Int J Clin Exp Pathol 2014;7(9):5527-5537 www.ijcep.com /ISSN:1936-2625/IJCEP0001443

Original Article Remodeling of the thoracic aorta after bone marrow cell transplantation Alyne Felix1, Nemesis Monteiro1, Vinícius Novaes Rocha1, Genilza Oliveira2, Alan Cesar Moraes1, Cherley Andrade1, Ana Lucia Nascimento1, Laís de Carvalho2, Alessandra Thole2, Jorge Carvalho1 Laboratory of Ultrastructure and Tissue Biology, 2Laboratory of Cell Culture, Institute of Biology Roberto Alcantara Gomes, State University of Rio de Janeiro, Rio de Janeiro, Brazil 1

Received July 17, 2014; Accepted August 23, 2014; Epub August 15, 2014; Published September 1, 2014 Abstract: Stem cells are characterized by their ability to differentiate into multiple cell lineages and display the paracrine effect. The aim of this work was to evaluate the effect of therapy with bone marrow cells (BMCs) on blood glucose, lipid metabolism and aortic wall remodeling in mice through the administration of a high fat diet and subsequent BMCs transplantation. C57BL/6 mice were fed a control diet (CO group) or an atherogenic diet (AT group). After 16 weeks, the AT group was divided into four groups: an AT 14 days group and AT 21 days group, that were given an injection of vehicle and sacrificed at 14 and 21 days after, respectively; AT-BMC 14 days group and AT-BMC 21 days group that was given an injection of BMCs and sacrificed at 14 and 21 days after. The CO group was sacrificed along with other groups. The BMCs transplant had reduced blood glucose, triglycerides and total cholesterol. The Qa (1/mm2) was quantitatively reduced in AT 14 days group, AT 21 days group and was high in AT-BMC 21 days group. The AT 21 days group exhibited increased tunica media and elastic system fibers. The immunolabeling for α-SMA and VEGF showed less immunolabeling in transplanted groups with BMCs. The immunostaining for PCNA seems to be more expressive in the group AT-BMC 21 days group. To conclude, our results support the concept that in mice, the injection of BMCs improve glucose levels, lipid metabolism and remodeling of the aortic wall in animals using atherogenic diet. Keywords: Bone marrow cells, aorta remodeling, atherogenic diet

Introduction Atherosclerosis is a multifactorial vascular disorder initiated by endothelial dysfunction that develops progressively and is characterized by [1] by enhanced extracellular lipid content as well as necrotic cells in the subendothelial layer. The atherosclerotic plaque then undergoes fissure, erosion or rupture, leading to thrombosis of the plaque surface [1, 2]. Evidence suggests that endothelial dysfunction/injury is triggered by several factors such as increased blood pressure, obesity and shear stress in blood vessels [3, 4]. The difficulty of detecting atheroma plaques in humans via non-invasive methods further complicates our understanding of the development of this disease [5]. An unbalanced diet associated with a sedentary lifestyle favors the development of dyslipidemia, obesity, inflammation and vascular

injury [6, 7]. High intake of saturated fatty acids contributes to the development of hypercholesterolemia and atherosclerosis [8, 9]. Therefore, several animal models have been developed to better understand plaque development, one of which is a mouse model fed a high-fat diet with cholic acid and cholesterol [10, 11]. An atherogenic diet consisting of 15% fat, 1.25% cholesterol and 0.5% cholic acid is able to trigger atherosclerotic plaque formation in mouse aortas after 15 weeks [12]. Cholic acid is a bile acid that facilitates the absorption of fat and cholesterol excretion. When added to mice chow, it reduces plasma concentration of the apolipoprotein E (Apo E), affecting reverse cholesterol transport, which is an important mechanism that transports cholesterol from peripheral tissues to the liver, where it is excreted through the bile duct [13, 14]. Without this mechanism, the lipids accumulate in the plasma and may lead to the development of atherosclerosis.

Aorta remodeling in BMCs transplantation Bone marrow cells (BMCs) are the main source for cell transplantation therapies, and studies have shown that they are capable of remodeling activity in atherosclerosis [15]. In the bone marrow, there is a population of heterogeneous cells including blood lineage cells, hematopoietic stem cells, mesenchymal stem cells and endothelial progenitor cells [16-19]. Evidence has emerged suggesting that the vascular homing of endothelial progenitor cells (EPCs) contributes to endothelial recovery, thus limiting neointima formation after arterial injury. In patients with coronary artery disease, the number and function of EPCs have been linked with improved endothelial function or regeneration but have been inversely correlated with cardiovascular risk [20]. It is thought that endothelial progenitor cells can repair and renew arteries under specific physiological conditions. For instance, progenitor cells stabilize the atherosclerotic plaque in pathological conditions [21], and studies have demonstrated that infusion of bone marrow cells is neuroprotective after permanent cerebral ischemia [22]. Conversely, other studies have shown that endothelial progenitor cells contribute to the formation of the atherosclerotic lesions [23]. Thus, whether BMCs would further increase the injury or be beneficial is still controversial [15, 24]. The low number of circulating endothelial progenitor cells (EPCs), one type of progenitor cell, has emerged as a biomarker of cardiovascular risk in humans adults [25]. Because concern regarding morbidity and mortality due to obesity-associated atherosclerosis is growing, it is important to investigate the potential benefits or hazards associated with cell therapy. Therefore, this work aims to evaluate the effect of therapy with BMCs on lipid metabolism and aortic wall remodeling in C57Bl/6 mice through the administration of a high fat diet and subsequent BMCs transplantation. Materials and methods Animals and diet The study was performed in accordance with the Guide for Care and Use of Laboratory Animals 2011 from the National Academies Press, Washington, DC. All protocols were approved by the local ethics committee (CEUA/010/2011). Male C57BL/6 mice (n = 60, 10 weeks old, 27 g ± 1.5) were obtained from the Research 5528

