The development of the mature cardiovascular system is one of the most captivating stories in embryonic development. The heart is the first organ to form in embryogenesis, and is functional early in development to perfuse the embryo with blood supplying oxygen and the nutrients required for organogenesis. The structural changes in heart development required for formation of the mature four chambered heart are under tight molecular regulation. Severe defects manifest as gross structural malformations of the valves, septa, or vessels that result in physiological consequences that my include hypertension, arrhythmia, or heart failure and may ultimately lead to lethality. According to the American Heart Association, cardiovascular disease is the leading cause of mortality worldwide. A more detailed understanding of the origin of congenital heart defects is necessary for improving prediction, diagnosis, and treatment of cardiovascular disease. Derived from the epicardium, coronary vessel formation relies on growth factor as well as extracellular matrix (ECM) influences on cells of the epicardium that regulate proliferation, motility, invasion and differentiation. The Transforming Growth Factor β (TGFβ) family of receptors have been well described in regulating cardiovascular development. The Type III TGFβ receptor (TGFβR3) has been shown to be required for development of the coronary vessels. Mouse embryos lacking TGFβR3 exhibit inhibited invasion of epicardially derived cells (EPDCs) into the myocardium. This delay of cell invasion of EPDCs and formation of coronary vessels is lethal at E 14.5. Relative to Tgfbr3+/+ cells, epicardial cells lacking TGFβR3 are hypo-proliferative, deficient in cell invasion, and deficient in executing TGFβ ligand and High-Molecular Weight Hyaluronan (HMWHA) stimulated cell invasion. Hyaluronan (HMWHA) is a glycosaminoglycan unmodified sugar extracellular matrix (ECM) molecule synthesized by the Hyaluronan Synthase (Has) family of enzymes. Mouse embryos lacking Hyaluronan Synthase 2 (Has2) are lethal at E 9.5 as a result of severely blocked cardiogenesis due to insufficient endocardial EMT. HA serves structural and bioactive functions in its capacity to stimulate signal transduction pathways required for EMT. Src kinase is a non-receptor tyrosine kinase well characterized to function in growth factor as well as ECM signal transduction, but its role in epicardial cell biology is unclear. Our hypothesis is that Src kinase is a critical regulator of TGFβ and Hyaluronan induced epicardial cell invasion, differentiation and migration during coronary vessel development. Our studies reveal that Src activity is required for TGFβ2-induced synthesis of HA in epicardial cells. We show Src is required for TGFβ2-induced vascular smooth muscle differentiation as well as TGFβ2-induced EMT, cell invasion, and filamentous actin polymerization. Src activity is sufficient to drive epicardial activation of EMT, but not vascular smooth muscle differentiation. These data show that Src is required in the context of TGFβ2-stimulated invasion and differentiation, and sufficient to drive activation of EMT. Next we demonstrate that TGFβR3 and Src are required for HMWHA induced cell invasion and filamentous actin polymerization in epicardial cells. HMWHA induces activation of Src kinase in Tgfbr3+/+ epicardial cells, but not Tgfbr3-/- epicardial cells. siRNA knockdown of TGFβR3 in Tgfbr3+/+ epicardial cells subsequently stimulated with HMWHA phenocopy this deficit in Src activation. Tgfbr3-/- epicardial cells fail to activate Rac1 or RhoA GTPases in the presence of HMWHA. Finally, we demonstrate stimulus independent activation of TGFβR3 is sufficient to activate Src. Taken together, these constitute novel findings establishing TGFβR3 as an HMWHA responsive receptor that is upstream of Src signal transduction. Migration of the epicardium to cover the looped and functioning heart tube is an early step required for development of the coronary vessels. We demonstrate that Tgfbr3-/- epicardial cells are delayed in cell migration relative to Tgfbr3+/+ cells in a wound healing model of cell migration. Tgfbr3-/- cells lack expression of BMP2 mRNA, we found that exogenous BMP2 is sufficient to drive Tgfbr3-/- (but not Tgfbr3+/+) cell migration to levels comparable to unstimulated Tgfbr3+/+ epicardial cells, without enhancing cell proliferation. We demonstrate that Src is required for this BMP2 induced cell migration and filamentous actin polymerization in Tgfbr3-/- cells. These studies demonstrate mechanisms required for TGFβ ligand as well as HMWHA stimulated epicardial cell behavior changes have a common mediator in Src kinase, and provide novel insights into early events in the development of the cardiovascular system. The adult epicardium has been demonstrated to participate in repair of ischemic myocardium in mouse models of myocardial infarction. Expression of molecules required for coronary vessel development are re-expressed in this regeneration (as discussed in chapter 5). Elucidating these pathways will constitute important future targets in aiding in adult cardiovascular regeneration and cardioprotection in adult heart disease.
| Identifer | oai:union.ndltd.org:arizona.edu/oai:arizona.openrepository.com:10150/347226 |
| Date | January 2014 |
| Creators | Allison, Patrick Bartlett |
| Contributors | Camenish, Todd D., Camenish, Todd D., Vaillancourt, Richard R., Regan, John W., Runyan, Raymond B., Barnett, Joey V. |
| Publisher | The University of Arizona. |
| Source Sets | University of Arizona |
| Language | en_US |
| Detected Language | English |
| Type | text, Electronic Dissertation |
| Rights | Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. |
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