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Prepublished online as a Blood First Edition Paper on July 25, 2002; DOI 10.1182/blood-2002-03-0945.
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the First Department of Internal Medicine, Nagoya
University School of Medicine, Aichi Blood Disease Research Foundation,
Department of Medical Technology, Nagoya University School of Health
Sciences, and Nagoya National Hospital, Nagoya, Japan;
Department of Basic Gerontology, National Institute for Longevity
Sciences, Obu, Japan; and Department of Vascular Biology
(VB-3), The Scripps Research Institute, La Jolla, CA.
Hypercoagulability and thrombotic tendency are frequently induced
by a variety of stressors. Clinically, aged subjects and obese patients
are more susceptible to thrombotic diseases associated with stress, but
the underlying mechanisms are unknown. We investigated the expression
of a procoagulant gene, tissue factor (TF), in a mouse model of
restraint stress. Twenty hours of restraint stress to mice caused a
substantial induction of TF mRNA in several tissues. Importantly, the magnitude of induction of TF mRNA by restraint stress
was larger in aged mice compared with young mice. In situ hybridization
analysis of the stressed aged mice revealed that strong signals for TF
mRNA were localized to renal epithelial cells, smooth muscle cells,
adventitial cells, and adipocytes but not to vascular endothelial
cells. These observations suggest that restraint stress induces the TF
expression in a tissue-specific and cell type-specific manner.
Genetically obese mice were also hyperresponsive to restraint stress in
the induction of TF gene, especially in their livers and adipose
tissues. Stress-induced microthrombi formation was pronounced in renal
glomeruli and within the vasculature in adipose tissues of aged mice.
Tumor necrosis factor- The stress response is thought to be required
for the maintenance of homeostasis and is characterized by rapid
changes in the gene expression of stress proteins.1 This
response is best demonstrated in the neuro-endocrine system (eg, the
hypothalamic-pituitary-adrenal axis)2 and, in this setting,
may be mediated by the activation of the glucocorticoid
cascade3 and of the sympathetic nervous system.4,5 The restraint stress model often has been used to investigate the stress response experimentally in terms of pharmacologic, physiologic, or pathologic phenomena in
vivo.6 For example, restraint stress induces the
expression of heat shock protein, a typical stress protein, in the
rat,2,4 and this induction may be mediated by the
sympathetic nervous system.4,5 The induction of stress
proteins may contribute to the development of a number of clinically
relevant phenomena, including tissue and organ damage, and the immune
response.7 Hypercoagulability in the blood and thrombotic
tendency also may be induced by physical8,9 and mental
stress.10 In this context, aged animals2-5
and/or obese mice11,12 may have lower tolerance to stress
insults. Clinically, older and obese individuals are susceptible to the stress-mediated pathologic changes, including thrombotic
complications,13,14 possibly because of the stress-induced
imbalance in the coagulation and fibrinolytic system.
Tissue factor (TF) is the primary cellular initiator of the coagulation
protease cascade and serves as a specific cofactor for plasma factors
VII/VIIa.15 TF is constitutively expressed by several
extravascular cell types (eg, epithelial cells, adventitial cells,
adipocytes) and is inducibly expressed by monocytes and endothelial
cells within the vasculature. Cis-acting regulatory elements
within the human TF promoter would control constitutive and inducible
expression in various cell types.16 TF gene expression appears to be regulated by a variety of transcription factors (eg,
nuclear factor- We previously reported that plasminogen activator inhibitor-1 (PAI-1)
and/or antithrombin was involved in the development of renal glomerular
thrombosis induced by restraint stress.23,24 In the
present study, we analyzed the gene expression of another key molecule
for thrombosis, TF, in a murine model of restraint stress in vivo.
