Regulation of Endothelial Cell Growth and Development
Cardiovascular disease is one of the leading causes of morbidity and mortality in the United States. Vascular injury, hypoxia, and inflammation activate the normally quiescent endothelial cell (EC) to undergo several physiological changes in response to growth factors and cytokines that are released during these pathophysiological events. Therefore, to develop therapeutic strategies for vascular diseases it is necessary to: i) identify key growth factors, cytokines and their receptors involved in normal EC behavior as well as in pathologic conditions, and ii) to identify the molecular mechanisms by which these growth factors and receptors regulate cellular behavior. The angiogenic factors FGF2 and VEGF promote the proliferation, migration, and differentiation of endothelial cells (ECs) through the activation of their cognate receptor tyrosine kinases (RTKs). Dysregulation of either the FGF or VEGF signaling pathways leads abnormal endothelial cell function. We have found that members of a family of proteins called Sprouty (Spry) regulate endothelial cell proliferation, migration and adhesion, in part through regulating the expression of cell adhesion molecules. In particular, Spry4 impairs EC adhesion in part though down regulating integrin proteins (Figure 1). Identify the mechanism of the regulation of integrins and other adhesion receptors and their interactions with RTKs by Spry are a major focus of the lab.
Figure 1. Spry inhibits endothelial cell adhesion to ECM in part through post-transcriptional down regulation of integrins β1 and β3. A) Spry1 and Spry4 inhibit adhesion to gelatin, collagen I, and vitronectin. B) Immunoblot analysis shows that Spry4 decreases integrin β1 and β3 protein levels. C) RT-PCR shows no change in β1 and β3 transcript levels upon over expression of Spry1 or Spry4.
Regulation of Vascular Smooth Muscle Cell Phenotypic Switching
Vascular injury such as occurs in angioplasty, stenting, or atherosclerosis results in a transition of vascular smooth muscle cells (VSMC) from a quiescent, contractile phenotype to a proliferative, synthetic phenotype, a process called phenotypic switching. In normal vessels, VSMC express a unique repertoire of contractile genes such as smooth muscle myosin heavy chain (SM-MHC), SM actin alpha (SMA), and SM22α. Upon VSMC injury, VSMC decreases expression of contractile genes and migrates from the medial layer into the intima and proliferate, resulting in formation of a neointima. The VSMC phenotypic switch is regulated in part by growth factors that activate receptor tyrosine kinases. Although the stimuli are diverse, two key nodes of signal propagation are the phosphatidylinositol-3 kinase (PI3K/AKT) and mitogen-activated protein kinase (MAPK/ERK) pathways. In general, MAPK/ERK activation stimulates VSMC proliferation, migration and inhibits the VSMC contractile phenotype. PI3K/Akt signal promotes VSMC proliferation and, paradoxically, differentiation. The net effect of these signaling pathways on VSMC phenotype likely depends on the strength and the ratio between PI3K/Akt and MAPK/ERK signaling. However, the mechanisms behind this regulation are not fully understood. The Sprouty (Spry) genes were identified as feedback regulators of tyrosine kinase receptor (RTK) signaling pathways. Sprys are reported to affect various components of RTK pathways, and these differences are likely to be receptor or cell type specific. Our recent data indicate that Spry1 is necessary for maintaining the differentiated state of VSMC in vitro whereas Spry4 plays a role in regulating VSMC proliferation. Further study on role of Spry proteins in the VSMC response to injury will identify new targets for treatment of vascular disease.
Figure 2. SM22α-Cre mediated VSMC over-expression of Sprys in vivo. (A) CAGGFP-Spry1 or CAGGFP-Spry4 transgenic mice were mated with SM22α-Cre transgenic mice to obtain double transgenic mice. PCR for GFP and Cre was used to confirm genotype. Arteries were dissected from both control and double transgenic mice, and fixed in 4% PFA for 2 hours. OCT embedded arteries were sectioned at 5~7 μM. Sections were stained using anti-SM22α and Cy3-anti-Myc, and followed by FITC-anti-rabbit secondary antibody, nuclei were stained by DAPI. (B) Spry4;SM22α-Cre bitransgenic male mice and their littermates control were subjected to left carotid artery ligation for 28 days, HE staining shows over-expression of Spry4 inhibited injury-induced neointima formation in vivo relative to control mice.
