© 2000 American Heart Association, Inc.
Molecular Medicine |
Development of a Smooth Muscle–Targeted Cre Recombinase Mouse Reveals Novel Insights Regarding Smooth Muscle Myosin Heavy Chain Promoter Regulation
From the Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Va. C.P.R.’s present address is Department of Genomic Pharmacology, Vertex Pharmaceuticals, Cambridge, Mass.
Correspondence to Gary K. Owens, PhD, Department of Molecular Physiology and Biological Physics, 1300 Jefferson Park Ave, PO Box 800736, Charlottesville, VA 22908. E-mail gko{at}virginia.edu
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Abstract |
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Abstract—The use of genetically modified mice has been an important model system to study gene function in cardiovascular development and under pathophysiological conditions. Although conventional gene knockout studies have provided important insights into gene function in the cardiovascular system, they may be limited by upregulation of compensatory pathways and the inability to differentiate direct versus indirect functions in vivo. As a first step in developing systems that can target gene activation or inactivation specifically to smooth muscle cells (SMCs), we coupled the smooth muscle myosin heavy chain (SMMHC) promoter to the cre recombinase gene and generated transgenic mice that express cre in SMCs. In addition, we used these mice to address whether the heterogeneous staining observed in SMMHC-LacZ mice was due to subsets of SMCs that required different regulatory cassettes of the promoter or if it reflected episodic expression of the transgene. To address both the feasibility of SMC targeting and the apparent heterogeneous expression, we bred SMMHC-cre mice to indicator mice containing a cre-activated LacZ gene. Results showed high-level expression in SMCs at various embryonic time points and in adult tissues. Because breeding of SMMHC-cre mice to an indicator line provided an integration of cre activity over time, results of this study revealed that expression of the SMMHC promoter fragment more closely resembled the expression of the endogenous gene, both with respect to the onset of activation during development and uniformity of staining among individual cells within tissues. Overall, these mice will provide a powerful tool to researchers to study gene function in vascular development/disease by using cre/lox technology to direct smooth muscle–specific gene activation or inactivation in vivo.
Key Words: cre recombinase • smooth muscle myosin heavy chain • cardiovascular development • gene targeting • vascular biology
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Introduction |
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The generation of gene knockout mice by conventional gene targeting has contributed substantially to elucidating the potential function of many genes in cardiovascular development and in response to pathophysiological stimuli, such as atherosclerosis and hypertensive cardiovascular remodeling.1 2 3 4 5 6 7 Although conventional knockout mice have provided novel insights into the function of a variety of genes in cardiovascular development, this approach has several potential limitations that may impair elucidating the direct function of specific genes with respect to cardiac and vascular biology. First, conventional knockout approaches that abolish gene expression in all cell types throughout the entire development of the animal may activate a variety of pathways that compensate for the loss of the gene and thus obscure the direct function of the candidate gene.8 Second, ablation of gene function in all cells may make analysis of vascular effects difficult if gene inactivation causes embryonic lethality, because of its role in the development of structures outside the cardiovascular system, or if the gene of interest is expressed in both the heart and the vasculature. For example, many of the genes identified through conventional knockout approaches that have been shown to cause lethal defects in cardiac development1 2 3 9 often have shown some defect in vascular development as well.4 5 6 However, because the gene of interest has been deleted in all cells of the embryo, it is difficult to determine whether the vascular effects are independent of the cardiac defects or if defects in cardiac development have caused changes in early hemodynamic parameters that then, secondarily, affect vascular development.
The advent of cre/lox technology has provided an important tool to manipulate gene expression in mice by allowing researchers to specifically activate or inactivate genes in a cell type–specific manner, thereby significantly alleviating several limitations of conventional knockout approaches.7 10 Although the development of mice that express cre in a cardiac-specific manner11 to inactivate or overexpress genes has provided important insights into cardiac development and function,12 13 there have been no reports of gene targeting to smooth muscle cells (SMCs). A main problem in targeting cre expression to SMCs is that many smooth muscle promoters are transiently expressed in other cell types, such as the heart and somites, during development.14 15 Because the transient expression of cre will cause activation or inactivation of the gene of interest in these tissues, these promoters may be of limited value to dissect the in vivo functions of many candidate SMC genes.
