty New Zealand rabbits received doxorubicin for the induction of
33
DCM and were divided into four groups according to treatment: saline, MSC, PRP and
34
MSC+ RP. Treatment agents were injected directly into the left ventricular myocardium
35
following a thoracoscopy. Rabbits were assessed through echocardiographic and
36
electrocardiographic examinations, as well as serum cardiac troponin I measurements at
37
baseline, after the induction of DCM and 15 days after treatment. Animals were
38
euthanased
39
histopathological analyses.
following
the
last
assessment,
and
hearts
were
collected
for
40 41
The MSC group showed improvements in all parameters assessed, while the
42
PRP group showed significantly impaired heart function. Histopathology of the heart
43
revealed that the MSC group displayed the lowest number of lesions, while rabbits in 2 Page 2 of 20
3 44
the MSC+PRP, saline and PRP groups had steadily advancing lesions. These results
45
suggest that MSC transplantation can improve heart function in rabbits with DCM, and
46
underscore the need for further studies of the effects of PRP on the myocardium.
47 48
Keywords: Cardiology; Cell therapy; Scaffold; Troponin I; Stem cell
3 Page 3 of 20
4 49
Introduction
50
In spite of significant advances in medical and surgical care, congestive heart
51
failure is still one of the main causes of cardiovascular morbidity and mortality. Dilated
52
cardiomyopathy (DCM) is a primary myocardial disease of unknown aetiology,
53
characterized by a loss of cardiomyocytes and an increase in fibroblasts, as well as a
54
common cause of heart failure. Although both myocyte mitosis and cardiac precursor
55
cells have been found in adult hearts, the death of a large number of cardiomyocytes can
56
still result in heart failure. As such, restoring cardiomyocyte levels may be an adequate
57
treatment strategy for DCM (Nagaya et al., 2005).
58 59
Mesenchymal stem cells (MSC) help repair damaged myocardial tissues through
60
several mechanisms, the main one being the production of repair factors, which increase
61
the local expression of growth factors and cytokines. MSC may also suppress local
62
inflammation, repair damaged cells and contribute to the creation of a favourable
63
environment for endogenous tissue repair. These findings have established MSC as a
64
promising new treatment approach for several cardiovascular conditions (Souza et al.,
65
2010).
66 67
MSC are generally applied to an aqueous medium, from which they can be
68
easily isolated, especially when they come into direct contact with the circulatory
69
system. Stem cells are combined with scaffolds to optimize the patency of the implanted
70
grafts (Huang et al., 2005). Platelet-rich plasma (PRP) is an autologous blood product
71
which contains a high concentration of growth factors. PRP has been widely used in the
72
healing of skeletal muscle, for which it has proven to be a safe and effective treatment.
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In spite of growing evidence of the safety and efficacy of PRP, few studies have
74
analysed its effects on cardiovascular tissues (Mischra et al., 2010).
75 76
Therefore, the goal of the present study was to assess myocardial regeneration in
77
rabbits with doxorubicin-induced DCM treated with adipose MSC, either with or
78
without the use of PRP as a scaffold.
79 80
Materials and methods
81
Animals
82
Twenty-one New Zealand rabbits (Oryctolagus cuniculus), comprising a male
83
donor and twenty females, aged between 3 and 4 months, weighing 2 to 3.5 kg, were
84
used in this study. The animals received doxorubicin to induce heart failure and were
85
divided into four groups containing five rabbits each, which were labelled according to
86
the treatment received: MSC resuspended in PRP (MSC+PRP group), PRP (PRP
87
group), MSC suspended in a culture medium (MSC group) and 0.9% sodium chloride
88
solution (NaCl group). The number of MSC used was in the order of 106 cells/animal.
89
PRP-treated animals received a 1 mL of the substance.
90 91
Rabbits are particularly susceptible to the cardiotoxicity of doxorubicin and this
92
experimental model pioneered studies on the pathophysiology of heart failure. This
93
experimental model develop lesions similar to those described in humans, including
94
cytoplasmic vacuolization, interstitial edema and myofibrillar rupture. All animals were
95
housed and handled according to Brazilian Animal Experimentation Code and Animal
96
Research Ethics Committee guidelines, based on the law 11.794, of October 8, 2008.
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This study was approved by the Research Ethics Committee of the Porto Alegre Clinical
98
Hospital (HCPA) (protocol number 11-0279).
99 100
Adipose tissue collection
101
A rabbit was premedicated with tramadol chlorhydrate 5 mg/kg (União
102
Química), midazolam 1 mg/kg (Dormonid, Roche) and ketamine 20 mg/kg (Cetamin,
103
Syntec), administered intramuscularly (IM), followed by isoflurane (Isoforine, Cristália)
104
to facilitate orotracheal intubation, and maintained with isoflurane and 100% oxygen.
105
The animal was placed in a sternal decubitus position and, after antisepsis, adipose
106
tissue was collected from the interscapular region. The skin was then sutured.
107 108
Adipose MSC isolation
109
The adipose tissue was placed in type I collagenase solution (1 mg/mL) in
110
Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) containing 9 mM HEPES,
111
for 1 h at 37 °C to facilitate tissue digestion. After digestion, the collagenase was
112
inactivated by dilution with DMEM containing 10% foetal bovine serum (FBS,
113
Invitrogen). Once isolated, cells were cultivated in a low-glucose DMEM medium
114
supplemented with 9 mM HEPES, 20% FBS and an antibiotic solution containing 100
115
U/mL penicillin and 100 mg/mL streptomycin. The culture was kept at a constant
116
temperature of 37 °C, in an atmosphere of 5% CO2 and 100% humidity. After 24 h, the
117
culture medium was aspirated and replaced by fresh medium. When the culture reached
118
80% confluence, the adherent cells were removed from the dishes by the addition of
119
0.5% trypsin/EDTA (Gibco). The material was then placed in DMEM supplemented
120
with 10% FBS (complete medium).
121
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In the second passage, MSCs were added to FBS supplemented with 5%
123
dimethylsulfoxide (DMSO) and stored in a freezer at -80 °C. Approximately one week
124
before the transplant, the cells were thawed and expanded. The transplants were
125
performed using cells between the third and fourth passages. MSCs were classified
126
using in vitro morphology and differentiation into chondrogenic, osteogenic and
127
adipogenic lineages.
128 129
Doxorubicin-induced dilated cardiomyopathy
130
DCM was induced using doxorubicin chlorhydrate (Glenmark), which was
131
applied once a week at a dose of 2 mg/kg in the first two weeks and a dose of 3 mg/kg
132
in the last two weeks, reaching a cumulative dose of 10 mg/kg. Rabbits were first
133
sedated with midazolam (2 mg/kg) and ketamine (10 mg/kg) IM, after which
134
doxorubicin was injected intravenously (IV). The animals were assessed before and
135
after the induction of DCM as well as 15 days after treatment using echocardiography
136
(Mylab 30 Vet bi-dimensional echocardiography equipment), electrocardiograms (TEB
137
PC Vet electrocardiograph) and serum cardiac troponin I concentration (Boehring
138
Diagnostics Opus Plus Kit). Electrocardiography (heart rate, QRS morphology and
139
duration) and echocardiography (systolic left ventricular diameter, shortening and
140
ejection fractions) results were recorded as the mean of three measurements made by an
141
examiner blind to group assignment.
