- Email: [email protected]
The role of clotting factor IX in the development of atherosclerosis
Accepted Manuscript The role of clotting factor IX in the development of atherosclerosis
Laís Ívina Ívina Silva de Paula, Aline Urban, Devanira Souza Paixão Costa, Erich V. de Paula, Joyce M. Annichino-Bizzacchi PII: DOI: Reference:
S0049-3848(17)30305-5 doi: 10.1016/j.thromres.2017.04.025 TR 6641
To appear in:
Thrombosis Research
Received date: Revised date: Accepted date:
17 October 2016 20 April 2017 25 April 2017
Please cite this article as: Laís Ívina Ívina Silva de Paula, Aline Urban, Devanira Souza Paixão Costa, Erich V. de Paula, Joyce M. Annichino-Bizzacchi , The role of clotting factor IX in the development of atherosclerosis, Thrombosis Research (2017), doi: 10.1016/j.thromres.2017.04.025
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.
ACCEPTED MANUSCRIPT The role of clotting factor IX in the development of atherosclerosis Laís Ívina Ívina Silva de Paula; Aline Urban, PhD; Devanira Souza Paixão Costa; Erich V de Paula; Joyce M Annichino-Bizzacchi
IP
T
Universidade Estadual de Campinas, Hemocentro, Cidade Universitária Zeferino Vaz Barão Geraldo, Campinas - SP, 13083-, 13083-878 Campinas, Brazil
US
CR
Dear Editors,
Atherosclerotic occlusive diseases (AOD) are the major cause of death worldwide.
AN
Acute episodes of AOD occur when slow-developing atherosclerotic plaques undergo acute rupture, followed by local deposition of platelets and fibrin, leading
M
to vessel obstruction [1]. The critical role of hemostasis in the acute phase of AOD
ED
is well demonstrated by the benefits of antithrombotic agents (targeting both
PT
platelets and thrombin generation) in these patients [2]. Atherosclerosis is a complex multifatorial process, and early studies suggested that
CE
mortality caused by AOD is lower in hemophiliacs and hemophilia carriers compared with the general population [3]. More recently, studies detecting
AC
atherosclerotic lesions by non-invasive methods in hemophiliacs suggested that the development of atherosclerotic lesions is similar when compared to normal individuals [4]. Accordingly, whether differences in cardiovascular mortality in hemophiliac patients result from decreased atherosclerosis, or from decreased thrombin generation at the time of plaque rupture remain to be determined. We have previously demonstrated that FVIII deficiency is consistent with normal
ACCEPTED MANUSCRIPT atherosclerotic lesion development in mice with LDL receptor (LDLR) deficiency, but not in a different atherosclerosis-prone model (apoE deficient mice). These results highlight the heterogenous effect of FVIII in different mice models [5]. Here we used a similar approach to investigate the role of coagulation factor IX factor in
IP
T
the initiation and progression of atherosclerosis.
CR
Material and methods
Animals were obtained by cross between mice of different initial genotypes,
US
comprising the following groups: 21 control animals deficient in apolipoprotein E
AN
(NL/APOE -/-) and 21 hemophilic B animals deficient in apolipoprotein E (HB/APOE -/ -). The knockout mouse for apoE was developed to represent a study
M
model for atherosclerosis. The gene encoding apoE was inactivated in the
ED
embryonic stem cells of a healthy mouse, and as a consequence these cells were inserted into blastomers of C57 mice. In 1997, using the plug-socket method of
PT
gene manipulation, grew up in a murine model of FIX deficient knockout, replacing
CE
the exon 3 promoter makes the factor IX gene by neoHPRT, which is a Functional gene, plus a partially deleted mini-hypoxanthine phosphoribosyl transferase gene.
