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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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