Skeletal Growth and Homeostasis
Craniofacial and skeletal disorders are among the most common birth defects in humans, and skeletal strength declines as we age. Therefore, to develop therapeutic strategies for skeletal diseases it is necessary to: i) identify key growth factors, cytokines and their receptors involved in normal bone growth and development as well as in pathologic conditions of bone, and ii) to identify the molecular mechanisms by which these growth factors and receptors regulate bone cell behavior. The fibroblast growth factors (FGF) and their receptors (FGFR) promote the proliferation and differentiation osteoblasts and chondrocytes, the two major cell types in bone. The discovery that several clinically distinct types of craniosynostosis and dwarfism are caused by activating mutations in FGFR1, FGFR2 and FGFR3 supports the notion that FGF signaling must be tightly regulated for normal skeletal development and homeostasis. The identification of feedback inhibitors that tightly control FGF signaling has added to the complexity to regulation of FGF signaling. Among these FGF signaling pathway inhibitors are members of the Sprouty family and a transmembrane protein called Sef or IL17RD. We have shown that Spry1 plays critical roles in skeletal development and mesenchymal lineage allocation. We recently discovered that Sef null mice have increased cortical and trabecular bone density and that osteoblasts from Sef-/- mice in culture show enhanced expression of osteoblast differentiation genes and increased matrix mineralization. A major goal of my laboratory is to unravel the complex regulatory pathways, in mechanistic detail, that control proper temporal regulation of FGF signaling during skeletal growth and development.
Figure 3. Sef null mice have increased cortical bone thickness. A) Eight week-old Sef-/- (n=13) or Sef+/+ (n=12) mice were euthanized, femurs removed and scanned using a MicroCT40 (Scanco Medical) to evaluate mid-shaft cortical thickness and distal femur trabecular bone thickness and volume. B) MicroCT images of mid-shaft cortical bone thickness of Sef+/+ (control) and Sef-/- (KO) mice. C) Hematoxylin and eosin staining of sections of decalcified 8 week-old femurs from Sef+/+ (control) and Sef-/- (KO) mice.
Yang, X., Liaw, L., Prudovsky, I., Brooks, P.C., Vary, C.P., Oxburgh, L., Friesel, R. Fibroblast Growth Factor Signaling in the Vasculature. Current Atherosclerosis Reports, 17:509, 2015.
He, Q., Yang, X., Gong, Y., Kovalenko, D., Canalis, E., Rosen, C.J., and Friesel, R.E. Deficiency of Sef in mice is associated with increased postnatal cortical bone mass by regulating Runx2 activity. J. Bone Miner. Res., 29(5):1472-1431, 2014. PMCID: PMC3984377
Yang, X., Gong, Y., Tang, Y., Li, H., He, Q., Gower, L., Liaw, L., and Friesel, R.E. Spry1 and Spry4 differentially regulate human aortic smooth muscle cell phenotype via Akt/FoxO/myocardin signaling. PLoS One, 8(3):e58746, 2013. PMCID: PMC3598808
Gong, Y., Yang, X., He, Q., Prudovsky, I., Vary, C.P.H., Brooks, P.C., and Friesel, R.E. Sprouty4 regulates endothelial cell adhesion and migration by regulating c-Src activation and integrin b3 stability. Angiogenesis, 16:861-875, 2013. PMCID: PMC3790316
Contois, L.W., Nugent, D.P., Caron, J.M., Cretu, A., Tweedie, E., Akalu, A., Liebes, L., Friesel, R., Rosen, C., Vary, C., and Brooks, P.C. Insulin-like growth factor binding protein-4 (IGFBP-4) differentially inhibits growth factor induced angiogenesis. J Biol Chem., 287(3):1779-1789, 2012. PMCID: PMC3265860
Brown, A., Adams, D., de Caestecker, M., Yang, X., Friesel, R., and Oxburgh, L. FGF/EGF signaling regulates the renewal of early nephron progenitors during embryonic development. Development, 138:5099-5112, 2011. PMCID: PMC3210493
Tang, Y., Yang, X., Friesel, R.E., Vary, C.P.H., Liaw, L. Mechanisms of TGFb induced differentiation in human vascular smooth muscle cells. J Vascular Research, 48(6):485-494, 2011. PMCID: PMC3169366
Yang, X., Gong, Y., and Friesel, R. Spry1 is expressed in hemangioblasts and negatively regulates primitive hematopoiesis and endothelial cell function. PLoS One, 6(4):e18374, 2011. PMCID: PMC3069969
Urs, S., Venkatesh, D., Tang, Y., Henderson, T., Yang, X., Friesel, R.E., Rosen, C.J., and Liaw, L. Sprouty1 is a critical regulatory switch of mesenchymal stem cell lineage allocation. FASEB J., 24:3264-3273, 2010. PMCID: PMC2923355
Yang, X., Harkins, L.K., Zubanova, O., Harrington, A., Kovalenko, D., Nadeau, R.J., Chen, P.Y., Toher, J.L., Lindner, V., Liaw, L., and Friesel, R. Overexpression of Spry1 in Chondrocytes Causes Attenuated FGFR Ubiquitination and Sustained ERK Activation Resulting in Chondrodysplasia. Developmental Biology, 321:64-78, 2008. PMCID: PMC2548288