142 143
PRP preparation
144
Prior to the surgical procedure, 10 mL blood was collected from the central ear
145
artery of each animal, placed in a plastic tube containing sodium citrate and centrifuged
146
at 300 g for 10 min. A total of 500 L was then collected from the top layer of the
7 Page 7 of 20
8 147
plasma and placed in a separate sterile 15 mL tube labelled with the letter A, containing
148
150 L calcium gluconate. The remaining plasma in the upper and intermediate layers
149
was placed in another sterile plastic tube, labelled with the letter B. Both tubes were
150
centrifuged at 640 g for 10 min.
151 152
After centrifugation, half of the material in tube B was discarded, and the
153
remaining solution was homogenized. Two mL of the contents of tube B and 1 mL of
154
the material drawn from tube A were then placed in a 2:1 ratio (2 mL PRP:1 mL
155
thrombin) in another sterile plastic tube labelled with the letter C. One mL of the
156
material in tube C was then placed in a microtube, at which point the PRP, with or
157
without the MSC, was prepared for implantation into the myocardium.
158 159
Video-assisted thoracoscopy
160
Rabbits were premedicated with pethidine chlorhydrate 3 mg/kg (União
161
Química), midazolam 0.7 mg/kg and ketamine 14 mg/kg, administered IM. Anaesthesia
162
was induced using an isoflurane mask and maintained by orotracheal intubation with
163
vaporized isoflurane in 100% oxygen. After antisepsis, the chest cavity was accessed
164
through an incision made above the sixth left intercostal space, 5 cm ventral to the
165
costovertebral region, and a 5 mm trocar was used to perforate the pleura for the
166
insertion of a rigid endoscope. In the fourth intercostal space, between the costochondral
167
and sternal regions, a 13x4 mm hypodermic needle attached to a 1 mL syringe was used
168
to inject the treatment agents into the least vascularized region of the left ventricular
169
wall. The incision was closed and negative intrathoracic pressure was then
170
reestablished. In the postoperative period, animals were treated with tramadol
8 Page 8 of 20
9 171
chlorhydrate 3 mg/kg subcutaneously (SC) and enrofloxacin 10 mg/kg (Zelotril,
172
Agener) IM for 72 h.
173 174
After the 15-day assessment period, all rabbits were euthanased and their hearts
175
were collected for examination. These were fixed in 10% buffered formalin for 24 h,
176
after which they were embedded in paraffin wax and stained with haematoxylin and
177
eosin (H&E). The hearts were investigated histopathologically for sarcoplasmic
178
vacuolization, myofiber necrosis and fibrosis.
179 180
Statistical analysis
181
Results were expressed as mean +_ standard deviation (SD). Statistical analyses
182
were performed using the SPSS software (v.18.0), and P <0.05 was considered
183
statistically significant. Electrocardiography and echocardiography data were subjected
184
to a three-factor repeated measures analysis of variance (ANOVA). Troponin
185
concentrations were assessed through a two-way repeated measures ANOVA assuming
186
a symmetrical component correlation matrix between assessments. Statistically
187
significant analyses were followed by Tukey post-hoc tests. Histopathological data were
188
assessed through non-parametric Kruskal-Wallis tests, followed by Bonferroni post-hoc
189
tests.
190 191
Results
192
The troponin I, echocardiography, electrocardiography and histology data
193
indicated that DCM was successfully induced in all rabbits. The same exams were used
194
to assess the results of the treatments administered.
195
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Rabbits showed an increase in serum troponin I over time (Fig. 1). The most
197
pronounced increase in troponin I at euthanasia was observed in the PRP group,
198
followed by the saline group, the MSC+PRP group and, lastly, the MSC group. The
199
presence of heart lesions was determined by the presence of troponin I concentrations >
200
0.05 ng/mL (Alvarez et al., 2012).
201 202
The
electrocardiography results
showed
no
significant
between-group
203
differences in QRS configuration, although its duration increased throughout the
204
experiment in the saline and MSC+PRP groups, while the remaining groups displayed
205
an increase in this value followed by a decrease. Nonetheless, all QRS values were
206
within the expected physiological range for the species (Fig. 2 A).
207 208
Fig. 2 B illustrates the changes in systolic left ventricular (LV) diameter
209
observed in each treatment group throughout the experiment. At the pre-euthanasia
210
assessment, LV diameter was found to have increased in the saline group, but decreased
211
in the other treatment groups. The ejection fraction decreased over time in the
212
MSC+PRP group, and was found to first decrease, then increase in the other three
213
treatment groups, with this pattern being more pronounced in the MSC group (Fig. 2 C).
214
Fig. 2 D shows a decrease in the shortening fraction following the induction of
215
cardiomyopathy by doxorubicin, and an increase in this value following surgery in the
216
MSC, MSC+PRP and PRP groups. The saline group displayed a decrease in the SF over
217
the course of the study.
218 219
Histological analysis showed extensive sarcoplasmic vacuolisation with isolated
220
myofiber necrosis and diffuse myocardial fibrosis (Fig. 3). Furthermore, in several
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11 221
animals in all groups, locally extensive myocardial fibrosis, attributed to the impact of
222
the intracardiac injection, was also observed. The PRP group had statistically more
223
lesions than the saline group. The MSC group had the best histological results out of all
224
analysed groups. Data regarding between-group differences in histological findings (P =
225
0.010) can be found in Table 1. Significantly more histological alterations were found in
226
the myocardium of PRP-treated rabbits than in MSC (P = 0.007) and MSC+PRP-
227
treated (P = 0.007) animals (Table 2).
228 229
Discussion
230
The present study demonstrated the following findings: (1) doxorubicin was
231
successful in inducing cardiomyopathy in rabbits; (2) the MSC transplant attenuated the
232
degree of DCM and improved heart function; (3) treatment with PRP worsened the
233
condition of rabbits with DCM.
234 235
After a 4-week period of doxorubicin treatment, echocardiography results
236
showed a decrease in systolic heart function and a reduction in shortening and ejection
237
fractions associated with LV dilation, all of which are indicative of DCM. This was
238
corroborated by an increase in serum troponin I concentrations and the histopathological
239
results (sarcoplasmic vacuolisation, myofiber necrosis and diffuse myocardial fibrosis)
240
(Silva and Camacho, 2005).
241 242
In the present study, a decrease in serum troponin I concentrations, QRS
243
duration and systolic LV diameter were observed 15 days after rabbits with
244
doxorubicin-induced DCM were subjected to a MSC transplant. These findings,
245
associated with an increase in EF and SF values and a decrease in the number of lesions
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12 246
detected by histological analysis, suggest improvements in heart function and that the
247
MSC transplant had a positive effect on cardiomyocytes. One possible explanation for
248
these findings is the differentiation of MSC into cardiomyocytes, which has been
249
reported in previous studies involving rat models of DCM. These studies found that
250
some transplanted MSCs stained positive for troponin T and desmin (Nagaya et al.,
251
2005). Some of the transplanted MSCs were also positive for an endothelial vascular
252
marker, suggesting their possible role in vessel formation and in increasing myocardial
253
capillary density (Tokunaga et al., 2004; Nagaya et al., 2005).