AC
In a later study, Kung et al.demonstrated success in the use of C57B1 / 6 mice FIX in their experiments, with time correction of activated partial thromboplastin (aPTT) and hemorrhagic symptoms, using factor IX purified from human plasma [12]. Both FVIII knockout animals, as well as FIX knockout, did not show activity of the deficient factor, demonstrated by PTTA. [6]. The animals were kept on a normal diet until the age of 8 weeks and were then subjected to a Western type diet (21% fat and 0.15% cholesterol), prepared and
ACCEPTED MANUSCRIPT provided by the company PRAGSOLUÇÕES Bioscience based on the AIN-93. The crosses between the different models were carried out according to the techniques and standards of the central laboratory of UNICAMP (CEMIB), approved by ecological experimentation commission (EAEC) of the Institute of Biology of
T
Unicamp (number 1393-1). Animals were sacrificed after 8 or 22 weeks on
IP
Western diet. The animals were opened longitudinally and perfused slowly through
CR
a puncture in the left ventricle with 10 ml of ice cold PBS. The aorta was then dissected, removed and maintained in 10% buffered formalin for later analysis. The
US
heart was removed and embedded in OCT medium (Optimum cutting temperature
AN
- Sakura Finetek, Torrance, CA, USA), frozen on dry ice, and stored in a freezer 80 ° C for histological sections.
M
Quantitative analysis and distribution pattern of atherosclerotic injuries
ED
A staining with Sudan IV was performed to indicate the atherosclerotic plaques (Fig.1.a). The images were obtained and captured with the aid of a magnifying
PT
glass (Leica M205 FA) from Department of Histology of the Institute of Biology /
CE
Unicamp. The images were computer analyzed with the Image J program, and the results were obtained counting the percentage of stained areas on the total area of
AC
the aorta. A quantitative analysis was also performed in the aortic root, using hearts frozen in Tissue-Tek OCT medium and cut in a cryostat with 8 µm thick cuts. Initial zone of the aorta was used for the histological analysis of cardiac tissue. For each animal, 6 slides were analyzed after oil red-O and hematoxylin staining, and images were captured by a camera. The captured images were evaluated with the program Image J, and accounted in mm². Qualitative analysis of atherosclerotic lesions
ACCEPTED MANUSCRIPT We also carried out immunofluorescence analysis with the material obtained from the aortic root, using CF 4/80 (macrophage marker), and anti-α-actin (smooth muscle cell marker) antibodies. For each analysis, 3 to 5 animals per group were evaluated with AxioCam image captured by the system and subsequently analyzed
T
by Image J program. Antibody F4/80 was used for labeling aortic root sections of
IP
animals subjected to 8 weeks of diet, and the α-actin antibody were used for aortic
CR
root sections of animals at 22 weeks of diet. Determination of lipid profile
US
At the time of sacrifice, retro-orbital blood samples were collected with heparinized
AN
capillary, and cholesterol and triglycerides were measured in plasma by enzymatic method with Bioclin kit (Quibasa - MG / Brazil).
M
Statistical analysis
ED
The results of the experiments are presented as mean ± standard deviation. Statistical significance of the differences between the medians was evaluated by
PT
Mann-Whitney test for nonparametric variables. Differences were considered
AC
Results
CE
statistically when the p value was ≤ 0.05.
Cholesterol and triglyceride levels were not statistically different between HB/APOE-/- and NL/APOE -/- animals treated with high-cholesterol diet, as shown in Tables 1. (Table 1 Cholesterol and Triglycerides of disabled animals of apoE (NL / APOE - / -) and hemophilia B deficient apoE (HB / APOE - / -) on a high-cholesterol diet. Values represent the mean ± standard deviation.)
ACCEPTED MANUSCRIPT HB/APOE-/- and NL/APOE -/- animals presented similar aortic lesions after 8 weeks (1.8% and 1.6% respectively, p = 0.74) and 22 weeks of diet (8.1% and 7.2% respectively, p = 0.64). Data are represented in figure 1(b and c).