Institute of the National Cancer Institute (INCA) and housed at the Animal Care Facility in the Laboratory of Histology and Embryology of the State University of Rio Janeiro (UERJ), Brazil. The room was both temperature and humidity controlled (temperature 21 ± 2°C, humidity 60 ± 10%, dark/light cycle 12 h/12 h). The male C57BL/6 mice were randomly allocated into two groups: the control group (CO group, n = 20 animals) was fed a standard diet (AIN 93 M) [26] for 16 weeks, and the atherogenic diet group (AT group, n = 40) was fed a modified atherogenic diet that consisted of 60% of fat (10% soy oil plus 50% lard), cholic acid (0.5%) and cholesterol (1.25%) for 16 weeks [11, 27]. After 16 weeks on the atherogenic diet (6 months old), the AT group (fed with a modified atherogenic diet) was randomly allocated to four groups: the AT 14 days group (n = 10); the AT-BMC 14 days group (n = 10); the AT 21 days group (n = 10); the AT-BMC 21 days group (n = 10). The AT 14 days group and AT 21 days group were fed a modified atherogenic diet for 16 weeks and at the end of the 16th week, the animals received 0.1 ml of PBS (PhosphateBuffered Saline) in the tail vein, besides they were sacrificed after 14 and 21 days respectively, after injection into the tail vein. The AT-BMC 14 days group and AT-BMC 21 days group were fed a modified atherogenic diet for 16 weeks and at the end of the 16th week, the animals received a transplant of BMCs (bone marrow cells) in 0.1 ml of PBS in tail vein, and they were sacrificed after 14 and 21 days respectively. During the entire experiment, all animals continued to receive the modified atherogenic diet. The animals in the CO group were sacrificed at the same time as those in the other groups (14 and 21 days). Water was offered ad libitum during the entire experiment. The body mass was measured at each stage of the study (g). Isolation of bone marrow cells (BMCs) and BMCs transplantation BMCs were obtained from male C57BL/6 mice (n = 10, 10 weeks old, 27 g ± 1.5, INCA-Rio de Janeiro). Mice were anaesthetized with pentobarbital (150 mg/kg), and BMCs were isolated from the tibias and femurs [22, 28]. The medul-

Int J Clin Exp Pathol 2014;7(9):5527-5537

Aorta remodeling in BMCs transplantation Table 1. Body mass and blood biochemistry CO Parameter Body mass, g Blood glucose, mg/dL (sacrifice day) Triglycerides, mg/dL Total cholesterol, mg/dL

AT 14 days

29.6 ± 1.0 37.34±1.6a 105.4 ± 9.1 200.6± 6.9a 61.1 ± 1.3 103.8± 9.9a 63.6 ± 10.6 130.4±12.4a

Experimental groups AT-BMC 14 days AT 21 days 34.4±31.1a 112±7.1b 53.16±2.8b 90.2±11.0

38.3 ± 2.5a 200.6 ± 7.9a,c 86.0 ± 8.2a,c 124.6 ± 13.1a

AT-BMC 21 days 36.6 ± 1.2a 167 ± 8.2a 50.0 ± 6.8b,c,d 59.6 ± 8.3b,d

Data are shown as mean ± standard error of the mean. Symbols represent statistical difference (P < 0.05) with: avs. CO group; bvs. AT 14 days group; cvs. AT-BMC 14 days group; dvs. AT 21 days group. Abbreviations: CO, mice fed with the standard diet during the experiment; AT 14 days group and AT 21 days group, mice fed with the atherogenic diet during 16 weeks, received vehicle into tail vein and sacrificed at 14 and 21 days after respectively; AT-BMC 14 days group and AT-BMC 21 days, mice fed with the atherogenic diet during 16 weeks and that received a single injection of BMCs (1×106 cells) into the tail vein at the 16th week and sacrificed at 14 and 21 days after respectively.

lar cavities of the bones were exposed and flushed with DMEM (Dulbecco’s Modified Eagle’s Medium, Sigma Aldrich-St. Louis, MO, USA), pH 7.2 [29]. The BMCs were resuspended in a red blood cell lysis buffer (10 mM NaHCO3, 150 mM NH4Cl, 0.4% EDTA, pH 7.4) for 10 minutes at 37°C. Then, the cells were washed and counted in a Neubauer chamber and adjusted to 106 cells per experiment for transplantation (BD Ultra-Fine II 0.5 ml syringe) into the tail vein. This pool of cells comprised bone marrow mononuclear cells [19]. Blood biochemistry Before the beginning of the experiment, the blood glucose levels were examined. The same glucose levels were examined in the day of the death. The mice were fasted for 6 hours and then anesthetized with pentobarbital (150 mg/ kg). The thoracic wall was opened through a median incision; then, blood was collected by cardiac puncture through the right atrium, centrifuged at 1500 rpm for 15 minutes and stored at -80°C. Blood glucose, triglycerides (TG) and total cholesterol (TC) were analyzed by an enzymatic colorimetric assay (Bioclin System II, Quibasa, Belo Horizonte, MG, Brazil). Histochemistry and immunohistochemistry The thoracic aorta and the adjacent aortic arch were isolated from the surrounding tissue. Nonconsecutive aortic rings (approximately 5 mm long) were immersed in freshly prepared 4% w/v formaldehyde (0.1 M phosphate buffer pH 7.2 for 48 h) for light microscopy and sectioned according to the vertical section method. Samples were processed according to routine histo5529

