Nigel Mackman, Division of Hematology/Oncology, 917 Mary Ellen Jones Bldg, CB #7035, Chapel Hill, NC 27599, USA. Tel.: +1 919 843 3961; fax: +1 919 966 7639; e-mail: email@example.com
Summary. Background: The tissue-specific pattern of tissue factor (TF) expression suggests that it plays a major role in the hemostatic protection of specific organs, such as the heart and lung. In support of this notion, we found that mice expressing very low levels of TF exhibit hemostatic defects in the heart and lung. Hemosiderosis and fibrosis are observed in the hearts of all low TF mice as early as 3 months of age. In contrast, TF+/– mice expressing ∼50% of wild-type levels of TF had no detectable hemostatic defects. Objective and methods: The objective of this study was to determine the threshold of TF that is required to maintain hemostasis under normal and pathologic conditions, and to investigate the specific role of cardiac myocyte TF in heart hemostasis using mice with altered levels of TF expression in cardiac myocytes. Results: First, we found that mice with 20% of wild-type levels of TF activity in their hearts had hemosiderosis and fibrosis by 6 months of age. Secondly, mice with a selective deletion of the TF gene in cardiac myocytes had a mild hemostatic defect under normal conditions but exhibited a significant increase in hemosiderosis and fibrosis after challenge with isoproterenol. Finally, we showed that cardiac myocyte-specific overexpression of TF abolished hemosiderin deposition and fibrosis in the hearts of low TF mice. Conclusions: Taken together, our results indicate that TF expression by cardiac myocytes is important to maintain heart hemostasis under normal and pathologic conditions.
Tissue factor (TF) is the primary cellular activator of blood coagulation and binds factor VII/factor VIIa (FVII/VIIa) . Its expression by adventitial fibroblasts, pericytes, and vascular smooth muscle cells provides a hemostatic envelope around blood vessels . In addition, TF is expressed by cardiac myocytes in the heart, astrocytes in the brain, and alveolar type II cells in the lung [2–4]. These data suggest that TF limits hemorrhage from damaged blood vessels, particularly in specific organs, such as the heart, lung and brain .
Deficiencies in various coagulation factors, such as FVIII and FIX, are associated with hemophilia . In contrast, TF deficiency in humans has not been reported. Consistent with this observation, inactivation of the murine TF gene leads to 100% lethality either during embryonic development or in the perinatal period [6–8]. These results strongly suggest that TF plays an essential role in hemostasis.
TF+/– mice express ∼50% of wild-type levels of TF and exhibit no detectable hemostatic defects [6–8]. We have previously reported that expression of a low level of TF [∼1% of wild-type levels from a human TF (hTF) transgene] is sufficient to rescue TF null embryos and to produce viable mice . However, these so-called ‘low TF’ mice are prone to spontaneous hemorrhages in the lung, heart, brain, uterus and placenta . We speculate that the lung and heart, because of their mechanical nature, contain blood vessels that require additional hemostatic protection. Indeed, the lungs of low-TF mice contain hemosiderin-filled macrophages , which is commonly associated with hemorrhage. In addition, the hearts of low-TF mice also have hemosiderin deposits and areas of fibrosis, which are likely to be due to hemorrhage from capillaries . A similar heart phenotype was observed in mice expressing low levels of murine FVII but not in mice lacking FVIII or FIX [12,13]. These results suggest that TF and FVIIa are required for normal hemostasis in the heart.
Cardiac myocytes occupy as much as 75% of cardiac mass but constitute only about 30% of the total cell numbers in the heart . The majority of the remaining cells are interstitial cardiac fibroblasts. These cells provide structural support for cardiac myocytes by regulating the extracellular matrix and are a source of growth factors. To date, TF expression by cardiac fibroblasts has not been reported. However, connective tissue fibroblasts surrounding blood vessels express high levels of TF, and cultured fibroblasts constitutively express TF [2,15]. Therefore, it is likely that cardiac fibroblasts also express TF.
In this study, we determined the threshold of TF that is required to maintain heart hemostasis in mice under normal and pathologic conditions, and the role of cardiac myocyte TF in heart hemostasis.