T
Fig.1.: (A) Histological cross-section of the aortic root deficient apoE animal (APOE - / -) and hemophilia B animal (HB / APOE - / -) subjected to 08 weeks of a 0.15% cholesterol diet, stained with Oil Red. (A1) Quantification of atherosclerosis in
IP
the aortic root of animals receiving the diet for 8 weeks, hemophilia B deficient apoE (HB / APOE -/-) and apoE deficient
CR
mice only (NL/APOE -/-). (A2) Quantification of atherosclerosis in the aortic root of animals receiving the diet for 22 weeks, hemophilia B deficient in apoE (HB/APO - /-), and apoE deficient mice only (NL / APOE - / -). (B) Entire aorta of HB/APOE - / - deficient mice subjected to 22 weeks of diet with 0.15% cholesterol, stained with Sudan IV. Reddish areas represent the
US
extensive atherosclerotic plaques (B.1) Quantification of atherosclerosis in the animals on a 8 week diet with 0.15% cholesterol in apoE-deficient mice (NL / APOE - / -) or hemophilia B / apoE deficient mice (HB / APOE - / -) (B.2) and
AN
quantification of atherosclerotic disease in animals at 22 weeks.
M
Aortic root showed no significant difference in the quantification of atherosclerotic
ED
disease among animals NL/APOE -/- and HB/APOE-/-, after 8 weeks (0.047 mm² and 0.053 mm², respectively, p = 0.22) and 22 weeks of diet (0.426 mm² and 0.423
CE
PT
mm², respectively; p = 0.64).
The patterns between both groups (HB/APOE -/- and NL/APOE - / -), in the
AC
qualitative evaluation related to smooth muscle (α-actin) (5.59% and 7.62%, respectively, p = 017), and the macrophages (F4/80) (6.74% and 8.46%, respectively p = 0.30) did not differ.
Discussion At the end of the last century some population studies showed that hemophilia patients had lower mortality rates resulting from ischemic heart disease compared
ACCEPTED MANUSCRIPT to the general population, even in the presence of risk factors such as high blood pressure [3, 6]. The assumptions to justify this protection were based on the lower survival of these patients, and lower thrombin generation and thrombus formation inability on the atheromatous plaque. Another possibility was that these patients
T
had a lower degree of arteriosclerosis, since there is an interrelationship between
IP
hemostasis and inflammation. However, more recent studies have shown
CR
conflicting results, with the description of increased mortality rates resulting from cardiovascular disease in hemophilic patients, when compared to the normal
US
population [8, 9, 10]. Recently our research group evaluated the role of severe
AN
deficiency of FVIII in the development of atherosclerosis in apoE deficient murine model (APOE -/-) [7]. We showed a protective effect of this deficiency in the
M
formation of atherosclerotic plaque. Thus, these results are very challenging, as
ED
they are not consistent with the clinical findings published more recently. Severe FIX deficiency also produces the same degree of hypocoagulability observed in
PT
animals with hemophilia A. In this study, using the same dyslipidemic model, we
CE
investigated the interaction of coagulation factor IX in the development of atherosclerosis, from the earliest stages until the middle stage of the disease.
AC
Supposing the protective effect observed in animals with hemophilia A could also to be found in hemophilia B model, the direct role of hypocoagulability on the atherosclerotic process would be supported. To this end, we crossed apoE deficient animals with hemophilia B. We observed that the two groups developed atherosclerotic plaques, and similarly along the entire length of the aorta and in the aortic root. Therefore, the severe deficiency of FIX did not protect the animals from the development of atherosclerotic plaques in the aortic areas evaluated after
ACCEPTED MANUSCRIPT periods of 8 and 22 weeks. The difference of our results in relation to those of previous studies with hemophilic A mice conducted by our group, can be related to the effect of factor VIII on other systems, not directly associated to blood clotting [6]. FVIII is one of PCa substrate, presents independent coagulation cell effects,
T
mediated in part by ligation to PAR-1 receptors. Through these pathways, PCa
IP
exerts multiple cytoprotective effects, such as anti-inflammatory, anti-apoptotic
CR
activity, control of genetic expressions of proteins of barrier endothelial function [11]. When there is a significant reduction of FVIII, a high availability of PCa can
US
cause increase in the anti-inflammatory and cytoprotective effects. In the case of
AN
FIX deficiency, this mechanism is not present.