logical procedures, embedded in Paraplast plus (Sigma-Aldrich, St. Louis, USA), and cut into 5 µm sections. The sections were stained with Weigert resorcin fuchsin (with pre-oxidation with potassium peroxymonosulfate [oxone]) for visualization and quantification of elastic fibers [30, 31]. The following proteins were identified by immunohistochemical procedures: Vascular Endothelial Growth Factor (VEGF, 1:100, SC-7269, Santa Cruz Biotechnology), Smooth Muscle alpha-Actin (α-SMA, 1:100, SC-53142, Santa Cruz Biotechnology), Proliferating Cell Nuclear Antigen (PCNA, 1:100, ab-2426, Abcam). The reaction was amplified with a biotin-streptavidin complex system (K0679, Kit LSAB, Universal DakoCytomation, Glostrup, Denmark), and the immunoreactivity was determined after incubation with 3, 3’-diaminobenzidine tetrachloride (K3466, Universal DakoCytomation, Glostrup, Denmark). Sections were stained with hematoxylin to identify cell nuclei, and then the slides were mounted and analyzed. Morphometry Five non-consecutive digital images from aortic rings were acquired (TIFF format, 36-bit color) with an LC evolution camera and an Olympus BX51 light microscope. The tunica media (TM) was defined as the region enclosed by the internal and external elastic lamina. To estimate the intima and media thickness (TM thickness), four measurements per image were obtained at 0°, 90°, 180°, and 270° [32]. An area was delimited using the irregular AOI tool, and the area occupied by the white was quantified using an image histogram tool and expressed as percentage. This evaluation will be perInt J Clin Exp Pathol 2014;7(9):5527-5537

Aorta remodeling in BMCs transplantation

Figure 1. Quantitative analysis of cell nuclei number (Qa 1/mm2). Data are shown as mean ± standard error of the mean. Symbols represent statistical difference (P < 0.05) with: [a] vs. CO group; [b] vs. AT 14 days group; [c] vs. AT-BMC 21 days group; [d] vs. AT 21 days. Figure 3. Quantitative analysis of the percentage of elastic fibers within Tunica media. Abbreviations: CO, AT 21 days group, AT-BMC 21 days group. Data are shown as mean ± standard error of the mean. Symbols represent statistical difference (P < 0.05) with: [a] vs. CO group; [b] vs. AT 21 days group.

Figure 2. Quantitative analysis of thoracic aorta showing TM thickness. Abbreviations: CO, AT 21 days group, AT-BMC 21 days group. Data are shown as mean ± standard error of the mean. Symbols represent statistical difference (P < 0.05) with: [a] vs. CO group; [b] vs. AT 21 days group.

formed only in post-transplant 21 days groups for the final observation thickness. The quantification of the cell nuclei number of was performed starting from a test area of 36 points and 11.2 cm2 (Qa 1/mm2). Statistical analysis Data are shown as the mean and standard error of the mean (SEM). Differences among groups were analyzed by one-way ANOVA and a post-hoc Bonferroni test. A p value of 0.05 was considered statistically significant (Graph Pad® Prism version 5.0, San Diego, USA). Results Body mass and blood biochemistry The atherogenic diet was successful in promoting overweight and metabolic changes in C57Bl/6 mice. 5530

Body mass of the control group at the end of 16 weeks were 29.6 ± 1.0 g. The body mass of the AT group were 38.3 ± 2.5 g (P = 0.0027). The body mass were 37.34 ± 1.6 g in the AT 14 days group. The body mass were 34.4 ± 31.1 g in the AT-BMC 14 days group. The body mass were 38.3 ± 2.5 g in the AT 21 days group. The body mass were 36.6 ± 1.2 g in the AT-BMC 21 days group (Table 1). At the day of death, the blood glucose levels were 105.4 ± 9.1 mg/dL in CO group. In the AT 14 days group was 200.6 ± 6.9 mg/dL. In the AT-BMC 14 days group was 112 ± 7.1 mg/dL. In the AT 21 days group was 200.6 ± 7.9 mg/dL. In the AT-BMC 21 days group was 167 ± 8.2 mg/dL (Table 1). The TG levels were 61.1 ± 1.3 mg/dL in CO group. In the AT 14 days group was 103.8 ± 9.9 mg/dL. In the AT-BMC 14 days group was 53.16 ± 2.8 mg/dL. In the AT 21 days group was 86.0 ± 8.2 mg/dL. In the AT-BMC 21 days group was 50.0 ± 6.8 mg/dL (Table 1). The TC levels was 63.6 ± 10.6 mg/dL in CO group. In the AT 14 days group was 130.4 ± 12.4 mg/dL. In the AT-BMC 14 days group was 90.2 ± 11.0 mg/dL. In the AT 21 days group was 124.6 ± 13.1 mg/dL. In the AT-BMC 21 days group was 59.6 ± 8.3 mg/dL (Table 1). Aortic wall remodeling The Qa mean observed is 3751.56 1/mm2 in CO group. In the AT 14 days group was 2885.96 Int J Clin Exp Pathol 2014;7(9):5527-5537

Aorta remodeling in BMCs transplantation

Figure 4. Photomicrographs of the thoracic aorta stained for anti VEGF. The negative control of the immunohistochemical reaction is shown in (D). Groups: CO (A); AT 14 days group (B); AT-BMC 14 days group (C); AT 21 days group (E); AT-BMC 21 days group (F). All images are in same scale (bar = 50 µm). Arrows indicate markings.