Materials and methods
Mice were analyzed between 6 and 12 months of age. Low-TF mice express very low levels of hTF+ (1%) from an hTF minigene on a mouse TF null (mTF–/–) background . Mice expressing the Cre recombinase under the control of the myosin light chain 2v (MLC2v) promoter were purchased from Dr K. Chien (UCSD, La Jolla, CA, USA) . Transgenic mice expressing the Cre recombinase under the control of the ubiquitously expressed adenovirus EIIa promoter, and mice expressing the flipase recombinase under the control of the human β-actin promoter, were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). All studies were approved by The Scripps Research Institute Animal Care and Use Committee and comply with the National Institute of Health guidelines.
Primers used to characterize embryonic cell (ES) clones and genotype mice
The presence or absence of the loxP sites was determined using primer pairs (5′loxP site): 5′-ATGAGGAGCTGTGTTAAAGGGTCGCAGAA-3′ and 5′-TGCAGTAAATGCACGTGTCTGCCAT-3′, and (3′loxP site) 5′-CCACATCTACCCAGTGCTGAGTTGA-3′ and 5′-CTCGTGCTTTACGGTATCGC-3′. Mice containing the αMHC-TF transgene were identified by PCR of genomic DNA using primers for human growth hormone poly A sequence: forward primer, 5′-AACCAAGCTGGAGTGCAGTGGCAC-3′, and reverse primer, 5′-AAGGAGGGTAGATGACCTGAGATT-3′.
Tissues were fixed with 10% zinc-formalin solution and embedded in paraffin. Hemosiderin was detected by staining with Prussian blue and fibrosis was assessed using Masson’s Trichrome .
Levels of mouse and hTF mRNAs were determined by northern blotting using radiolabeled probes. Bands were visualized by autoradiography. Loading was monitored by re-probing the blot for glyceraldehyde 3-phosphate dehydrogenase (GAPDH).
Single-stage clotting assay
Procoagulant activity (PCA) was determined using a single-stage clotting assay with mouse plasma (Sigma-Aldrich, St Louis, MO, USA) as previously described . Diluted heart homogenates were mixed with equal volumes of mouse pool plasma and 20 mm CaCl2 and the clot time determined using a STart4 clotting machine (Diagnostica Stago, Parsippany, NJ, USA). Clotting times were converted to activity units using a standard curve generated with a mouse brain extract.
Preparation of neonatal mouse ventricular cardiac myocytes and cardiac fibroblasts
Neonatal mouse cardiac myocytes and cardiac fibroblasts were prepared as described . Cardiac myocytes were separated from non-myocytes by discontinuous Percoll density gradient centrifugation.
Reverse transcription PCR and real time PCR
Total RNA was extracted from mouse cardiac myocytes using Trizol reagent. cDNA was made from 1 μg of total RNA using a SuperScript First-Strand Synthesis System for RT-PCR kit (Invitrogen Corp., Carlsbad, CA, USA). Levels of mouse TF mRNA in cardiac myocytes were determined by real time PCR using standard Taqman® technology (Applied Biosytems, Foster City, CA, USA) with the following primers: forward primer 5′-CATGGAGACGGAGACCAACT-3′, reverse primer 5′-CCATCTTGTTCAAACTGCTGA-3′ and probe 5′-FAM-TTACCGAGACACAAACCTTGGACAGC-TAMRA-3’. Levels of GAPDH mRNA were determined using a Taqman Rodent GAPDH Control Reagents Kit (Applied Biosystems). Primers for both mouse TF and GAPDH mRNA were run in the same PCR reaction.
Twelve-week-old TFflox/flox/MLC2v-Cre and TFflox/flox mice received daily intraperitoneal injections of isoproterenol (15 mg kg–1) dissolved in saline for 2 weeks. At the end of that period, mice were euthanized and heart weight to body weight ratio (HW:BW) was calculated by dividing the heart weight (in mg) by body weight (in g).
All data are presented as mean ± SD. Comparisons between the different groups were performed using Student’s t-test for unpaired data or chi-squared test with 10 d.f. Statistical differences were considered significant for P-values < 0.05.
Generation of human TF mice
Mice expressing hTF from a chromosome vector have been described . These mice (mTF+/+/HTF+) express detectable levels of hTF in the brain, kidney and intestine . We generated HTF mice that express hTF from the chromosome vector in the absence of mouse TF (mTF–/–/HTF+) in two sttif, by first crossing mTF+/+/HTF+ mice with mTF+/– mice and then crossing mTF+/–/HTF+ mice with mTF+/– mice. To increase the number of mTF–/–/HTF+ mice that were generated, mTF–/–/HTF+ male mice were crossed with mTF+/– female mice. From this breeding, mTF–/–/HTF+ mice with one copy of the chromosome vector were generated at 58% of the expected frequency (100%). This is consistent with the previously reported transmission frequency of the chromosome vector (62% of the expected frequency) in wild-type mice .