Of the smooth muscle–specific or –selective genes, smooth muscle myosin heavy chain (SMMHC) appears to be the most specific marker of smooth muscle lineage identified to date.16 17 Our laboratory has recently described sufficient regions of the SMMHC promoter to direct smooth muscle–specific expression throughout development and in adult transgenic mice.18 However, an apparent limitation of this promoter fragment for use in directing cre-mediated SMC-specific alterations in gene function was that many SMC tissues showed heterogeneous staining for the LacZ transgene. Our previous studies described 2 categories of heterogeneous transgene expression, as follows: one in which cells within certain tissues, such as the aorta and intestine, showed variable transgene expression (ie, microheterogeneity), and one in which the transgene expression was completely absent in certain vascular beds, such as the renal and pulmonary circulation (ie, regional heterogeneity).18 There is considerable evidence suggesting the presence of SMC heterogeneity based on immunohistochemical studies19 20 and transgenic analysis using fragments of SMC-specific/selective promoters.15 However, a limitation of such studies is the inability to discern actual heterogeneity from such issues as assay sensitivity and/or periodicity of endogenous gene/transgene expression.
Therefore, the goals of the present study were 2-fold, as follows: (1) to test the feasibility of targeting cre recombinase to achieve SMC-specific alterations in gene function by using the SMMHC promoter fragment and (2) to test the hypothesis that certain smooth muscle tissues consist of heterogeneous populations of SMCs, some of which require additional regulatory elements to activate the SMMHC promoter transgene. To address both aims, we have generated several SMMHC-cre transgenic lines and assayed cre expression by crossing these mice to mice that express ß-galactosidase (Bgal) from the ROSA26 locus only on cre-mediated recombination (R26R mice).21 A key issue in these crosses is to ensure that the indicator strain has the potential to express LacZ in all cells on cre-mediated activation. Indeed, the ROSA26 locus has previously been shown to drive ubiquitous and uniform expression of Bgal at all developmental and postnatal times, and the activity of the locus has no apparent sensitivity to genetic background.21 22 23 These crosses enabled us to determine the specificity of cre activity throughout development, and in adult mice, by assaying for expression of cre-dependent LacZ activity. Furthermore, because breeding of SMMHC-cre mice to the R26R line provided an integration of cre activity over time, this system enabled us to address whether heterogeneous populations of SMCs existed within a given tissue as defined by the ability to express the SMMHC promoter fragment. Overall, our results demonstrated that, unlike our previous report describing the activity of this promoter fragment in vivo,18 the SMMHC promoter fragment was indeed active in all SMCs of most smooth muscle–containing tissues. Furthermore, our results demonstrate the feasibility of successful targeting of cre to SMCs. As such, these mice should provide an extremely valuable tool for researchers to track smooth muscle lineage, as well as to assess the function of candidate genes in vivo through SMC-specific activation or inactivation of genes of interest.
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Materials and Methods |
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Generation of Transgenic Mice
Transgenic founder lines were generated by standard microinjection techniques24 in collaboration with the University of Virginia transgenic mouse core facility. Four founder lines were generated and bred to C57/BL6 mice to produce subsequent generations. Mice from each SMMHC-cre founder line were bred to R26R indicator mice, which demonstrate cre-activated, cytoplasmic LacZ staining.21 With this system, cre-dependent activation of the LacZ gene will occur in all cells in which the SMMHC-cre transgene is actively being expressed or has been expressed (at a level sufficient to direct cre-mediated DNA excision) at any time before the assay. Conversely, areas where there was no cre expression or where expression was too low to produce sufficient enzyme to excise loxP-flanked DNA will show no staining for Bgal. To determine the genotype of embryos and adult mice, DNA was isolated from a piece of the embryo or tail and assayed for cre11 and LacZ 14 18 genes by PCR.
Bgal Staining and Histology
To determine Bgal staining during development, pregnant mice
were anesthetized at various times (10.5 to 16.5 days)
postcoitum (PC) with pentobarbital (100 mg/kg). Embryo and adult
mouse tissue was harvested, stained, and visualized as described
previously.14 18 Select adult tissues were processed for
routine histology, sectioned at 4 µm, and counterstained with
eosin. No background Bgal staining, as determined by staining mice
containing only the R26R gene, was observed at any embryonic time point
or in any adult tissues examined (data not shown).