254 255
Some authors speculate that the positive effects of MSCs on cardiomyopathy are
256
more likely attributable to their paracrine actions rather than their differentiation
257
abilities. The transplantation of MSCs into ischemic myocardial tissues has been found
258
to lead to an increase in the production of angiogenic factors and a decrease in apoptosis
259
(Tang et al., 2005). These results appeared to be better explained by the secretion of
260
paracrine factors than by the differentiation of MSCs. The cardioprotective role of
261
paracrine MSC secretions may be associated with the actions of exosomes (Lai et al.,
262
2010).
263 264
Consistent with literature reports, increased fibrosis was observed in rabbits with
265
doxorubicin-induced DCM (Klimtova et al., 2002; Aupperle et al., 2007). An
266
investigation using doxorubicin-induced DCM in rabbits also found an improvement in
267
heart function following MSC transplantation (Aupperle et al., 2007). In contrast, the
268
injection of culture medium had no beneficial effects on heart function, and led to
269
higher levels of myocardial fibrosis. Since the MSC could no longer be identified after 4
12 Page 12 of 20
13 270
weeks, the authors concluded that their results were most likely attributable to the
271
paracrine effects of these cells (Aupperle et al., 2007).
272 273
An important paracrine effect of MSCs is their anti-inflammatory activity. A
274
study involving MSC transplantation in acute myocarditis found that, when this
275
procedure was conducted 1 week after treatment with myosin, heart function improved
276
and pathological alterations, such as myocardial inflammation, decreased, suggesting
277
that these cells may have a cardioprotective paracrine effect (Ohnishi et al., 2007).
278 279
Recent studies have demonstrated that both autologous and allogeneic MSCs
280
have a strong suppressant effect on the proliferation of T lymphocytes (Di Nicola et al.,
281
2002; Tse et al., 2003). Cultivated MSCs have also been found to have a
282
cardioprotective effect and secrete large quantities of angiogenic and anti-apoptotic
283
factors, such as vascular endothelial growth factor (VEGF), hepatocyte growth factor
284
(HGF), insulin-like growth factor (IGF) and adrenomedullin (Gnecchi et al., 2005;
285
Nagaya et al., 2005). Therefore, only a small number of the implanted MSCs
286
differentiate into endothelial cells or cardiomyocytes, and the contribution of
287
differentiated MSCs to the improvement of heart function is likely insignificant
288
(Ohnishi et al., 2007).
289 290
The decision of treating animals with both MSCs and PRP was made because
291
the factors secreted by the PRP may enrich the MSC medium, increase the cellular
292
regenerative capacity, as well as promote organ recovery. The fibrous matrix and
293
interconnected pores found in PRP act as a scaffold for heart tissue regeneration,
294
allowing for cell penetration and growth factor secretion, which promote angiogenesis,
13 Page 13 of 20
14 295
as well as anti-apoptotic and chemotactic factors (Mishra et al., 2009; Chimenti et al.,
296
2010). However, the histopathological analysis showed that the hearts of PRP-treated
297
animals displayed more extensive lesions than those of the remaining rabbits, including
298
those in the saline group. Similar result were obtained with regard to the increase in
299
troponin I levels following treatment, which was found to be greater in the PRP group.
300
PRP appeared to cause significant damage to the heart in animals with doxorubicin-
301
induced DCM. Studies suggest that platelets can contribute to inflammation in patients
302
with chronic heart failure (CHF) through the induction of MCP-1 expression, thus
303
aggravating the condition (Cheng et al., 2012). Therefore, the fact that rabbits in the
304
MSC+PRP group displayed less tissue damage than those in the PRP group may be
305
explained by a beneficial effect of the MSCs, which inhibited the inflammatory effects
306
of PRP.
307 308
The last decade has seen an increase in the evidence supporting a role of
309
immunological and inflammatory responses in the pathology of CHF. Several studies
310
have found that patients with CHF have elevated inflammatory cytokine levels,
311
suggesting that the production of these substances is directly associated with the
312
severity of the disease (Yndestad et al., 2006). Studies have shown that the expression
313
of MCP-1 is regulated in the ischemic myocardium and is responsible for the
314
recruitment of mononuclear cells into the damaged tissue. These cells produce cytokines
315
and other factors required for the growth and proliferation of fibroblasts, which leads to
316
tissue repair and scar formation. Therefore, in cases of acute myocardial infarction or
317
reperfusion ischemia, MCP-1 expression is likely to be beneficial (Kakio et al., 2000).
318
However, in patients with CHF, there appears to be a significant increase in the plasma
319
levels of MCP-1. Patients with advanced CHF and low EFs show the highest levels of
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15 320
MCP-1. Likewise, circulating platelets are markedly activated in patients with CHF and
321
may induce endothelial MCP-1 secretion, contributing to the increase in the levels of
322
this chemokine (Stumpf et al., 2008). Although several studies have shown increased
323
platelet activity in CHF, little is known about the role of these cells as modulators of the
324
pro-inflammatory responses involved in CHF (Frangogiannis et al., 2002). However,
325
evidence suggests that platelets can contribute to inflammatory conditions through the
326
secretion of MCP-1 (Stumpf et al., 2008).
327 328
This study is mainly observational, and as such, there are some inherent
329
limitations due to the design. Importantly, the current investigation was not designed to
330
elucidate the mechanism or combination of mechanisms behind the functional
331
alterations detected. Further investigations are required to assess the mechanisms
332
underlying improved heart function following MSC transplantation, and the aggravation
333
of this condition following treatment with PRP. In the MSC+PRP group, the MSCs may
334
have protected the myocardium from the inflammatory actions of PRP and doxorubicin.
335
These results should also be further studied in connection with histological findings and
336
biological markers both in vitro and in vivo.
337 338
Conclusion
339
MSC transplantation improved heart function in rabbits with doxorubicin
340
induced-DCM and may be a promising treatment for DCM. In contrast, PRP should be
341
further studied before it can be considered a viable therapeutic option for this condition.
342 343
Conflict of interest statement
15 Page 15 of 20
16 344
None of the authors of this paper has a financial or personal relationship with
345
other people or organisations that could inappropriately influence or bias the content of
346
the paper.
347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387
Acknowledgements This study was supported by the Research Incentive Fund (FIPE/HCPA).
