Nature - USA (2020-02-13)

(Antfer) #1
Nature | Vol 578 | 13 February 2020 | 291

transcription factors that are known to be upregulated by atheropro-
tective shear stress^8 ,^9. We found that knockdown of PLXND1 attenu-
ated flow-induced upregulation of both of these genes compared with
control mouse ECs (Fig. 1a). We then investigated whether PLXND1
could mediate the endothelial response to disturbed shear stress.


We subjected mouse ECs to atheroprone flow for 24 h and examined
mRNA levels of the pro-inflammatory genes monocyte chemoat-
tractant protein-1 (Mcp1 (also known as Ccl2)) and vascular cell adhe-
sion molecule-1 (Vcam1)^10. We noted that knockdown of PLXND1 in
ECs treated with siRNA significantly reduced the upregulation of
both genes in response to atheroprone shear stress (Fig. 1b). Taken
together, these data demonstrate that PLXND1 is a critical mediator
of key shear-stress responses in ECs.
To explore the biological relevance of our findings, we used a trans-
genic mouse model to enable the endothelial-specific inducible dele-
tion of PLXND1 (Plxnd1iECKO) (Extended Data Figs. 1b, 7b). Confocal
imaging of actin filaments in ECs and staining for the junctional marker
β-catenin revealed a reduction in the elongation of ECs and a reduced
intensity of actin stress fibres in the absence of PLXND1 (Fig. 1c)—con-
sistent with in vitro observations (Extended Data Fig. 2b).
Given the decrease in the expression of inflammatory genes in
response to atheroprone shear stress in vitro that was observed with
loss of PLXND1 (Fig. 1b), we assessed the role of endothelial PLXND1
in a pathophysiogical setting. Atherosclerotic lesions are known
to occur in regions of the vasculature with low or disturbed blood
flow, flow reversal and other complex spatiotemporal flow patterns^1.
Systemic risk factors, such as hypercholesterolaemia, interact with
local biomechanical factors to initiate and advance the deposition
of atherosclerotic plaques. To assess whether endothelial deletion
of PLXND1 affected atherosclerosis in vivo, we crossed Plxnd1fl/fl
and Plxnd1iECKO mice with hypercholesterolaemic apolipoprotein-E
deficient (Apoe−/−) mice^11 and fed them a high-fat diet for 10 weeks.
Although the body weights and lipid levels of the mice were unaf-
fected by the loss of PLXND1 (Extended Data Fig. 4a), quantification
of oil-red-O-stained aortic samples revealed a significant decrease
in the plaque burden of both the whole aorta and the aortic arch in
Plxnd1iECKOApoe−/− mice (Fig. 1d, e). To explore these differences fur-
ther, we examined the expression of inflammatory markers in the
inner curvature of the aortic arch. Immunostaining and quantita-
tive (q)PCR analysis showed reduced levels of MCP-1 and VCAM-1 in
Plxnd1iECKOApoe−/− compared with Plxnd1fl/flApoe−/− mice (Fig. 1f and
Extended Data Fig. 4b). Given the atheroprotective role of laminar
shear stress and the reduced alignment of ECs with loss of PLXND1, we
examined the effects in the atheroprotected descending aorta. After
a high-fat diet for an extended period of 20 weeks, we observed an
increase in the plaque burden in the descending aortas of Plxnd1iECKO
Apoe−/− mice (Extended Data Fig. 5); these plaques also appeared to
correlate with intercostal branch points that have flow disturbances.
Together, these results show that endothelial PLXND1 is required for
the endothelial response to fluid shear stress and the site-specific
distribution of atherosclerosis.
The requirement of PLXND1 in flow-mediated responses in vitro
and in vivo prompted us to investigate whether this is because
PLXND1 is simply a player in mechanochemical signalling cascades
or functions as a mechanoreceptor that is capable of detecting
mechanical force. We applied tensional forces, using a magnetic
system^12 , to paramagnetic beads coated with an antibody that rec-
ognizes the extracellular domain of PLXND1 and examined force
responses using four different readouts. First, force on PLXND1
induced activation of the same signalling cascades (ERK1/2, Akt
and VEGFR2) (Fig. 2a), as those induced by shear stress^13 (Extended
Data Fig. 2a). Second, we observed a robust transient increase in
intracellular calcium levels in ECs when force was applied on PLXND1
(Fig. 2b), similar to the response observed for other recently dis-
covered mechanosensors^14 ,^15. Third, we examined cytoskeletal
responses^12 : ECs responded to the application of force on PLXND1
by exhibiting a robust increase in both vinculin-positive focal
adhesions (Fig. 2c) and ligated integrin β1 staining (Extended
Data Fig. 6a). Notably, the mechanotransduction response was not
restricted to the vicinity of the magnetic bead under tension, but