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Results |
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We generated 4 SMMHC-cre transgenic founder lines (5013, 5770, 5790, and 6195), 3 of which (5013, 5770, and 6195) transmitted the transgene in the predicted Mendelian frequency and thus were chosen for further study. A key rationale for this experimental approach is that once cre expression has been induced and excision of the loxP-flanked stop sequence of the R26R gene occurs, Bgal expression is then under control of a ubiquitous promoter and is independent of further cre expression. As such, even transient expression of cre at a level sufficient to mediate DNA excision will result in activation of LacZ. Initial studies focused on determining cre activity at various embryonic time points by breeding each SMMHC-cre line to the R26R indicator line and assaying for Bgal expression. In general, the 3 lines demonstrated a similar pattern of cre-dependent excision. Whereas the cre expression in both embryos and adults seemed weaker in line 6195 (data not shown), cre-dependent LacZ staining followed the same pattern as in the other 2 lines. This is likely a result of transgene insertion site. It is interesting to note that the insertion site only affected the amount of staining within smooth muscle–containing tissues and not the tissues in which cre-dependent LacZ staining occurred. The other 2 lines, 5013 and 5770, showed nearly identical staining patterns, both in location and approximate number of SMCs showing cre-dependent LacZ staining, suggesting that the expression of cre in these 2 lines was primarily a function of the transgene construct rather than the integration site.
As can be seen in Figure 1
, cre-dependent
Bgal expression was evident in the umbilical artery, bronchi, and aorta
of embryos at 12.5 days PC (Figure 1A). Previous studies using a
SMMHC promoter-LacZ transgene failed to show expression in the aorta
and umbilical vessels until much later in development (Figures 1D through 1F).18 Because multiple independent
founder lines were examined here and in our original studies, the
earlier detection of SMMHC transgene activity is likely a function of
the increased sensitivity of the binary cre/lox detection system rather
than a function of differing insertion sites or copy number.
Significantly, the current data agree more closely with the time at
which endogenous SMMHC transcript is detected in the
developing aorta by in situ hybridization.17
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LacZ expression was also detected in a small number of cardiac
myocytes. It is possible that this staining represents ectopic
expression of cre due to transgene integration site. However, a similar
myocardial staining pattern was seen in all 3 lines studied. Therefore,
the myocardial staining is probably specific, because it is unlikely
that ectopic transgene expression would be so consistent across
multiple founder lines. The limited myocardial cell staining was also
seen at 10.5 days PC, suggesting that the cre-dependent excision event
occurred very early in cardiovascular development
(Figure 2).
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Analysis of successively later embryonic time points showed
that cre-dependent LacZ staining was observed in an increasingly
greater number of smooth muscle tissues. This result is
consistent with earlier in situ studies of the
endogenous transcript.17 By 14.5 days PC,
expression was clearly evident in the entire gastrointestinal tract as
well as numerous medium-sized arteries and a few arterioles (Figure 1B
). In addition, the amount of LacZ staining at 14.5 days PC
was qualitatively increased in the aorta, bronchi, and umbilical
arteries as compared with 12.5 days PC. By 16.5 days PC, staining had
significantly increased in the peripheral vasculature
(Figure 1C). Interestingly, we observed no significant increase
in myocardial staining at later embryonic time points, suggesting that
SMMHC-cre was only transiently activated early in development
in a subset of cells that became cardiac myocytes.
Activity of SMMHC-cre transgene was also examined in adult tissues
(Figure 3), which represents an
integral of cre activity from development through the experimental end
point. We assayed a variety of SMC and non-SMC tissues and found, with
the exception of the limited myocardial staining mentioned earlier,
that Bgal staining was completely restricted to SMCs. Whole-mount
examination of the thoracic aorta and carotid arteries (Figures 3A and 3B, respectively) suggested that all SMCs were labeled.
When examined histologically, the aorta (Figure 4A2) as well as the carotid artery
(Figure 4-A1) showed complete staining of the medial wall. This
homogenous staining is in marked contrast to the patchy,
heterogeneous staining observed in multiple founder lines
when the SMMHC promoter fragment was directly coupled to LacZ (Figures 3-A1 [SMMHC-crexR26R] versus 3-A2
[SMMHC-LacZ]).18 Figure 3B also demonstrated
staining of tracheal smooth muscle, and as can be seen in Figure 4C, the staining was localized to tracheal smooth muscle.
