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The Ras-t of the Story (Cardiology 2011)


Presentation on Ras-opothies (LEOPARD syndrome & NF1) and novel approaches for treatment. Presented at Cardiology 2011, 15th Annual Update on Pediatric and Congenital Cardiovascular Disease.

Presentation on Ras-opothies (LEOPARD syndrome & NF1) and novel approaches for treatment. Presented at Cardiology 2011, 15th Annual Update on Pediatric and Congenital Cardiovascular Disease.

Published in Health & Medicine
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  • The mouse model of Neurofibromatosis, in which the disease gene, Nf1, has been “knocked-out”, demonstrates embryonically lethal cardiovascular defects in the homozygous mutant state. They die in mid-gestation from a series of cardiovascular defects, arising in large part from defects involving endothelial tissues. The cardiovascular failure, demonstrated by the large pericardial effusion in panel B (*), is a manifestation of this series of defects. Most notable from among these abnormalities are enlarged endocardial cushions resulting from abnormal endothelial to mesenchymal transformation, (dashed line, panel B), ventricular septal defects (arrows, panel D) and malrotation of the cardiac outflow tract, and a thinned, non-compacted ventricular myocardium (arrowheads, panel F).
  • NF1 encodes neurofibromin, a large protein expressed ubiquitously throughout development. The bulk of neurofibromin is of unknown function, but a small domain, known as the GAP-related domain (or GRD) shares homology with several Ras-GTPase activating proteins, or GAPs. GAPs downregulate the activity of Ras protooncogenes.
  • We constructed a “knock-in” mouse in which a cre-dependant NF1 GRD is knocked into the Rosa26 locus. In this construct, the GRD is preceded by a STOP signal flanked by loxP sites. DNA sequences between loxP sites are removed by cre-recombinase activity. This “floxed” STOP sequence would prevent the expression of the NF1 GRD, except if removed by cre. In this set of experiments, mice expressing cre ubiquitously (under the control of a CMV promoter) would have global expression of the GRD protein throughout development.


  • 1.
    • Fraz Ahmed Ismat, MD
    • Clinical Associate in Pediatrics, Division of Cardiology
    • The Children’s Hospital of Philadelphia
    The Ras -t of the story… The role of Neurofibromin & Ras in cardiovascular development & disease
  • 2.  
  • 3. Nf1 -/- embryos die of cardiovascular failure Jacks T. et al., Nat Gen 1994
  • 4. Nf1 -/- hearts have a lethal series of defects wild type e13.5 Nf1 -/- e13.5 Gitler, et al., Nat Gen 2003
  • 5.  
  • 6. Loss of endothelial Nf1 recapitulates Nf1 -/- heart defects myocardium AV cushion Outflow tract Gitler, et al., Nat Gen 2003
  • 7. Disease-causing mutations occur throughout NF1 … but only the NF1 G AP- R elated D omain (GRD) has a well-described function
  • 8. Expressing of GRD without Nf1 ( A ) Targeting strategy of Rosa26-HA-GRD knock-in. ( B ) Southern blot of wild type ( +/+ ) and knock-in ( k/+ ) mice. ( C ) Western blot of cre-dependent HA-GRD from transfected cells ( Cos7 ) and newborn pups ( mice ). Ismat et al., JCI 2006
  • 9. NF1 GRD downregulates activated Ras Ismat et al., JCI 2006
  • 10.  
  • 11. GRD expression rescues Nf1 -/- embryonic heart defects NF1 GRD expression rescues pericardial effusion ( ), enlarged endocardial cushion ( arrows ), and myocardial thinning (arrowheads). * Ismat et al., JCI 2006
  • 12. GRD expression rescues Nf1 -/- embryonic lethality Genotype Mendelian Expected Observed Genotypes of 208 P0 pups from Nf1 +/- , GRD k/+ × Nf1 +/- , CMV-cre + . Ismat et al., JCI 2006 Nf1 +/+ 13 17 27 Nf1 +/+ , GRD k/+ 13 17 13 Nf1 +/+ , CMV-cre + 13 17 15 Nf1 +/+ , GRD k/+ , CMV-cre + 13 17 17 Nf1 +/- 26 35 22 Nf1 +/- , GRD k/+ 26 35 40 Nf1 +/- , CMV-cre + 26 35 37 Nf1 +/- , GRD k/+ , CMV-cre + 26 35 32 Nf1 -/- 13 0 0 Nf1 -/- , GRD k/+ 13 0 0 Nf1 -/- , CMV-cre + 13 0 0 Nf1 -/- , GRD k/+ , CMV-cre + 13 0 5*
  • 13.  
  • 14. Hypertrophic occlusive vasculopathy in NF1 Kanter et al., J Peds 2006
  • 15. “ Normal” arteries in Nf1 smooth muscle knock-out ( Nf1 smKO) No anatomical differences seen in elastin ( A , B ) and smooth muscle α - actin staining ( C , D ) between wild type and Nf1 smKO arteries. Xu & Ismat et al., Circ 2007
  • 16. Elevated blood pressures in Nf1 smKO mice Junwang Xu & Tao Wang
  • 17. Nf1 smKO injury-induced intimal hyperplasia rescued by GRD Xu & Ismat et al., Circ 2007
  • 18. Cardiac enlargement in adult Nf1 smKO mice Junwang Xu
  • 19. Strong SM-22 α transgene activity in cardiac precursors Lepore et al., Genesis 2005
  • 20. Generation of Nf1 myocardial “knock-out” ( Nf1 mKO)
  • 21. Late mortality in Nf1 mKO n=23 n=20
  • 22. Progressive cardiac hypertrophy & fibrosis in Nf1 mKO
  • 23. Cardiac dilation & reduced shortening in Nf1 mKO
  • 24. Rescue of Nf1 mKO cardiac hypertrophy with NF1 GRD
  • 25. Tidyman & Rauen Current Opinion in Genetics & Development 2009, 19:230–236 (PTPN11)
  • 26. Acknowledgements Past support: a Young Investigator Award from the Children’s Tumor Foundation , a Physician Scientist Development Award (K08) from the NHLBI ; Current support: a Basil O’Connor scholarship from the March of Dimes Foundation , a cardiovascular research grant from the W.W. Smith Charitable Trust , and grants from the CHOP Cardiac Center Scientific Review Committee . Ismat Lab Almedia McCoy Timothy Macatee MCRC Core Facilities Histology Core (MM Lu) Transgenic Core (D Zhu) Physiology Core (T Wang*) Penn Core Facilities Flow Cytometry & Cell Sorting Small Animal Imaging Microarray Core Facility Collaborators & Others Jonathan Epstein (Penn) Aaron Gitler (Penn) Elizabeth Goldmuntz (CHOP) Tyler Jacks (MIT) Maria Kontardis (BIDH-HMS) Thomas Look (DFCI) Arun Padmanabhan (Penn) Luis Parada (UTSW) Nancy Ratner (Cincinnati) Xin Zhang (IUPUI) Yuan Zhu (Michigan)