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Cardiovascular Research 2003 59(3):573-581; doi:10.1016/S0008-6363(03)00511-X
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Copyright © 2003, European Society of Cardiology

Arteriogenesis is associated with an induction of the cardiac ankyrin repeat protein (carp)

Kerstin Boengler*, Frederic Pipp, Borja Fernandez, Tibor Ziegelhoeffer, Wolfgang Schaper and Elisabeth Deindl

Max-Planck-Institute for Physiological and Clinical Research, Department of Experimental Cardiology, Benekestr. 2, D-61231 Bad Nauheim, Germany

k.boengler{at}kerckhoff.mpg.de

* Corresponding author. Tel.: +49-6032-705-404; fax: +49-6032-705-419.

Received 15 January 2003; accepted 30 May 2003


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Collateral artery growth (arteriogenesis) can be induced in rabbit and mice by occlusion of the femoral artery. We aimed to identify genes that are differentially expressed during arteriogenesis. Methods: 24 h after femoral ligation or sham operation collateral arteries were isolated from New Zealand white rabbits, mRNAs were extracted and amplified using the SMART technique. cDNAs were subjected to suppression subtractive hybridization. The differential expression was confirmed by Northern blot, Real time PCR and Western blot. Additionally, the gene expression was modulated in vivo by application of cytokines via osmotic minipumps. Results: We found the cardiac ankyrin repeat protein (carp) mRNA to be upregulated at 24 h and already at 6 h and 12 h after surgery as shown by Northern blot hybridization and real time PCR. The carp mRNA was also increased in our mouse model of arteriogenesis. Western blot results on nuclear extracts of rabbit collaterals 24 h after surgery indicated that carp, which we showed to be expressed in endothelial cells and smooth muscle cells of collateral arteries by immunohistochemistry, was also upregulated on the protein level. We infused MCP-1, TGF-β1 or doxorubicin for 24 h in rabbits and found that only TGF-β1 led to an additional increase of carp mRNA. Overexpression of carp in cos-1 cells resulted in a 3.7-fold increase of the immediate early gene egr-1. Conclusions: Our results implicate that carp is associated with the initiation and regulation of arteriogenesis.

KEYWORDS Arteries; Collateral circulation; Cytokines; Gene expression; Remodeling

This article is referred to in the Editorial by A.R. Strauch (pages 532–533) in this issue.


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Vascular occlusive diseases are a major cause of death in industrialized countries. To attenuate the consequences of the diseases, there is a need to develop strategies for the growth of large vessels, which function as natural bypasses. Arteriogenesis, the growth of collateral arteries, can be induced by occlusion of the femoral artery in rabbits and mice [1,2]. Collateral vessels are growing e.g. in the m. quadriceps, a tissue not associated with ischemia or hypoxia in this model [3]. The inductor of arteriogenesis is likely to be fluid shear stress. It is elevated in collaterals as a consequence of femoral ligation, and influences gene expression in the vessel wall. The immediate early gene egr-1 is one of the genes quickly upregulated in response to shear stress in vitro and in vivo [4,5] and can itself target a variety of other genes [6]. In vivo, shear stress induces an activation of the endothelium, resulting in the adherence and invasion of monocytes supplying growth factors and cytokines promoting arteriogenesis [7]. The development by growth of the pre-existing collateral arteries includes the proliferation of endothelial as well as of smooth muscle cells and can already be detected 3 days after occlusion of the femoral artery [1]. The process of arteriogenesis is enhanced by the infusion of cytokines and growth factors like the monocyte chemoattractant protein 1 (MCP-1), the granulocyte macrophage colony stimulating factor (GM-CSF), or the transforming growth factor β1 (TGF-β1) into the collateral arteries [8–10].

Whereas the time-course of arteriogenesis and the morphology of the developing collateral arteries are well described [8,11], the molecular mechanisms regulating this process are less defined.