Whole-mount staining of the heart revealed staining of the
coronary arteries as well as discrete patches of staining in
the myocardium (Figure 3C). When the heart was cut
into smaller pieces before staining and then examined
histologically, smaller intramyocardial arteries showed
Bgal staining (Figure 4-B1). As stated earlier, staining of a
small fraction of myocardial cells (Figure 3C) was observed in
all 3 SMMHC-cre lines studied. This staining was restricted to discrete
patches of cardiac myocytes in the adult (Figure 4-B2),
suggesting that the SMMHC-cre construct was not active in cardiac
myocytes at any later stage of development. The whole-mount staining of
lung (Figure 3D) showed staining of bronchioles, but it was
difficult to determine whether the pulmonary vasculature was
stained. On histological examination (Figure 4D), it is clear that both the bronchiolar and vascular smooth
muscle were stained for Bgal activity. In our initial studies in which
the promoter was directly coupled to Bgal, we were unable to detect
pulmonary vasculature staining across multiple founder
lines.18 However, the current approach revealed transgene
promoter activity in vascular beds previously unidentified, namely the
pulmonary circulation. A variety of other
peripheral vessels also expressed cre throughout
development, as shown in Figure 3. In skeletal muscle, very
small arterioles stained highly for Bgal activity with no staining in
skeletal muscle (Figures 3E and 4E). It is important to
note the significant staining of even very small arteries/arterioles in
the mesenteric arcade (Figure 3F) and particularly in the
cerebral vessels (Figure 3G). Such staining was rarely observed
in SMMHC-LacZ transgenic mice.18
Histological examination of these vessels also revealed
that staining was restricted to smooth muscle within the medial wall
(data not shown). Also, it is interesting to note that the skeletal
muscle vein (Figure 4E) appeared unstained. In fact, a number of
small veins did not appear to be stained. However, large veins, such as
the vena cava and pulmonary vein, did show staining. This may
reflect the following: (1) there is a limitation in visualization
of the Bgal reaction product in such a thin-walled vessel, (2)
venous SMCs express lower levels of SMMHC and expression of the
SMMHC-cre transgene may be below the level necessary for detection,
and/or (3) additional promoter elements are needed to drive efficient
expression in venous smooth muscle.
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In addition to smooth muscle–containing cardiopulmonary
tissues, the SMMHC-cre construct was active in all other smooth muscle
tissues. For example, Figure 3 shows complete staining of
intestinal wall (H1) smooth muscle. Histologically,
homogenous staining was restricted to the circumferential and
longitudinal smooth muscle layers (Figure 4F). As mentioned
earlier in reference to the aortic and carotid staining, staining of
gastrointestinal smooth muscle was also homogenous, in contrast to
earlier reports using this promoter fragment (Figures 3H1
[SMMHC-crexR26R] versus 3H2 [SMMHC-LacZ]).18
Moreover, in contrast to our previous report,18 the smooth
muscle layer of the esophagus was also stained for Bgal activity
(Figure 4G). In the genitourinary tract, cre-mediated LacZ
expression was observed in all of the SMCs of the bladder (Figure 3I) and ureter (Figure 4H).
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Discussion |
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In this study, we have described the first report of a cre-based system that can be used to target gene inactivation or overexpression to SMCs throughout development and in adult mice. By limiting gene inactivation or overexpression to SMCs, the cre-based system provides a number of significant advantages over traditional gene-targeting approaches, including the following: (1) reducing (although not eliminating) activation of potential compensatory pathways due to gene ablation in multiple cell types, (2) negating effects of gene manipulation on the development of structures unrelated to SMC development, and (3) having the ability to discern the direct role of genes that are expressed in both the developing heart and vasculature4 5 6 on blood vessel development/maturation by specific gene targeting to SMCs. Multiple SMMHC-cre lines were bred to a previously described cre-activated LacZ indicator line21 to assay for cre-dependent DNA excision throughout development and in adult tissues. Results showed that the SMMHC promoter fragment drove cre expression specifically in SMCs throughout development.
Previously, SMMHC-LacZ mice demonstrated heterogeneous expression of LacZ in a wide array of embryonic and adult SMC tissues.18 Results from the SMMHC-LacZ mice18 showed heterogeneous expression of the transgene within a given smooth muscle tissue (ie, microheterogeneity) and among various smooth muscle–containing tissues (ie, regional heterogeneity). The previously observed patchy staining pattern suggested either heterogeneous populations of SMCs that required further regulatory elements to activate this fragment or periodicity of transgene expression of which the staining pattern reflected episodic expression.18 Because SMMHC-dependent cre expression can be assayed as an integral over time by using the binary cre/lox system, this system provided a model to address issues of SMC heterogeneity, as defined by the ability of the cells to activate the SMMHC promoter fragment.