References Alvarez, I., Uribe, A., Duarte, S., 2012. Biomarcadores de la falla cardíaca em pequeños animales. Revista de Medicina Veterinaria 24, 59-70. Aupperle, H., Garbade, J., Schubert, A., Barten, M., Dhein, S., Schoon, H.A., Mohr, F.W., 2007. Effects of autologous stem cells on immunohistochemical patterns and gene expression of metalloproteinases and their tissue inhibitors in doxorubicin cardiomyopathy in a rabbit model. Veterinary Pathology 44, 494-503. Cheng, K., Malliaras, K., Shen, D., Tseliou, E., Ionta, V., Smith, J., Galang, G., Sun, B., Houde, C., Marbán, E., 2012. Intramyocardial injection of platelet gel promotes endogenous repair and augments cardiac function in rats with myocardial infarction. Journal of the American College of Cardiology 59, 256-264. Chimenti, I., Smith, R.R., Li, T.S., Gerstenblith, G., Messina, E., Giacomello, A., Marbán, E., 2010. Relative roles of direct regeneration versus paracrine effects of human cardiosphere-derived cells transplanted into infarcted mice. Circulation Research 106, 971-980. Di Nicola, M., Carlo-Stella, C., Magni, M., Milanesi, M., Longoni, P.D., Matteucci, P., Grisanti, S., Gianni, A.M., 2002. Human bone marrow stromal cells suppress Tlymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli. Blood Journal 99, 3838–3843. Frangogiannis, N.G., Smith, C.W., Entman, M.L., 2002. The inflammatory response in myocardial infarction. Cardiovascular Research 53, 31–47. Gnecchi, M., He, H., Liang, O.D., Melo, L.G., Morello, F., Mu, H., Noiseux, N., Zhang, L., Pratt, R.E., Ingwall, J.S., Dzau, V.J., 2005. Paracrine action accounts for marked protection of ischemic heart by Akt-modified mesenchymal stem cells. Nature Medicine 11, 367–368. Huang, N.F., Yu, J., Sievers, R., Li, S., Lee, R.J., 2005. Injectable Biopolymers Enhance Angiogenesis after Myocardial Infarction. Tissue Engineering 11, 18601866. 16 Page 16 of 20
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Kakio, T., Matsumori, A., Ono, K., Ito, H., Matsushima, K., Sasayama, S., 2000. Roles and relationship of macrophages and monocyte chemotactic and activating factor/monocyte chemoattractant protein-1 in the ischemic and reperfused rat heart. Laboratory Investigation 80, 1127–1136. Klimtova, I., Simunek, T., Mazurova, Y., Hrdina, R., Gersl, V., Adamcova, M., 2002. Comparative study of chronic toxic effects of daunorubicin and doxorubicin in rabbits. Human and Experimental Toxicology 21, 649–657. Lai, R.C., Arslan, F., Lee, M.M., 2010. Exosome secreted by MSC reduces myocardial ischemia/reperfusion injury. Stem Cell Research 4, 214-222. Mishra, A., Woodall, J., Vieira, A., 2009. Treatment of tendon and muscle using platelet-rich plasma. Clinical Journal of Sports Medicine 28, 113–25. Mischra, A., Velotta, J., Brinton, T.J., Wang, X., Chang, S., Palmer, O., Sheikh, A., Chung, J., Yang, P.C., Robbins, R., Fischbein, M., 2010. RevaTen platelet-rich plasma improves cardiac function after myocardial injury. Cardiovascular Revascularization Medicine 12, 158-163. Nagaya, N., Kangawa, K., Itoh, T., Iwase, T., Murakami, S., Miyahara, Y., Fujii, T., Uematsu, M., Ohgushi, H., Yamagishi, M., Tokudome, T., Mori, H., Miyatake, K., Kitamura, S., 2005. Transplantation of mesenchymal stem cells improves cardiac function in a rat model of dilated cardiomyopathy. Circulation 112, 11281135. Ohnishi, S., Yanagawa, B., Tanaka, K., Miyahara, Y., Obata, H., Kataoka, M., Kodama, M., Ishibashi-Ueda, H., Kangawa, K., Kitamura, S., Nagaya, N., 2007. Transplantation of mesenchymal stem cells attenuates myocardial injury and dysfunction in a rat model of acute myocarditis. Journal of Molecular and Cellular Cardiology 42, 88-97. Silva, C.E.V., Camacho, A.A., 2005. Alterações ecocardiográficas em cães sob tratamento prolongado com doxorrubicina. Arquivo Brasileiro de Medicina Veterinária e Zootecnia 57, 300-306. Souza, C.F., Napoli, P., Han, S.W., Lima, V.C., Carvalho, A.C.C., 2010. CélulasTronco Mesenquimais: Células Ideais para a Regeneração Cardíaca? Revista Brasileira de Cardiologia Invasiva 18, 344-353. Stumpf, C., Lehner, C., Raaz, D., Yilmaz, A., Anger, T., Daniel, W.G., Garlichs, C.D., 2008. Platelets contribute to enhanced MCP-1 levels in patients with chronic heart failure. Heart 94, 65-69. Tang, Y.L., Zhao, Q., Qin, X., Shen, L., Cheng, L., Ge, J., Phillips, M.I., 2005. Paracrine action enhances the effects of autologous mesenchymal stem cell transplantation on vascular regeneration in rat model of myocardial infarction. The Annals Thoracic Surgery 80, 229–236.
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Tokunaga, N., Nagaya, N., Shirai, M., Tanaka, E., Ishibashi-Ueda, H., Harada-Shiba, M., Kanda, M., Ito, T., Shimizu, W., Tabata, Y., Uematsu, M., Nishigami, K., Sano, S., Kangawa, K., Mori, H., 2004 Adrenomedullin gene transfer induces therapeutic angiogenesis in a rabbit model of chronic hind limb ischemia: benefits of a novel nonviral vector, gelatin. Circulation 109, 526-531. Tse, W.T., Pendleton, J.D., Beyer, W.M., Egalka, M.C., Guinan, E.C., 2003. Suppression of allogeneic T-cell proliferation by human marrow stromal cells: implications in transplantation. Transplantation 75, 389–397. Yndestad, A., Kristian, D.J., Oie, E., Ueland, T., Gullestad, L., Auskrust, P., 2006. Systemic inflammation in heart failure - the whys and wherefores. Heart Failure Reviews 11, 83–92.
18 Page 18 of 20
19 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468
Figure legends Fig.1. Mean serum troponin I concentrations and standard deviations (SD) in New Zealand rabbits with dilated cardiomyopathy (DCM) induced by treatment group (n = 5/subgroup). P < 0.05. Fig.2. The electrocardiography results measured in New Zealand rabbits with dilated cardiomyopathy (DCM) induced with doxorubicin chlohydrate in each treatment group. (A) Mean and standard deviation (SD) of QRS. (B) Mean and SD of LV profiles. (C) Mean and SD of ejection fractions (EFs). (D) Mean and SD of shortening fraction (SF) values. Data are mean +_ S.D (n = 5/subgroup). P < 0.05. Fig.3. Histological analysis of New Zealand rabbit hearts with dilated cardiomyopathy (DCM) induced with doxorubicin chlohydrate. (A) Mild myocardial lesion. (B) Moderate myocardial lesion. (C) Severe myocardial lesion. The arrow indicates the presence of sarcoplasmic vacuolisation. Haematoxylin-Eosin, 40x.