HTF mice express 20% of wild-type levels of TF activity and have impaired heart hemostasis under normal conditions
During the characterization of HTF mice we noticed that they express only 20% of the expected 50% level of PCA in their hearts from a single copy of a chromosome vector (Fig. 1A). In contrast, these mice express close to wild-type levels of PCA in their lungs and kidneys, and higher levels of PCA than expected in the brain (Fig. 1A). Next, we analyzed hTF and mTF mRNA levels in mTF+/–/HTF+ mice so that we could compare the levels of each in the same mice. Levels of hTF mRNA were lower than expected in the heart, higher in the brain, and at close to the expected level in the kidney when compared with levels of mTF mRNA in the different tissues (Fig. 2B).
Because of the reduced levels of TF in the hearts of HTF mice, we investigated heart hemostasis in these mice. HTF mice showed no abnormalities in their hearts up to 4 months of age (n =7), but mice older than 6 months of age all exhibited some degree of hemosiderosis and heart fibrosis (n =7) (Fig. 1C). HTF mice had no signs of any hemostatic defect in all other tissues examined, including the lung, kidney and brain, and had normal life spans.
Generation and characterization of mice containing a floxed TF gene
A TF gene-targeting vector was made using a targeting vector based on pDelBoy (a kind gift from Dr F. Naya) (http://gttf.uchc.edu/Infodelboy.html) and contains two loxP sites flanking the mouse TF promoter and exon 1, and two frt sites flanking the neomycin gene (Fig. 2A). The vector was linearized and transfected into ES cells. Gancyclovir and G418 were used to select clones containing the targeting vector. One hundred and forty-four clones were screened by Southern blotting using a radiolabeled DNA probe located upstream of the TF gene (5′ probe) (Fig. 2B). Six clones demonstrated a targeted cross-over event that was upstream of the TF gene. Each of these six clones was re-screened by Southern blotting using a radiolabeled DNA probe located in intron 2 (3′ probe) to confirm that they were correctly targeted (Fig. 2B). The presence of the 5′ and 3′loxP sites was determined by PCR with pairs of primers that were located on either side of the respective loxP site and three correctly targeted clones with both loxP sites were identified. Representative Southern blots of clone 67 are shown in Fig. 2C. Each of the three clones was injected into blastocysts (SV129 background) to generate chimeric mice but only one line of chimeric mice (line 67) was obtained that exhibited germ line transmission of the targeted TF gene (Fig. 2D).
This mouse line was bred with C57Bl/6 mice to generate TF+/floxneo mice. The neomycin gene was removed by crossing TF+/floxneo mice with mice expressing the flipase recombinase under the control of the human β-actin promoter. TF+/flox mice were then intercrossed to generate TFflox/flox mice, which were viable and were born at a frequency of 25.8%, which was close to the expected frequency of 25%. To confirm that the loxP sites were functional, TFflox/flox mice were crossed with mice that express the Cre recombinase under the control of the ubiquitously expressed adenovirus EIIa promoter (EIIa-Cre). We did not recover any TFflox/flox/EIIa-Cre mice at wean (0 out of 99) from intercrosses of TFflox/+/EIIa-Cre mice. In addition, we observed a dead, bloodless TFflox/flox/EIIa-Cre embryo at embryonic day 9.5 that resembled the phenotype of TF–/– embryos (Fig. 2E).
Cardiac myocyte-specific deletion of the TF gene has minimal effect on heart hemostasis under normal conditions but is associated with enhanced fibrosis under pathologic conditions
To analyze the role of cardiac myocyte TF in heart hemostasis, we determined the effect of deleting the TF gene in cardiac myocytes. The TF gene was selectively deleted in cardiac myocytes by crossing TFflox/flox mice with mice expressing the Cre recombinase under the control of the cardiac myocyte-specific promoter MLC2v . TFflox/flox/MLC2v-Cre mice were viable and were born at a frequency of 18.3% from crosses between TFflox/flox and TFflox/+/MLC2v-Cre mice, which is close to the expected frequency of 25%. We determined the level of TF expression in the hearts of TFflox/flox/MLC2v-Cre mice. TFflox/flox littermate mice were used as controls in these experiments because they express similar levels of TF as wild-type mice (data not shown). TF mRNA expression and PCA in the hearts of TFflox/flox/MLC2v-Cre mice were 28% and 31%, respectively, compared with TFflox/flox controls (Fig. 3A,B).
