Investigating the role of fibulin-5 in linking tissue stiffness with inflammation

Colin JamoraIFOM-inStem Joint Research Laboratory

Center for Inflammation and Tissue HomeostasisBangalore, India

University of Salento, Nov. 2014

Probing fibroblast biology in the mammalian skin

Activated Fibroblasts in Tissue Repair and Disease

Wound healing

Adapted from: Kalluri and Zeisberg 2006

Koontongkaew 2013 Tissue Fibrosis

Hyperactive

Myofib

roblast

Tumor Stroma

CAF

Inflammation Proliferation Remodeling

Scar formation in wounded Snail transgenic skin

Human skin Mouse skin

Snail is involved in development and disease

K14Snail

(Hoot K. E. et al. J. Clin. Invest 2008)

Human cutaneous SCC

Snail expression in cancer & fibrotic disease

Du et al., Cancer Research 2010

in collaboration with John Varga (Northwestern)

Figure 5. Expression of Snail in human sclerotic skin. Immunohistochemistry of normal and sclerotic (Ssc) skin using an antibody specific for human Snail.

scleroderma

RESEARCH ARTICLEAssociation of the Autoimmune Disease Scleroderma with an Immunologic Response to CancerChristine G. Joseph1,*, Erika Darrah2,*, Ami A. Shah2,*, Andrew D. Skora1,*, Livia A. Casciola-Rosen2, Fredrick M. Wigley2, Francesco Boin2, Andrea Fava2, Chris Thoburn3, Isaac Kinde1, Yuchen Jiao1, Nickolas Papadopoulos1, Kenneth

W. Kinzler1,†, Bert Vogelstein1,†, Antony Rosen2,†

Science 10 January 2014

Snail transgenic skin displays features of fibrosis

WT Snail Tgepi

der

der

epi

WT Snail Tg

Mechanical characterization of the Snail transgenic skin

At higher strain

Avg

. Eta

n m

odul

us (k

Pa)

0

200

400

600

800

1000*

WT Snail Tg

K5/

colla

gen

Collagen:WT Snail Tg 0.0 0.1 0.2 0.3 0.4

0

30

60

90

120

Strain

Str

ess

(kP

a)

0.0 0.1 0.2 0.30

30

60

90

120

Strain

Str

ess (

kP

a)

Stress-Strain curve

WT

Snail Tg

Collaborator: Shyni Varghese (UCSD)

Avg

. Eta

n m

odul

us (k

Pa)

At lower strain

0

30

60

90

120

150

*

WT Snail Tg

Elastic Fibers:

Elastic fiber staining (Verhoeff-Van Gieson Stain)

WT Snail Tg

Kielty, Journal of Cell Science, 2002

Fibulin-5 in elastic fiber assembly

2.0

WT Snail Tg 0.0

0.5

1.0

1.5

Rel

ativ

e ex

pres

sion

*

WT Snail Tg

Fibu

lin-5

/ K

5

100 µm

Fibulin-5 affects elastic but not collagen fiber formation

WT

Fibulin

-5 K

O

Snail Tg

Snail Tg/

Fibulin

-5 K

O

0

200

400

600

800

1000

*

*E

lastin c

onte

nt (μ

g)

*

Effect on Elastic fibers:

WT

Fibulin

-5 KO

Snail T

g0

20

40

60

80

100 **

Hyd

roxy

prol

ine

cont

ent (μ

g)

Snail T

g/

Fibulin

-5 KO

0

50

100

150

200

250

Snail Tg/

Fibulin

-5 K

O

Snail Tg

At higher strain

Avg

. E

tan m

od

ulu

s (

kP

a)

Effect on Collagen fibers:

Can fibulin-5 regulate tissue inflammation to impact fibrosis?