469 470
Table 1
471
Results of the histological and comparative analyses of the rabbit hearts in each
472
treatment group (n = 5/group). Severity of the myocardial lesions: mild, +; moderate,
473
++; severe, +++. P = 0.010 Treatment group Stem cells + Histopathology Saline
Stem cells
PRP
Total
P
PRP n %
n %
n %
n %
n
%
+
0 0.0
2 40.0
0 0.0
2 40.0
4
20.0
++
5 100.0
3 60.0
1 20.0
3 60.0
12
60.0
+++
0 0.0
0 0.0
4 80.0
0 0.0
4
20.0
Total
5 100
5 100
5 100
5 100
20
100
0.010
474 475 476
19 Page 19 of 20
20 477
Table 2
478
Comparative analyses of histological findings between subgroup pairs (n = 5/subgroup). Comparison
Z-value
P
Saline vs MSC
0.90
0.369
Saline vs PRP
-1.80
0.072
Saline vs. MSC+PRP
0.90
0.369
MSC vs. PRP
-2.70
0.007
MSC vs. MSC+PRP
0.00
1.000
PRP vs MSC+PRP
2.70
0.007
479
20 Page 20 of 20
S1090-0233(15)00480-3 http://dx.doi.org/doi: 10.1016/j.tvjl.2015.11.009 YTVJL 4707
To appear in:
The Veterinary Journal
Accepted date:
15-11-2015
Please cite this article as: P.D. Mörschbächer, T.N.A. Garcez, A.H. Paz, A.B. Magrisso, H.F. Mello, V.M. Rolim, E.B. Neuwald, D. Driemeier, E.A. Contesini, E. Cirne-Lima, Treatment of dilated cardiomyopathy in rabbits with mesenchymal stem cell transplantation and platelet-rich plasma, The Veterinary Journal (2015), http://dx.doi.org/doi: 10.1016/j.tvjl.2015.11.009. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Treatment of dilated cardiomyopathy in rabbits with mesenchymal stem cell transplantation and platelet-rich plasma
P.D. Mörschbächer a,b*, T.N.A. Garcez a, A.H. Paz b, A.B. Magrisso b, H.F. Mello b, V.M. Rolim a, E.B. Neuwald a, D. Driemeier a, E.A. Contesini a, E. Cirne-Lima a,b a
Graduate Program in Veterinary Science, Federal University of Rio Grande do Sul, Av. Bento Gonçalves 9090 CEP 91540-000, Porto Alegre, Brazil b Laboratory of Embryology, Porto Alegre Clinical Hospital, Porto Alegre, Brazil
*
Corresponding author. Tel: +55 51 8121630. E-mail address: [email protected] (P.D. Mörschbächer)
16
1 Page 1 of 20
2 17
Highlights
18 19
A rabbit dilated cardiomyopathy model was used.
20 21
Mesenchymal cell (MSC) transplantation may be promising for dilated cardiomyopathy.
22 23
Platelet-rich plasma (PRP) should be further studied before its use in dilated cardiomyopathy.
24 25
Further investigations are required to understand the mechanisms of MSC transplantation.
26
Abstract
27
Dilated cardiomyopathy (DCM) is a major cause of cardiovascular mortality and
28
morbidity, and there is evidence to suggest that stem cell transplantation may be a
29
viable treatment option for this condition. Therefore, the goal of the present study was
30
to assess myocardial regeneration in rabbits with doxorubicin-induced DCM treated
31
with adipose mesenchymal stem cells (MSC) alone or in combination with platelet-rich
32
plasma (PRP). Twenty New Zealand rabbits received doxorubicin for the induction of
33
DCM and were divided into four groups according to treatment: saline, MSC, PRP and
34
MSC+ RP. Treatment agents were injected directly into the left ventricular myocardium
35
following a thoracoscopy. Rabbits were assessed through echocardiographic and
36
electrocardiographic examinations, as well as serum cardiac troponin I measurements at
37
baseline, after the induction of DCM and 15 days after treatment. Animals were
38
euthanased
39
histopathological analyses.
following
the
last
assessment,
and
hearts
were
collected
for
40 41
The MSC group showed improvements in all parameters assessed, while the
42
PRP group showed significantly impaired heart function. Histopathology of the heart
43
revealed that the MSC group displayed the lowest number of lesions, while rabbits in 2 Page 2 of 20
3 44
the MSC+PRP, saline and PRP groups had steadily advancing lesions. These results
45
suggest that MSC transplantation can improve heart function in rabbits with DCM, and
46
underscore the need for further studies of the effects of PRP on the myocardium.
47 48
Keywords: Cardiology; Cell therapy; Scaffold; Troponin I; Stem cell
3 Page 3 of 20
4 49
Introduction
50
In spite of significant advances in medical and surgical care, congestive heart
51
failure is still one of the main causes of cardiovascular morbidity and mortality. Dilated
52
cardiomyopathy (DCM) is a primary myocardial disease of unknown aetiology,
53
characterized by a loss of cardiomyocytes and an increase in fibroblasts, as well as a
54
common cause of heart failure. Although both myocyte mitosis and cardiac precursor
55
cells have been found in adult hearts, the death of a large number of cardiomyocytes can
56
still result in heart failure. As such, restoring cardiomyocyte levels may be an adequate
57
treatment strategy for DCM (Nagaya et al., 2005).
58 59
Mesenchymal stem cells (MSC) help repair damaged myocardial tissues through
60
several mechanisms, the main one being the production of repair factors, which increase
61
the local expression of growth factors and cytokines. MSC may also suppress local
62
inflammation, repair damaged cells and contribute to the creation of a favourable
63
environment for endogenous tissue repair. These findings have established MSC as a
64
promising new treatment approach for several cardiovascular conditions (Souza et al.,
65
2010).
66 67
MSC are generally applied to an aqueous medium, from which they can be
68
easily isolated, especially when they come into direct contact with the circulatory
69
system. Stem cells are combined with scaffolds to optimize the patency of the implanted
70
grafts (Huang et al., 2005). Platelet-rich plasma (PRP) is an autologous blood product
71
which contains a high concentration of growth factors. PRP has been widely used in the
72
healing of skeletal muscle, for which it has proven to be a safe and effective treatment.
4 Page 4 of 20
5 73
In spite of growing evidence of the safety and efficacy of PRP, few studies have
74
analysed its effects on cardiovascular tissues (Mischra et al., 2010).
75 76
Therefore, the goal of the present study was to assess myocardial regeneration in
77
rabbits with doxorubicin-induced DCM treated with adipose MSC, either with or
78
without the use of PRP as a scaffold.
79 80
Materials and methods
81
Animals
82
Twenty-one New Zealand rabbits (Oryctolagus cuniculus), comprising a male
83
donor and twenty females, aged between 3 and 4 months, weighing 2 to 3.5 kg, were
84
used in this study. The animals received doxorubicin to induce heart failure and were
85
divided into four groups containing five rabbits each, which were labelled according to
86
the treatment received: MSC resuspended in PRP (MSC+PRP group), PRP (PRP
87
group), MSC suspended in a culture medium (MSC group) and 0.9% sodium chloride
88
solution (NaCl group). The number of MSC used was in the order of 106 cells/animal.
89
PRP-treated animals received a 1 mL of the substance.
90 91
Rabbits are particularly susceptible to the cardiotoxicity of doxorubicin and this
92
experimental model pioneered studies on the pathophysiology of heart failure. This
93
experimental model develop lesions similar to those described in humans, including
94
cytoplasmic vacuolization, interstitial edema and myofibrillar rupture. All animals were
95
housed and handled according to Brazilian Animal Experimentation Code and Animal
96
Research Ethics Committee guidelines, based on the law 11.794, of October 8, 2008.
5 Page 5 of 20
6 97
This study was approved by the Research Ethics Committee of the Porto Alegre Clinical
98
Hospital (HCPA) (protocol number 11-0279).
99 100
Adipose tissue collection
101
A rabbit was premedicated with tramadol chlorhydrate 5 mg/kg (União
102
Química), midazolam 1 mg/kg (Dormonid, Roche) and ketamine 20 mg/kg (Cetamin,
103
Syntec), administered intramuscularly (IM), followed by isoflurane (Isoforine, Cristália)
104
to facilitate orotracheal intubation, and maintained with isoflurane and 100% oxygen.
105
The animal was placed in a sternal decubitus position and, after antisepsis, adipose
106
tissue was collected from the interscapular region. The skin was then sutured.
