(( chappell--vhl talk-v01 )) · 10/11/2018 1 john c. chappell, phd assistant professor, virginia...
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John C. Chappell, PhDAssistant Professor, Virginia Tech Carilion Research InstituteCenter for Heart and Regenerative Medicine, VTCRIDept of Biomedical Science Education, Va Tech Carilion School of MedicineDept of Biomedical Engineering and Mechanics, Va TechVa Tech-Wake Forest Univ School of Biomedical Engineering and Sciences
Vascular Abnormalities with VHL Mutations
PECAM-1PDGFR
DAPI
Adult Mouse Brain Vasculature
Growth factorproduction
Blood vesselformation
Tumor growth & Metastasis
Tumor
modified from Genentech
Blood Vessels in Health and Disease
Blindness
Diabetic RetinopathyVascular Defects
Edema & Hemorrhage
Hypoxia & Vessel Growth
ProliferativeRetinopathy
Abbott Nature 2015
NeonatalIntracranialHemorrhage
Weill Cornell Medical Center
Live Imaging of Blood Vessel Formation during Mouse Embryonic Stem Cell Differentiation
Vasculogenesis –Differentiationand assembly
Angiogenesis –Proliferationand morphogenesis
Day 0
Day 8
Day 3
Wild-Type Embryonic Tissue Culture Assay - Untreated
Wild-Type Embryonic Tissue Culture Assay – VEGF Treated (100ng/mL)
Endothelial Cell Reporter:Flk‐1‐eGFP
PericyteReporter:NG2‐DsRed
XDoubleReporter:Flk‐1‐eGFPNG2‐DsRed
Angiogenesis –Proliferation andmorphogenesis
Day 0Live Imaging of Blood Vessel Formation during Mouse Tissue Culture
Darden et al. Angiogenesis 2018
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Developing Retinal Vessels in Postnatal Mice.
Vitreous
Blood Vessels (IsolectinB4)Smooth Muscle Cells (SMA)
P8‐9
Modified from Gerhardt et al. J Cell Biol 2003
Postnatal Day 1 (P1)
VEGF
sFlt‐1
VEGF‐A
sFlt1 2
3
1
2
3sFlt
4
sFltsFlt
4
Flt-1/VEGF-A coordinates endothelial cell heterogeneity during several stages of blood vessel formation
Chappell et al. 2009Hashambhoy et al. 2011
Walpole et al. 2015
Chappell et al. 2013
Chappell et al. 2016Nesmith et al. 2017
Walpole et al. 2017
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Arreola et al. 2018
In Vivo Inducible Genetic Mutations – Experimental Setup
loxP loxPVhl Exon 1
X
CreER
Cre-recombinase fused to humanestrogen receptor ligand bindingdomain
Wide‐spread: Human ubiquitin C (UBC) promoterCreERT2
Tamoxifen
Exon1
ANDConditional loss of Vhl (Vhlfl)
Exon2 Exon3
Vhl 2B Knock-In (Vhl2B)
Possible genotypes:1– UBC+/+; Vhlfl/fl2– UBC+/+; Vhlfl/2B
3– UBCCreER ; Vhlfl/fl
4– UBCCreER ; Vhlfl/2B
Vhl mutations induce distinct morphological
changes in capillary plexus formation
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Vhl mutations induce distinct morphological
changes in capillary plexus formation
Vhl mutations give rise to vessel maturation defects including ectopic expression of SMA in pericytes
Vhl mutations give rise to vessel maturation defects including ectopic expression of SMA in pericytes Vhl null and 2B mutations yield distinct vascular defects
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Vhl retinas display unique transcriptional signatures Vhl retinas display unique transcriptional signatures
Vhl retinas display unique transcriptional signatures Vhl null and 2B mutations induce changes in VEGF-A and Claudin-5
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Future Directions
Continue mining RNA Seq data for unique transcriptional profiles for the null and 2B mutations
Induce low-level, mosaic mutations in adult mice and observe vascular changes in retinas, brain, and kidneys
Acknowledgements
R00‐HL105779R56‐HL133826
NSF CAREER Award
Chappell LabSarah Taylor (Lab manager)Laura Beth Payne (Postdoctoral fellow)Jordan Darden (TBMH grad student)Huaning Zhao (BEAM grad student)David McGuire (TBMH grad student)Tim Nguyen (VTC SoMM4)Clifton Jenkins‐Houk (VTC SoMM4)Anisha Chada (VTC SoMM3)
Malek Bouzaher (VTC SoMM3)Harsh Patolia (VTC SoMM3)Morgan Julian (VTC SoMM2)Justin Davis (VT Biology undergrad) Christina Compton (VT HNFE undergrad)Karan Paralkar (VT Post‐Baccalaureate)Ella Higgins (Roanoke Governor’s High School)Nathalie Lemon (Roanoke Governor’s High School)Abigail Ingram (Roanoke Governor’s High School)Logan Dunkenberger (Roanoke Governor’s High School)
ReagentsVictoria Bautch (UNC‐CH)
Jamie Smyth (VTCRI)Guo‐Hua Fong (U Conn)
Scott Baldwin (Vanderbilt)Janet Rossant (Toronto)Andras Nagy (Toronto)
UVA‐VTC NeuroscienceSeed Grant
Va Tech ICTAS JuniorFaculty Award Collaborators
Shayn Peirce‐Cottler (UVA)Michael Rikkard (UVA)
Kim Rathmell (Vanderbilt)
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