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Cardiovascular Research 1999 43(4):1040-1048; doi:10.1016/S0008-6363(99)00173-X
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Copyright © 1999, European Society of Cardiology

The 2.3 kb smooth muscle myosin heavy chain promoter directs gene expression into the vascular system of transgenic mice and rabbits1,2,

Wolfgang M Franza,*, Oliver J Muellera, Michaela Fleischmannb, Philip Babijc, Norbert Freya, Matthias Muellera,b, Urban Besenfelderb, Antoon F.M Moormand, Gottfried Bremb and Hugo A Katusa

aMedizinische Klinik II, University of Lübeck, Lübeck, Germany
bInstitut für Tierzucht und Genetik, University of Veterinary Medicine, Vienna, Austria
cWyeth-Ayerst Research, Princeton, NJ, USA
dDepartment of Anatomy and Embryology, University of Amsterdam, Amsterdam, The Netherlands

* Corresponding author. Tel.: +49-451-500-2732; fax: +49-451-500-6437 franz{at}medinf.mu-luebeck.de

Received 13 January 1999; accepted 9 April 1999


   Abstract
Top
 Abstract
1 Introduction
 3 Results
 4 Discussion
 References
 
Background: Smooth muscle cells (SMC) are a preferential target for gene therapeutic approaches in atherosclerosis and restenosis. However, the undesirable expression of putative therapeutic genes in tissues other than the vascular wall is a considerable safety limitation for clinical trials, thus requiring the identification of a smooth-muscle-specific promoter sequence. Since the 2.3 kb rabbit Smooth Muscle Myosin Heavy Chain (SMHC) promoter was shown to be transcriptionally active in primary vascular but not visceral or other non-SMC in vitro, this fragment was chosen for in vivo analysis. Methods and Results: Transgenic mice and rabbits were established expressing a luciferase reporter gene under control of the 2.3 kb rabbit SMHC promoter. In contrast to the endogenous expression pattern of the SMHC gene both species revealed light emission predominantly in the arterial system including coronary arteries. Low activities were measured in large veins and the gastrointestinal system. In situ hybridization of murine embryos using a luciferase riboprobe confirmed reporter gene expression in large arteries with no detectable mRNA in the viscera. Unlike adult animals, ectopic luciferase activities were found in ventricular myocardium during murine development ceasing 1 week post partum. Conclusions: In two animal species, the 2.3 kb SMHC promoter appeared to be effective in discriminating between the pathways regulating vascular and visceral smooth muscle gene expression. The vascular-specific expression profile of the 2.3 kb SMHC promoter suggests that the 2.3 kb SMHC promoter contains the regulatory elements necessary for selective gene targeting into vascular SMC of large arteries including coronary arteries in vivo.

KEYWORDS Gene expression; Gene therapy; Smooth muscle; Arteries; Coronary circulation


   1 Introduction
Top
 Abstract
 1 Introduction
3 Results
 4 Discussion
 References
 
Proliferation of smooth muscle cells (SMC) plays a central role in atherosclerosis and restenosis following balloon angioplasty [1]. Therefore vascular SMC are a preferential target for therapeutic approaches. In contrast to various pharmacological substances failing to prevent restenosis after PTCA in human clinical trials [2], numerous gene therapeutic approaches have shown promising effects in animal models of atherosclerosis and restenosis [3]. However, the undesirable expression of putative therapeutic genes in tissues other than the vascular wall is currently a considerable safety limitation for clinical applications. Therefore a regulatory sequence needs to be identified, which allows smooth-muscle-specific gene expression in vivo.

Suitable candidates for further in vivo characterization are 5' regulatory elements of genes with a smooth-muscle-specific expression pattern. Predominant expression in smooth muscle tissue could be shown for several contractile and cytoskeletal genes such as {alpha}-1 integrin [4], smooth muscle {alpha}-actin [5], calponin [6,7], SM22{alpha} [8], telokin [9] and smooth muscle myosin heavy chain (SMHC) [10]. Unlike other smooth-muscle-specific genes, which are expressed in non-smooth muscle tissues during embryogenesis, SMHC mRNA has been shown to be a specific marker for SMC [10]. Furthermore, the 2.3 kb rabbit SMHC promoter was shown to be transcriptionally active in primary vascular but not visceral or other non-SMC cultures [11].

