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Graduate School of Biomedical Sciences

The Robert Friesel Lab

Molecular Mechanisms of Cardiovascular Disease

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 cells of the vasculature 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 endothelial and vascular smooth muscle cell 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 adherens junctions and vascular permeability, migration and adhesion, in part through regulating the expression of cell adhesion molecules. Specifically, endothelial cell specific knock out of Spry4 in mice results in increased VEGF-induced vascular permeability in Miles assay (Figure 1). Conversely, endothelial cell specific transgenic overexpression of Spry4 in mice resulted in decreased vascular permeability (not shown). In vitro studies reveal that Spry4 regulates the stability of endothelial adherens junctions through c-Src dependent tyrosine phosphorylation of VE-cadherin. Additional studies are aimed at the role of Spry proteins in regulating the vascular response to inflammation.

Friesel Fig 1

Figure 1. Spry4 regulates vascular permeability. A) Conditional deletion of Spry4 in endothelial cells in vivo was achieved by crossing Spry4f/f (Spry4+/+)mice with Tie2-promoter transgenic mice to generate Spry4ECKO mice (Spry4-/-) . B) Evans blue dye (100 μl, 1.0 %) was injected retro-orbitally into wild type, or Spry4ECKO mice. After 10 min, 50 µl PBS with or without 50 ng VEGF-A was injected intradermally into a shaved patch of skin on the dorsal flank. After 20 min mice were euthanized and the area of injected skin was excised and Evan’s blue dye quantified at 620 nm.

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 injury, VSMC decrease expression of contractile genes and migrate from the medial layer into the intima and proliferate, resulting in formation of a neointima resulting in occluded blood flow. 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 regulate various nodes of RTK signaling 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. In a carotid artery ligation model of vascular injury, we recently reported that deletion of Spry1 resulted in reduced neointima formation in response to injury, suggested it as a target for future therapeutic strategies to treat cardiovascular disease.

Friesel Fig 2

Figure 2. Loss of Spry1 attenuates injury induced neointima formation by decreasing VSMC proliferation in vivo. A) Representative H & E stained images of ligated carotid artery sections from VSMC condition Spry1 null mice (Spry1VSMC-/-) and controls (Spry1f/f). B) Quantification of neointima thickness 21 days after injury from experiment in panel A. C) Representative H & E staining images of ligated carotid artery sections from global Spry1 null and wild type controls. D) Quantification of neointima thickness 21 days after injury from the experiment in panel C.

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 interleukin-17 receptor D (IL17RD). Our previous work showed that Spry1 plays critical roles in skeletal development and mesenchymal lineage allocation. We recently discovered that IL17RD null mice have increased cortical bone thickness at weeks of age, and that osteoblasts from Il17rd-/- mice show enhanced expression of osteoblast differentiation genes and increased matrix mineralization in vitro. A major goal of my laboratory is to unravel the complex regulatory pathways, in mechanistic detail, that control proper temporal regulation of signaling by FGFs and other cytokines during skeletal growth and homeostasis. To gain additional insight into the physiological role of IL17RD in the skeleton, we performed ovariectomy on wild type or Il17rd deficient female mice, which results in an estrogen deficient state that mimics post-menopausal osteoporosis. Ovariectomized Il17rd-/- mice where shown to lose less bone than their wild type counter parts. Studies are ongoing to reveal the molecular mechanisms that result in protection from bone loss in Il17rd deficient mice.

Friesel Fig 3

Figure 3. Loss of Il17rd ameliorates OVX-induced bone loss. A) Il17rd-/- mice gain body weight upon OVX similar to Il17rd+/+ mice. B) OVX results in uterine atrophy similarly between Il17rd+/+ and Il17rd-/-. C & D) DEXA scanning of femoral and lumbar BMD. E & F) CT images of femoral trabecular region show reduction in bone loss in the IL17rd-/- relative to Il17rd+/+ after OVX. G-J) Quantification shows OVX decreased trabecular bone volume and increased femur trabecular bone separation, and loss of Il17rd diminishes these effects. *: p<0.05; **: p<0.01.