Furthermore, we investigated the effects of aging and obesity on the
stress-induced TF expression and tissue thrombosis by using extremely
aged (12- and 24-month-old) mice and genetically obese (C57BL/6J ob/ob)
mice. Overnight restraint stress to mice substantially induced TF mRNA
expression in some tissues. More importantly, the stress-induced TF
expression and/or microvascular thrombosis were pronounced in aged mice
and in obese mice. Finally, endogenous TNF- Restraint stress
In separate experiments, 8-week- and 24-month-old male C57BL/6J mice
were pretreated intraperitoneally either with control (nonimmune)
hamster immunoglobulin G (IgG; 25 µg/mouse; Genzyme, Cambridge, MA)
or with the IgG fraction of a neutralizing hamster monoclonal antibody
(mAb) to mouse TNF- RNA extraction and quantitative RT-PCR assay
Statistical analysis Statistical analysis of all quantitative RT-PCR results was performed by using the unpaired Student t test. Differences were not considered significant when P > .05 (group size, n = 6 or 8).Determination of TNF-  antigen in plasma (picogram per milliliter was
measured using the ELISA-view kit (BioSource International, Camarillo, CA).
In situ hybridization In situ hybridization was performed using 35S-labeled antisense riboprobes, as described previously.26 After hybridization, the slides were dehydrated by immersion in a graded alcohol series containing 0.3 M NH4Ac and dried. Then the slides were coated with NTB2 emulsion (Kodak, Rochester, NY; 1:2 in water), and exposed in the dark at 4°C for 8 to 12 weeks. The slides were developed for 2 minutes in D19 developer (Kodak), fixed, washed in water, and counterstained with hematoxylin/eosin. No specific hybridization signal could be detected in parallel sections using 35S-labeled sense probes in each experiment (not shown).Preparation of antimouse TF antibody and Western blot analysis Polyclonal rabbit antiserum specific for mouse TF (2 mg/mL, 1:2000 dilution in Tris (tris(hydroxymethyl)aminomethane)-buffered saline (TBS) containing 0.1% Tween-20 (TBS-T) was kindly provided by Drs K. Enjyoji and H. Kato, National Cardiovascular Center and Research Institute, Osaka, Japan. This specific antibody was raised in rabbits by the direct introduction of the encoding cDNA of mouse TF cloned into the pcDNA3 plasmid as described previously.27,28 Serum titers and the specificity of the antibody were determined by standard Western blot analysis using protein extracts from COS-7 cells expressing mouse TF and recombinant mouse TF fused to glutathione-S-transferase protein (Amersham Pharmacia Biotech, Tokyo, Japan) (not shown). TF antigen in the lysates of adipose tissues obtained from obese and lean mice after 0, 2, and 20 hours of restraint stress was determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis using the enhanced chemiluminescent (ECL) detection system (Amersham International, Buckinghamshire, United Kingdom). Briefly, each 2 µg of the tissue lysates was electrophoresed under reduced conditions on an 8% SDS-PAGE and transferred to polyvinylidene diflouride (PDVF) membranes (Bio-Rad Laboratories, Hercules, CA). The membranes were soaked in TBS containing 5% nonfat milk and 0.1% Tween-20 for 1 hour at room temperature to block additional protein binding sites and washed 3 times (15 minutes/wash) in TBS-T. The membranes were then incubated with antimouse TF, washed 4 times in TBS-T, and incubated for 1 hour with peroxidase-linked donkey antirabbit antibody (Amersham). After 3 washes in TBS-T, the membranes were developed with the ECL detection kit according to manufacturer's instructions.
Induction of TF mRNA in tissues of the restraint-stressed mice Initially, the effects of short and long exposure to restraint stress on the expression of TF mRNA in tissues were investigated in 8-week-old male C57BL/6J mice by using quantitative RT-PCR technique (Figure 1). Short duration (2 hours) of stress to mice did not substantially increase TF mRNA in all tissues examined. In contrast, a substantial induction of TF mRNA was detected in kidneys (6-fold), small intestines (2-fold), aortas (2-fold), and adipose tissues (2-fold) after 20 hours of restraint stress. Unexpectedly, little or no responses of TF gene to restraint stress were observed in livers, lungs, hearts, and brains. These results suggest that the induction of TF gene by restraint stress occurred in a tissue-specific manner.