Since fundamental differences may exist between a permanent in vivo expression in viable tissue and a transient in vitro expression in cultured cells [12,13], we generated independent transgenic mouse and rabbit lines expressing a luciferase reporter under control of the 2.3 kb rabbit SMHC promoter. In our study luciferase was used as a reporter in order to detect even low ectopic or unspecific promoter activity with high sensitivity [14]. Expression of the reporter gene was predominantly measured in the arterial system including coronary arteries of both species. Low expression was found in large veins and the gastrointestinal system.

2 Materials and methods
2.1 Generation and breeding of transgenic mice and rabbits
Cloning of the rabbit SMHC promoter was described previously [11]. We used a 2.301 kb promoter fragment ranging from –2305 bp to –4 bp upstream of the transcription start site, isolated with SalI and BssHII (Fig. 1A). The sequence was flanked with HindIII linkers and subcloned into the HindIII site of the luciferase reporter vector pGL2basic (Promega, Mannheim, FRG). The 5.0 kb SMHC-luciferase fusion gene (2.3SMHC-luc) was excised for microinjection with SalI (Fig. 1B). One picoliter containing 500–1000 copies of the transgene in 10 mmol/l Tris–HCL (pH 7.5) and 0.2 mmol/l EDTA was microinjected into male pronuclei of fertilized mouse (HimOF1) or rabbit oocytes (ZIKA® hybrid rabbits) according to established procedures [15–18]. HimOF1 mice are a well characterized strain and allow microinjection efficiencies comparable to NMRI mice [15]. Generation of transgenic animals, breeding and tail clipping were performed following the institutional guidelines of the University of Veterinary Medicine, Vienna, which conform with the ‘Guide for the Care and Use of Laboratory Animals’ published by the US National Institute of Health (NIH Publication No. 85-23, revised 1996). For identification of transgenic founders and their offspring, DNA was analyzed by PCR-amplification and Southern blot hybridization. A 1.8 kb SmaI-EcoNI luciferase probe was labeled with Digoxigenin-11-dUTP (Boehringer-Mannheim, Mannheim, FRG). For PCR, the following primers were used: 5'-AAGAGCAGCGTCCGAGGC-3' (SMHC-1-forward), and 5'-TTCCATTTTACCAACAGTACC-3' (LUC-10-reverse). For studies of ontogeny, homozygous F2 males were crossbred with wild-type females. Gestational age of the mouse embryos was determined by counting the morning of plug observation as embryonic day (ED) 0.5.


Figure 1
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  		Fig. 1 Generation of the microinjection construct. (A) shows the genomic structure of the 5'-region including the first two exons of the rabbit smooth muscle myosin heavy chain (SMHC) gene. The 2.301 bp 5' regulatory region contains the TATA box, two CCTCCC sequences, three CArG elements, a vascular-specific enhancer (VSE), an A/T-rich MEF2-like region (AT-r), a negative regulatory element (NRE) overlapping the enhancer and a repressing G/C-rich (GC-r) element close to CarG-2. Black boxes indicate exon 1 and 2; dark hatched boxes symbolize positive regulatory elements (VSE, A/T-rich and CCTCCC sequences); light hatched boxes represent negative regulatory regions (NRE and G/C-r).

 
2.2 Luciferase assay
Tissue samples of heterozygous mice and rabbits were excised and immediately frozen in liquid nitrogen. Vascular structures were removed carefully from parenchymal organs and visceral smooth muscle tissues. For analyses of developmental regulation, tissues derived from embryonic and neonatal mice were pooled from at least five littermates. For each developmental stage tissues from four independent preparations were analyzed. Luciferase analyses were performed according to established procedures with minor modifications [13]. 100 µl of luciferin solution was added to 50 µl of protein extract. Light emission was recorded over a period of 20 s with a Lumat LB 9501 Luminometer (Berthold, Bad Wildbad, FRG). Enzyme activities were related to the amount of total protein extract in milligrams determined by the bicinchoninic acid method (Pierce, Rockford, IL). For quantitation of luciferase expression a standard curve was established as previously described [13]. Light emission was in a linear range between 10 fg and 3 ng.