The residual TF expression observed in these hearts may be due to either an incomplete deletion of the TF gene in cardiac myocytes or TF expression by other cell types, such as cardiac fibroblasts. To distinguish between these possibilities, we analyzed TF mRNA expression in isolated cardiac myocytes. We found that TF mRNA expression in cardiac myocytes from TFflox/flox/MLC2v-Cre neonates was 1–2% of the levels in cardiac myocytes from TFflox/flox in two independent experiments that had minimal contamination of cardiac fibroblasts. In contrast, there was no difference in TF mRNA expression in the non-myocyte cell fraction, which consists mostly of cardiac fibroblasts (data not shown). These results indicate that TF is efficiently deleted in the cardiac myocytes and that the residual PCA in whole hearts is due to TF expression by other cell types, such as cardiac fibroblasts.
Next, we investigated the effect of cardiac myocyte-specific deletion of the TF gene on heart hemostasis. The hearts of 6-month-old TFflox/flox/MLC2v-Cre (n =5) mice were normal with no signs of hemosiderin deposition or fibrosis (data not shown). However, we observed hemosiderin and fibrosis in 3 out of 12 hearts from 9-month-old TFflox/flox/MLC2v-Cre mice (Fig. 3C). As expected, no hemosiderin or fibrosis was observed in 9-month-old TFflox/flox control mice (n =5; data not shown). Together, these data suggest that a selective deletion of the TF gene in cardiac myocytes has a mild effect on heart hemostasis under normal conditions.
We further explored the role of cardiac myocyte TF in heart hemostasis under pathologic conditions. We used isoproterenol to activate the β-adrenergic receptor and induce stress in the heart. This treatment is associated with heart hypertrophy. Cardiac myocyte-specific deletion of the TF gene had no effect on isoproterenol-induced heart hypertrophy, measured by an increase of HW:BW ratio (Table 1). However, TFflox/flox/MLC2v-Cre mice exhibited a significant increase in hemosiderin deposition and fibrosis in their hearts compared with TFflox/flox mice (Table 1). These data indicate that cardiac myocyte TF plays an important role in maintaining heart hemostasis under pathologic conditions.
Table 1. Heart weight:body weight (HW:BW) ratio, hemosiderin deposition and fibrosis with or without isopreterenol
14 days of isopreterenol treatment
Tissue factor (TF)flox/floxn =5
TFflox/flox MLC2v-Cre n =5
TFflox/flox MLC2v-Cre n =12
Values are mean ± SD. *Comparison between baseline and 14 days within genotype. †Comparison between isopreterenol treated groups.
4.63 ± 0.32
4.52 ± 0.45
5.46 ± 0.23*
5.53 ± 0.29*
Over-expression of TF in cardiac myocytes restores heart hemostasis in low TF mice
We hypothesized that the hemostatic defect in low-TF mice is due to reduced levels of TF in cardiac myocytes. To test this hypothesis, we determined if selective overexpression of TF on cardiac myocytes would restore normal hemostasis in the hearts of low-TF mice. We used the α-myosin heavy chain (αMHC) promoter (kindly provided by Dr F. Naya) to overexpress TF in cardiac myocytes . This promoter is also expressed in cells within the walls of pulmonary veins and venules of the lung . Three transgenic lines were generated. We found that TF mRNA was overexpressed in the hearts of all three transgenic lines (4, 69, and 70) (Fig. 4A,B). Lines 4 and 69 also exhibited a slight increase in TF expression in the lung (Fig. 4A,B). We then analyzed the PCA of heart homogenates from the three αMHC-TF transgenic lines and found that they expressed between fourteen- and sixtyfold higher levels of PCA than wild-type littermate controls (Fig. 4C). Despite overexpression of TF in the heart, mice from all three transgenic lines developed normally up to 12 months of age and had no abnormalities in their hearts (data not shown).