1/mm2. In the AT-BMC 14 days was 3238.7 1/ mm2. In the AT 21 days group was 2636.12 1/ mm2. In the AT-BMC 21 days group was 5232.4 1/mm2 (Figure 1). The TM thickness mean was 48.9953 µm in CO group. In the AT 21 days group was 66.6183 µm. In the AT-BMC 21 days group was 57.7585 µm (Figure 2). The % of the elastic fiber mean was 55.754 in CO group. In the AT 21 days group was 63.868%. In the AT-BMC 21 days group was 56.364% (Figure 3). There was no difference in the number of elastic lamellae between among all study groups at any time during the study. Immunohistochemistry revealed that VEGF and α-SMA in the intima and media tunics were noticeably. The AT-BMC 14 days group and AT-BMC 21 days group shows equal and lower immunostaining than the AT 14 days group and AT 21 days group (Figures 4, 5). The immunostaining for PCNA showed increased labeling of nuclei in AT-BMC 21 days group (Figure 6). Discussion The data concerning the effect of BMCs on the physiopathology of several diseases, including 5531

atherosclerosis, are still inconclusive. We demonstrated the beneficial effects of BMCs transplantation on lipid metabolism and arterial wall remodeling in a mice model of atherogenic diet. It is important to explain that much of the effects caused by stem cell transplant does not relate the differentiation of these cells into the tissue cells similar but the paracrine effect that these cells are capable of generating in an organism transplanted. Cell therapy with endothelial progenitor cells is an emerging therapeutic option to promote angiogenesis or������ endothelial repair. Although the release of angiogenic paracrine factors is known to contribute to their therapeutic effect, little is known about their release of proinflammatory factors and expression of proinflammatory adhesion molecules [33]. BMCs transplantation successfully decreased blood glucose, triglycerides, total cholesterol and reduced media thickness in the aorta of overweight mice previously fed an atherogenic diet. The blood glucose revealed a reduction in their serum levels of animals subjected to injection of cells compared to groups that did not receive these cells. This reduction was 43% in AT-BMC 14 days group compared to the AT 14 days group and 16% in AT-BMC 21 days group compared to the AT 21 days group (P < 0.0001). The reduction of glucose is more efficient in the first weeks after injection of cells (observed in the AT-BMC 14 days group) Int J Clin Exp Pathol 2014;7(9):5527-5537

Aorta remodeling in BMCs transplantation

Figure 5. Photomicrographs of the thoracic aorta stained for anti α-SMA. The negative control of the immunohistochemical reaction is shown in (D). Groups: CO (A); AT 14 days group (B); AT-BMC 14 days group (C); AT 21 days group (E); AT-BMC 21 days group (F). All images are in same scale (bar = 50 µm). Arrows indicate markings.

Figure 6. Photomicrographs of the thoracic aorta immunohistochemistry for anti PCNA (A-C). The negative control of the immunohistochemical reaction is shown in (D). Groups: CO (A); AT 21 days group (B); AT-BMC 21 days group (C). The negative control of the immunohistochemical reaction is shown in (D). All images are in same scale (bar = 50 μm). Arrows indicate immunolabeling.

and does not display significant difference from the CO group. This is not observed in the AT-BMC 21 days group data when compared to CO group. The glucose levels in AT-BMC 14 days group was similar compared to the CO group, though the AT-BMC 21 days group showed no such similarity. The explanation for this finding is that during the whole experiment, animals continued receiving fat diet and the chemotactic effect of transplanted cells would not be enough so long after the injection to modify related to the levels of blood glucose mechanisms. Recent data show that diabetes increases both the number of bone marrow-derived smooth muscle-like cells in atherosclerotic lesion and the differentiation of mononuclear cells towards smooth muscle-like cells, which may contribute to accelerated atherosclerotic 5532

plaque formation in diabetic apoE(-/-) mice [34]. Another explanation for this finding is that even after transplantation with bone marrow cells, the stimulus for the development of atherosclerotic plaques is maintained, perhaps due to diabetes, because glucose levels remained high in our study. In addition, hyperglycemia promotes myelopoiesis and impairs the resolution of atherosclerosis [35]. The initial evaluation of triglyceride and total cholesterol was necessary to demonstrate the homogeneity and homocedasticity of the sample the beginnings of the study and after the division among groups. TC and TC were first assessed in the groups studied separately for further comprehensive assessment. The TC increased by 104% (P = 0.0189) in AT-14 days Int J Clin Exp Pathol 2014;7(9):5527-5537