2.3 In situ hybridization
Tissues derived from 14.5-days-old mouse embryos were fixed in freshly prepared phosphate-buffered saline (PBS) containing 4% paraformaldehyde at 4°C overnight and embedded in Paraplast Plus (Monoject) as previously described [19,20].

Labeling of riboprobes and hybridization were performed according to established procedures with the following modifications [20]: to increase sensitivity, RNA probes were double labeled with 5 µmol/l 35S-UTP and 5 µmol/l 35S-CTP. Probes used to detect luciferase were obtained by in vitro transcription of linearized pGEM-LUC (Promega) using T7 polymerase. Luciferase sense probes were derived by transcribing with SP6 polymerase. For detection of SMHC, a 387 bp cDNA fragment was used containing exon 1 and 2 [10]. The probe was obtained by in vitro transcription with T3 polymerase. As a control, an SMHC sense probe was generated transcribing with T7 polymerase. All probes were hybridized to tissue sections following RNAse treatment.


   3 Results
Top
 Abstract
 1 Introduction
 3 Results
4 Discussion
 References
 
3.1 Generation of transgenic lines
Transgenic mice and rabbits were generated for analysis of the tissue-specificity of the 2.3 kb rabbit SMHC promoter. Three independent transgenic mouse lines (2127, 2128, 1785) and one rabbit line (2196) transmitted the transgene to offspring. A further founder rabbit (2195) harboring 2.3SMHC-luc died before breeding. Therefore, only tissue of the founder animal could be analyzed.

3.2 Expression pattern of the 2.3 kb SMHC promoter in transgenic mice and rabbits
To analyze promoter activity in adult transgenic 2.3SMHC-luc mice and rabbits, luciferase expression was determined in tissue lysates of representative organs (Fig. 2). In transgenic mouse line 2127, highest luciferase activities were measured in large arteries including mesenteric vessels. Low levels of expression were observed in large veins and the gastrointestinal system. Occasionally minor luciferase activities were found in trachea, lung, spleen, kidney, uterus and bladder. In atrial or ventricular myocardium no luciferase activities were detected, which is also true for liver, esophagus, skeletal muscle, and brain. These findings were confirmed in the other two transgenic mouse lines (2128, 1785). Transgenic rabbit line 2196 revealed up to four times higher luciferase values in the arterial vasculature compared to transgenic mice (Fig. 2B). Low levels of expression were observed in large veins, esophagus, intestines and bladder. In contrast to the transgenic mouse lines, no visceral activities were found in trachea, lung, spleen, kidney and uterus. Expression levels in the transgenic rabbit founder 2195 were in a similar range.


Figure 2
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  		Fig. 2 Luciferase expression in adult transgenic mice and rabbits. Mean values and standard deviations of luciferase activities (light units [LU] per milligram protein) are shown representatively for (A) transgenic mouse line 2127 and (B) transgenic rabbit line 2196. Tissues were obtained from 10 heterozygous adult mice and 5 rabbits.

 
To address the question whether the 2.3 kb SMHC promoter also directs reporter gene expression to coronary vessels, the proximal part of both coronary arteries was dissected from surrounding myocardial and aortic tissue. In comparison to transgenic mice, rabbits showed a five times higher light emission in coronary vessels. Myocardial tissue of both species revealed only background activity (Fig. 3). These findings demonstrate that the 2.3 kb SMHC promoter sequence contains cis-elements also sufficient for directing gene expression into coronary arteries of different species.


Figure 3
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  		Fig. 3 Luciferase expression in coronary arteries of adult transgenic animals. Mean values and standard deviations of luciferase activities (light units [LU] per milligram protein) in coronary arteries and ventricles of (A) transgenic mouse line 2127 and (B) transgenic rabbit line 2196 show specific reporter gene activity in coronary arteries.

