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