Stress-induced TF mRNA expression in young and aged mice Experiments were performed to investigate the effect of aging on the induction of TF expression by restraint stress, using young (8-week-old) and extremely aged (12- and 24-month-old) mice (Figure 2). Basal (before stress) levels of TF mRNA expression in the tissues were slightly elevated in aged mice, but the differences were not substantial. Importantly, the magnitude of induction of TF mRNA expression in kidneys, small intestines, aortas, and adipose tissues substantially increased as animals age (Figure 2). Only in kidneys, TF mRNA expression was more increased by stress in 12-month-old mice than in 24-month-old mice. Again, no substantial induction of TF mRNA by 20 hours of restraint stress was observed in livers, lungs, hearts, and brains both in young and aged mice (not shown).
Cellular localization of TF mRNA in tissues of the restraint-stressed mice To localize the TF mRNA induced by restraint stress in each tissue, in situ hybridization analysis was performed by using tissue sections from the control and stressed mice. Although there was no detectable signal for TF mRNA in kidneys of the unstressed aged mice (Figure 3 A,C), the epithelial cells of proximal and distal tubules (Figure 3B,D) in the stressed mice expressed abundant TF mRNA. Moreover, signals for TF mRNA in renal tubular epithelial cells were dramatic in the stressed aged mice compared with young mice (Figure 3B,D). In small intestines of the stressed mice, several cell types, including smooth muscle cells and inflammatory cells, in the villous core, in lamina propria, and in muscularis mucosae, showed strong signals for TF mRNA (Figure 3F,H) although these cells occasionally expressed TF mRNA in the unstressed mice (Figure 3E,G). Again, the increased signals for TF mRNA by stress in the intestinal cells were dramatic in aged mice (compare Figure 3F with 3H). These results are consistent with the data obtained by quantitative RT-PCR assay (Figure 2).
In aortas, only focal signals for TF mRNA were detected in the
unstressed young and aged mice (Figure 4
A,C). However, increased signals for TF
mRNA were observed in adventitial cells of the vascular wall and in
surrounding adipose tissues after restraint stress, and the signals
were stronger in aged mice than young mice (Figure 4B,D). In control
epididymal fat tissues, a few adipocytes were positive for TF mRNA both
in young and aged mice (Figure 4E,G). In epididymal fat tissues of the
stressed mice, more numbers of adipocytes specifically expressed
considerable amounts of TF mRNA, and, again, the adipocyte-specific
signals for TF mRNA were stronger in aged mice than those in young mice
(Figure 4F,H). We observed no specific hybridized signals for TF mRNA
in vascular endothelial cells in any organs examined in the stressed
mice (data not shown).
Induction of TF mRNA and antigen by restraint stress in obese mice To investigate the effect of obesity on the stress-induced TF expression, we performed restraint experiments by using genetically obese mice and their lean counterparts and then analyzed TF mRNA expression in the tissues (Figure 5). Interestingly, 20 hours of restraint stress caused a substantial increase in TF mRNA level in livers of obese mice, which revealed degeneration to fatty liver but not in livers of lean mice. In kidneys and guts, differences in the induction of TF mRNA by stress between obese and lean mice were not so dramatic. In adipose tissues, the basal expression level of TF mRNA was 2-fold higher in obese mice than in lean mice. More importantly, the magnitude of induction of TF mRNA by stress was larger in adipose tissues of obese mice than in those of lean mice. The stress-induced TF expression in adipose tissues was analyzed at antigen level as well as by Western blotting using polyclonal rabbit antimouse TF antibody (Figure 6). Although TF antigen in the lysates of adipose tissues from lean mice was at an undetectable level, a specific band of 46 kDa corresponding to TF was detected in those of obese mice. The amount of TF antigen produced in adipose tissues dramatically increased after 20 hours of restraint stress in obese mice (Figure 6, lane 6), which was consistent with the data at mRNA level (Figure 5). This increase in the adipose-derived TF production may result in the higher procoagulant potential in obese mice than in lean mice because obese mice carry abundant adipose mass.