Aorta remodeling in BMCs transplantation group and 96% (P = 0.05) in AT 21 days group when compared to CO. The TG increased by 69% (P = 0.05) in AT 14 days group and 40% (P = 0.05) in the AT 21 days group compared to CO group. When assessing the TG, the groups that received the cell injection, we observed no significant difference between these groups with the CO group, showing that transplantation was able to maintain the levels of normality this parameter. The same occurs in relation to the TC levels. No difference was observed in these two parameters versus time elapsed cell injection, either 14 or 21 days. Regarding the improvement of triglycerides and total cholesterol, our results demonstrate that the adverse remodeling that occurred in the aorta maintained vascular endothelial function. A whole and free remodeling of endothelium are less susceptible to dyslipidemia. With this endothelium hardly oxidized LDL molecules would be retained on the wall of the aorta, returning to the vessel by reverse cholesterol transport to the liver and be taken. This would be the explanation for the reduced levels of total cholesterol and in consequence with interlocking mechanisms, reducing triglyceride. Recent advances in understanding of how the bone marrow responds to endothelial injury now suggest that multiple bone marrow cell populations, including both endothelial progenitor cells and a novel group of cells called early outgrowth cells, promote endothelial repair and regeneration through different, yet complementary, mechanisms. Moreover, certain subsets of bone marrow-derived cells also appear to have novel, potent, angiogenesis-independent tissue-protective properties [36]. The key to reducing these levels would be the greatest integrity of the endothelium, as already seen in previous studies that bone marrow cells would be able to promote endothelial repair and regeneration. This would be extremely important since the cholesterol reverse transport and the removal of oxidized LDL in the vessel wall requires the integrity of the endothelial wall and to deliver these molecules being disposed in a pathway in the liver. The C57Bl/6 mouse is a useful strain for studying chronic diseases such as obesity, hypertension, insulin resistance, and dyslipidemia when saturated fat acids are added to the diet [37, 38]. When a high-fat diet is further supplemented with cholic acid and cholesterol, it becomes

5533

a useful model of atherosclerosis [11]. The cholic acid is a bile acid that, together with apolipoprotein E (ApoE), is involved in reverse cholesterol transport. This transport removes cholesterol from peripheral tissues to the liver. ApoE is mainly produced in the liver, though it can also be synthesized by other cells such as macrophages [39]. Our goal was to evaluate the role of BMCs in the prevention, rather than treatment, of an established lesion. The atherogenic diet successfully altered the lipid metabolism, as has already been shown by other researchers using similar high-fat diets [37-39]. It is still controversial, however, whether progenitor cell therapy would improve these metabolic changes as well as inhibit the progression of atherosclerosis [15]. The present study suggests that BMCs can positively influence lipid metabolism after transplantation. The reduction in total cholesterol might be associated with increased production of ApoE and thus increased reverse cholesterol transport. Additionally, the reduction in total cholesterol might have helped to normalize serum triglycerides. The Qa observed significantly reduced in AT 14 days group (P < 0.05) and AT 21 days group (P = 0.01) when compared to CO. Contrary to these groups, the AT-BMC 14 days group (P > 0.05) has shown the same cell number as well the CO and the AT-BMC 21 days group (+ 39%) becomes excessively increased (P < 0.0001). These cell nucleus station further increased when more time elapsed after transplantation of cells. Convincing evidence accumulated that dysregulation of apoptosis, the programmed cell death, of endothelial cells is involved in the pathophysiology of atherosclerosis [40]. This explains the decrease Qa in the groups receiving the atherogenic diet in additional to adverse remodeling of the vessel sustained by continuous exposure to a noxious stimulus. The increase in Qa in relation to AT-BMC 21 days group for all groups was observed and reviewed this increase was performed immunohistochemistry for PCNA. The PCNA is an auxiliary protein of DNA polymerase delta and is involved in the control of eukaryotic DNA replication by increasing the polymerase’s processibility during elongation of the leading strand. It was demonstrated that positively stained cells were peripheral blood monocytes that differentiated in the atherosclerotic lesion [41]. The immunohistochemistry for PCNA suggested that the cell prolifera-

Int J Clin Exp Pathol 2014;7(9):5527-5537

Aorta remodeling in BMCs transplantation tion in the arterial wall in the AT-BMC 21 days group was due to a paracrine effect from cells derived from bone marrow after being transplanted and was not due to the presence of inflammatory cells such as macrophages. These results should be further elucidated in future studies. The atherogenic diet induced adverse remodeling in aortic wall. The thoracic aortas of the AT 21 days group were thicker (35%, P < 0.001) and had a higher elastic fiber content (14%, P < 0.01) compared with the CO group. The tunica media of the AT-BMC 21 days group was 12% thinner (P < 0.001), and the percentage of elastic fibers in the media layer decreased by 13% compared to the AT 21 days group (P < 0.01). Aortic wall remodeling occurs due to physiological or pathological stimuli, such as hypertension and atherosclerosis [32, 42]. In this study, as expected, we observed increased thickness of the aortic wall and the elastic fiber system in the AT 21 days group due to the atherosclerotic stimulus within the aorta wall. The fact that remodeling did not occur in the aorta of the transplanted group suggested that there was no stimulus since it the reduced levels of circulating total cholesterol and triglycerides have been observed. The number of elastic lamellae did not vary between the groups because the initial production of elastic fibers occurred after the deposition of elastic lamelae. Alpha smooth muscle actin (α-SMA) is an important marker of fibrous plaque development in atherosclerosis. Bone marrow derived fibrocytes can acquire smooth muscle characteristics, including α-SMA expression [43]. Increasing evidence indicates that mechanical forces, cytokines and other factors influence the differentiation of stem cells into smooth muscle cells and endothelial cells [44]. However, the exact mechanism by which these cells improve the disease as well as the stage at which this improvement would occur is poorly understood. Positive α-SMA staining was observed in previous works that reported its role in the formation of fibrous plaques in atherosclerosis. Recently, the endothelial to mesenchymal cell transition, a newly recognized type of cellular transdifferentiation, revealed that endothelial cells lose their specific markers and acquire a mesenchymal or myofibroblastic phenotype, such as the α-SMA phenotype, in pathological fibro-

