vascular smooth muscle cells (vsmcs) in vascular disease · vascular smooth muscle cells originate...

Embryonic Origins of Vascular Smooth Muscle Cells and Implications for Disease

Dr Sanjay Sinha

BHF Senior Clinical Research Fellow

University of Cambridge

Brexit means Brexit

A SMC means a

SMC?

Vascular Smooth Muscle Cells Originate from Multiple Embryonic Lineages

Neural crest

Somites

Secondary heart field

Epicardium

Neuroectoderm

Lateral plate mesoderm

Paraxial mesoderm

1. Development informs disease

2. Practical examples of using stem cells to model human development and disease

Site-Specific Manifestation of Atherosclerosis

Internal mammary

Descending thoracic aorta

Vanderlaan et al. ATVB 2004;24: 12-22

LDLR -/- mouse

Site-specific manifestation of vascular diseases

Atherosclerosis

Haimovici & Maier. Arch Surg 1964 Majesky ATVB 2007

Henry C. McGill, Jr et al. ATVB 2000;20:836-845 & ATVB

2000;20:1998-2004.

Early Athero: Diverse Regional Responses to Risk factors

Aorta RCA

• ♀ → Abdo Aorta• ♂ → RCA

• Smoking → Abdo Ao

• Gly Hb → RCA

Pathobiological Determinants of Atherosclerosis in Youth (PDAY)

Vascular SMC Pathologies: Aortic Aneurysm & Dissection

Cystic medial necrosisSMC death

ECM breakdown

Genetic syndrome – Marfans, Loeys-DietzNon-syndromic TAAD

Site-specific manifestation of vascular diseases

Aortic Aneurysms & Dissections

Vascular Smooth Muscle Cells Originate from Multiple Embryonic Lineages

Neural crest

Somites

Secondary heart field

Epicardium

Neuroectoderm

Lateral plate mesoderm

Paraxial mesoderm

“It is not birth, marriage, or death, but gastrulation which is truly the most important time in your life.” – Lewis Wolpert (1986)

Ectoderm

Intermediate

mesoderm

Lateral plate

mesoderm

Notochord –

axial mesoderm

Endoderm

Neural tube

Paraxial

mesoderm

(somites)

Neural crest

HPSCs

LM-SMC

Cross-section of a human embryo at week 4 In vitro derivation of origin-specific SMCs

MMP9+

TIMP1+++

MMP9++

TIMP1++

MMP9+++

TIMP1+

IL-1β

Somatic (body cavity & limbs)Splanchnic (cardiovascular/gut)

Dias MS, and McLone DG. 2001

Development of Embryonic Lineage-Specific Vascular Smooth Muscle Cells from Human Pluripotent Stem Cells

Neural crest

Somites

Secondary heart field

Epicardium

Neuroectoderm

Lateral plate mesoderm

Paraxial mesoderm

ChristineCheung

Human ESCs / iPSCs

Early mesoderm

PDGF-BB + TGF-b1Neuroectoderm

Lateral plate mesoderm

Paraxial mesoderm

FGF2 + Ly294002

FGF2 + BMP4

FGF2 + SB431542

Cheung et al. Nature Biotech 2012;30:165-73Cheung et al. Nature Protocols 2014;9:929-38

(PDGF-BB + TGF-b1)

Generation of SMCs from Embryonic Lineages

• ~90% SMCs• hESCs & iPSCs• Chemically defined

Lineage-Specific Developmental and Functional Differences Between VSMC

Neuroectoderm

Lateral plate mesoderm

Paraxial mesoderm

siRNA MRTF-B TGF-b induced proliferation

++

-

-

Cheung et al. Nature Biotech 2012;30:165-73

SMC subtypes predict origin-dependent MMP9 and TIMP1 activation

Aortic

root

Aortic

arch

Thoracic

descending

aorta

LM-SMC

NE-SMC

PM-SMC

SMC subtypes exhibit differential proteolytic ability

Ectoderm

Intermediate

mesoderm

Lateral plate

mesoderm

Notochord –

axial mesoderm

Endoderm

Neural tube

Paraxial

mesoderm

(somites)

Neural crest

HPSCs

LM-SMC

Cross-section of a human embryo at week 4 In vitro derivation of origin-specific SMCs

MMP9+

TIMP1+++

MMP9++

TIMP1++

MMP9+++

TIMP1+

IL-1β

Origin-specific SMCs may contribute to the preferential sites of aortic dissection

Cheung et al. Nature Biotech 2012;30:165-73Cheung et al. Nature Protocols 2014;9:929-38

‘Regionality’ and development of VSMC: insights into atheroscerosis?