107 108
Adipose MSC isolation
109
The adipose tissue was placed in type I collagenase solution (1 mg/mL) in
110
Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) containing 9 mM HEPES,
111
for 1 h at 37 °C to facilitate tissue digestion. After digestion, the collagenase was
112
inactivated by dilution with DMEM containing 10% foetal bovine serum (FBS,
113
Invitrogen). Once isolated, cells were cultivated in a low-glucose DMEM medium
114
supplemented with 9 mM HEPES, 20% FBS and an antibiotic solution containing 100
115
U/mL penicillin and 100 mg/mL streptomycin. The culture was kept at a constant
116
temperature of 37 °C, in an atmosphere of 5% CO2 and 100% humidity. After 24 h, the
117
culture medium was aspirated and replaced by fresh medium. When the culture reached
118
80% confluence, the adherent cells were removed from the dishes by the addition of
119
0.5% trypsin/EDTA (Gibco). The material was then placed in DMEM supplemented
120
with 10% FBS (complete medium).
121
6 Page 6 of 20
7 122
In the second passage, MSCs were added to FBS supplemented with 5%
123
dimethylsulfoxide (DMSO) and stored in a freezer at -80 °C. Approximately one week
124
before the transplant, the cells were thawed and expanded. The transplants were
125
performed using cells between the third and fourth passages. MSCs were classified
126
using in vitro morphology and differentiation into chondrogenic, osteogenic and
127
adipogenic lineages.
128 129
Doxorubicin-induced dilated cardiomyopathy
130
DCM was induced using doxorubicin chlorhydrate (Glenmark), which was
131
applied once a week at a dose of 2 mg/kg in the first two weeks and a dose of 3 mg/kg
132
in the last two weeks, reaching a cumulative dose of 10 mg/kg. Rabbits were first
133
sedated with midazolam (2 mg/kg) and ketamine (10 mg/kg) IM, after which
134
doxorubicin was injected intravenously (IV). The animals were assessed before and
135
after the induction of DCM as well as 15 days after treatment using echocardiography
136
(Mylab 30 Vet bi-dimensional echocardiography equipment), electrocardiograms (TEB
137
PC Vet electrocardiograph) and serum cardiac troponin I concentration (Boehring
138
Diagnostics Opus Plus Kit). Electrocardiography (heart rate, QRS morphology and
139
duration) and echocardiography (systolic left ventricular diameter, shortening and
140
ejection fractions) results were recorded as the mean of three measurements made by an
141
examiner blind to group assignment.
142 143
PRP preparation
144
Prior to the surgical procedure, 10 mL blood was collected from the central ear
145
artery of each animal, placed in a plastic tube containing sodium citrate and centrifuged
146
at 300 g for 10 min. A total of 500 L was then collected from the top layer of the
7 Page 7 of 20
8 147
plasma and placed in a separate sterile 15 mL tube labelled with the letter A, containing
148
150 L calcium gluconate. The remaining plasma in the upper and intermediate layers
149
was placed in another sterile plastic tube, labelled with the letter B. Both tubes were
150
centrifuged at 640 g for 10 min.
151 152
After centrifugation, half of the material in tube B was discarded, and the
153
remaining solution was homogenized. Two mL of the contents of tube B and 1 mL of
154
the material drawn from tube A were then placed in a 2:1 ratio (2 mL PRP:1 mL
155
thrombin) in another sterile plastic tube labelled with the letter C. One mL of the
156
material in tube C was then placed in a microtube, at which point the PRP, with or
157
without the MSC, was prepared for implantation into the myocardium.
158 159
Video-assisted thoracoscopy
160
Rabbits were premedicated with pethidine chlorhydrate 3 mg/kg (União
161
Química), midazolam 0.7 mg/kg and ketamine 14 mg/kg, administered IM. Anaesthesia
162
was induced using an isoflurane mask and maintained by orotracheal intubation with
163
vaporized isoflurane in 100% oxygen. After antisepsis, the chest cavity was accessed
164
through an incision made above the sixth left intercostal space, 5 cm ventral to the
165
costovertebral region, and a 5 mm trocar was used to perforate the pleura for the
166
insertion of a rigid endoscope. In the fourth intercostal space, between the costochondral
167
and sternal regions, a 13x4 mm hypodermic needle attached to a 1 mL syringe was used
168
to inject the treatment agents into the least vascularized region of the left ventricular
169
wall. The incision was closed and negative intrathoracic pressure was then
170
reestablished. In the postoperative period, animals were treated with tramadol
8 Page 8 of 20
9 171
chlorhydrate 3 mg/kg subcutaneously (SC) and enrofloxacin 10 mg/kg (Zelotril,
172
Agener) IM for 72 h.
173 174
After the 15-day assessment period, all rabbits were euthanased and their hearts
175
were collected for examination. These were fixed in 10% buffered formalin for 24 h,
176
after which they were embedded in paraffin wax and stained with haematoxylin and
177
eosin (H&E). The hearts were investigated histopathologically for sarcoplasmic
178
vacuolization, myofiber necrosis and fibrosis.
179 180
Statistical analysis
181
Results were expressed as mean +_ standard deviation (SD). Statistical analyses
182
were performed using the SPSS software (v.18.0), and P <0.05 was considered
183
statistically significant. Electrocardiography and echocardiography data were subjected
184
to a three-factor repeated measures analysis of variance (ANOVA). Troponin
185
concentrations were assessed through a two-way repeated measures ANOVA assuming
186
a symmetrical component correlation matrix between assessments. Statistically
187
significant analyses were followed by Tukey post-hoc tests. Histopathological data were
188
assessed through non-parametric Kruskal-Wallis tests, followed by Bonferroni post-hoc
189
tests.
190 191
Results
192
The troponin I, echocardiography, electrocardiography and histology data
193
indicated that DCM was successfully induced in all rabbits. The same exams were used
194
to assess the results of the treatments administered.
195
9 Page 9 of 20
10 196
Rabbits showed an increase in serum troponin I over time (Fig. 1). The most
197
pronounced increase in troponin I at euthanasia was observed in the PRP group,
198
followed by the saline group, the MSC+PRP group and, lastly, the MSC group. The
199
presence of heart lesions was determined by the presence of troponin I concentrations >
200
0.05 ng/mL (Alvarez et al., 2012).
201 202
The
electrocardiography results
showed
no
significant
between-group
203
differences in QRS configuration, although its duration increased throughout the
204
experiment in the saline and MSC+PRP groups, while the remaining groups displayed
205
an increase in this value followed by a decrease. Nonetheless, all QRS values were
206
within the expected physiological range for the species (Fig. 2 A).
207 208
Fig. 2 B illustrates the changes in systolic left ventricular (LV) diameter
209
observed in each treatment group throughout the experiment. At the pre-euthanasia
210
assessment, LV diameter was found to have increased in the saline group, but decreased
211
in the other treatment groups. The ejection fraction decreased over time in the
212
MSC+PRP group, and was found to first decrease, then increase in the other three
213
treatment groups, with this pattern being more pronounced in the MSC group (Fig. 2 C).
214
Fig. 2 D shows a decrease in the shortening fraction following the induction of
215
cardiomyopathy by doxorubicin, and an increase in this value following surgery in the
216
MSC, MSC+PRP and PRP groups. The saline group displayed a decrease in the SF over
217
the course of the study.
