Reelin Amplifies Glycoprotein VI Activation and AlphaIIb Beta3 Integrin Outside-In Signaling via PLC Gamma 2 and Rho GTPases

Objective: Reelin, a secreted glycoprotein, was originally identified in the central nervous system, where it plays an important role in brain development and maintenance. In the cardiovascular system, reelin plays a role in atherosclerosis by enhancing vascular inflammation and in arterial thrombosis by promoting platelet adhesion, activation, and thrombus formation via APP (amyloid precursor protein) and GP (glycoprotein) Ib. However, the role of reelin in hemostasis and arterial thrombosis is not fully understood to date. Approach and Results: In the present study, we analyzed the importance of reelin for cytoskeletal reorganization of platelets and thrombus formation in more detail. Platelets release reelin to amplify alphaIIb beta3 integrin outside-in signaling by promoting platelet adhesion, cytoskeletal reorganization, and clot retraction via activation of Rho GTPases RAC1 (Ras-related C3 botulinum toxin substrate) and RhoA (Ras homolog family member A). Reelin interacts with the collagen receptor GP (glycoprotein) VI with subnanomolar affinity, induces tyrosine phosphorylation in a GPVI-dependent manner, and supports platelet binding to collagen and GPVI-dependent RAC1 activation, PLC gamma 2 (1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase gamma-2) phosphorylation, platelet activation, and aggregation. When GPVI was deleted from the platelet surface by antibody treatment in reelin-deficient mice, thrombus formation was completely abolished after injury of the carotid artery while being only reduced in either GPVI-depleted or reelin-deficient mice. Conclusions: Our study identified a novel signaling pathway that involves reelin-induced GPVI activation and alphaIIb beta3 integrin outside-in signaling in platelets. Loss of both, GPVI and reelin, completely prevents stable arterial thrombus formation in vivo suggesting that inhibiting reelin-platelet-interaction might represent a novel strategy to avoid arterial thrombosis in cardiovascular disease.

99mTc-DTPA-bis-c(RGDfK) a potential alpha(v)beta3 integrin based homobivalent radioligand for imaging neoangiogenesis in malignant glioma and melanoma

A new99mTc-labeled bivalent DTPA-bis-c(RGDfK) conjugate has been developed and successfully synthesized. Promising results have been obtained for its preclinical evaluation on human glioma and melanoma tumor expressing αvβ3targets.

Epidermal Growth Factor-like Domain 7 Promotes Hematopoietic Stem Cell Expansion and Increases Myeloid-Megakaryocytic Lineage Priming through beta3 Integrin

Interactions between stem cells and their surrounding microenvironment, or niche, are critical for the establishment and maintenance of stem-cell properties. Niche cells within the bone marrow (BM) are contacted by the hematopoietic stem cells (HSCs), which retain their stem-cell character through the direct association with these niche cells. Within the BM microenvironment, an adhesion-dependent or -independent niche system regulates HSC function. Here we show that the extracellular matrix protein epidermal growth factor-like domain protein 7 (Egfl7) induced HSCs to enter the cell cycle and to undergo myelo-megakaryocytic differentiation both under steady state conditions and after myelosuppression in vivo. Mechanistically, we show that Egfl7 binds to the beta3 integrin expressed on HSCs, thereby activating the Akt pathway in HSCs. In beta3-/- mice, Egfl7-mediated HSC expansion, cell cycle progression and myelo-megakaryocytic differentiation did not occur. We propose that the ECM protein Egfl7 recruits dormant HSCs into active cell cycle, and can govern stress-induced hematopoiesis by beta3 integrin-dependent anchoring to the stem cell niche. Disclosures No relevant conflicts of interest to declare.

Abstract 317: Beta3 Integrin-mediated CTGF Expression in Cardiac Hypertrophy

Connective Tissue Growth Factor (CTGF/CCN2) is a fibrotic mediator overexpressed in human atherosclerotic lesions, myocardial infarction and hypertension. CTGF regulates ECM deposition, fibrosis, wound repair and angiogenesis. Our preliminary studies using wild type (WT) and global 3 integrin knockout (3-/-) mice show for the first time that pressure overload (PO) by transverse aortic constriction (TAC) induces both CTGF and 3 integrin expression in WT mice and that 3 integrin is a prerequisite for the PO-induced CTGF expression. Since both the major cells, cardiomyocyte (CM) and cardiac fibroblast (CFb) have been shown to express CTGF, exploring the primary source for the 3 integrin mediated CTGF expression is expected to identify potential targets for fibrosis. Therefore, the main focus of this proposed study is to use CM specific β3 integrin KO mice with preserved β3-integrin function in CFb as well as CFb specific β3 integrin KO mice with preserved β3-integrin function in CM and evaluate which heart cell type contributes to β3-integrin-mediated PO-induced CTGF production and ECM accumulation. Furthermore, using cells isolated from these mice, we will perform in vitro studies to explore the mechanism by which β3-integrin mediates CTGF secretion by analyzing specific nonreceptor tyrosine kinases and transcription factors.