In this study we analyzed the differential gene expression in growing collateral arteries of rabbits by suppression subtractive hybridization (SSH) [12] and identified the cardiac ankyrin repeat protein (carp) mRNA. The upregulation of carp, which was associated with increased protein levels as shown by Western blot analysis, was confirmed by independent techniques like Northern Blot and quantitative real time PCR in rabbits and mice. For the first time we show by immunohistochemistry that carp is expressed in vascular endothelial cells and smooth muscle cells in vivo. In addition, we provide evidence for the induction of the carp mRNA expression by TGF-β1 in collateral arteries. Furthermore, our data showed that the overexpression of carp in cos-1 cells led to an induction of the immediate early gene egr-1.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Animal models
New Zealand white rabbits were anesthetized with an intramuscular injection of ketamine hydrochloride (4–8 mg/kg body weight) and xylazine (8–9 mg/kg body weight), and the right femoral artery was ligated with two ligatures 1.5 cm apart for 6, 12 and 24 h as described previously [13]. For reasons of control the left femoral arteries were sham operated, n=5 per time point. BALB/c mice (n=5) were also anesthetized with a mixture of ketamine hydrochloride and xylazine, the right femoral arteries were ligated for 12 h and the left femoral arteries sham operated as described previously [2]. The investigation conforms with the Guide for Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publication No. 85-23, revised 1996).

2.2 Isolation of collateral vessels
To visualize the collateral arteries of experimental and sham operated rabbits in the m. quadriceps, a contrast medium based on gelatine and barium was infused into the rabbit hindlimbs [13] (Fig. 1A). The arteries were dissected without contaminating muscle from the surrounding tissue (Fig. 1B). Additionally, samples of various rabbit organs were collected. Mice adductor muscles harboring collateral arteries were also harvested. All tissue samples were snap frozen in liquid nitrogen and stored at –80°C until further investigations.


Figure 1
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  		Fig. 1 (A) Arteriolar connections and collateral arteries 1 week after sham operation in the m. quadriceps of a rabbit. (B) Excision of collateral arteries from the m. quadriceps without contaminating tissue.

 
2.3 Application of MCP-1, TGF-β1 or doxorubicin in rabbits
Right femoral arteries of rabbits (n=3 per group) were ligated with two ligatures about 1.5 cm apart and were randomly assigned to receive either monocyte chemoattractant protein 1 (MCP-1) (0.43 µg/kg; Peprotech), doxorubicin (0.29 mg/kg; Sigma) or TGF-β1 (0.48 µg/kg; Peprotech) at constant flow-rates of 10 µl/h via osmotic minipump (2 ML-1, Alza) for 24 h. Pumps were connected to the proximal stump of the ligated femoral artery by a catheter pointing upstream —with the tip of the catheter positioned distal to the branching of the arteria circumflexa femoris—and subcutaneously fixed in the flank. The left femoral arteries were sham operated.

2.4 RNA isolation and cDNA synthesis
Total RNA was isolated from the tissue samples according to the protocol of Chomczynski and Sacchi [14]. A 10-µg amount of total RNA of rabbit collateral arteries 24 h after occlusion of the femoral artery or sham operation was DNase treated and the mRNA extracted using the Oligotex mini kit (Qiagen). The mRNA was reverse transcribed (Superscript II, Invitrogen) and amplified using the SMART technique (Smart PCR cDNA Synthesis Kit, BD-Clontech).

2.5 Suppression subtractive hybridization
Suppression subtractive hybridization was performed with the rabbit Smart cDNA using the PCR Select cDNA subtraction kit (BD-Clontech). For the forward hybridization cDNA derived from collateral arteries 24 h after femoral ligation served as tester and the cDNA of the 24 h sham operated animals as driver cDNA (and vice versa for the reverse subtraction). The subtractions were carried out according to the manufacturer’s protocol. The resulting PCR products were cloned in pGEM-Teasy (Promega), analysed for differential expression by hybridization with forward and reverse subtracted cDNAs, and sequenced on an A.L.F. DNA sequencer using the AutoRead 200 sequencing kit (Amersham Biosciences).

2.6 Northern blot hybridization
Northern hybridization studies (n=6) were carried out according to standard procedures [15]. The blot was hybridized with a 32P-dCTP random-primed 850 bp cDNA probe specific for rabbit carp (rediprime II, Amersham Biosciences), which was isolated from the forward subtracted library. The 18S rRNA, which was used for normalization, was detected by hybridization of a specific oligonucleotide as described by Deindl [16].