5534

sis in multisystemic diseases [43]. Our work showed a reduction in α-SMA in the AT-BMC 14 days group and AT-BMC 21 days group compared to the groups that received atherogenic diet without the injection of bone marrow cells (AT 14 days group and AT 21 days group, respectively), possibly due to reduced exposure of the aortic wall to cholesterol and triglycerides. Because this exposure is essential for the development of atherosclerosis, transplantation of BMCs would delay (or even prevent) the development of atherosclerotic plaques, resulting in less fibrosis and reduced transdifferentiation of endothelial cells. A reduction in the endothelial lesion could lead to better morphological organization in the transplantation group. There are some reports supporting the hypothesis that the smooth muscle and endothelial progenitors may act as a double-edged sword in the pathogenesis of atherosclerosis [15]. The smooth muscle cells forming the neointima would be derived from progenitor cells within the vessel wall and blood circulation [45]. The injection of smooth muscle cell progenitors may have a beneficial effect by reducing the progression of atherosclerotic plaques in mice [46]. Endothelial dysfunction stimulates the proliferation and migration of smooth muscle cells and increases the synthesis of extracellular matrix components, which explains the characteristic thickening of the tunica intima and thus the arterial wall. These smooth muscle cells may be recruited from circulating precursors or may derive from the adjacent tunica media [47]. These data support our results that showed an increase in immunolabeling for α-SMA and increased thickness of the aortic wall in the AT 21 days group. The BMC transplant reduced the α-SMA immunolabeling in the AT-BMC 14 days group and AT-BMC 21 days group as well as thickness in AT-21 days group. Vascular endothelial growth factor (VEGF) is a member of the superfamily of growth factors, and it has two main receptors in blood vessels: VEGFR-1 and VEGFR-2. In physiological condition, VEGF induces new vessel formation in processes such as tissue healing and scarring. Previous studies have shown that pulmonary hypertension increases VEGF [48] and that VEGF is present in atherosclerotic lesions during coronary artery disease [49]. The cellular cardiomyoplasty technique has proved effective in patients with transmural infarction [50]

Int J Clin Exp Pathol 2014;7(9):5527-5537

Aorta remodeling in BMCs transplantation after mononuclear cells from bone marrow aspirated from the ileum of 10 patients were injected onto the infarct zone. This approach resulted in significant benefit to patients in terms of a decrease in the perfusion defect [51]. As in the aforementioned studies, the increased expression of VEGF in the AT 14 days group and AT 21 days group was expected. The lighter VEGF stain in the AT-BMC 14 days group and AT-BMC 21 days group suggests that those animals aortas were less injured and therefore required less VEGF. In conclusion, the present study demonstrated that BMCs therapy improves blood glucose, lipid metabolism and the adverse aortic remodeling in C57Bl/6 mice fed an atherogenic diet. Although the data regarding beneficial effects of BMCs on atherosclerosis are controversial, our results contribute to the current knowledge favoring cell therapy. Acknowledgements The authors are grateful to the Brazilian Council of Science and Technology (CNPq), the Rio de Janeiro State Foundation for Scientific Research (FAPERJ), the Council for the Improvement of Graduate Personnel (CAPES), and the State University of Rio de Janeiro (UERJ). Address correspondence to: Dr. Alyne Souza Felix, Laboratory of Ultrastructure and Tissue Biology, Institute of Biology Roberto Alcantara Gomes, State University of Rio de Janeiro (UERJ), Bouvelard 28 de Setembro Avn, 77, Vila Isabel, Cep 20.551-030 - Rio de Janeiro-RJ, Brazil. E-mail: felixfonseca.alyne@ gmail.com

References [1]

[2] [3]

[4]

Mudau M, Genis A, Lochner A and Strijdom H. Endothelial dysfunction: the early predictor of atherosclerosis. Cardiovasc J Afr 2012; 23: 222-231. Lahoz C and Mostaza JM. Atherosclerosis as a systemic disease. Rev Esp Cardiol 2007; 60: 184-195. Beltowski J. Leptin and the regulation of endothelial function in physiological and pathological conditions. Clin Exp Pharmacol Physiol 2012; 39: 168-178. Meng, W, Yu F, Chen H, Zhang J, Zhang E, Guo Y and Shi Y. Relationship between concentration difference of different density lipoproteins and shear stress in atherosclerosis. Comput Math Methods Med 2012; 2012: 135256.

5535

[5]

[6]

[7] [8]

[9] [10]

[11]

[12]

[13] [14]

[15] [16] [17]

[18]

