Targeting of ±v integrin identifies a core molecular ... Pdgfrb-Cre HSCs cultured with and...

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  • Targeting of v integrin identifies a core molecular pathway that regulates

    fibrosis in several organs

    Neil C Henderson*, Thomas D Arnold, Yoshio Katamura, Marilyn M Giacomini, Juan D

    Rodriguez, Joseph H McCarty, Antonella Pellicoro, Elisabeth Raschperger, Christer

    Betsholtz, Peter G Ruminski, David W Griggs, Michael J Prinsen, Jacquelyn J Maher, John

    P Iredale, Adam Lacy-Hulbert, Ralf H Adams, Dean Sheppard*

    *Address correspondence to: Neil Henderson (e-mail: Neil.Henderson@ed.ac.uk) or Dean

    Sheppard (e-mail: Dean.Sheppard@ucsf.edu).

    Nature Medicine: doi:10.1038/nm.3282

  • Nature Medicine: doi:10.1038/nm.3282

  • ALT

    (IU/L

    1

    03)

    cb Control v Cre

    Oil

    20

    864

    j Controlv cre

    CD31

    (% a

    rea)

    50

    201510

    Oil CCl4

    Control v Cre

    NS

    f Control v CreO

    il

    Neut

    / po

    rtal t

    ract

    Controlv cre

    Chr

    onic

    CC

    l 4

    Oil CCl4

    NS

    g

    50

    201510

    NS

    Oil

    Acu

    te C

    Cl 4

    Control v Cre

    Oil

    Chr

    onic

    CC

    l 4

    h

    Oil CCl4

    20

    864

    Controlv cre

    F4/8

    0 (%

    are

    a)

    i

    NSNS

    NS

    NS

    Oil

    Chr

    onic

    CC

    l 4

    Controlv cre

    v SMAv / SMA

    / DAPI

    a

    10

    d

    Desm

    in (%

    are

    a)

    Cont v Cre

    50

    25201510

    NS

    PDG

    FR

    (% a

    rea)

    Cont v Cre

    e

    50

    25201510

    NS

    CCl4

    Supplementary Figure 2: Hepatic myofibroblasts express v integrin in human fibrotic liver tissue, and v integrin depletion on murine HSCs does not affect initial liver injury after CCl4 treatment, baseline HSC number, inflammatory infiltrates or the vasculature of itgavflox/flox;Pdgfrb-Cre livers.(a) Representative immunofluorescence micrographs of human fibrotic liver tissue stained for v integrin (red), SMA (green) and DAPI (blue) (n = 5 cases). Arrow (right panel) indicates v integrin and SMA double-positive cell. Scale bar, 10 m. (b) Micrographs of hematoxylin / eosin stained liver sections from control or itgavflox/flox;Pdgfrb-Cre (v Cre) mice 24 h after a single i.p. injection of olive oil or CCl4 (n = 6 male mice per group). Scale bar, 200m. (c) Serum alanine aminotransferase (ALT) levels. (d,e) Digital image analysis of desmin and PDGFR staining in uninjured control and itgavflox/flox;Pdgfrb-Cre livers (n = 6 male mice per group). (f) Gr1 immunohistochemistry of liver tissue after control or chronic CCl4 treatment of control and itgav

    flox/flox;Pdgfrb-Cre mice. (n = 8 male mice per group). Scale bar, 50m. (g) Neutrophil counting. (h) F4/80 immunohistochemistry of liver tissue after control or chronic CCl4 treatment of control and itgavflox/flox;Pdgfrb-Cre mice. Scale bar, 200m. (i) Digital image analysis quantification of F4/80 staining. (j) CD31 immunohistochemistry of liver tissue (left) after control or chronic CCl4 treatment of control and itgavflox/flox;Pdgfrb-Cre mice. Scale bar, 100m. Digital image analysis quantification of CD31 staining (right). Data are mean s.e.m. (Students t test).

    Nature Medicine: doi:10.1038/nm.3282

  • Hep LSEC Kupffer HSC

    -actin

    v

    Cre

    Con

    trol

    v

    Cre

    Con

    trol

    v

    Cre

    Con

    trol

    v

    Cre

    Con

    trol

    PDGFR

    v

    b

    - TML

    C

    - TML

    C

    + TML

    C

    + TML

    C

    TMLC

    c d

    SM

    A (fo

    ld c

    hang

    e)

    Col

    1A1

    (fold

    cha

    nge)

    e

    Contr

    ol

    Contr

    ol

    + TGF

    + TGF

    NS

    NS Controlv CreTMLC

    0

    100

    200

    300

    0

    200

    400

    600

    - TML

    C

    - TML

    C

    + TML

    C

    + TML

    C

    TMLC

    TGF

    1 (p

    g/m

    l) NSNS

    Controlv Cre

    0

    1

    2

    3NS

    NS

    * **

    Contr

    ol

    Contr

    ol

    + TGF

    + TGF

    0

    1

    2

    3

    TGF

    1 (p

    g/m

    l)

    a

    Supplementary Figure 3: v integrin and PDGFR expression in purified hepatic cell populations from itgavflox/flox;Pdgfrb-Cre mice, total TGF-1 concentrations in control and itgavflox/flox;Pdgfrb-Cre HSCs, and addition of TGF-1 to itgavflox/flox;Pdgfrb-Cre HSCs rescues pro-fibrotic gene expression. (a) Western blotting of v integrin and PDGFR expression in purified hepatocytes (Hep), liver sinusoidal endothelial cells (LSECs), kupffer cells and HSCs from control and itgavflox/flox;Pdgfrb-Cre (v Cre) male mice 5 days post isolation. (b) Total TGF-1 concentrations measured by ELISA in cell lysates from control and itgavflox/flox;Pdgfrb-Cre HSCs cultured with and without TMLCs (c) Total TGF-1 concentrations measured by ELISA in supernatants from control and itgavflox/flox;Pdgfrb-Cre HSCs cultured with and without TMLCs. (d) qPCR analysis of SMA expression in control and itgavflox/flox;Pdgfrb-Cre (v Cre) HSCs treated with control or recombinant TGF-1 (1ng ml-1) for 24 h. (e) qPCR analysis of Col1A1 expression in control anditgavflox/flox;Pdgfrb-Cre (v Cre) HSCs treated with control or recombinant TGF-1 (1ng ml-1) for 24 h. Data are mean s.e.m. *P < 0.05, **P < 0.01 (Students t test).