To determine whether the heterogeneous staining pattern observed in SMMHC-LacZ transgenic animals was consistent with the idea of SMC heterogeneity, we assayed LacZ staining in adult mice containing both the SMMHC-cre transgene and the R26R gene. We reasoned that if tissues contained a heterogeneous population of SMCs, as defined by their differing abilities to activate the SMMHC promoter fragment, we would be unable to obtain SMC tissues that showed complete LacZ staining (ie, 100% positive cells). Data from the current study demonstrated that the SMMHC-cre transgene mediated recombination in all SMCs of certain tissues, including the aorta and intestine. Therefore, the promoter fragment was sufficient to drive expression in all of these cells, consistent with the idea that the original heterogeneous expression noted in these tissues18 was likely due to episodic changes in expression of the transgene combined with limits in assay sensitivity. However, it is important to emphasize that our results do not distinguish between whether expression of the SMMHC promoter fragment is episodic in adult SMC-containing tissues and whether the promoter may be permanently inactivated in a subset of SMCs in adult tissues. Indeed, distinguishing between these possibilities will require the development of assays that can assess real-time promoter activity. Finally, results of the current study also revealed that the promoter fragment was active in previously unidentified vascular beds, such as the pulmonary circulation, demonstrating that the promoter also contained sufficient regions to be activated in this population of SMCs.
It is interesting to note that although the cre/lox system provides an
integrated assay of promoter activity over time, there were still
vessels or portions of vessels that remained negative for Bgal
activity, most notably very small terminal arterioles and the majority
of the renal circulation. It is possible that the SMMHC-cre transgene
may be either constitutively or episodically active in these cells but
at a very low level, so cre protein may never attain sufficient levels
to effectively mediate recombination of loxP-flanked DNA. However, it
is also possible that further regulatory elements may be required to
drive expression from this promoter fragment in certain subpopulations
of SMCs similar to reports of the SM22
promoter.15
Interestingly, results of the present study also revealed
expression of the SMMHC promoter fragment in developing aortic and
airway SMCs as early as 12.5 days PC. In later developmental time
points, we detected cre activation in an increasingly greater number of
small vessels and other SMC-containing tissues in comparison with the
staining seen in the SMMHC-LacZ mice (compare Figures 1A through
1C versus 1D through 1F). Taken together, these results are
consistent with the idea that the binary cre/lox assay system
used here demonstrated greater sensitivity in detecting promoter
activity than direct coupling the promoter to the Bgal
gene.18 Moreover, our results demonstrate the general
utility of cre/lox-type assay systems for purposes of rigorous
assessment of cell specificity/selectivity of transgene expression
patterns in vivo.
A potentially intriguing result of our analysis of the SMMHC-cre mice was the appearance of labeling of a subset of myocardial cells. This myocardial staining was limited to a small number of cells, occurred at least as early as 10.5 days PC, and was consistent across multiple founder lines. Binary cre/lox systems are increasingly being used to perform fate-mapping studies in mice.25 26 Our data suggest there may be cells that invest the myocardium that transiently expressed SMMHC at an early stage of development. It is possible that this slight staining represents ectopic expression of the transgene, but this is difficult to reconcile with the fact that we obtained similar staining patterns in multiple founder lines. It is not unreasonable to speculate that a population of cells, which ultimately become cardiac myocytes, may transiently activate the SMMHC gene at early developmental time points. First, the heart is known to transiently express other genes that are normally restricted to smooth muscle.27 28 Furthermore, because the heart arises from a similar population of cells that give rise to portions of the vasculature, such as neural crest and splanchnic mesoderm,29 30 it is possible that some cells in this area may transiently express SMMHC and subsequently become incorporated into the developing heart. Clearly our observations will require further studies that use cre/lox approaches in combination with specific antibodies and probes to SMMHC.