2.7 Quantitative PCR
A 1-µg amount of DNase treated total RNA was reverse transcribed using random nonamers (Amersham Biosciences). Quantitative PCR was performed on an iCycler (Bio-Rad) using Platinum Taq Polymerase (Invitrogen), Sybr Green I (Molecular probes), 100 nM of each primer (carp forward: 5'-TGCCGACCTCACCATCAAG-3', carp reverse: 5'-ACGTAGCTATCGCAGAGGTTTTG-3', 18S forward: 5'-GGACAGGATTGACAGATTGATAG-3', 18S reverse: 5'-CTCGTTCGTTATCGGAATTAAC-3') and 1 µl first strand RT reaction from a serial 100–10–4 dilution in a total volume of 20 µl. Three independent RT reactions were used and for each dilution four replicates were amplified. The protocol was 3 min 95°C initial denaturation, 30 s 95°C, 1 min 60°C, 45 cycles. Melt curve analysis was performed to ensure that only one specific product was amplified.

2.8 Cell culture and transient transfection assays
Cos-1 cells were maintained in modified DMEM (Invitrogen) and transfected at 50% confluency with 2 µg of a carp expression plasmid, containing the whole rabbit carp coding sequence in pCMS–EGFP (BD-Clontech), or with the vector pCMS–EGFP using FUGENE6 (Roche). The cells were harvested 48 h after transfection (n=6).

2.9 Western blot analysis
Nuclear protein extracts of collateral vessels isolated 24 h after occlusion or sham operation (n=4) were prepared as previously described [17]. Briefly, collateral arteries were homogenized in buffer A (20 mM Tris–HCl, 250 mM sucrose, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol (DTT), 0.1 mM sodium orthovanadate, 10 mM NaF and 0.5 mM PMSF, pH 7.4) and centrifuged at 14 000 g for 30 min at 4°C. The sediments were resuspended in buffer B (20 mM Tris–HCl, 1 M sucrose, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.1 mM sodium orthovanadate, 10 mM NaF, 10 mM KCl, 0.1 mM PMSF, pH 7.4) and centrifuged at 10 000 g for 30 min at 4°C. The resulting sediments were resuspended in buffer C (10% glycerol, 20 mM Tris–HCl, 400 mM KCl, 1 mM EGTA, 1 mM DTT, 0.1 mM sodium orthovanadate, 10 mM NaF, 0.5 mM PMSF, and 0.1% Triton X-100, pH 7.4) and sonicated. Nuclear proteins of transfected cos-1 cells were isolated as described by Schreiber et al. [18]. A 20-µg amount of protein was electrophoretically separated under reducing conditions on a 4–12% Bis–Tris gel (Invitrogen) and immunoreactive bands were visualized using an ECL detection system (Amersham Biosciences) and polyclonal rabbit anti-murine carp (1:500, kindly provided by Siegfried Labeit, Mannheim, Germany) or polyclonal rabbit anti-human egr-1 antisera (1:1000, Santa Cruz), respectively.

2.10 Immunohistochemistry
Cryosections (5 or 10 µm) of mouse collateral arteries 2 days after femoral occlusion or sham operation were fixed with 4% paraformaldehyde, and washed with phosphate buffered saline (PBS). After quenching of the endogenous peroxidase activity with 3% hydrogen peroxide, the sections were washed in PBS, immersed in blocking solution (0.1% bovine serum albumin+0.4% glycine in PBS) and incubated with an anti-carp antibody in the same solution. The sections were then incubated with a peroxidase-conjugated anti-rabbit IgG, followed by 1 mg/ml 3,3'-diaminobenzidine+0.3 µl/ml H2O2 in PBS. The sections were counterstained with hematoxilin and eosin (H.E.). Photomicrographs were obtained with a Leica DC 200 video camera connected to a computer system.

2.11 Quantification and statistical analysis
Signals were quantified using a Storm PhosphorImager (Molecular Dynamics) using IMAGEQUANT software. Results were expressed as mean±S.E.M. For statistical analysis the unpaired Student’s t-test was used. Data were considered to be statistically significant (*) at a value of P<0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
To characterize the gene expression during the early phase of arteriogenesis, we amplified mRNA from rabbit collateral arteries 24 h after occlusion of the femoral artery and after sham operation by means of the SMART technique. The resulting cDNA was subjected to suppression subtractive hybridization using the cDNA from collaterals after occlusion as tester and the cDNA derived from the sham operated control vessels as driver. The screening procedure for differentially expressed genes was performed by hybridizing the clones from the forward subtracted library to forward and reverse subtracted cDNA libraries. In this assay we identified the cardiac ankyrin repeat protein (carp) mRNA.

