| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online May 8, 2002 as doi:10.1096/fj.01-0986fje. |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Johannes-Müller-Institut für Physiologie and
* Klinik für Innere Medizin I, Humboldt-Universität, Charité, 10117 Berlin, Germany
3Correspondence: Johannes-Müller-Institut für Physiologie, Medizinische Fakultät Charité, Humboldt-Universität zu Berlin, Tucholskystrasse 2, 10117 Berlin, Germany. E-mail: holger.scholz{at}charite.de
SPECIFIC AIMS
Expression ofthe Wilms’ tumor gene Wt1 in the developing epicardium is critical for cardiac growth during embryogenesis. Since revival of a ‘fetal-type’ gene expression program is a common theme in the growth of adult hearts, we reasoned that Wt1 could be involved in cardiac hypertrophy. To test this hypothesis, we analyzed the expression of Wt1 in the normal and hypertrophied left ventricular myocardium of rats.
PRINCIPAL FINDINGS
1. Wt1 mRNA in normal and hypertrophied rat hearts
RNase protection assay was used to measure Wt1 mRNA in normal rat hearts and in the hypertrophied left ventricles of rats with 1) spontaneous hypertension (SHR), 2) activated renin-angiotensin system due to transgenic (over)expression of the human renin and angiotensinogen genes (TGR), and 3) postinfarct remodeling of the heart after ligation of the left coronary artery (LAD). No differences in Wt1 transcript levels were detected between the hearts of normal rats and the hypertrophied left ventricular myocardium of SHR and TGR. However, Wt1 mRNA was increased > twofold in the left ventricles of rats 6 wk after LAD ligation.
2. Kinetics of Wt1 expression in the heart after myocardial infarction
To study Wt1 expression in the infarcted rat hearts in more detail, we measured its mRNA at different time intervals after LAD ligation. Compared with sham-operated rats, Wt1 mRNA was increased 2.5- to 3-fold in the left ventricular myocardium 24 h after ligation of the LAD (Fig. 1
A, B). Wt1 transcripts remained elevated for 9 wk after myocardial infarction. No significant differences in Wt1 mRNA were detected between the noninfarcted right ventricles of the LAD-ligated and sham-operated rats at variable time points after cardiac surgery. (Fig. 1C
).
|
3. Localization of Wt1 mRNA and protein in the infarcted rat hearts
Immunohistochemistry and mRNA in situ hybridization were performed to identify the cellular sites of Wt1 expression in the hearts of sham-operated rats and of rats with LAD ligation. As shown by other groups, Wt1 was detected in the epicardial layer but not in the myocardium of the sham-operated animals. However, Wt1 was expressed in coronary vessels located in close proximity to the infarcted tissue in rats with LAD ligation (Fig. 2
b, c, e, f). Wt1 was not expressed in the vasculature of the noninfarcted right ventricles.
|
4. Identification of the Wt1-expressing cell type(s) in the infarcted myocardium
To further characterize the cell type(s) that expressed Wt1 in the coronary vesselsafter myocardial infarction, we used a double-immunofluorescent labeling technique. Staining of vascular smooth muscle cells (VSMCs) was performed with a polyclonal antibody against smooth muscle 
-actin; vascular endothelial cells were marked with antibody against platelet/endothelial cell adhesion molecule 1 (PECAM-1). Overlapping expression of these proteins indicated that vascular smooth muscle cells and vascular endothelial cells in the border zone of the infarcted myocardium were capable of Wt1 expression.
5. Wt1 colocalizes with cell proliferation markers and VEGF in the ischemic rat hearts
The overlapping distribution of PECAM-1 and Wt1 is remarkable, as PECAM-1 has been implicated in vascular development. This raises the possibility that local up-regulation of Wt1 in the coronary vasculature was related to vascular cell proliferation and the formation of new blood vessels in response to regional tissue ischemia. This notion is supported by our findings that Wt1 and proliferating cell nuclear antigen (PCNA) shared a strikingly similar expression pattern in the vasculature of the infarcted rat hearts (Fig. 2b
, 2c
). In the coronary vasculature of the ischemic myocardium, Wt1 was also colocalized with vascular endothelial growth factor (VEGF), which is a potent stimulus of vascular endothelial cell proliferation and migration (Fig. 2e
, 2f
). Wt1 and VEGF immunoreactivity was not detected in the nonischemic right ventricles of rats with coronary artery ligation.