Haidari M, Ali M, Gangehei L, Chen M, Zhang W and Cybulsky MI. Increased oxidative stress in atherosclerosis-predisposed regions of the mouse aorta. Life Sci 2010; 87: 100-110. Pasanisi F, Contaldo F, de Simone G and Mancini M. Benefits of sustained moderate weight loss in obesity. Nutr Metab Cardiovasc Dis 2001; 11: 401-406. Lesi C, Giaquinto E, Valeriani L and Zoni L. [Diet prescription in obese patients]. Monaldi Arch Chest Dis 2005; 64: 42-44. Seo T, Qi K, Chang C, Liu Y, Worgall TS, Ramakrishnan R and Deckelbaum RJ. Saturated fatrich diet enhances selective uptake of LDL cholesteryl esters in the arterial wall. J Clin Invest 2005; 115: 2214-2222. Getz GS and Reardon CA. Diet and murine atherosclerosis. Arterioscler Thromb Vasc Biol 2006; 26: 242-249. Paigen B, Morrow A, Holmes PA, Mitchell D and Williams RA. Quantitative assessment of atherosclerotic lesions in mice. Atherosclerosis 1987; 68: 231-240. Schreyer SA, Wilson DL and Leboeuf RC. C57BL/6 mice fed high fat diets as models for diabetes-accelerated atherosclerosis. Atherosclerosis 1998; 136: 17-24. Qiao JH, Xie PZ, Fishbein MC, Kreuzer J, Drake TA, Demer LL and Lusis AJ. Pathology of atheromatous lesions in inbred and genetically engineered mice. Genetic determination of arterial calcification. Arterioscler Thromb 1994; 14: 1480-1497. Glomset JA. The plasma lecithins: cholesterol acyltransferase reaction. J Lipid Res 1968; 9: 155-167. Stein O, Dabach Y, Hollander G, Ben-Naim M, Halperin G and Stein Y. Effect of atherogenic diet on reverse cholesterol transport in vivo in atherosclerosis susceptible (C57BL/6) and resistant (C3H) mice. Atherosclerosis 2001; 156: 307-313. Campagnolo P, Wong MM and Xu Q. Progenitor cells in arteriosclerosis: good or bad guys? Antioxid Redox Signal 2011; 15: 1013-1027. Dotsenko O. Stem/Progenitor cells, atherosclerosis and cardiovascular regeneration. Open Cardiovasc Med J 2010; 4: 97-104. Janeczek Portalska K, Leferink A, Groen N, Fernandes H, Moroni L, Van blitterswijk C and De Boer J. Endothelial differentiation of mesenchymal stromal cells. PLoS One 2012; 7: e46842. Thole AA, Rodrigues-Cunha AC, Carvalho SN, Garcia-Souza EP, Cortez E, Stumbo AC, Carvalho L and Moura AS. Progenitor cells and TNFalpha involvement during morphological changes in pancreatic islets of obese mice. Tissue Cell 2012; 44: 238-248.

Int J Clin Exp Pathol 2014;7(9):5527-5537

Aorta remodeling in BMCs transplantation [19] Carvalho S, Da Cunha Lira D, Costa Cortez EA, De Andrade DC, Thole AA, Stumbo AC and De Carvalho L. Bone marrow cell transplantation is associated with fibrogenic cells apoptosis during hepatic regeneration in cholestatic rats. Biochem Cell Biol 2013; 91: 88-94. [20] Hristov M, Zernecke A, Schober A and Weber C. Adult progenitor cells in vascular remodeling during atherosclerosis. Biol Chem 2008; 389: 837-844. [21] Xu Q. Stem cells and transplant arteriosclerosis. Circ Res 2008; 102: 1011-1024. [22] Jiang C, Wang J, Yu L, Ou C, Liu X, Zhao X and Wang J. Comparison of the therapeutic effects of bone marrow mononuclear cells and microglia for permanent cerebral ischemia. Behav Brain Res 2013; 250: 222-229. [23] Fadini GP, Agostini C, Sartore S and Avogaro A. Endothelial progenitor cells in the natural history of atherosclerosis. Atherosclerosis 2007; 194: 46-54. [24] Jevon M, Dorling A and Hornick PI. Progenitor cells and vascular disease. Cell Prolif 2008; 41 Suppl 1: 146-164. [25] Glowinska-Olszewska B, Moniuszko M, Hryniewicz A, Jeznach M, Rusak M, Dabrowska M, Luczynski W, Bodzenta-Lukaszyk A and Bossowski A. Relationship between circulating endothelial progenitor cells and endothelial dysfunction in children with type 1 diabetes: a novel paradigm of early atherosclerosis in high-risk young patients. Eur J Endocrinol 2013; 168: 153-161. [26] Reeves PG, Nielsen FH and Fahey GC Jr. AIN93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet. J Nutr 1993; 123: 1939-1951. [27] Fraulob JC, Ogg-Diamantino R, FernandesSantos C, Aguila MB and Mandarim-de-Lacerda CA. A Mouse Model of Metabolic Syndrome: Insulin Resistance, Fatty Liver and NonAlcoholic Fatty Pancreas Disease (NAFPD) in C57BL/6 Mice Fed a High Fat Diet. J Clin Biochem Nutr 2010; 46: 212-223. [28] Fukushima S, Varela-Carver A, Coppen SR, Yamahara K, Felkin LE, Lee J, Barton PJ, Terracciano CM, Yacoub MH and Suzuki K. Direct intramyocardial but not intracoronary injection of bone marrow cells induces ventricular arrhythmias in a rat chronic ischemic heart failure model. Circulation 2007; 115: 2254-2261. [29] Carvalho SN, Lira DC, Oliveira GP, Thole AA, Stumbo AC, Caetano CE, Marques RG and Carvalho L. Decreased collagen types I and IV, laminin, CK-19 and alpha-SMA expression after bone marrow cell transplantation in rats

5536

[30]

[31]

[32]

[33]

[34]

[35]

[36]

[37]

[38]