To confirm our results and to define the window of carp upregulation, we performed Northern blot analysis on RNA isolated from rabbit collateral arteries 6, 12 and 24 h after femoral ligation or sham operation. Furthermore, we investigated the expression level of the carp mRNA in a second model of arteriogenesis, in mice (Fig. 2). Results in rabbit showed a significant upregulation of the carp transcript in collaterals 6, 12 and 24 h after femoral occlusion compared to sham operation, although a slight upregulation of the carp mRNA 12 h after sham operation was detected. In the mouse model of arteriogenesis we investigated the carp mRNA level exemplary at 12 h after femoral occlusion and demonstrated an enhanced expression (180%) of the carp transcript. Results on rabbit organs, which were analyzed for reasons of control, showed that carp is expressed in heart and lung, but not in brain, liver, kidney and uterus. The upregulation of the carp mRNA in growing rabbit collaterals was further confirmed and quantified using real time PCR. The carp mRNA significantly increased to 230, 260 and 330% at 6, 12 and 24 h, respectively, after occlusion of the femoral artery compared to the value of the sham operated animals, which were set as 100% (Fig. 3).


Figure 2
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  		Fig. 2 Northern blot analysis of the carp mRNA level in in rabbit organs and in growing collateral arteries of rabbit and mice. Total RNA from rabbit collateral vessels at 6, 12 and 24 h after femoral artery occlusion (occ) or after sham operation, from the mouse adductor muscles harboring the collateral arteries, and from different rabbit organs were hybridized with a carp specific cDNA probe (top). To control RNA loading, the blot was rehybridized with an 18S rRNA specific probe (bottom).

 

Figure 3
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  		Fig. 3 Real time PCR analysis of the carp mRNA expression level. The level of the carp mRNA in rabbit collateral arteries 6, 12 and 24 h after occlusion of the femoral artery (occ) was analyzed by real time PCR and normalized to the expression of the 18S rRNA. The values of the sham operated animals were set as 100%.

 
Western blot analysis on nucleic extracts of rabbit collateral arteries showed that carp was elevated on the protein level about 2.3-fold 24 h after occlusion of the femoral artery compared to sham operated animals (Fig. 4).


Figure 4
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  		Fig. 4 Carp protein expression 24 h after surgical procedure. (A) Western blot analysis of the carp expression in nuclear extracts of rabbit collateral arteries 24 h after occlusion of the femoral artery (occ) or after sham operation. To demonstrate equal loading, a representative ponceau S stained protein band is shown. (B) Quantification of the carp protein expression. The values of the sham operated animals were set as 100%.

 
3.1 Localization of the carp protein in growing collateral arteries
Mouse collateral arteries 2 days after femoral occlusion were analyzed by immunohistochemistry for the localization of the carp protein. Carp expression was detected in both endothelial cells and smooth muscle cells of collateral arteries. Some nuclei of striated myocytes also stained positive for carp. In collateral arteries carp expression was not restricted to the nucleus, but was distributed throughout the cytoplasm of endothelial and smooth muscle cells (Fig. 5) as displayed by hematoxilin–eosin counterstaining of the same section.


Figure 5
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  		Fig. 5 Localization of the carp protein in different cell types. Immunoperoxidase localization of carp protein (A) and hematoxilin–eosin counterstaining (B) of a collateral artery 2 days after femoral artery occlusion. Positive signals were found in the cytoplasm of endothelial cells (small arrows) and smooth muscle cells (big arrows). Nuclei of striated myocytes (big arrowheads) and some perivascular cells (small arrowheads) were also positive. Note the perinuclear localization of carp in some endothelial cells (green arrowheads) and smooth muscle cells (green arrows).

 
3.2 Carp mRNA expression is induced by infusion of TGF-β1 in vivo
In order to investigate the induction of carp we infused MCP-1, TGF-β1 (both known to promote arteriogenesis) or doxorubicin, shown to suppress the carp mRNA expression in vitro, via osmotic minipumps in the rabbit collateral arteries and measured the carp mRNA expression after 24 h in comparison to occlusion alone or sham operation by Northern blot hybridization (Fig. 6A).