Depleting fibulin-5: Blocking inflammation:

Fibulin-5 is required for a robust inflammatory response

WT

Fibulin

-5 K

O

Snail Tg

Snail Tg/

Fibulin

-5 K

O

0

20

40

60

80*** *

Ma

c1

-po

sitiv

e c

ells/m

m

K5/M

ac-1

WT Snail TgFibulin-5 KO Snail Tg/Fibulin-5 KO

K5/C

D3

0

20

40

60

80

100** *

CD

3-p

ositiv

e c

ells/m

m

WT

Fibulin

-5 K

O

Snail Tg

Snail Tg/

Fibulin

-5 K

O

WT Snail TgFibulin-5 KO Snail Tg/Fibulin-5 KO

adapted from Kalluri and Zeisberg 2006

How can fibulin-5 regulate the inflammatory response?

Activated fibroblast/CAF

?

Fibulin-5 accentuates activation of dermal fibroblasts in vivo

WT Snail TgFibulin-5 KO Snail Tg/Fibulin-5 KO

α-S

MA

SMA SPARC CTGF

0

2

4

6

8

10

***

##*

**

##

Rel

ativ

e fo

ld e

xpre

ssio

n

WT

Fibulin-5 KO

Snail Tg

Snail Tg/Fibulin-5 KO

adapted from Kalluri and Zeisberg 2006

How can fibulin-5 regulate the inflammatory response?

Activated fibroblast/CAF

?

Fibulin-5 promotes inflammatory chemokine production from dermal fibroblasts in vivo

CXCL1 CXCL5 CXCL10 CXCL160

10

20

30

40WT

Fibulin-5 KO

Snail Tg

Snail Tg/Fibulin-5 KO

***##

Re

lative

fo

ld e

xp

ressio

n

####

### ***

**

***

Activated fibroblast/CAF

Intercellular crosstalk in the tumor stroma

Mindin

adapted from Kalluri and Zeisberg 2006

CXCL1 CXCL5 CXCL10 CXCL160

5

10

15

20

252 kPa WT CM2 kPa Snail CM5 kPa WT CM5 kPa Snail CM

* ***

**

**

Rel

ativ

e fo

ld e

xpre

ssio

nStiffer substrate accentuates the inflammatory response

2 kPa buffer 2 kPa mindin 5 kPa buffer 5 kPa mindin

Summary

•  Snail transgenic skin is a model of tissue/tumor fibrosis

•  Elastin binding protein (Fibulin-5) is elevated in transgenic dermis

•  Deletion of Fibulin-5 decreases elastin (collagen remains elevated), dermal thickness, and corresponding tissue stiffness

•  Deletion of Fibulin-5 decreases inflammation in the fibrotic skin

•  Small increase in substrate rigidity is able to enhance a proinflammatory response in dermal fibroblasts

Acknowledgements

Manando Nakasaki MD, PhD

Tuan-Lin Tan, PhD Subhasri Ghosh, PhD

Tanay Bhatt Sunny Kataria Neha Pincha

Federica Centonze Syed Abrar Rizvi

Edries Hajam Isha Rana

Bhavya Byantri Ajai Pulianmackal

Prof. Tomoyuki Nakamura (Kansai Medical Univ) Prof. Steven Briggs (UCSD)

Prof. Shyni Varghese (UCSD) Yongsung (Joshua) Hwang, PhD

Active fibroblasts are not a product of an EMT

Guarino et al., Human Pathology 2009

 

 

Figure 2. Lineage tracing of the activated fibroblasts. Epithelial cells in the skin of wild type (WT) or Snail transgenic mice (K14-Snail) were labeled in blue with X-gal staining when these respective mice were mated with ROSA26/K14-Cre mice. Activated dermal fibroblasts were labeled with an antibody recognizing

αSMA (brown). 