[39] [40]

with liver fibrosis. Histochem Cell Biol 2010; 134: 493-502. Junqueira LC, Montes GS, Toledo OM, Bexiga SR, Gordilho MA and Brentani RR. Evidence for collagen molecular orientation in basement membranes. Histochem J 1983; 15: 785-794. Felix AS, Rocha VN, Nascimento AL and De Carvalho JJ. Carotid body remodelling in lNAME-induced hypertension in the rat. J Comp Pathol 2011; 146: 348-356. Moraes-Teixeira Jde A, Felix A, FernandesSantos C, Moura AS, Mandarim-de-Lacerda CA and de Carvalho JJ. Exercise training enhances elastin, fibrillin and nitric oxide in the aorta wall of spontaneously hypertensive rats. Exp Mol Pathol 2010; 89: 351-357. Zhang Y, Ingram DA, Murphy MP, Saadatzadeh MR, Mead LE, Prater DN and Rehman J. Release of proinflammatory mediators and expression of proinflammatory adhesion molecules by endothelial progenitor cells. Am J Physiol Heart Circ Physiol 2009; 296: H167582. Fledderus JO, Van Oostrom O, De Kleijn DP, Den Ouden K, Penders AF, Gremmels H, De Bree P and Verhaar MC. Increased amount of bone marrow-derived smooth muscle-like cells and accelerated atherosclerosis in diabetic apoE-deficient mice. Atherosclerosis 2013; 226: 341-347. Nagareddy PR, Murphy AJ, Stirzaker RA, Hu Y, Yu S, Miller RG, Ramkhelawon B, Distel E, Westerterp M, Huang LS, Schmidt AM, Orchard TJ, Fisher EA, Tall AR and Goldberg IJ. Hyperglycemia promotes myelopoiesis and impairs the resolution of atherosclerosis. Cell Metab 2013; 17: 695-708. Yuen DA, Gilbert RE and Marsden PA. Bone marrow cell therapies for endothelial repair and their relevance to kidney disease. Semin Nephrol 2012; 32: 215-23. Gallou-Kabani C, Vige A, Gross MS, Rabes JP, Boileau C, Larue-Achagiotis C, Tome D, Jais JP and Junien C. C57BL/6J and A/J mice fed a high-fat diet delineate components of metabolic syndrome. Obesity (Silver Spring) 2007; 15: 1996-2005. Catta-Preta M, Martins MA, Cunha Brunini TM, Mendes-Ribeiro AC, Mandarim-de-Lacerda CA and Aguila MB. Modulation of cytokines, resistin, and distribution of adipose tissue in C57BL/6 mice by different high-fat diets. Nutrition 2012; 28: 212-219. Stein O and Stein Y. Atheroprotective mechanisms of HDL. Atherosclerosis 1999; 144: 285-301. Schmidt-Lucke C, Fichtlscherer S, Rossig L, Kamper U and Dimmeler S. Improvement of

Int J Clin Exp Pathol 2014;7(9):5527-5537

Aorta remodeling in BMCs transplantation

[41]

[42]

[43]

[44] [45]

[46]

endothelial damage and regeneration indexes in patients with coronary artery disease after 4 weeks of statin therapy. Atherosclerosis 2010; 211: 249-254. Katsuda S, Coltrera MD, Ross R and Gown AM. Human atherosclerosis. IV. Immunocytochemical analysis of cell activation and proliferation in lesions of young adults. Am J Pathol 1993; 142: 1787-1793. Larifla L, Deprez I, Pham I, Rideau D, Louzier V, Adam M, Eloit M, Foucan L, Adnot S and Teiger E. Inhibition of vascular smooth muscle cell proliferation and migration in vitro and neointimal hyperplasia in vivo by adenoviral-mediated atrial natriuretic peptide delivery. J Gene Med 2012; 14: 459-467. Piera-Velazquez S, Li Z and Jimenez AS. Role of endothelial-mesenchymal transition (EndoMT) in the pathogenesis of fibrotic disorders. Am J Pathol 2011; 179: 1074-1080. Xu Q. The role of stem cells in atherosclerosis. Arch Mal Coeur Vaiss 2005; 98: 672-676. Hu Y, Mayr M, Metzler B, Erdel M, Davison F and Xu Q. Both donor and recipient origins of smooth muscle cells in vein graft atherosclerotic lesions. Circ Res 2002; 91: e13-20. Zoll J, Fontaine V, Gourdy P, Barateau V, Vilar J, Leroyer A, Lopes-Kam I, Mallat Z, Arnal JF, Henry P, Tobelem G and Tedgui A. Role of human smooth muscle cell progenitors in atherosclerotic plaque development and composition. Cardiovasc Res 2008; 77: 471-480.

5537

[47] Semenza GL. Vasculogenesis, angiogenesis, and arteriogenesis: mechanisms of blood vessel formation and remodeling. J Cell Biochem 2007; 102: 840-7. [48] Taraseviciene-Stewart L, Kasahara Y, Alger L, Hirth P, MC Mahon G, Waltenberger J, Voelkel NF and Tuder RM. Inhibition of the VEGF receptor 2 combined with chronic hypoxia causes cell death-dependent pulmonary endothelial cell proliferation and severe pulmonary hypertension. FASEB J 2001; 15: 427-438. [49] Inoue M, Itoh H, Ueda M, Naruko T, Kojima A, Komatsu R, Doi K, Ogawa Y, Tamura N, Takaya K, Igaki T, Yamashita J, Chun TH, Masatsugu K, Becker AE and Nakao K. Vascular endothelial growth factor (VEGF) expression in human coronary atherosclerotic lesions: possible pathophysiological significance of VEGF in progression of atherosclerosis. Circulation 1998; 98: 2108-2116. [50] Strauer BE, Brehm M, Zeus T, Kostering M, Hernandez A, Sorg RV, Kogler G and Wernet P. Repair of infarcted myocardium by autologous intracoronary mononuclear bone marrow cell transplantation in humans. Circulation 2002; 106: 1913-1918. [51] Reed DM, Foldes G, Harding SE and Mitchell JA. Stem cell-derived endothelial cells for cardiovascular disease: a therapeutic perspective. Br J Clin Pharmacol 2013; 75: 897-906.

Int J Clin Exp Pathol 2014;7(9):5527-5537