Stress-induced thrombosis in aged mice and in obese mice Microscopic examination of tissue sections revealed that renal glomerular thrombus developed in the 20-hour restraint aged (24-month-old) mice (Figure 7B) but not in the stressed young (8-week-old) mice (Figure 7A). Stress-induced thrombi formation was also observed in the microvasculature in epididymal adipose tissues in aged mice (Figure 7E) but not in adipose tissues of the stressed young mice (Figure 7D). Quantitative analysis of the stress-induced thrombi was performed by counting positive glomeruli for thrombi and the number of thrombi within the microvasculature in epididymal adipose tissues. Although renal glomerular thrombi were detected in less than 5% glomeruli in only 1 of 8 restraint mice of 8 weeks old, all (n = 8) of the stressed aged mice showed glomerular thrombi. The percentage of positive glomeruli for thrombi increased 10% to 27% (Figure 7C).23 Similarly, we observed occasional thrombi formation in adipose tissues in 6 of 8 restraint mice 24 months old, although no thrombi were detected in adipose tissues of the stressed young mice (n = 8) (Figure 7F). Meanwhile, rare microthrombi were observed within the vasculature in adipose tissues in only 2 of 8 stressed obese mice, and none of the stressed lean mice showed thrombi formation in their adipose tissues (not shown). No stress-induced thrombi were detected in livers, hearts, intestines, and aorta of the aged mice and obese mice (not shown).
Stress-mediated changes in plasma TNF- antigen in plasma of young and aged
mice, and of obese and lean mice, by restraint stress were analyzed (Figure 8 A,D). The basal level of plasma
TNF- was already higher in aged mice versus young mice and in obese
mice versus their lean counterparts. After 20 hours of restraint
stress, a substantial elevation of TNF- antigen in plasma was
detected both in young and aged mice, and the magnitude of this
stress-mediated induction of TNF- was larger in aged mice (10-fold)
versus young mice (4-fold). Similarly, substantial increases in plasma
TNF- level were observed in stressed obese (2.5-fold) and lean mice
(4-fold).
To investigate the role of TNF-
Although hypercoagulability and thrombotic diseases appear to be induced by a variety of stress,8-10 little information is available about underlying mechanisms. In this report, we observed that restraint stress to mice substantially induced the expression of TF gene (Figure 1). The stress-induced TF mRNA was localized primarily to renal tubular epithelial cells, adventitial cells, smooth muscle cells, and adipocytes but not to vascular endothelial cells (Figures 3-4), indicating that the induction of TF gene by stress occurs in a cell type-specific manner. It has been reported that these cells are a principal source of TF in vivo under physiologic29 and/or pathologic conditions.26,30 The constitutive production of TF in these cell populations suggests that TF forms a hemostatic envelope that activates the coagulation system when vascular integrity is disrupted by a variety of stressors. Another important source of TF in vivo is the adipose tissue/adipocytes.30 The adipose tissue is a highly vascularized organ, and the adipocytes appear to be in intimate contact with vascular beds. Under stressful conditions, the integrity of the circulation is important for the lipid utilization, and TF may help to maintain this integrity in the adipose tissue. The incidence of thrombotic diseases is increasing in aged individuals. Aged subjects may have lower tolerance to stress2-5 and be more susceptible to thrombotic diseases caused by a variety of stressors than the young.13,14 We previously showed that the expression of PAI-1 was also induced by restraint stress and that this response was exacerbated by aging.23 In this study, we demonstrated the induction of TF mRNA in several tissues by restraint stress was substantially higher in aged mice than in young mice (Figures 2-4). Renal glomerular thrombosis and microthrombi formation in adipose tissues were markedly induced by stress in aged mice (Figure 7),23 suggesting that the stress-mediated TF induction contributes to the elevation of regional procoagulant activity and to the development of thrombosis. Thus, the increased expression of TF gene in response to stress may lead to a prothrombotic state in elderly individuals who are exposed to stress. Obesity is associated with increased incidence of thrombotic diseases and accelerated atherosclerosis. We investigated the effect of obesity on the stress-induced TF expression and observed that TF mRNA was substantially increased by restraint stress in livers and adipose tissues of obese mice (Figures 5-6). This observation leads to a speculation that obese adipocytes and degenerative hepatocytes containing abundant lipids may show an increased response to stress in the expression of TF gene. Adipocytes/adipose tissues are the principal sites of TF production in obesity30; thus, obese animals may have a large potential of TF synthesis in response to stress, leading to an increase in the systemic and/or regional procoagulant activity. However, the induction of TF expression by stress in livers and adipose tissues of obese mice did not directly contribute to regional thrombi formation, implying that the process of obesity-linked thrombosis includes multifactorial and complex possibilities. The expression of inflammatory cytokines could be altered by stress, as
shown in the regulation of interleukins.31,32 We observed
that the basal level of TNF- In conclusion, we demonstrate that restraint stress induces the TF
expression in a tissue- and cell type-specific manner in the mouse and
that aging and/or obesity enhance the stress-mediated induction of TF
gene. The stress-induced endogenous TNF-
We thank T. Thinnes, E. Yamafuji, K. Kaneko, and K. Sakakura for their expert technical assistance; and Drs K. Enjyoji and H. Kato (National Cardiovascular Center and Research Institute, Osaka, Japan) for providing rabbit antimouse TF antibody.