218 219
Histological analysis showed extensive sarcoplasmic vacuolisation with isolated
220
myofiber necrosis and diffuse myocardial fibrosis (Fig. 3). Furthermore, in several
10 Page 10 of 20
11 221
animals in all groups, locally extensive myocardial fibrosis, attributed to the impact of
222
the intracardiac injection, was also observed. The PRP group had statistically more
223
lesions than the saline group. The MSC group had the best histological results out of all
224
analysed groups. Data regarding between-group differences in histological findings (P =
225
0.010) can be found in Table 1. Significantly more histological alterations were found in
226
the myocardium of PRP-treated rabbits than in MSC (P = 0.007) and MSC+PRP-
227
treated (P = 0.007) animals (Table 2).
228 229
Discussion
230
The present study demonstrated the following findings: (1) doxorubicin was
231
successful in inducing cardiomyopathy in rabbits; (2) the MSC transplant attenuated the
232
degree of DCM and improved heart function; (3) treatment with PRP worsened the
233
condition of rabbits with DCM.
234 235
After a 4-week period of doxorubicin treatment, echocardiography results
236
showed a decrease in systolic heart function and a reduction in shortening and ejection
237
fractions associated with LV dilation, all of which are indicative of DCM. This was
238
corroborated by an increase in serum troponin I concentrations and the histopathological
239
results (sarcoplasmic vacuolisation, myofiber necrosis and diffuse myocardial fibrosis)
240
(Silva and Camacho, 2005).
241 242
In the present study, a decrease in serum troponin I concentrations, QRS
243
duration and systolic LV diameter were observed 15 days after rabbits with
244
doxorubicin-induced DCM were subjected to a MSC transplant. These findings,
245
associated with an increase in EF and SF values and a decrease in the number of lesions
11 Page 11 of 20
12 246
detected by histological analysis, suggest improvements in heart function and that the
247
MSC transplant had a positive effect on cardiomyocytes. One possible explanation for
248
these findings is the differentiation of MSC into cardiomyocytes, which has been
249
reported in previous studies involving rat models of DCM. These studies found that
250
some transplanted MSCs stained positive for troponin T and desmin (Nagaya et al.,
251
2005). Some of the transplanted MSCs were also positive for an endothelial vascular
252
marker, suggesting their possible role in vessel formation and in increasing myocardial
253
capillary density (Tokunaga et al., 2004; Nagaya et al., 2005).
254 255
Some authors speculate that the positive effects of MSCs on cardiomyopathy are
256
more likely attributable to their paracrine actions rather than their differentiation
257
abilities. The transplantation of MSCs into ischemic myocardial tissues has been found
258
to lead to an increase in the production of angiogenic factors and a decrease in apoptosis
259
(Tang et al., 2005). These results appeared to be better explained by the secretion of
260
paracrine factors than by the differentiation of MSCs. The cardioprotective role of
261
paracrine MSC secretions may be associated with the actions of exosomes (Lai et al.,
262
2010).
263 264
Consistent with literature reports, increased fibrosis was observed in rabbits with
265
doxorubicin-induced DCM (Klimtova et al., 2002; Aupperle et al., 2007). An
266
investigation using doxorubicin-induced DCM in rabbits also found an improvement in
267
heart function following MSC transplantation (Aupperle et al., 2007). In contrast, the
268
injection of culture medium had no beneficial effects on heart function, and led to
269
higher levels of myocardial fibrosis. Since the MSC could no longer be identified after 4
12 Page 12 of 20
13 270
weeks, the authors concluded that their results were most likely attributable to the
271
paracrine effects of these cells (Aupperle et al., 2007).
272 273
An important paracrine effect of MSCs is their anti-inflammatory activity. A
274
study involving MSC transplantation in acute myocarditis found that, when this
275
procedure was conducted 1 week after treatment with myosin, heart function improved
276
and pathological alterations, such as myocardial inflammation, decreased, suggesting
277
that these cells may have a cardioprotective paracrine effect (Ohnishi et al., 2007).
278 279
Recent studies have demonstrated that both autologous and allogeneic MSCs
280
have a strong suppressant effect on the proliferation of T lymphocytes (Di Nicola et al.,
281
2002; Tse et al., 2003). Cultivated MSCs have also been found to have a
282
cardioprotective effect and secrete large quantities of angiogenic and anti-apoptotic
283
factors, such as vascular endothelial growth factor (VEGF), hepatocyte growth factor
284
(HGF), insulin-like growth factor (IGF) and adrenomedullin (Gnecchi et al., 2005;
285
Nagaya et al., 2005). Therefore, only a small number of the implanted MSCs
286
differentiate into endothelial cells or cardiomyocytes, and the contribution of
287
differentiated MSCs to the improvement of heart function is likely insignificant
288
(Ohnishi et al., 2007).
289 290
The decision of treating animals with both MSCs and PRP was made because
291
the factors secreted by the PRP may enrich the MSC medium, increase the cellular
292
regenerative capacity, as well as promote organ recovery. The fibrous matrix and
293
interconnected pores found in PRP act as a scaffold for heart tissue regeneration,
294
allowing for cell penetration and growth factor secretion, which promote angiogenesis,
13 Page 13 of 20
14 295
as well as anti-apoptotic and chemotactic factors (Mishra et al., 2009; Chimenti et al.,
296
2010). However, the histopathological analysis showed that the hearts of PRP-treated
297
animals displayed more extensive lesions than those of the remaining rabbits, including
298
those in the saline group. Similar result were obtained with regard to the increase in
299
troponin I levels following treatment, which was found to be greater in the PRP group.
300
PRP appeared to cause significant damage to the heart in animals with doxorubicin-
301
induced DCM. Studies suggest that platelets can contribute to inflammation in patients
302
with chronic heart failure (CHF) through the induction of MCP-1 expression, thus
303
aggravating the condition (Cheng et al., 2012). Therefore, the fact that rabbits in the
304
MSC+PRP group displayed less tissue damage than those in the PRP group may be
305
explained by a beneficial effect of the MSCs, which inhibited the inflammatory effects
306
of PRP.
307 308
The last decade has seen an increase in the evidence supporting a role of
309
immunological and inflammatory responses in the pathology of CHF. Several studies
310
have found that patients with CHF have elevated inflammatory cytokine levels,
311
suggesting that the production of these substances is directly associated with the
312
severity of the disease (Yndestad et al., 2006). Studies have shown that the expression
313
of MCP-1 is regulated in the ischemic myocardium and is responsible for the
314
recruitment of mononuclear cells into the damaged tissue. These cells produce cytokines
315
and other factors required for the growth and proliferation of fibroblasts, which leads to
316
tissue repair and scar formation. Therefore, in cases of acute myocardial infarction or
317
reperfusion ischemia, MCP-1 expression is likely to be beneficial (Kakio et al., 2000).
318
However, in patients with CHF, there appears to be a significant increase in the plasma
319
levels of MCP-1. Patients with advanced CHF and low EFs show the highest levels of
14 Page 14 of 20
15 320
MCP-1. Likewise, circulating platelets are markedly activated in patients with CHF and
321
may induce endothelial MCP-1 secretion, contributing to the increase in the levels of
322
this chemokine (Stumpf et al., 2008). Although several studies have shown increased
323
platelet activity in CHF, little is known about the role of these cells as modulators of the
324
pro-inflammatory responses involved in CHF (Frangogiannis et al., 2002). However,
325
evidence suggests that platelets can contribute to inflammatory conditions through the
326
secretion of MCP-1 (Stumpf et al., 2008).