Figure 6
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  		Fig. 6 Modulation of the carp mRNA expression in vivo. (A) Northern blot analysis of the carp mRNA expression 24 h after infusion of doxorubicin (dox), MCP-1 or TGF-β1 in the rabbit collateral arteries. As control served collateral arteries 24 h after occlusion of the femoral artery as well as the collaterals from the sham operated contralateral hindlimb (top). To control equal RNA loading, the blot was rehybridized with an 18S rRNA specific probe (bottom). (B) Quantification of the carp mRNA expression. The carp mRNA level 24 h after occlusion and after occlusion plus infusion of dox, MCP-1 or TGF-β1 was normalized to the 18S rRNA level. n=3 per each substance.

 
The infusion of MCP-1 as well as doxorubicin led to no significant change of the carp expression level. TGF-β1 infusion, however, enhanced the carp mRNA by about factor 3.3 compared to occlusion alone (Fig. 6B).

3.3 Overexpression of carp in cos-1 cells induces the egr-1 protein
Cos-1 cells were transiently transfected with an expression plasmid coding for the rabbit carp protein (pCMS–CARP) or with the expression vector pCMS–EGFP as control. The increased amount of the carp protein in the nuclear fraction after the overexpression was confirmed by Western blot analysis (Fig. 7A). Furthermore, we monitored the expression level of egr-1 after overexpression of carp. Results on nuclear fractions of cos-1 cells showed an about 3.7-fold increase of the egr-1 protein 2 days after carp overexpression compared to the control (Fig. 7B).


Figure 7
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  		Fig. 7 Influence of carp overexpression on the egr-1 protein level in vitro. (A) Cos-1 cells were transfected with a carp expression plasmid (pCMS–CARP) and with the vector pCMS–EGFP as control. The protein level of carp and egr-1 2 days after transfection were monitored in the nuclear fractions by Western blot analysis. To demonstrate equal loading, a representative ponceau S stained protein signal is shown. (B) Quantification of the egr-1 expression. The values of the control vector pCMS–EGFP were set as 100%. Each transfection was performed six times independently.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
In this study we analyzed the differential gene expression in early stages of arteriogenesis and identified the carp mRNA to be upregulated in rabbit on mRNA as well as on protein level. Furthermore, we showed an increase of the carp transcript in a mouse model of collateral artery growth. For the first time we demonstrated a localization of carp in endothelial and smooth muscle cells of collateral arteries in vivo. Our data showed that TGF-β1 is capable of inducing carp mRNA expression in rabbit. Overexpressing carp in cos-1 cells resulted in increased expression of the immediate early gene egr-1.

This study aimed to identify and characterize genes that are differentially expressed at the early phase of arteriogenesis. We compared the gene expression in collateral arteries 24 h after occlusion of the femoral artery with collateral vessels after sham operation by suppression subtractive hybridization and identified the cardiac ankyrin repeat protein (carp). Northern blot results showed a significant increase of the carp mRNA in rabbit collaterals 6, 12 and 24 h after occlusion of the femoral artery. However, 12 h after sham operation the carp mRNA expression in collateral arteries was enhanced to a minor extend compared to the other sham operated control arteries, indicating an influence of the surgery itself on the transcript level of carp. The results obtained by Northern blot hybridization were further confirmed by quantitative PCR. In order to investigate the carp mRNA expression in other animal models of arteriogenesis we analyzed the carp mRNA expression in mice after femoral ligation and demonstrated an upregulation of the transcript 12 h after surgery.

The carp cDNA was cloned from rabbit heart in 1999 [19]. The corresponding 36-kDa protein consists of a PEST-like sequence, which targets proteins for rapid destruction [20] and four ankyrin repeats, which are involved in protein–protein interactions [21]. Furthermore, the carp amino acid sequence revealed a nuclear localization signal. We analyzed the carp protein level in nuclear protein fractions of rabbit collateral arteries by Western blot analysis. Results displayed an about 2.3-fold increase of the carp protein 24 h after femoral ligation compared to the control. The upregulation of carp represents an early event in the process of arteriogenesis, assuming a role for carp in the initiation of collateral artery growth. By in situ hybridization former studies displayed a localization of the carp mRNA predominantly in the heart as well as after denervation in skeletal muscle [22,23]. Our immunohistochemical data showed for the first time a localization of the carp protein in endothelial cells and smooth muscle cells of collateral arteries as well as in the nuclei of skeletal myocytes. These findings are in accordance with previous in vitro studies demonstrating a carp mRNA expression in endothelial and smooth muscle cell lines [24,25]. However, in contrast to the described nuclear restricted expression of carp, our immunohistochemical data localized the carp protein throughout the whole cell of collateral arteries, whereas in striated myocytes only the nuclei were positive for carp.