 
For determination of promoter strength, we quantitated luciferase expression in our model by using a standard curve as previously described [13]. Adult transgenic 2.3SMHC-luc mice revealed up to 0.6 pg, rabbits 8 pg luciferase per mg total protein, which is comparable to the MLC-2v promoter directing cardiac-specific gene expression [13,21,22].

3.3 Developmental expression pattern of the 2.3 kb SMHC promoter in transgenic mice
Endogenous SMHC is known to be expressed specifically in vascular and visceral smooth muscle tissues throughout embryogenesis [10]. To determine whether the vascular specific property of the 2.3 kb SMHC promoter is also preserved during development, in situ hybridization was performed at ED 11.5 and 14.5 either with a 35S-labeled luciferase- or a murine SMHC-riboprobe. In transverse sections of the lower abdomen (ED 14.5) luciferase-mRNA was clearly present in the wall of renal arteries (Fig. 4A). However, expression in the abdominal aorta was close to background. Although endogenous SMHC expression shows a higher intensity in visceral than in vascular smooth muscle tissues (Fig. 4B), no luciferase-mRNA was detected in organs containing visceral SMC such as gut (Fig. 4A) and bladder (data not shown). Positive hybridization signals using luciferase and SMHC-probes were observed in arteries of pelvis and upper limb (data not shown). No specific staining was observed using a sense riboprobe (Fig. 4C).


Figure 4
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  		Fig. 4 Distribution of luciferase- and SMHC-mRNA in abdominal sections of a transgenic mouse embryo at ED 14.5. In situ hybridization with a 35S-labeled luciferase antisense riboprobe (A) revealed specific expression in renal arteries (ra) with minor expression in the abdominal aorta (a). Hybridization with a murine SMHC-riboprobe (B) revealed an additional positive signal in visceral smooth muscle tissue of gut (g). No hybridization was observed using a sense luciferase riboprobe (C). Bar: 100 µm.

 
Thoracic sections of embryos at ED 14.5 showed a strong staining pattern of luciferase expression in right and left ventricular myocardium (Fig. 5A) compared to endogenous SMHC expression (Fig. 5B). An intense cardiac signal was also observed in murine embryos at ED 11.5 hybridized with the luciferase probe (data not shown). Both luciferase and SMHC expression were found in the dorsal aorta. In concordance with the study of Miano et al., no SMHC signal could be detected in the developing gut at this developmental stage [10].


Figure 5
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  		Fig. 5 Expression of luciferase- and SMHC-mRNA in the heart of a transgenic mouse embryo at ED 14.5. (A) shows in situ hybridization with a 35S-labeled luciferase antisense riboprobe resulting in a clear staining of the right (rv) and left ventricle (lv). Only background staining was observed in the right (ra) and left atrium (la). When hybridized with a murine SMHC-riboprobe, no cardiac signal was detected (B). Bar: 400 µm.

 
Ectopic cardiac luciferase expression was confirmed during development measuring luciferase activities in representative organs of newborn mice (Fig. 6). Transgenic 2.3SMHC-luc mouse lines revealed predominant luciferase expression in the arterial system represented by the descending part of the thoracic aorta, the abdominal aorta, and the iliac artery. Marginal light activities were detected in lung, esophagus, intestine, uterus, and bladder. Therefore, the expression pattern in newborn mice resembles that observed in adult mice except for ectopic cardiac activity. Absolute luciferase activities are higher in the neonatal than in adult samples, suggesting an increased promoter activity during development. The time course of ectopic luciferase expression during cardiogenesis was analyzed at different developmental stages. In line 2127, luciferase activities declined approximately eight-fold from 2.0x107 LU/mg at ED 14.5 to 2.5x106 LU/mg protein at birth. A similar decrease in luciferase activity was observed in the low expression line 1785. In both transgenic mouse lines, no cardiac luciferase activity was detected after the first postnatal week.