M
Conclusions
ED
Our results showed that atherosclerosis develops in hemophilic B animals. With the improvements in treatment for haemophilia and prolonged survival and life
PT
expectancy, atherosclerosis and cardiovascular disease would certainly be on the
AC
References
CE
rise in these patients
[1] Conlan MG, Folsom AR, Finch A, Davis CE, Sorlie P, Marcucci G, Wu KK. Associations of factor VIII and von Willebrand factor with age, race, sex, and risk factors for atherosclerosis. The Atherosclerosis Risk in Communities (ARIC) Study. Thromb Haemost 1993; 70: 380–5.
ACCEPTED MANUSCRIPT [2] Plump AS, Smith JD, Hayek T, Aalto KS, Walsh A, Verstuyft JG, Rubin EM, Breslow JL. Severe hypercholesterolemia and atherosclerosis in apolipoprotein
T
Edeficient mice created by homologous in ES cells. Cell 1992; (71): 343-353.
IP
[3] Sramek A, Kriek M, Rosendaal FR. Decreased mortality of ischaemic heart
CR
disease among carriers of haemophilia. Lancet 2003; 362: 351–4.
US
[4] Rosendaal FR, Varekamp I, Smit C, Brocker-Vriends AH, van Dijck H,
AN
Vandenbroucke JP, Hermans J, Suurmeijer TP, Briet E. Mortality and causes of
M
death in Dutch haemophiliacs, 1973–86. Br J Haematol 1989; 71: 71–6.
ED
[5] Fabri DR, De Paula EV, COSTA D S P, Annichino-bizzacchi J M, Arruda V R. Novel insights into the development of atherosclerosis in hemophilia A mouse
CE
PT
models. Journal of Thrombosis and Haemostasis.2011; 9: 1556–1561.
[6] Lin HF, Maeda N, Smithies O, Straight DL, Stafford DW: A coagulation factor IX
AC
deficient mouse model for human hemophilia B. Blood 1997; (90): 3962- 3966.
[6] Vancine, S. M., S. K. Picoli-Quaino, D. S. Costa, S. A. Montalvao, M. C. Ozelo, J. M. Annichino-Bizzacchi and E. V. de Paula. "Evaluation of the host response to endotoxemia of FVIII and FIX deficient mice." Haemophilia. 2011; 17(5): 800-807.
ACCEPTED MANUSCRIPT [7] Biere-Rafi S, Zwiers M, Peters M, et al. The effect of haemophilia and von Willebrand disease on arterial thrombosis: a systematic review. Neth J Med. 2010; 68(5):207-214.
T
[8] L¨ovdahl S, Henriksson KM, Baghaei F, et al. Incidence, mortality rates and
IP
causes of deaths in haemophilia patients in Sweden. Haemophilia. 2013;
CR
19(3):362-369.
US
[9] Sharathkumar AA, Soucie JM, Trawinski B, Greist A, Shapiro AD. Prevalence
AN
and risk factors of cardiovascular disease (CVD) events among patients with haemophilia: experience of a single haemophilia treatment centre in the United
ED
M
States (US). Haemophilia. 2011;17(4): 597-604.
[10] Levi M, van der Pollt, Buller HR. Bidirectional relation between inflammation
CE
PT
and coagulation. Circulation. 2004; 109 (22): 2698-704.
[11] Mosnier LO, Zlokovic BV, Griffin JH. The cytoprotective protein C pathway.
AC
Blood 2007 (109): 3161-3172.
[12] Kung S-H, Hagstrom JN, Cass D, Tai SJ, Lin H-F, Stafford DW, High KA. Human factor IX corrects the bleeding diathesis of mice with hemophilia B. Blood 1998; (91): 784-790.
