Personnel
Calvin Vary
http://www.mmcri.org/cmm/vary.html
Center for Molecular Medicine, Maine Medical Center Research Institute
Application of 4Pi Confocal Microscopy, and Image Correlation Spectroscopy, in elucidation of mechanisms in vascular disease
Role of LIM Domain Proteins in TGFB Receptor Signaling. The TGFB receptor signaling pathway is of central importance in angiogenesis and vessel wound repair, and is mediated by TGF? ligands binding to types I (TBRI: Alk1, Alk5), II (TBRII), and III (endoglin and betaglycan) receptors. The role of endoglin (END) in the vessel wall is poorly understood though its significance is underscored by its requirement for the developmental process of angiogenesis, and as the target gene responsible for hereditary hemorrhagic telangiectasia type 1 (HHT1). We have shown that endoglin expression in vitro results in inhibition of cell proliferation and migration, and have reported a novel mechanism underlying migration inhibition that requires endoglin's cytosolic domain, a site of serine/threonine (S/T) phosphorylation by the TGFB receptors. The endoglin cytosolic domain interacts with the focal adhesion-associated LIM domain proteins zyxin (Conley et al., 2004) and ZRP1 (Sanz-Rodriguez et al., 2004). These two interactions function to effect p130(Cas)/CrkII subcellular localization and actin fiber formation, respectively. We have recently obtained data indicating that endoglin threonine phosphorylation may provide a functional distinction between Alk1 and Alk5, and control endoglin-dependent changes in focal adhesion composition in response to TGFB or other signaling pathways. We wish to evaluate the hypothesis that: endoglin is a SMAD-independent lateral signaling device for the TGFB receptors, which is important for establishment of the proper balance of chemotactic and inhibitory stimuli that regulate cell migration via changes in focal adhesion composition. Our studies seek to understand how endoglin's function is affected by S/T phosphorylation by the TGFB receptors, specifically Alk1, Alk5 and TBRII, and to elucidate the downstream consequences for the interaction of endoglin with the LIM proteins, zyxin and ZRP1, as well as novel potential LIM-domain mediators including Ajuba, Enigma, Crp2 and LPP. 4Pi confocal microscopy, FRET and Image Correlation Spectroscopy (FICS) may be extensively exploited to examine the dynamics of the receptors within sites of focal adhesion and other structures.
Function of the BMPRII/Alk1/Endoglin in Vascular Disease Phenotypes. Though endoglin, Alk1 and the bone morphogenetic protein type II receptor BMPRII have been implicated in HHT1/2 and pulmonary arterial hypertension (PAH) respectively, the unifying mechanism by which mutations in BMPRII, Alk1 or endoglin may contribute to the overlapping but distinct pathological phenotypes encompassing these vascular diseases is lacking. Using conditional endoglin expression targeted to embryonic vascular smooth muscle we have shown that vascular malformations occur that are strikingly reminiscent of the HHT phenotype, and that E10.5 embryos expressing a cytosolic domainless endoglin mutant which is not phosphorylated by the BMPRII/Alk1 complex, has a pronounced phenotype exhibiting not only dilatated vessels, but also pericardial hemorrhage and perturbation of cardiac structure. We propose the hypothesis that: Alterations in individual BMPRII/Alk1/endoglin receptor components produce overlapping but distinct vascular phenotypes. This work seeks to understand the consequences of specific mutations in these receptor components in terms of transcriptional and physiological responses and including matrix metalloproteinase activity and collagen degradation using in vitro and in vivo approaches. These studies will focus on BMPRII/Alk1/endoglin-mediated signal transduction complexes and will lead to a better understanding the relationship of BMP receptor signaling in vascular development and the distinct vascular pathologies of PPH/HHT, enable a better understanding of interactive signaling between different TGFB receptor complexes, and ultimately contribute to a rational basis for the treatment of these diseases. 4Pi confocal microscopy, and FICS will be used to examine the colocalization of Alk1, BMPRII, and wild-type and mutant endoglin within the cell.
Molecular Imaging and Tracking of Vascular Smooth Muscle Cell Precursors in vivo. We have previously demonstrated that quantifiable age related changes occur in the dosage sensitivity of neural crest stem cells (NCSCs) to instructive neurogenic and gliogenic differentiation factors as NCSCs self-renew in vivo. In these studies, we propose to study the age-related changes in the dosage sensitivity of NCSCs to the instructive myogenic differentiation factor, TGF?1. Recent data indicate that END is a functional marker of hematopoietic and mesenchymal stem cells. We present preliminary evidence that indicates that as early as E10 in rat development, NCSCs can be isolated into two populations with distinct differentiation profiles based on the expression of END. We have shown that endoglin-positive (END+) NCSCs display tripotent potential, differentiating into autonomic neurons, Schwann cells and smooth muscle-like myofibroblasts, while END- NCSCs do not possess myogenic differentiation potential and are restricted to neuronal and glial differentiation outcomes. In the sciatic nerve, a reservoir of NCSCs throughout life, the ratio of END+ to END- NCSCs declines with age as does the dosage sensitivity of NCSCs to TGF?1-instructive myogenic differentiation signals. In vitro serial subcloning experiments suggest that as END+ NCSC self-renew, END- NCSC are generated. Moreover, ectopic expression of END into END- NCSCs restores myogenic differentiation potential, suggesting that the loss of END expression is a determining factor in the restriction of fate outcomes of NCSCs with age. This proposal seeks to examine the hypothesis that END is a causal determinant in the age-related differences in NCSC myogenic differentiation potential. To address this hypothesis we have initiated studies to: 1) Determine the role of endoglin during myogenic differentiation of NCSCs, and 2) Determine the molecular mechanisms that regulate endoglin expression in NCSCs, with emphasis on Notch and Hedgehog signaling events. An important question is whether NCSCs contribute to a circulating SMC progenitor pool that participates in myofibroblast populations in the healing or injured vascular wall. Tracking studies using MRI, two-photon, confocal and other imaging modalities, with transgenic GFP-bearing NCSC populations will enable new insights into this issue. These studies should provide novel and functional insights into the consequences of END expression in NCSCs, the control of TGF? signaling and its impact on smooth muscle cell specification. These insights should enable a better understanding of the age-dependent capacity of NCSCs to form smooth muscle, and may lead to a better understanding of age-related vascular phenotypes including atherosclerosis and HHT.
Relevant Recent Lab References
Conley, B. A., Koleva, R., Smith, J. D., Kacer, D., Zhang, D., Bernabeu, C., and Vary, C. P. H. (2004). Endoglin Controls Cell Migration and Composition of Focal Adhesions: FUNCTION OF THE CYTOSOLIC DOMAIN. J Biol Chem 279, 27440-27449.
Sanz-Rodriguez, F., Guerrero-Esteo, M., Botella, L.-M., Banville, D., Vary, C. P. H., and Bernabeu, C. (2004). Endoglin Regulates Cytoskeletal Organization through Binding to ZRP-1, a Member of the Lim Family of Proteins. J Biol Chem 279, 32858-32868.
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