Figure 6
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  		Fig. 6 Luciferase activities (in light units [LU] per mg protein) in newborn transgenic mice. Average values and standard deviations are shown for representative organs of newborn mice of transgenic line 2127. Tissue was pooled from at least five heterozygous littermates. Altogether four independent litters were analyzed.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 3 Results
 4 Discussion
References
 
In the present study, we have analyzed the ability of the rabbit SMHC promoter to selectively target a luciferase reporter gene to vascular smooth muscle tissue of transgenic mice. In contrast to the visceral and vascular-specific expression pattern of a recently described rat 15.8 kb SMHC sequence [23], the truncated 2.3 kb rabbit SMHC promoter revealed predominant reporter gene expression in the arterial system including coronary arteries of independent transgenic mouse and rabbit lines. Low expression was observed in large veins and the gastrointestinal system. Transgenic rabbits revealed an up to four-fold higher luciferase activity in the arterial vasculature compared to transgenic mice. The vascular-specific property of the 2.3 SMHC promoter was further confirmed by developmental studies. Analyses at ED 11.5, 14.5 and in newborn mice revealed expression of luciferase mRNA in the arterial system and an ectopic activity in the ventricular myocardium ceasing in the first postnatal week. These findings suggest that the activity of the 2.3 kb SMHC promoter corresponds to the expression pattern of the endogenous gene in tissue containing vascular, but not visceral SMC.

4.1 Vascular-specific activity of the 2.3 kb SMHC promoter in transgenic mice and rabbits
Several promoters with a smooth-muscle-specific expression pattern have been characterized in transgenic animals. Some regulatory sequences, such as SMC {alpha}-actin or telokin, reflect the tissue distribution of the endogenous genes [9,24]. In contrast to the endogenous expression pattern, the 2.3 kb SMHC promoter directed reporter gene expression only into the vascular system in vivo. Using luciferase as reporter gene, we cannot directly exclude activity in endothelium or adventitia. However, the activity in the vascular system is considered to be due to reporter expression in vascular SMC as veins containing much lower amounts of SMC, but similar amounts of endothelium and adventitial cells revealed marginal luciferase activities compared to arteries. Further evidence derives from previous tissue culture studies demonstrating specific activity of the 2.3 kb SMHC promoter in primary SMC culture [11].

Similar to the 2.3 kb SMHC promoter, a 445 bp SM22{alpha} regulatory sequence targets reporter gene expression to SMC of the arterial system except for coronary arteries [25,26]. Therefore it seems to be most likely that transcriptional activity to visceral SMC is not encoded in these truncated promoter fragments. This hypothesis may be further strengthened by a recently published transgenic study using the regulatory sequence of the rat SMHC gene spanning from –4.2 kb to +11.6 kb of the first intron [23]. These 4.2SMHC-Intron-lacZ transgenic mice expressed the reporter both in vascular and visceral smooth muscle, suggesting that the extended 15.8 kb fragment contains sufficient regulatory sequence to allow transcription in both phylogenetically different cell types.

For the 2.3 kb SMHC promoter the vascular-specific expression pattern was confirmed in transgenic rabbits. As the expression pattern of rabbit line 2196 corresponds to that of founder 2195 and three independent mouse lines, the vascular-specific activity of the 2.3 kb SMHC promoter in line 2196 by a genomic integration effect can therefore be excluded. Differences in expression levels between mice and rabbits may be interpreted as higher transcriptional activity of the rabbit 2.3SMHC promoter in the rabbit background. A positional effect accounting for the enhanced gene expression in rabbits is less likely, since the rabbit line 2196 and the founder 2195 displayed elevated reporter activities compared to transgenic mice. The elements located on the rabbit SMHC promoter are highly conserved between mouse, rat, and rabbit [27]. Therefore, the existence of different expression patterns between the rat 15.8 kb and the rabbit 2.3 kb regulatory sequences in transgenic animals suggests that distinct regulatory pathways for vascular and visceral SMC have evolved.

Several cis-acting elements within the 2301 bp region of the SMHC promoter are known, which may be responsible for gene expression in vascular SMC. Those highly conserved DNA elements include CArG boxes, CCTCCC sequences, a MEF2-like A/T-rich region, a putative negative regulatory element, and a GC-rich repressor element as well as a vascular-specific enhancer (VSE) [11,27–30]. The VSE represents a unique regulatory region spanning 107 bp (–1332 to –1225) of the rabbit SMHC 5'-region. This sequence was shown to confer enhancer-type activity in primary vascular SMC, when fused to other promoters [11]. In the context of the presently known regulatory elements, the VSE region seems to be most likely crucial for the vascular-specific expression pattern. However, further in vivo studies may be required to prove this hypothesis.