ACCEPTED MANUSCRIPT Table 1 Diet time Genotype
n
(weeks) 8
HB/APOE 10
Triglycerides p
Cholesterol p
(mg/dL)
(mg/dL) 0.62
329±150
877±59
0.14
08
311±52
804±94
IP
NL/APOE
T
-/-
328±120
0.14
424±165
US
HB/APOE 11 -/11
AN
NL/APOE
CE
PT
ED
M
-/-
AC
22
CR
-/-
781±159
864±24
0.10
CR
IP
T
ACCEPTED MANUSCRIPT
AC
CE
PT
ED
M
AN
US
Fig. 1
ACCEPTED MANUSCRIPT Highlights
T IP CR US AN M ED PT
CE
The severe deficiency of FIX did not protect the animals from the development of atherosclerotic plaques in the aortic areas evaluated after periods of 8 and 22 weeks. In the qualitative evaluation related to smooth muscle (a-actin), and macrophages did not differ between the control and experimental groups. With the improvements in treatment for haemophilia and prolonged survival and life expectancy, atherosclerosis and cardiovascular disease would certainly be on the rise in these patients.
AC
Laís Ívina Ívina Silva de Paula, Aline Urban, Devanira Souza Paixão Costa, Erich V. de Paula, Joyce M. Annichino-Bizzacchi PII: DOI: Reference:
S0049-3848(17)30305-5 doi: 10.1016/j.thromres.2017.04.025 TR 6641
To appear in:
Thrombosis Research
Received date: Revised date: Accepted date:
17 October 2016 20 April 2017 25 April 2017
Please cite this article as: Laís Ívina Ívina Silva de Paula, Aline Urban, Devanira Souza Paixão Costa, Erich V. de Paula, Joyce M. Annichino-Bizzacchi , The role of clotting factor IX in the development of atherosclerosis, Thrombosis Research (2017), doi: 10.1016/j.thromres.2017.04.025
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.
ACCEPTED MANUSCRIPT The role of clotting factor IX in the development of atherosclerosis Laís Ívina Ívina Silva de Paula; Aline Urban, PhD; Devanira Souza Paixão Costa; Erich V de Paula; Joyce M Annichino-Bizzacchi
IP
T
Universidade Estadual de Campinas, Hemocentro, Cidade Universitária Zeferino Vaz Barão Geraldo, Campinas - SP, 13083-, 13083-878 Campinas, Brazil
US
CR
Dear Editors,
Atherosclerotic occlusive diseases (AOD) are the major cause of death worldwide.
AN
Acute episodes of AOD occur when slow-developing atherosclerotic plaques undergo acute rupture, followed by local deposition of platelets and fibrin, leading
M
to vessel obstruction [1]. The critical role of hemostasis in the acute phase of AOD
ED
is well demonstrated by the benefits of antithrombotic agents (targeting both
PT
platelets and thrombin generation) in these patients [2]. Atherosclerosis is a complex multifatorial process, and early studies suggested that
CE
mortality caused by AOD is lower in hemophiliacs and hemophilia carriers compared with the general population [3]. More recently, studies detecting
AC
atherosclerotic lesions by non-invasive methods in hemophiliacs suggested that the development of atherosclerotic lesions is similar when compared to normal individuals [4]. Accordingly, whether differences in cardiovascular mortality in hemophiliac patients result from decreased atherosclerosis, or from decreased thrombin generation at the time of plaque rupture remain to be determined. We have previously demonstrated that FVIII deficiency is consistent with normal
ACCEPTED MANUSCRIPT atherosclerotic lesion development in mice with LDL receptor (LDLR) deficiency, but not in a different atherosclerosis-prone model (apoE deficient mice). These results highlight the heterogenous effect of FVIII in different mice models [5]. Here we used a similar approach to investigate the role of coagulation factor IX factor in
IP
T
the initiation and progression of atherosclerosis.