β-ga

l/αSM

A

Primary Dermal fibroblasts

Control CM

Snail CM

WT CM

Snail CM

Assay to test for secreted factor capable of activating dermal fibroblasts

WT keratinocytes

Snail transgenickeratinocytes

What is in secreted by Snail expressing keratinocytes to activate dermal fibroblasts?

epid

erm

isde

rmis

derm

is

Snail? Collagen contraction activity

Induction of CTGF & αSMA

Concanavalin A sepharose columnCollagen contraction activityInduction of CTGF & αSMA

WT or Snail Tg Conditioned media

Anion exchange chromatography (Mono Q)

MassSpec

Candidate proteins mediating epithelial-mesenchymal crosstalk

Fibulin 5 on activated fibroblasts

Background on spondin-2 (aka mindin)

What is spondin-2/mindin?•  Member of the F-spondin family•  Secreted, associates with ECM•  Component of basal lamina (zebrafish)•  Ligand for integrins•  PRR – production of inflammatory cytokines•  Promotes outgrowth of hippocampal neurons

Wynn & Ramalingam 2012

Image of function of fibulin-5 (and interacting partners??)

Effect of fibulin-5 on vascular cells

The function of fibulin-5 in adult blood vessels wassuggested by the strong upregulation of Fbln5 in theneointima after balloon withdrawal injury or carotid arteryligation, and in activated endothelial cells of atheroscleroticplaques (Kowal et al. 1999; Spencer et al. 2005). Fbln5-nullSMCs displayed enhanced proliferation and migration inresponse to serum and PDGF. This enhancement wasinhibited by over-expression of Fbln5 (Spencer et al.2005). Forced expression of Fbln5 in primary humanendothelial cells improved cell attachment against contin-uous shear stress and enhanced cell retention on artificialgrafts subjected to pulsatile flow. However, the proliferationof endothelial cells was decreased in Fbln5-overexpressingcells compared to control cells (Preis et al. 2006). Humanprimary SMCs plated on recombinant fibulin-5 exhibitedPDGF receptors with undetectable phosphorylation, andpoorly phosphorylated EGF receptors, compared to SMCsplated on fibronectin (Lomas et al. 2007). Interestingly, thepresence of integrin β1-activating antibody in SMCs,plated on fibulin-5, restored phosphorylation of PDGF αand β receptors. Together with the observation that fibulin-5binds fibronectin receptors (α5β1 and α4β1) but fails toactivate downstream signaling, these data suggest a possi-bility that fibulin-5 may regulate vascular cell behavior byantagonizing fibronectin-mediated signaling. However,further investigation is required to establish the in vivorelevance of the fibulin-5-integrin binding during vesseldevelopment and pathological insults.

Fibulin-5 and angiogenesis

The effect of fibulin-5 on angiogenesis was first describedusing mouse brain microvascular MB114 endothelial cells,in which Fbln5 overexpression or incubation with recom-binant fibulin-5 inhibited sprouting, proliferation, and

invasion in matrigel (Albig and Schiemann 2004). Upre-gulation of thrombospondin-1 expression, as well asantagonization of VEGF165 -mediated signaling, includingactivation of P38 MAPK and ERK1/2, were suggested tobe the underlying causes of the inhibitory effect of fibulin-5. Fibulin-5 was also shown to reduce migration of humanmicrovascular HMEC-1 endothelial cells toward fibronec-tin, and overexpression of FBLN5 in MB114 cells down-regulated MMP-2 expression, as well as enzymatic activityduring tubulogenesis on collagen gels (Albig et al. 2006).Matrigel implantation experiments in the skin of wild-typemice showed that matrigel containing high or low doses offibulin-5 stimulated the invasion of bFGF-induced fibro-blasts, but inhibited vessel formation and angiogenesis,independent of RGD-integrin interactions. Consistent withthese observations, Fbln5-null mice exhibited increasedangiogenesis after physiological wound healing and PVAsponge implantation, without an effect on fibroblastinvasion (Sullivan et al. 2007; Zheng et al. 2006).