b


a


(^0) Scramble Plxnd1
50
100
150
Fold change



  • Mcp1
    (^0) Scramble Plxnd1
    50
    100
    150
    Vcam1
    Fold change


  • siRNA:Scramble Plxnd1
    siRNA:
    0
    50
    100
    150
    Klf2
    Fold change
    **
    (^0) Scramble Plxnd1
    50
    100
    150
    Klf4
    Fold change




    Phalloidin β-Catenin DAPI Merged




    Plxnd1
    fl/fl
    Plxnd1
    iECKO
    c
    0
    5
    10
    15
    0
    5
    10
    15
    20 *
    Atheroprotective ow
    (^40 30)
    (^20 10)
    Shear stress^0
    (dynes cm
    –2)
    –10
    Time (×100 ms)
    0 2 4 6 8
    Atheroprone ow
    (^40 30)
    (^20 10)
    –10^0
    Time (×100 ms)
    Elongation factor
    Plxnd1fl/fl Plxnd1iECKO
    Plxnd1fl/fl Plxnd1iECKO
    Fluorescence intensity (×100)
    d Plxnd1fl/flApoe–/– Plxnd1iECKOApoe–/– e
    Plxnd1fl/fl
    Apoe–/–
    Plxnd1iECKO
    Apoe–/–
    Plxnd1fl/fl
    Apoe–/–
    Plxnd1iECKO
    Apoe–/–
    Plxnd1fl/fl
    Apoe–/–
    Plxnd1iECKO
    Apoe–/–
    Plxnd1fl/fl
    Apoe–/–
    Plxnd1iECKO
    Apoe–/–
    0
    5
    10
    15
    20
    25
    Aortic arch
    **
    0
    2
    4
    6
    Whole aorta




  • Plaque area (%)
    Plaque area (%)
    f
    0
    50
    100
    150
    Mcp1




    0
    50
    100
    150
    200
    Vcam1




  • Shear stress(dynes cm
    –2)
    0 2 4 6 8
    Fold change Fold change
    Fig. 1 | PLXND1 mediates the EC response to f luid shear stress and regulates
    the site-specific distribution of atherosclerosis. a, b, Mouse ECs were
    transfected with either scrambled or Plxnd1 siRNA and exposed to either
    atheroprotective or atheroprone f low for 24 h, using a cone-and-plate
    viscometer. qPCR was performed to quantify the expression of Klf2 and Klf4 in
    samples subjected to atheroprotective f low, and expression of the
    inf lammatory markers Mcp1 (also known as Ccl2) and Vcam1 in samples
    subjected to atheroprone f low. n = 4 biological replicates. c, The descending
    thoracic aorta was isolated and prepared en face from Plxnd1fl/fl and Plxnd1iECKO
    mice and stained for β-catenin, phalloidin and DAPI to visualize the cell
    junctions, actin stress fibres and nuclei. Quantification of alignment and mean
    f luorescence intensity was performed using ImageJ; 3–5 images (each image
    has n ≤ 100 cells) taken from 3 regions along the length of the descending aorta
    were obtained from n = 5 mice of each genotype (exact sample numbers are
    provided in the Source Data). d, Representative en face preparations of whole
    aortas showing atherosclerosis in Plxnd1fl/flApoe−/− and Plxnd1iECKOApoe−/− mice
    after 10 weeks of high-fat diet feeding, visualized by oil-Red-O staining.
    e, Quantification of the lesion area in whole aortas and aortic arches from
    Plxnd1fl/flApoe−/− and Plxnd1iECKOApoe−/− mice. n = 8 mice. f, Aortic arches from
    Plxnd1fl/flApoe−/− and Plxnd1iECKOApoe−/− mice were isolated and qPCR was
    performed for expression of the inf lammatory markers Vcam1 and Mcp1. n = 5.
    Data are mean ± s.e.m. P values were obtained using two-tailed Student’s t-tests
    using GraphPad Prism. P < 0.05, P < 0.01, P < 0.001, ****P < 0.0001. Scale
    bar, 20 μm.



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