Carp is expressed in a segment-specific manner during embryonic heart development [26], and an increased carp mRNA expression was found to be a marker of cardiac hypertrophy [27,28]. The development of cardiac hypertrophy includes changes in the expression of a variety of genes, among them the immediate early gene egr-1 [29,30], which is—like carp—also expressed in endothelial cells and smooth muscle cells in vivo [31]. In this study we showed that the overexpression of carp in the cytosol of cos-1 cells results in a transport of the protein to the nucleus, where it presumably functions in the regulation of gene expression. Our results furthermore demonstrated an induction of the immediate early gene egr-1 after overexpression of carp in cos-1 cells, making it likely that carp is an upstream effector of the zinc-finger protein egr-1. In recent papers it was demonstrated that carp functions as an inhibitor of cardiac gene expression and that it was capable of decreasing DNA synthesis in C2/2 cells [25,32]. Using egr-1 as a target protein, we provide evidence for the ability of carp to influence protein expression positively. Both the carp and egr-1 mRNA have been shown to be upregulated in vitro in response to shear stress [33,34], the main stimulus of arteriogenesis. In a mouse model of collateral artery growth an induction of the egr-1 mRNA was displayed, making it likely that egr-1, which regulates the expression of target genes like TGF-β1 or basic fibroblast growth factor (bFGF) [35,36], is involved in the process of arteriogenesis [37]. Previous studies on bFGF demonstrated that application of the growth factor enhances the formation of collaterals as well as collateral blood flow [38,39]. Additionally, egr-1 was shown to bind to the promoter of the urokinase-type plasminogen activator (uPa) [40], a member of the plasminogen system, which contributes to the process of arteriogenesis by promoting the infiltration of monocytes [41]. We hypothesize that carp functions in the nucleus not only by inhibiting the transcription of genes, but also by inducing the expression of target genes like egr-1, which itself controls the mRNA levels of genes associated with the process of arteriogenesis.

In order to analyse the induction of the carp mRNA in collateral arteries, we infused TGF-β1, MCP-1 or doxorubicin in rabbits. Our results demonstrated an increase of the carp mRNA after application of TGF-β1 in vivo, indicating that carp represents a target gene of TGF-β1 during the early steps of arteriogenesis. In recent studies an induction of the carp mRNA in vitro in C2/2 cells by TGF-β1 was demonstrated. This effect was mediated by the SMAD proteins binding to the carp promoter [25]. Despite the functions TGF-β1 exerts during vasculogenesis, angiogenesis, and the stabilization of the vessel wall [42], exogenous application of TGF-β1 is also known to promote arteriogenesis by enhancing the number of collateral vessels as well as the conductance in the collateral circulation [10]. In contrast, MCP-1, which was also described to enhance collateral artery growth by stimulating the attraction of monocytes [8], did not significantly alter the expression of carp mRNA. Therefore, our results suggest that for the regulation of the arteriogenic process, in which both MCP-1 and TGF-β1 are involved, carp might be a specific downstream target of TGF-β1.

The application of doxorubicin, a widely used antineoplastic drug shown to downregulate carp mRNA in vitro [32,43], did not affect the carp mRNA expression in vivo in our experiments. Although this might have been due to the low dose of the infused doxorubicin and the short time of application (24 h), it is likely that the in vitro situation does not reflect the in vivo situation. This assumption is corroborated by previous studies showing that doxorubicin had no evident effect on carp mRNA expression level after application in the rabbit heart [19].

In summary, our data showed that carp is expressed in endothelial cells and smooth muscle cells of collateral arteries in vivo, where it is upregulated on the mRNA and protein level in a rabbit and mouse model of arteriogenesis. Carp, which was induced by TGF-β1 in vivo, enhanced the expression of egr-1 in vitro. TGF-β1, also induced during arteriogenesis, may therefore transduce its signals via carp, which possibly affects the expression of other genes like egr-1 involved in collateral artery growth. Taken together, these data suggest a role for carp in the initiation and regulation of collateral artery growth.

Time for primary review 49 days.


   Acknowledgements
 
We want to thank Elfriede Neubauer for excellent technical assistance.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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