CR
Material and methods
Animals were obtained by cross between mice of different initial genotypes,
US
comprising the following groups: 21 control animals deficient in apolipoprotein E
AN
(NL/APOE -/-) and 21 hemophilic B animals deficient in apolipoprotein E (HB/APOE -/ -). The knockout mouse for apoE was developed to represent a study
M
model for atherosclerosis. The gene encoding apoE was inactivated in the
ED
embryonic stem cells of a healthy mouse, and as a consequence these cells were inserted into blastomers of C57 mice. In 1997, using the plug-socket method of
PT
gene manipulation, grew up in a murine model of FIX deficient knockout, replacing
CE
the exon 3 promoter makes the factor IX gene by neoHPRT, which is a Functional gene, plus a partially deleted mini-hypoxanthine phosphoribosyl transferase gene.
AC
In a later study, Kung et al.demonstrated success in the use of C57B1 / 6 mice FIX in their experiments, with time correction of activated partial thromboplastin (aPTT) and hemorrhagic symptoms, using factor IX purified from human plasma [12]. Both FVIII knockout animals, as well as FIX knockout, did not show activity of the deficient factor, demonstrated by PTTA. [6]. The animals were kept on a normal diet until the age of 8 weeks and were then subjected to a Western type diet (21% fat and 0.15% cholesterol), prepared and
ACCEPTED MANUSCRIPT provided by the company PRAGSOLUÇÕES Bioscience based on the AIN-93. The crosses between the different models were carried out according to the techniques and standards of the central laboratory of UNICAMP (CEMIB), approved by ecological experimentation commission (EAEC) of the Institute of Biology of
T
Unicamp (number 1393-1). Animals were sacrificed after 8 or 22 weeks on
IP
Western diet. The animals were opened longitudinally and perfused slowly through
CR
a puncture in the left ventricle with 10 ml of ice cold PBS. The aorta was then dissected, removed and maintained in 10% buffered formalin for later analysis. The
US
heart was removed and embedded in OCT medium (Optimum cutting temperature
AN
- Sakura Finetek, Torrance, CA, USA), frozen on dry ice, and stored in a freezer 80 ° C for histological sections.
M
Quantitative analysis and distribution pattern of atherosclerotic injuries
ED
A staining with Sudan IV was performed to indicate the atherosclerotic plaques (Fig.1.a). The images were obtained and captured with the aid of a magnifying
PT
glass (Leica M205 FA) from Department of Histology of the Institute of Biology /
CE
Unicamp. The images were computer analyzed with the Image J program, and the results were obtained counting the percentage of stained areas on the total area of
AC
the aorta. A quantitative analysis was also performed in the aortic root, using hearts frozen in Tissue-Tek OCT medium and cut in a cryostat with 8 µm thick cuts. Initial zone of the aorta was used for the histological analysis of cardiac tissue. For each animal, 6 slides were analyzed after oil red-O and hematoxylin staining, and images were captured by a camera. The captured images were evaluated with the program Image J, and accounted in mm². Qualitative analysis of atherosclerotic lesions
ACCEPTED MANUSCRIPT We also carried out immunofluorescence analysis with the material obtained from the aortic root, using CF 4/80 (macrophage marker), and anti-α-actin (smooth muscle cell marker) antibodies. For each analysis, 3 to 5 animals per group were evaluated with AxioCam image captured by the system and subsequently analyzed
T
by Image J program. Antibody F4/80 was used for labeling aortic root sections of
IP
animals subjected to 8 weeks of diet, and the α-actin antibody were used for aortic
CR
root sections of animals at 22 weeks of diet. Determination of lipid profile
US
At the time of sacrifice, retro-orbital blood samples were collected with heparinized
AN
capillary, and cholesterol and triglycerides were measured in plasma by enzymatic method with Bioclin kit (Quibasa - MG / Brazil).
M
Statistical analysis
ED
The results of the experiments are presented as mean ± standard deviation. Statistical significance of the differences between the medians was evaluated by
PT
Mann-Whitney test for nonparametric variables. Differences were considered
AC
Results
CE
statistically when the p value was ≤ 0.05.