Submitted April 4, 2002; accepted July 11, 2002.
Prepublished online as Blood First Edition Paper, July 25, 2002; DOI 10.1182/blood-2002-03-0945.
Supported by grants-in-aid from the Ministry of Education, Science, Sports and Culture from the Ministry of Health and Welfare, by Funds for Comprehensive Research on Aging and Health, Japan, and by grant HL-47819 (D.J.L.) from the National Institutes of Health.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Koji Yamamoto, First Department of Internal Medicine, Nagoya University School of Medicine, 65 Tsurumai, Showa, Nagoya 466-8550, Japan; e-mail: kojiy{at}med.nagoya-u.ac.jp.
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© 2002 by The American Society of Hematology.
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  A. Csiszar, M. Wang, E. G. Lakatta, and Z. Ungvari Inflammation and endothelial dysfunction during aging: role of NF-{kappa}B J Appl Physiol, October 1, 2008; 105(4): 1333 - 1341. [Abstract] [Full Text] [PDF]
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 R. J. Westrick, M. E. Winn, and D. T. Eitzman Murine Models of Vascular Thrombosis Arterioscler Thromb Vasc Biol, October 1, 2007; 27(10): 2079 - 2093. [Abstract] [Full Text] [PDF]
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 H. Klement, B. St. Croix, C. Milsom, L. May, Q. Guo, J. L. Yu, P. Klement, and J. Rak Atherosclerosis and Vascular Aging as Modifiers of Tumor Progression, Angiogenesis, and Responsiveness to Therapy Am. J. Pathol., October 1, 2007; 171(4): 1342 - 1351. [Abstract] [Full Text] [PDF]
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A. Csiszar, N. Labinskyy, K. Smith, A. Rivera, Z. Orosz, and Z. Ungvari Vasculoprotective Effects of Anti-Tumor Necrosis Factor-{alpha} Treatment in Aging Am. J. Pathol., January 1, 2007; 170(1): 388 - 698. [Abstract] [Full Text] [PDF]
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 K. Yamamoto, K. Takeshita, T. Kojima, J. Takamatsu, and H. Saito Aging and plasminogen activator inhibitor-1 (PAI-1) regulation: implication in the pathogenesis of thrombotic disorders in the elderly Cardiovasc Res, May 1, 2005; 66(2): 276 - 285. [Abstract] [Full Text] [PDF]
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 A. Csiszar, Z. Ungvari, A. Koller, J. G. Edwards, and G. Kaley Proinflammatory phenotype of coronary arteries promotes endothelial apoptosis in aging Physiol Genomics, March 12, 2004; 17(1): 21 - 30. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
B [NF-
70°C. The tissues were collected and prepared
as described above. Meanwhile, 6-week-old male obese mice (C57BL/6J
ob/ob) and their lean counterparts (C57BL/6J +/?), both of
which were obtained from The Jackson Laboratories (Bar Harbor, ME),
were put into restraint tubes for 20 hours and then killed. The tissues
were removed and prepared for subsequent analysis as described earlier.
They were also pretreated intraperitoneally either with control hamster
IgG (1 µg/g) or with hamster antimouse TNF-
-actin, as described
previously.
indicates level before stress; 
, after 2 hours of
restraint stress; and 
, after 20 hours of restraint stress. The data
are represented as the means and SD (n = 8) in each amount of time
under stress, and the error bars represent interanimal
variation. *P < .05; **P < .02. Ki
indicates kidney; Li, liver; H, heart; SI, small intestine; Ao, aorta;
Adi, adipose (epididymal fat); Lu, lung; B, brain.