Although this study has clearly demonstrated the ability to target gene manipulation specifically to SMCs, it is important to emphasize that there are clearly some cells within smooth muscle tissues as well as entire vascular beds that did not express sufficient levels of cre to mediate recombination. The phenomenon of "incomplete" penetrance with cre-based systems is not uncommon.31 However, it obviously must be considered in interpretation of results of gene-targeting experiments, particularly results that rely on whole tissue/organ assay systems to define phenotypic changes associated with alterations in gene function. On the other hand, for purposes of direct assessment of gene function at the single-cell level, such incomplete penetrance or mosaicism of cre recombination can be advantageous. For example, to fully exploit this incomplete cre penetrance, cre-dependent overexpression transgenes that combine cre-induced gene activation with inactivation of a reporter gene (ie, enhanced green fluorescence protein), could be designed. It may also be feasible to integrate a reporter gene into floxed endogenous gene-targeting cassettes to facilitate identification of cells in which the floxed gene has been deleted. In essence, such systems would provide the equivalent of a tissue-specific chimera and would provide an extremely powerful means to directly assess the function of candidate genes in SMCs in vivo.
In summary, the results of the present studies demonstrate the feasibility of achieving cre-dependant SMC gene targeting in vivo. In addition, these results provide comprehensive characterization of 2 unique SMMHC-cre mouse lines that should have major utility for assessing gene function in vascular development/disease, as well as dissecting smooth muscle lineage through the use of binary cre/lox systems. Furthermore, the present studies have also provided novel insights regarding mechanisms of SMMHC promoter regulation in vivo, and the data clearly demonstrate that many factors, including assay method and assay sensitivity, must be taken into account when assessing the existence of SMC heterogeneity.
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Acknowledgments |
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This work was supported by grants from the NIH (RO1HL57353, RO1HL19242, and RO1HL38854 to G.K.O.), the Virginia Affiliate of the American Heart Association (AHA-F98255V to I.M.), and the American Physiological Society Fellowship in Physiological Genomics (to C.P.R.). We thank Dr Brian Duling; Kathy Day, MS (University of Virginia Cardiovascular Research Center); Angela Miller (University of Virginia Histology Core Facility); Jennifer Clatterbuck; and Diane Raines for technical support and/or helpful discussions.
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Footnotes |
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1 Both authors contributed equally to this study.
Received May 9, 2000; revision received July 10, 2000; accepted July 10, 2000.
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 A. D. Frutkin, H. Shi, G. Otsuka, and D. A. Dichek Targeted Rearrangement of Floxed Alleles in Smooth Muscle Cells in Vivo Circ. Res., December 7, 2007; 101(12): e124 - e125. [Full Text] [PDF]
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Y. Liao, C. P. Regan, I. Manabe, G. K. Owens, K. H. Day, D. N. Damon, and B. R. Duling Smooth Muscle-Targeted Knockout of Connexin43 Enhances Neointimal Formation in Response to Vascular Injury Arterioscler. Thromb. Vasc. Biol., May 1, 2007; 27(5): 1037 - 1042. [Abstract] [Full Text] [PDF]
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 S. Sinha, B. R. Wamhoff, M. H. Hoofnagle, J. Thomas, R. L. Neppl, T. Deering, B. P. Helmke, D. K. Bowles, A. V. Somlyo, and G. K. Owens Assessment of Contractility of Purified Smooth Muscle Cells Derived from Embryonic Stem Cells Stem Cells, July 1, 2006; 24(7): 1678 - 1688. [Abstract] [Full Text] [PDF]
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J. Zhang, W. Zhong, T. Cui, M. Yang, X. Hu, K. Xu, C. Xie, C. Xue, G. H. Gibbons, C. Liu, et al. Generation of an Adult Smooth Muscle Cell-Targeted Cre Recombinase Mouse Model Arterioscler. Thromb. Vasc. Biol., March 1, 2006; 26(3): e23 - e24. [Full Text] [PDF]
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 C. A. Burckle, A.H. Jan Danser, D. N. Muller, I. M. Garrelds, J.-M. Gasc, E. Popova, R. Plehm, J. Peters, M. Bader, and G. Nguyen Elevated Blood Pressure and Heart Rate in Human Renin Receptor Transgenic Rats Hypertension, March 1, 2006; 47(3): 552 - 556. [Abstract] [Full Text] [PDF]