327 328
This study is mainly observational, and as such, there are some inherent
329
limitations due to the design. Importantly, the current investigation was not designed to
330
elucidate the mechanism or combination of mechanisms behind the functional
331
alterations detected. Further investigations are required to assess the mechanisms
332
underlying improved heart function following MSC transplantation, and the aggravation
333
of this condition following treatment with PRP. In the MSC+PRP group, the MSCs may
334
have protected the myocardium from the inflammatory actions of PRP and doxorubicin.
335
These results should also be further studied in connection with histological findings and
336
biological markers both in vitro and in vivo.
337 338
Conclusion
339
MSC transplantation improved heart function in rabbits with doxorubicin
340
induced-DCM and may be a promising treatment for DCM. In contrast, PRP should be
341
further studied before it can be considered a viable therapeutic option for this condition.
342 343
Conflict of interest statement
15 Page 15 of 20
16 344
None of the authors of this paper has a financial or personal relationship with
345
other people or organisations that could inappropriately influence or bias the content of
346
the paper.
347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387
Acknowledgements This study was supported by the Research Incentive Fund (FIPE/HCPA).
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Kakio, T., Matsumori, A., Ono, K., Ito, H., Matsushima, K., Sasayama, S., 2000. Roles and relationship of macrophages and monocyte chemotactic and activating factor/monocyte chemoattractant protein-1 in the ischemic and reperfused rat heart. Laboratory Investigation 80, 1127–1136. Klimtova, I., Simunek, T., Mazurova, Y., Hrdina, R., Gersl, V., Adamcova, M., 2002. Comparative study of chronic toxic effects of daunorubicin and doxorubicin in rabbits. Human and Experimental Toxicology 21, 649–657. Lai, R.C., Arslan, F., Lee, M.M., 2010. Exosome secreted by MSC reduces myocardial ischemia/reperfusion injury. Stem Cell Research 4, 214-222. Mishra, A., Woodall, J., Vieira, A., 2009. Treatment of tendon and muscle using platelet-rich plasma. Clinical Journal of Sports Medicine 28, 113–25. Mischra, A., Velotta, J., Brinton, T.J., Wang, X., Chang, S., Palmer, O., Sheikh, A., Chung, J., Yang, P.C., Robbins, R., Fischbein, M., 2010. RevaTen platelet-rich plasma improves cardiac function after myocardial injury. Cardiovascular Revascularization Medicine 12, 158-163. Nagaya, N., Kangawa, K., Itoh, T., Iwase, T., Murakami, S., Miyahara, Y., Fujii, T., Uematsu, M., Ohgushi, H., Yamagishi, M., Tokudome, T., Mori, H., Miyatake, K., Kitamura, S., 2005. Transplantation of mesenchymal stem cells improves cardiac function in a rat model of dilated cardiomyopathy. Circulation 112, 11281135. Ohnishi, S., Yanagawa, B., Tanaka, K., Miyahara, Y., Obata, H., Kataoka, M., Kodama, M., Ishibashi-Ueda, H., Kangawa, K., Kitamura, S., Nagaya, N., 2007. Transplantation of mesenchymal stem cells attenuates myocardial injury and dysfunction in a rat model of acute myocarditis. Journal of Molecular and Cellular Cardiology 42, 88-97. Silva, C.E.V., Camacho, A.A., 2005. Alterações ecocardiográficas em cães sob tratamento prolongado com doxorrubicina. Arquivo Brasileiro de Medicina Veterinária e Zootecnia 57, 300-306. Souza, C.F., Napoli, P., Han, S.W., Lima, V.C., Carvalho, A.C.C., 2010. CélulasTronco Mesenquimais: Células Ideais para a Regeneração Cardíaca? Revista Brasileira de Cardiologia Invasiva 18, 344-353. Stumpf, C., Lehner, C., Raaz, D., Yilmaz, A., Anger, T., Daniel, W.G., Garlichs, C.D., 2008. Platelets contribute to enhanced MCP-1 levels in patients with chronic heart failure. Heart 94, 65-69. Tang, Y.L., Zhao, Q., Qin, X., Shen, L., Cheng, L., Ge, J., Phillips, M.I., 2005. Paracrine action enhances the effects of autologous mesenchymal stem cell transplantation on vascular regeneration in rat model of myocardial infarction. The Annals Thoracic Surgery 80, 229–236.
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Tokunaga, N., Nagaya, N., Shirai, M., Tanaka, E., Ishibashi-Ueda, H., Harada-Shiba, M., Kanda, M., Ito, T., Shimizu, W., Tabata, Y., Uematsu, M., Nishigami, K., Sano, S., Kangawa, K., Mori, H., 2004 Adrenomedullin gene transfer induces therapeutic angiogenesis in a rabbit model of chronic hind limb ischemia: benefits of a novel nonviral vector, gelatin. Circulation 109, 526-531. Tse, W.T., Pendleton, J.D., Beyer, W.M., Egalka, M.C., Guinan, E.C., 2003. Suppression of allogeneic T-cell proliferation by human marrow stromal cells: implications in transplantation. Transplantation 75, 389–397. Yndestad, A., Kristian, D.J., Oie, E., Ueland, T., Gullestad, L., Auskrust, P., 2006. Systemic inflammation in heart failure - the whys and wherefores. Heart Failure Reviews 11, 83–92.
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Figure legends Fig.1. Mean serum troponin I concentrations and standard deviations (SD) in New Zealand rabbits with dilated cardiomyopathy (DCM) induced by treatment group (n = 5/subgroup). P < 0.05. Fig.2. The electrocardiography results measured in New Zealand rabbits with dilated cardiomyopathy (DCM) induced with doxorubicin chlohydrate in each treatment group. (A) Mean and standard deviation (SD) of QRS. (B) Mean and SD of LV profiles. (C) Mean and SD of ejection fractions (EFs). (D) Mean and SD of shortening fraction (SF) values. Data are mean +_ S.D (n = 5/subgroup). P < 0.05. Fig.3. Histological analysis of New Zealand rabbit hearts with dilated cardiomyopathy (DCM) induced with doxorubicin chlohydrate. (A) Mild myocardial lesion. (B) Moderate myocardial lesion. (C) Severe myocardial lesion. The arrow indicates the presence of sarcoplasmic vacuolisation. Haematoxylin-Eosin, 40x.
469 470
Table 1
471
Results of the histological and comparative analyses of the rabbit hearts in each
472
treatment group (n = 5/group). Severity of the myocardial lesions: mild, +; moderate,
473
++; severe, +++. P = 0.010 Treatment group Stem cells + Histopathology Saline
Stem cells
PRP
Total
P
PRP n %
n %
n %
n %
n
%
+
0 0.0
2 40.0
0 0.0
2 40.0
4
20.0
++
5 100.0
3 60.0
1 20.0
3 60.0
12
60.0
+++
0 0.0
0 0.0
4 80.0
0 0.0
4
20.0
Total
5 100
5 100
5 100
5 100
20
100
0.010
474 475 476
19 Page 19 of 20
20 477
Table 2
478
Comparative analyses of histological findings between subgroup pairs (n = 5/subgroup). Comparison
Z-value
P
Saline vs MSC
0.90
0.369
Saline vs PRP
-1.80
0.072
Saline vs. MSC+PRP
0.90
0.369
MSC vs. PRP
-2.70
0.007
MSC vs. MSC+PRP
0.00
1.000
PRP vs MSC+PRP
2.70
0.007
479
20 Page 20 of 20