Although exogenous and endogenous fibulin-5 wasshown to antagonize angiogenesis, the exact mechanismbehind this effect has remained elusive. We recentlyobserved that fibulin-5 exerts its effect on endothelial cellsand angiogenesis by controlling integrin-induced productionof reactive oxygen species (ROS), which have pro-angiogenic properties (Schluterman et al. 2009). ROS,including O2

- and H2O2, are highly reactive moleculesproduced commonly as by products of aerobic respirationand the mitochondrial transport chain. ROS were originallyidentified as host defense molecules produced by neutrophilsvia NAD(P)H oxidases, and have a critical function ineliciting biological processes required for the initiation ofangiogenesis (Wu 2006). By functioning as signalingmolecules, ROS have been shown to activate pathways suchas proliferation, cell adhesion, motility and invasion inendothelial cells (Ushio-Fukai 2007).

Endothelial cells, treated with H2O2, produced higherlevels of VEGF, thereby increasing their proliferation and

Fig. 3 A model of elastic fiber assembly. a Secretion of tropoelastin(TE) from elastogenic cells (green). Tropoelastin undergoes self-aggregation and fibulin-5 (purple) mediates this process. Tropoealstinbinds both N-terminal (N) and C-terminal tropoelastin-binding

domains (square) of fibulin-5. b Fibulin-5 binds tropoelastin, micro-fibrils (blue), and lysyl oxidase-like enzyme (yellow) to aid in elasticfiber assembly. c Cross-linked insoluble elastin (grey) is polymerizedand organized into functional elastic fibers

Fibulin-5 in development and disease 343

PMC full text: J Cell Commun Signal. Dec 2009; 3(3-4): 337–347.Published online Oct 2, 2009. doi: 10.1007/s12079-009-0065-3Copyright/License ► Request permission to reuse

Table 1

Interacting partners of Fibulin-5

Interacting

proteins

Binding site(s) within Fibulin-5 Cellular and/or Biological functions Reference

Fibulin-5 ND Unknown Zheng et al. 2007

Tropoelastin cbEGF domains Jones et al. 2009

N-terminal cbEGF domains Elastic fiber assembly Zheng et al. 2007

C-terminal EB domain

Loxl-1 a. C-terminal fibulin domain > 1stcbEGF

Elastic fiber assembly a. Hirai et al. 2007a

b. C-terminal region (245-448) b. Liu et al. 2004

Loxl-2, 4 C-terminal fibulin domain Elastic fiber assembly Hirai et al. 2007a

Emilin-1 ND Elastic fiber assembly Zanetti et al. 2004

LTBP-2 6th cbEGF Elastic fiber assembly Hirai et al. 2007b

Fibrillin-1 ND Elastic fiber assembly Freeman et al. 2005

SOD3 C-terminal region (320-448) Superoxide scavenge Nguyen et al. 2004

α5β1, α4β1 N-terminal half containing RGD Cell attachment, Antagonize fibronectinfunction

Lomas et al. 2007

αvβ3, αvβ5 N-terminal half containing RGD Cell attachment Nakamura et al. 2002

α9β1 ND Cell attachment Nakamura et al. 2002

Lipoprotein(a) C-terminal domain (350-448) Unknown Kapetanopoulos et al.2002

ND not determined

Fibulin-5 accentuates the inflammatory response

WT

Fibulin

-5 K

O

Snail Tg

Snail Tg/

Fibulin

-5 K

O

0

20

40

60

80*** *

Ma

c1

-po

sitiv

e c

ells/m

m

K5/M

ac-1

WT Snail TgFibulin-5 KO Snail Tg/Fibulin-5 KO

K5/C

D3

0

20

40

60

80

100** *

CD

3-p

ositiv

e c

ells/m

m

WT

Fibulin

-5 K

O

Snail Tg

Snail Tg/

Fibulin

-5 K

O

WT Snail TgFibulin-5 KO Snail Tg/Fibulin-5 KO