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 A. Dikalova, R. Clempus, B. Lassegue, G. Cheng, J. McCoy, S. Dikalov, A. S. Martin, A. Lyle, D. S. Weber, D. Weiss, et al. Nox1 Overexpression Potentiates Angiotensin II-Induced Hypertension and Vascular Smooth Muscle Hypertrophy in Transgenic Mice Circulation, October 25, 2005; 112(17): 2668 - 2676. [Abstract] [Full Text] [PDF]
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S. Handa, M. A. Momen, A.-M. Sadi, T. Afroze, C. Wang, and M. Husain Troubles With a Transgene: Experiences With SM22{alpha}-tTA Mice Circ. Res., October 14, 2005; 97(8): e85 - e87. [Full Text] [PDF]
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T. Yoshida and G. K. Owens Molecular Determinants of Vascular Smooth Muscle Cell Diversity Circ. Res., February 18, 2005; 96(3): 280 - 291. [Abstract] [Full Text] [PDF]
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 H.-B. Xin, K.-Y. Deng, B. Shui, S. Qu, Q. Sun, J. Lee, K. S. Greene, J. Wilson, Y. Yu, M. Feldman, et al. Gene trap and gene inversion methods for conditional gene inactivation in the mouse Nucleic Acids Res., January 19, 2005; 33(2): e14 - e14. [Abstract] [Full Text] [PDF]
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 J. M. Miano, N. Ramanan, M. A. Georger, K. L. de Mesy Bentley, R. L. Emerson, R. O. Balza Jr., Q. Xiao, H. Weiler, D. D. Ginty, and R. P. Misra Restricted inactivation of serum response factor to the cardiovascular system PNAS, December 7, 2004; 101(49): 17132 - 17137. [Abstract] [Full Text] [PDF]
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 S. Sinha, M. H. Hoofnagle, P. A. Kingston, M. E. McCanna, and G. K. Owens Transforming growth factor-{beta}1 signaling contributes to development of smooth muscle cells from embryonic stem cells Am J Physiol Cell Physiol, December 1, 2004; 287(6): C1560 - C1568. [Abstract] [Full Text] [PDF]
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 G. K. Owens, M. S. Kumar, and B. R. Wamhoff Molecular Regulation of Vascular Smooth Muscle Cell Differentiation in Development and Disease Physiol Rev, July 1, 2004; 84(3): 767 - 801. [Abstract] [Full Text] [PDF]
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 F. Dandre and G. K. Owens Platelet-derived growth factor-BB and Ets-1 transcription factor negatively regulate transcription of multiple smooth muscle cell differentiation marker genes Am J Physiol Heart Circ Physiol, June 1, 2004; 286(6): H2042 - H2051. [Abstract] [Full Text] [PDF]
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 G. Ji, M. E. Feldman, K.-Y. Deng, K. S. Greene, J. Wilson, J. C. Lee, R. C. Johnston, M. Rishniw, Y. Tallini, J. Zhang, et al. Ca2+-sensing Transgenic Mice: POSTSYNAPTIC SIGNALING IN SMOOTH MUSCLE J. Biol. Chem., May 14, 2004; 279(20): 21461 - 21468. [Abstract] [Full Text] [PDF]
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 M. L. Allende, T. Yamashita, and R. L. Proia G-protein-coupled receptor S1P1 acts within endothelial cells to regulate vascular maturation Blood, November 15, 2003; 102(10): 3665 - 3667. [Abstract] [Full Text] [PDF]
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A. M. Hoggatt, G. M. Simon, and B. P. Herring Cell-Specific Regulatory Modules Control Expression of Genes in Vascular and Visceral Smooth Muscle Tissues Circ. Res., December 13, 2002; 91(12): 1151 - 1159. [Abstract] [Full Text] [PDF]
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 H.-B. Xin, K.-Y. Deng, M. Rishniw, G. Ji, and M. I. Kotlikoff Smooth muscle expression of Cre recombinase and eGFP in transgenic mice Physiol Genomics, September 3, 2002; 10(3): 211 - 215. [Abstract] [Full Text] [PDF]
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R. Holtwick, M. Gotthardt, B. Skryabin, M. Steinmetz, R. Potthast, B. Zetsche, R. E. Hammer, J. Herz, and M. Kuhn Smooth muscle-selective deletion of guanylyl cyclase-A prevents the acute but not chronic effects of ANP on blood pressure PNAS, May 14, 2002; 99(10): 7142 - 7147. [Abstract] [Full Text] [PDF]
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 A. D. Eckhart, T. Ozaki, H. Tevaearai, H. A. Rockman, and W. J. Koch Vascular-Targeted Overexpression of G Protein-Coupled Receptor Kinase-2 in Transgenic Mice Attenuates beta -Adrenergic Receptor Signaling and Increases Resting Blood Pressure Mol. Pharmacol., April 1, 2002; 61(4): 749 - 758. [Abstract] [Full Text] [PDF]
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