We realize that the levels of TF expression from the αMHC promoter were significantly higher than the physiological levels expressed in the hearts of wild-type mice. Nevertheless, the expression of proteins from this promoter is restricted to cardiac myocytes in the heart, and this allowed us to determine if selective expression in these cells restores heart hemostasis in low-TF mice. Female mTF+/+/αMHC-TF line 4 mice were crossed with low-TF male mice (mTF–/–/hTF+). Female mice with the following genotype mTF+/–/hTF+/αMHC-TF were then bred with low-TF males to generate low-TF mice with or without the αMHC-TF transgene. As expected, the presence of the αMHC-TF transgene dramatically increased the levels of PCA in the hearts of low-TF mice (data not shown). Next, we analyzed heart hemostasis in low-TF mice with or without the αMHC-TF transgene. Histological analysis of heart sections from low-TF mice without the transgene (n =11) showed extensive heart fibrosis in all mice at 6 months of age (Fig. 5A). In contrast, all low-TF/αMHC-TF mice (n =18) had normal hearts with no evidence of fibrosis (Fig. 5A). These data indicate that overexpression of TF in cardiac myocytes restores hemostasis in the hearts of low-TF mice.
Interestingly, PCA in the lung of low-TF/αMHC-TF line 4 mice (n =6) was significantly increased compared with the PCA in the lung of low-TF mice (n =4) (PCA arbitrary units; 11 ± 8.1 and 2.1 ± 0.2, respectively, P < 0.05). However, the presence of the αMHC-TF transgene did not affect hemosiderin deposition in the lungs of low-TF mice at 6 months of age (Fig. 5B) (hemosiderin was observed in 11/18 mice with the transgene vs. 7/11 mice without the transgene) (P =0.79 by chi-squared test). This result is likely to be due to increased TF expression in cell types that do not contribute to hemostasis.
The tissue-specific pattern of TF expression suggests that certain tissues, such as the brain, heart and lung, require additional hemostatic protection. For instance, the heart may express TF because blood vessels, particularly capillaries, are prone to damage because of the contraction of cardiac muscle. There are no reports of humans deficient in TF. Therefore, mouse models are needed to study the consequences of TF deficiency on hemostasis. Previous studies have shown that reducing TF levels to 50% of wild-type mouse levels (mTF+/– mice) in all tissues does not affect hemostasis during normal life . In contrast, we have shown that heart hemostasis is severely compromised in mice expressing very low levels of TF in their hearts (1% of wild-type levels) . These low-TF mice uniformly have hemosiderin deposition and fibrosis in their hearts by 3 months of age.
We extended these observations by analyzing heart hemostasis in mice with different levels of TF expression in their hearts. As expected, we observed a negative correlation between the level of TF expression in the heart and the extent and timing of hemosiderin deposition and fibrosis. A reduction of TF expression in the heart to 20% of wild-type levels results in hemosiderin deposition and fibrosis in the hearts of all HTF mice by 6 months of age under normal conditions. Mice with a selective deletion of TF in cardiac myocytes exhibited a mild hemostatic defect under normal conditions but a dramatic impairment of hemostasis after treatment of the mice with isoproterenol. Furthermore, overexpression of TF in cardiac fibroblasts restored heart hemostasis in low-TF mice. These results support the notion that TF expression, particularly in cardiac myocytes, mediates heart hemostasis.
Our study suggests that cardiac fibroblasts also express TF. Previous studies both in vitro and in vivo demonstrated that adventitial fibroblasts express high levels of TF [2,20]. Indeed, we found that isolated cardiac fibroblasts express higher levels of TF than isolated cardiac myocytes (data not shown). Because of their abundance in the heart, we suspect that cardiac fibroblasts may contribute as much as 30% of the total TF expression in the heart.
A variety of agents have been developed that target the TF:FVIIa complex, including anti-TF monoclonal antibodies . However, the long-term effects of inhibition of the TF:FVIIa complex are unknown. One study showed that administration of the thrombin inhibitor hirudin to rats induced heart fibrosis . Similarly, the long-term use of inhibitors of the TF:FVIIa complex may compromise hemostasis in various tissues, including the heart. Our mouse models allow us to investigate the role of TF in heart hemostasis under normal and pathologic conditions. Further studies are required to investigate the potential cardiac complications of anticoagulant therapies that target the TF:FVIIa complex.
We thank H. Stuhlmann for advising on embryonic stem cell culture. We also thank M. Szeto for mouse breeding and C. Johnson for preparing the manuscript. This work was supported by grants from the National Institutes of Health.
Disclosure of Conflict of Interests
The authors state that they have no conflict of interests.