Cholesterol and triglyceride levels were not statistically different between HB/APOE-/- and NL/APOE -/- animals treated with high-cholesterol diet, as shown in Tables 1. (Table 1 Cholesterol and Triglycerides of disabled animals of apoE (NL / APOE - / -) and hemophilia B deficient apoE (HB / APOE - / -) on a high-cholesterol diet. Values represent the mean ± standard deviation.)
ACCEPTED MANUSCRIPT HB/APOE-/- and NL/APOE -/- animals presented similar aortic lesions after 8 weeks (1.8% and 1.6% respectively, p = 0.74) and 22 weeks of diet (8.1% and 7.2% respectively, p = 0.64). Data are represented in figure 1(b and c).
T
Fig.1.: (A) Histological cross-section of the aortic root deficient apoE animal (APOE - / -) and hemophilia B animal (HB / APOE - / -) subjected to 08 weeks of a 0.15% cholesterol diet, stained with Oil Red. (A1) Quantification of atherosclerosis in
IP
the aortic root of animals receiving the diet for 8 weeks, hemophilia B deficient apoE (HB / APOE -/-) and apoE deficient
CR
mice only (NL/APOE -/-). (A2) Quantification of atherosclerosis in the aortic root of animals receiving the diet for 22 weeks, hemophilia B deficient in apoE (HB/APO - /-), and apoE deficient mice only (NL / APOE - / -). (B) Entire aorta of HB/APOE - / - deficient mice subjected to 22 weeks of diet with 0.15% cholesterol, stained with Sudan IV. Reddish areas represent the
US
extensive atherosclerotic plaques (B.1) Quantification of atherosclerosis in the animals on a 8 week diet with 0.15% cholesterol in apoE-deficient mice (NL / APOE - / -) or hemophilia B / apoE deficient mice (HB / APOE - / -) (B.2) and
AN
quantification of atherosclerotic disease in animals at 22 weeks.
M
Aortic root showed no significant difference in the quantification of atherosclerotic
ED
disease among animals NL/APOE -/- and HB/APOE-/-, after 8 weeks (0.047 mm² and 0.053 mm², respectively, p = 0.22) and 22 weeks of diet (0.426 mm² and 0.423
CE
PT
mm², respectively; p = 0.64).
The patterns between both groups (HB/APOE -/- and NL/APOE - / -), in the
AC
qualitative evaluation related to smooth muscle (α-actin) (5.59% and 7.62%, respectively, p = 017), and the macrophages (F4/80) (6.74% and 8.46%, respectively p = 0.30) did not differ.
Discussion At the end of the last century some population studies showed that hemophilia patients had lower mortality rates resulting from ischemic heart disease compared
ACCEPTED MANUSCRIPT to the general population, even in the presence of risk factors such as high blood pressure [3, 6]. The assumptions to justify this protection were based on the lower survival of these patients, and lower thrombin generation and thrombus formation inability on the atheromatous plaque. Another possibility was that these patients
T
had a lower degree of arteriosclerosis, since there is an interrelationship between
IP
hemostasis and inflammation. However, more recent studies have shown
CR
conflicting results, with the description of increased mortality rates resulting from cardiovascular disease in hemophilic patients, when compared to the normal
US
population [8, 9, 10]. Recently our research group evaluated the role of severe
AN
deficiency of FVIII in the development of atherosclerosis in apoE deficient murine model (APOE -/-) [7]. We showed a protective effect of this deficiency in the
M
formation of atherosclerotic plaque. Thus, these results are very challenging, as
ED
they are not consistent with the clinical findings published more recently. Severe FIX deficiency also produces the same degree of hypocoagulability observed in
PT
animals with hemophilia A. In this study, using the same dyslipidemic model, we
CE
investigated the interaction of coagulation factor IX in the development of atherosclerosis, from the earliest stages until the middle stage of the disease.