6. Stimulation of Wt1 expression in rat hearts by hypoxia
Since reduced tissue oxygenation is a major component of local ischemia, we examined whether expression of Wt1 in the ischemic hearts could be mimicked by hypoxia. The oxygen supply to the tissues was reduced by exposure of rats for 6 h to normobaric hypoxia (8% O2) or 0.1% carbon monoxide to mimic anemia. A > fourfold increase in cardiac Wt1 mRNA was detected in hypoxic (8% O2, 0.1% CO) compared with normoxic (20% O2) animals. Similar to the ischemic myocardium, Wt1 immunoreactivity was detected in the coronary vasculature of the hypoxic rat hearts.
CONCLUSIONS AND SIGNIFICANCE
Coronary artery disease is the most important risk factor for mortality in the industrialized countries. It is therefore a major challenge to identify the genes involved in the progression of coronary artery disease and those that may protect ischemic myocardium through induction of collateral vessel formation. Our findings suggest that the Wilms’ tumor transcription factor Wt1 is a novel candidate mediator in coronary neovascularization.
Contrary to our original hypothesis, the lack of response of Wt1 in the hypertrophied left ventricles of spontaneously hypertensive rats and rats with activated renin-angiotensin system does not support a close relationship between Wt1 expression and cardiac growth. Instead, our findings suggest that up-regulation of Wt1 in the coronary vasculature is part of the genomic response of the myocardium to local tissue ischemia.
Some of the most obvious questions that arise from our findings are 1) How can it be explained that coronary vascular cells are capable of expressing Wt1? 2) Which signals stimulate Wt1 expression in the coronary vasculature after myocardial infarction? 3) What is the functional significance of Wt1 expression in coronary vessels in response to cardiac ischemia? To find reasonable answers to these questions, it is helpful to summarize briefly the spatial and temporal expression of Wt1 in the developing heart.
During cardiogenesis in the mouse embryo, Wt1 is initially detected at gestational day 9 (E9) in the proepicardial mesenchymal villi on the cranial surface of the septum transversum. Subsequently, these Wt1-positive cells migrate across the pericardial cavity and spread over the surface of the myocardium to form the epicardial layer. Between E11.5 and E12.5, Wt1-expressing cells begin to delaminate from the epicardium into the subepicardial zone, where they form a layer of subepicardial mesenchymal cells (SEMCs). Wt1 is thought to play a crucial role in this process by enabling epicardial cells to flip between an epithelial and mesenchymal state. This assumption is based on the characteristic phenotype of mouse embryos with inactivated Wt1 gene, which exhibit severe defects in the epicardium and die in utero, presumably from heart failure. In wild-type mice around E12.5, Wt1-positive cells migrate from the SEMC zone into the myocardium, where they give rise to coronary vascular smooth muscle cells, perivascular and intermyocardial fibroblasts, and possibly vascular endothelial cells. Wt1 expression is switched off once these cells have become fully differentiated. Our findings suggest that epicardium-derived coronary vascular cells retain the capacity of Wt1 expression in adult hearts. Our observations also demonstrate that local tissue ischemia and hypoxia are potent stimuli of Wt1 expression in the coronary vasculature. The molecular signals that underlie activation of Wt1 during hypoxia remain to be determined. Two predicted consensus bindings motifs (5'-RCGTGV-3') for the hypoxia-inducible factor-1 (HIF-1) are contained in the proximal promoter of the mouse Wt1 gene (K.-D. Wagner et al., unpublished observations). A master regulator of hypoxia-dependent gene expression, HIF-1 is a potential candidate for transcriptional activation of Wt1 in the coronary vasculature during reduced tissue oxygenation.