HPSCs

LM-SMC

-

Embryonic origin-dependent differences in Homeobox gene expression

Trigueros-Motos et al. ATVB 2013;33:1248-1256

Developmentally programmed

Reciprocal negative regulation between NF-kB and HoxA9

Trigueros-Motos et al. ATVB 2013;33:1248-1256

Marfan Syndrome - Clinical Features

Fibrillin-1 mutation

Cystic medial necrosis

Robertson et al. Biochem J 2011

Increased TGF-b and ERK1/2 Activity in Pathogenesis of Aortic Disease in Fibrillin-1 Cys->Gly/+ Mutant Mouse

Habashi et al Science 2006

TARGET GENES

AT1R

Dietz et al. Science 2011

• Medical management limited. Beta blockade +/- surgery

slow progression (30-70% aneurysm recurrence)

• Pathology from mouse models

o Fibrillin-1 mutation -> TGF-b1 signalling??

• Location of disease?

Clinical Challenges in Marfan Syndrome

Marfan Syndrome: Disease Modelling

Shinya Yamanaka

Skin fibroblast

Induced pluripotent stem cell(iPSC)

Vascular cellsCardiomyocytes

Neuronsetc

Oct4 KLF4

Myc Sox2

Disease mechanismsDrug screening

(PDGF-BB + TGF-b1)

Cheung et al Nature Biotechnol 2012

Abnormal Fibrillin-1 Deposition & Distribution in Marfan iPSC-SMC

Alex Granata

Mis-sense mutations

Granata et al Nature Genetics 2017

Fibrillin-1

Abnormal Fibrillin-1 Deposition & Distribution in Marfan iPSC-SMC

WT

Granata et al Nature Genetics 2017

↑ TGF-b1 & MMP Levels & Activity in MFS iPSC-SMCs

Granata et al Nature Genetics 2017

CRISPR/Cas9 Marfan hiPSC correction

Felipe Serrano

Fibrillin-1 depositionTGFb levels

Granata et al Nature Genetics 2017

Fibrillin-1

Partial Rescue of Phenotype with Losartanco

ntr

ol

losa

rtan

MF cell death

WT CTL

WT losartan

MF CTL

MF losartan

Dissecting Specific TGF-b Pathways in Marfans: Key Role for p38

Granata et al Nature Genetics 2017

TARGET GENES

PD98059

SB431542

Validation of p38 Activation in vivo

Granata et al Nature Genetics 2017

Marfan micePatient-derived tissue

p38: a Novel Therapeutic Target in Marfans

β1

SMC Origin and Disease: Summary

HPSCs

LM-SMC

-

1. Developmental origin may influence SMC behaviour & disease development

2. Developmental origin – specific SMC can be modelled in vitro using hESC/hiPSC

3. Embryonic origin affects HoxA9/NFkB: predisposes to atherosclerosis susceptibility

5. SMC lineage matters!

4. Disease in a dish: the power of in vitro modelling & importance of the right kind of SMC

AcknowledgementsGroup members

Laure GambardellaFelipe SerranoMaria Colzani

Will BernardAishwariya Jacob

Johannes BargehrMadeline McNamara

Loukia YiangouPriya SastryLay Ping Oh

Sophie McManusEsther Tan

*Christine Cheung*Dharini Iyer

*Alex Granata

* = Alumni

CollaboratorsAndreia BernardoLudovic VallierRoger PedersenMatthew TrotterMartin BennettZiad MallatRoger BarkerCay KieltyLaura NiklasonJay HumphriesDianna Milewicz

Elena GalloTom QuertermousGary OwensEric Olson

SMC Origin and Disease: Summary

HPSCs

LM-SMC

-

1. Developmental origin may influence SMC behaviour & disease development

2. Developmental origin – specific SMC can be modelled in vitro using hESC/hiPSC

3. Embryonic origin affects HoxA9/NFkB: predisposes to atherosclerosis susceptibility

5. SMC lineage matters!

4. Disease in a dish - the importance of the right kind of SMC & many other aortic diseases