AC
Supposing the protective effect observed in animals with hemophilia A could also to be found in hemophilia B model, the direct role of hypocoagulability on the atherosclerotic process would be supported. To this end, we crossed apoE deficient animals with hemophilia B. We observed that the two groups developed atherosclerotic plaques, and similarly along the entire length of the aorta and in the aortic root. Therefore, the severe deficiency of FIX did not protect the animals from the development of atherosclerotic plaques in the aortic areas evaluated after
ACCEPTED MANUSCRIPT periods of 8 and 22 weeks. The difference of our results in relation to those of previous studies with hemophilic A mice conducted by our group, can be related to the effect of factor VIII on other systems, not directly associated to blood clotting [6]. FVIII is one of PCa substrate, presents independent coagulation cell effects,
T
mediated in part by ligation to PAR-1 receptors. Through these pathways, PCa
IP
exerts multiple cytoprotective effects, such as anti-inflammatory, anti-apoptotic
CR
activity, control of genetic expressions of proteins of barrier endothelial function [11]. When there is a significant reduction of FVIII, a high availability of PCa can
US
cause increase in the anti-inflammatory and cytoprotective effects. In the case of
AN
FIX deficiency, this mechanism is not present.
M
Conclusions
ED
Our results showed that atherosclerosis develops in hemophilic B animals. With the improvements in treatment for haemophilia and prolonged survival and life
PT
expectancy, atherosclerosis and cardiovascular disease would certainly be on the
AC
References
CE
rise in these patients
[1] Conlan MG, Folsom AR, Finch A, Davis CE, Sorlie P, Marcucci G, Wu KK. Associations of factor VIII and von Willebrand factor with age, race, sex, and risk factors for atherosclerosis. The Atherosclerosis Risk in Communities (ARIC) Study. Thromb Haemost 1993; 70: 380–5.
ACCEPTED MANUSCRIPT [2] Plump AS, Smith JD, Hayek T, Aalto KS, Walsh A, Verstuyft JG, Rubin EM, Breslow JL. Severe hypercholesterolemia and atherosclerosis in apolipoprotein
T
Edeficient mice created by homologous in ES cells. Cell 1992; (71): 343-353.
IP
[3] Sramek A, Kriek M, Rosendaal FR. Decreased mortality of ischaemic heart
CR
disease among carriers of haemophilia. Lancet 2003; 362: 351–4.
US
[4] Rosendaal FR, Varekamp I, Smit C, Brocker-Vriends AH, van Dijck H,
AN
Vandenbroucke JP, Hermans J, Suurmeijer TP, Briet E. Mortality and causes of
M
death in Dutch haemophiliacs, 1973–86. Br J Haematol 1989; 71: 71–6.
ED
[5] Fabri DR, De Paula EV, COSTA D S P, Annichino-bizzacchi J M, Arruda V R. Novel insights into the development of atherosclerosis in hemophilia A mouse
CE
PT
models. Journal of Thrombosis and Haemostasis.2011; 9: 1556–1561.
[6] Lin HF, Maeda N, Smithies O, Straight DL, Stafford DW: A coagulation factor IX
AC
deficient mouse model for human hemophilia B. Blood 1997; (90): 3962- 3966.
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ACCEPTED MANUSCRIPT Table 1 Diet time Genotype
n
(weeks) 8
HB/APOE 10
Triglycerides p
Cholesterol p
(mg/dL)
(mg/dL) 0.62
329±150
877±59
0.14
08
311±52
804±94
IP
NL/APOE
T
-/-
328±120
0.14
424±165
US
HB/APOE 11 -/11
AN
NL/APOE
CE
PT
ED
M
-/-
AC
22
CR
-/-
781±159
864±24
0.10
CR
IP
T
ACCEPTED MANUSCRIPT
AC
CE
PT
ED
M
AN
US
Fig. 1
ACCEPTED MANUSCRIPT Highlights
T IP CR US AN M ED PT
CE
The severe deficiency of FIX did not protect the animals from the development of atherosclerotic plaques in the aortic areas evaluated after periods of 8 and 22 weeks. In the qualitative evaluation related to smooth muscle (a-actin), and macrophages did not differ between the control and experimental groups. With the improvements in treatment for haemophilia and prolonged survival and life expectancy, atherosclerosis and cardiovascular disease would certainly be on the rise in these patients.
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