Proliferation of vascular endothelial and smooth muscle cells is a critical step in the formation of collaterals from preexisting coronary vessels. The remarkably similar expression patterns of Wt1 and PCNA, as well as PECAM-1, in the coronary vasculature of the ischemic myocardium suggest a role for Wt1 in the proliferative response of the myocardial vessels to local tissue ischemia. This idea is further supported by our finding that VEGF was detected in Wt1-positive coronary vessels in the ischemic myocardium close to the infarcted areas. Therefore, we hypothesize that Wt1 is involved in the control of vascular growth in the ischemic myocardium. In agreement with a more general concept on the function of Wt1, we propose that Wt1 may allow vascular coronary cells to regain a mesenchymal phenotype and to proliferate.
In summary, these findings demonstrate for the first time that Wt1 is activated in the coronary vasculature of ischemic myocardium. Our results indicate that de novo Wt1 expression in cardiac vessels is stimulated by reduced tissue oxygenation. We suggest that Wt1 has a role in the proliferation of coronary vascular cells that is part of the neovascularization process.
|
FOOTNOTES
1 1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0986fje; to cite this article, use FASEB J. (May 8, 2002) 
2 K.-D.W. and N.W. contributed equally to this work.
This article has been cited by other articles:
|
|
 |
|
  E.M. Winter, R.W. Grauss, B. Hogers, J. van Tuyn, R. van der Geest, H. Lie-Venema, R. V. Steijn, S. Maas, M.C. DeRuiter, A.A.F. deVries, et al. Preservation of Left Ventricular Function and Attenuation of Remodeling After Transplantation of Human Epicardium-Derived Cells Into the Infarcted Mouse Heart Circulation, August 21, 2007; 116(8): 917 - 927. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. W. Small, Z. Bolender, C. Bueno, C. O'Neil, Z. Nong, W. Rushlow, N. Rajakumar, C. Kandel, J. Strong, J. Madrenas, et al. Wilms' Tumor 1-Associating Protein Regulates the Proliferation of Vascular Smooth Muscle Cells Circ. Res., December 8, 2006; 99(12): 1338 - 1346. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. M. Kirschner, N. Wagner, K.-D. Wagner, S. Wellmann, and H. Scholz The Wilms Tumor Suppressor Wt1 Promotes Cell Adhesion through Transcriptional Activation of the {alpha}4integrin Gene J. Biol. Chem., October 20, 2006; 281(42): 31930 - 31939. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Wagner, K.-D. Wagner, H. Scholz, K. M. Kirschner, and A. Schedl Intermediate filament protein nestin is expressed in developing kidney and heart and might be regulated by the Wilms' tumor suppressor Wt1 Am J Physiol Regulatory Integrative Comp Physiol, September 1, 2006; 291(3): R779 - R787. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Wagner, K.-D. Wagner, H. Theres, C. Englert, A. Schedl, and H. Scholz Coronary vessel development requires activation of the TrkB neurotrophin receptor by the Wilms' tumor transcription factor Wt1 Genes & Dev., November 1, 2005; 19(21): 2631 - 2642. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. P. Lawley, F. Cerimele, S. W. Weiss, P. North, C. Cohen, H. P. W. Kozakewich, J. B. Mulliken, and J. L. Arbiser Expression of Wilms Tumor 1 Gene Distinguishes Vascular Malformations From Proliferative Endothelial Lesions Arch Dermatol, October 1, 2005; 141(10): 1297 - 1300. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Scholz and K. M. Kirschner A Role for the Wilms' Tumor Protein WT1 in Organ Development Physiology, February 1, 2005; 20(1): 54 - 59. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Wagner, K.-D. Wagner, Y. Xing, H. Scholz, and A. Schedl The Major Podocyte Protein Nephrin Is Transcriptionally Activated by the Wilms' Tumor Suppressor WT1 J. Am. Soc. Nephrol., December 1, 2004; 15(12): 3044 - 3051. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Nakamura, S. Hirohata, T. Murakami, T. Miyoshi, K. Demircan, T. Oohashi, H. Ogawa, K. Koten, K. Toeda, S. Kusachi, et al. Dynamic Induction of ADAMTS1 Gene in the Early Phase of Acute Myocardial Infarction J. Biochem., October 1, 2004; 136(4): 439 - 446. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K.-D. Wagner, N. Wagner, and A. Schedl The complex life of WT1 J. Cell Sci., May 1, 2003; 116(9): 1653 - 1658. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