    Nature Medicine: doi:10.1038/nm.3282

  • 0.0005

    0

    0.0010

    0.0015TG

    F1

    / 18S

    HSC

    Kupff

    erLS

    EC

    Hepa

    tocyte

    s

    c

    b

    dControl v Cre

    TUN

    EL

    Contr

    ol

    Fibron

    ectin

    Colla

    gen I

    Colla

    gen I

    V

    Lami

    nin

    Fibrin

    ogen

    0

    40

    30

    20

    10

    50

    % a

    dhes

    ion

    Controlv cre

    % m

    igra

    tion

    0

    40

    30

    20

    10

    Control FCS

    Controlv cre

    e

    TUNE

    L-po

    sitive

    nuc

    lei (

    %)

    0

    0.4

    0.3

    0.2

    0.1

    Controlv Cre

    Con v Cre

    NS

    NS

    NS

    NS

    NS

    NS

    NS

    NS

    a

    Supplementary Figure 4: TGF-1 expression in hepatic cell populations isolated from CCl4-induced fibrotic liver, and assessment of adhesion, migration and anoikis in control and itgavflox/flox;Pdgfrb-Cre HSCs.(a) qPCR analysis of TGFb1 mRNA expression in purified HSCs, hepatocytes, liver sinusoidal endothelial cells (LSECs) and kupffer cells isolated from CCl4-induced fibrotic livers (n = 4 male mice per group). (b) Adhesion assay of control and itgavflox/flox;Pdgfrb-Cre HSCs plated on different matrices. (c) Migration assay of control and itgavflox/flox;Pdgfrb-Cre HSCs (d) TUNEL immunofluorescence staining of control and itgavflox/flox;Pdgfrb-Cre HSCs. Arrows indicate TUNEL positive nuclei (green), DAPI (blue). (e) Quantitation of TUNEL-positive nuclei. Data are mean s.e.m. (Students t test).

    Nature Medicine: doi:10.1038/nm.3282

  • Cou

    nts

    Fluoresence

    Cou

    nts

    Cou

    nts

    a b

    c

    0 105102 103 104

    0 105102 103 104

    Control 5-PE

    Fluoresence

    Fluoresence

    v Cre 5-PE

    Control Iso-PE

    v Cre Iso-PEControl 1-PE

    v Cre 1-PE

    Control Iso-PE

    v Cre Iso-PE

    Control 1-PE

    v Cre 1-PE

    Control Iso-PE

    v Cre Iso-PE

    0 105102 103 104

    0

    50

    100

    25

    75%

    reco

    mbi

    natio

    n

    Desm

    inPD

    GFR

    PDG

    FR

    Desm

    in

    SM

    A

    Saline Bleomycin

    d

    Supplementary Figure 5: FACS analysis of 1, 5 and 1 integrin expression levels in control and itgavflox/flox;Pdgfrb-Cre HSCs, and recombination efficiency in lung pericytes and myofibroblasts in Ai14;Pdgfrb-Cre mice.FACS analysis of (a) 1, (b) 5 and (c) 1 integrin expression levels in control and itgavflox/flox;Pdgfrb-Cre HSCs cultured on tissue culture plastic for 5 days. (d) Analysis of recombination efficiency in lungs from saline treated (control) or bleomycin treated Ai14;Pdgfrb-Cre reporter mice (n = 4 male mice per group) stained for desmin, PDGFR or SMA. Data are mean s.e.m.

    Nature Medicine: doi:10.1038/nm.3282

  • Reporter / DAPI SMA / DAPIb

    d

    Lung

    sK

    idne

    ys

    Mergeda-actin-SMA

    c-actin-SMA

    Salin

    eBl

    eoSa

    line

    Bleo

    Sham

    UUO

    Sham UU

    OReporter / DAPI SMA / DAPI Merged

    e v-actin

    v CreControlSaline

    g

    v-actin

    v CreControlBleomycin

    fv

    -actin

    v CreControlOil

    v-actin

    v CreControl

    CCl4

    h i v-actin

    v CreControlSham

    v-actin

    v CreControlUUO

    j

    Control v Cre

    Control v Cre Control v Cre

    CC

    l 4O

    il

    UU

    OS

    ham

    Ble

    oS

    alin

    e

    Supplementary Figure 6: Myofibroblast induction in Ai14-Td tomato positive cells sorted from lungs and kidneys of Ai14;Pdgfrb-Cre mice, and analysis of v and 6 integrin expression in uninjured and fibrotic lung, liver and kidney in control and itgavflox/flox;Pdgfrb-Cre mice.(a) Western blotting of SMA in freshly sorted Td tomato positive cells from saline or bleomycin treated (28 days post instillation) Ai14;Pdgfrb-Cre mice (n = 4 male mice per group). (b) Immunofluoresence staining of Td tomato positive cells sorted from the uninjured lungs of Ai14;Pdgfrb-Cre mice and plated on tissue culture plastic for 7 days. Endogenous report (red), SMA (green), DAPI (blue). Scale bar, 100m. (c) Western blotting of SMA in freshly