ECM and Mechanical Signals How to build a blood vessel MOVIES Origins of Vascular Smooth Muscle Cells Wnt1Cre/R26R Majesky, Arterioscler. Thromb. Vasc. Biol. 27:1248 (2007) Jiang et al., Develop. 127:1607 (2000) Cre-loxP Cre (“causes recombination”) Recombinase: Catalyzes recombination between two LoxP sites loxP site: 34 base pair DNA sequence Location and orientation of loxP sites determines: • Deletion (same chromosome, same orientation) • Inversion (same chromosome, opposite orientation) • Translocation (different chromosomes) Cre-loxP: Gene Deletion Cre-Lox Basics - Generating Knockout Mice. The Jackson Laboratory Download from class website FLP-FRT System Flipase recombinase (FLP) recognizes a FRT sequences Inducible Cre Models: CreER CreERT2 Kohan, Kidney Int 74:427 (2008) Doxycyline-regulated Gene Expression Kohan, Kidney Int 74:427 (2008) Mfap2 Knockout Allele Lineage Tracing Reporter Line and Cre Drivers mTmG reporter mouse line CRE Drivers Smooth Muscle Cells Secondary Heart Field Neural Crest Endothelial Cells Endothelial Cells Sm22αCre-mediates recombination in VSMC Sm22αCre;Elnf/+ Sm22αCre;Elnf/+ P14 AscAo mT/mG DescAo SM22+ (SMC) lineage Cdh5-Cre & Tie2-Cre mediate recombination in EC Cdh5-Cre;Elnf/+ AscAo DescAo mT/mG Endothelial Cell lineage Isl1-Cre (Secondary Heart Field) Isl1Cre;Elnf/+ P20 AscAo SMC (SHF) lineage Cells from SHF lineage are major contributors to neointima Isl1-Cre;Elnf/f SMC (SHF) lineage Three Requirements of Elastic Vessels • Highly resilient wall • Nonlinear Elasticity • Low hysteresis Elastic Artery! Elastic Artery! Muscular Artery! Establishment of SMC Layers is Complete by ~E14 in the Mouse Aorta In Adult Mouse Ascending Aorta: ~8 SMC layers Descending Aorta: ~5 SMC layers ~E9-10: Heart begins to beat ~E12: Heart is chambered-- Pulsatile flow ~E14: Separation of systemic and E12 pulmonary circulations Physiology and Gene Expression Analysis Blood pressure, vascular compliance, and • Gene Expression analysis in aortic tissue. cardiac output on mice from E14-Adult . • Extracellular Matrix Expression in the Vessel Wall • Early stages (<E12) of wall formation, ECM is enriched in proteoglycans (especially HA) and adhesive proteins (eg., fibronectin). • As SMCs begin to form tight layers, enhanced expression of proteoglycanases and basement membrane proteins. • With the onset of pulsatile flow and increases in blood pressure, expression of structural matrix proteins (elastin, fibrillar collagens, etc.) predominates. Correlation Between Changes in Blood Pressure and ECM Expression P0 Wagenseil and Mecham, Physiol. Rev. 2009 Elastic Modulus as a Function of Blood Pressure Shadwick, J. Exp. Biol. 202:3305 (1999) Elastic Modulus = ~0.4 (0.3- 1.0) MPa for all vessels Characteristic wave velocity = ~2-4 m/s Unanswered Questions • How do cells sense mechanical forces? • How do cells know which matrix molecules to produce and when/where to secrete them? • How do cells secrete and assemble the complex biopolymers of the ECM? • How do cells interpret ECM complexity to regulate gene expression and cellular differentiation? Learning Objectives • Cell movement and differentiation is greatly influenced by the ECM (chemotaxis and haptotaxis) • Know how Cre-loxP systems work: • 1) to inactivate/activate genes • 2) to lineage trace cells in tissues • 3) as conditional and inducible systems • 3) with FLP-FRT system • Understand why the extracellular environment is important in regulating gene expression. • Know that cells sense mechanical forces in their microenvironment. • Know which ECM molecules cells use to impart needed mechanical properties to tissues .