Promoter activities different from the endogenous expression pattern have previously been observed in other transgenic models of the cardiovascular system [12]. The endogenous myosin light chain-2 (MLC-2) gene, for example, is known to be expressed in ventricular myocardium as well as slow-twitch skeletal muscle. The 2.1 kb MLC-2 promoter fragment, however, targets gene expression only to the ventricular myocardium [13]. Therefore deletion of distinct regulatory sequences may generate truncated promoters with the ability to target gene expression predominantly into a subset of the original cell types.

4.2 Activity of the 2.3 kb SMHC promoter in coronary arteries and veins
In contrast to the SM22{alpha} model, the 2.3SMHC-luc transgenic mice and rabbits revealed additional luciferase activities in coronary arteries and large veins. These differences in the vascular expression pattern may be explained by distinct regulatory programs in SMC derived from a different developmental origin. It is known that vascular SMC arise from either the local mesoderm or the cervical neural crest. The origin of the proximal large vessels such as aortic arch, thoracic aorta, common carotids and pulmonary trunk is thought to be the neural crest, while more distal vessels like abdominal aorta and carotid arteries may contain a mixture of both neural crest-ectomesenchymal and mesoderm-derived SMC [31,32]. Coronary arteries are of mesodermal origin [33,34]. The origins of venous SMC are not yet well characterized, but these SMC are thought to be derived from mesodermal cells [35]. As the SMHC promoter is also active in coronary arteries and to a minor extend in veins arising from SMC of mesodermal origin, it may be hypothesized that the 2.3 kb promoter contains the necessary cis-elements required for expression in mesodermal as well as neuroectodermal derived SMC.

4.3 Ectopic activity of the 2.3 kb SMHC promoter in the developing heart
Smooth muscle-specific genes such as {alpha}1-integrin, SMC {alpha}-actin, calponin, and SM22{alpha} are known to be transiently expressed in the heart tube and the myotomal compartment of the somites, but become restricted to smooth muscle cells in adult tissue [4–8]. However, endogenous SMHC gene expression was not detected in the myocardium during murine development before [10]. In situ hybridization studies demonstrated that the 2.3 kb SMHC promoter fragment targeted reporter gene expression to the developing ventricular myocardium of transgenic mice. Newborn mice revealed high luciferase activities in the myocardium confirming the in situ hybridization data. No luciferase expression could be detected after the first postnatal week. This temporary ectopic activity cannot be explained by a positional effect of the transgene since luciferase expression was detectable in the developing heart of independent transgenic mouse lines. The myocardial luciferase expression may therefore reflect the absence of a putative repressor element in the 2.3 kb SMHC promoter. Artificial truncation of such a negative regulatory sequence may have unmasked a common developmental pathway of cardiomyocytes and vascular smooth muscle cells.

In summary these findings demonstrate that the 2.3 kb rabbit SMHC promoter contains regulatory cis-elements sufficient for targeting gene expression into the arterial system in vivo. Moreover, expression levels in adult rabbits are comparable to other household promoters. Therefore, the 2.3 kb SMHC promoter may enable the generation of new transgenic models of vascular disease. Finally, further studies investigating the 2.3 kb SMHC-promoter activity under pathological conditions will elucidate its role as a candidate for future studies of somatic gene therapy approaches in the vascular system.

Time for primary review 28 days.


   Acknowledgements
 
We thank Yvonne Müller for excellent technical assistance, Petra Hornsberger for skillful animal care, Piet de Boer for introduction into the in situ hybridization technology and Dr Joe Miano for the murine SMHC cDNA. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 320/B6 and Ka 493/3-1) and the Deutsche Forschungsanstalt für Luft- und Raumfahrt (01 KV 9560).


   Notes
 
1 Franz, Mueller, both authors contributed equally. Back

2 Brem, Katus, both Institutions contributed equally. Back


   References
Top
 Abstract
 1 Introduction
 3 Results
 4 Discussion
 References
 

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