Protective Role of Instant Membrane Resealing in NLRP3 Inflammmasome Activation of
Mouse Endothelial Cells Yang Chen, Ming Yuan, Min Xia, Pin-Lan Li
Department of Pharmacology, Virginia Commonwealth University School of Medicine, Richmond, VA 23298
Figure 1. Representative fluorescent images (panel A and B) show that both
LCWE and saponin (a membrane injury agent) increased Ca2+ (from green to
red color) and PI entry into cells (red). These changes in Ca2+ and PI
fluorescence were recorded, as shown by panel C and D. Given that PI cannot
be dissociated from their binding to DNA for red fluorescence, the df/dt of PI
was calculated to represent the speed of PI entry into cells, which is used as an
indicator of cell membrane injury/repair (E and F).
RESULTS
Simultaneous measurement of Ca2+ and PI production in MVECs. Plasmic
membrane damage and intracellular Ca2+ response to different stimulants were
determined by using Propidium iodide (PI) as an indicator of membrane open
and resealing and by fura-2, a Ca2+-sensitive fluorescent dye, respectively. A
fluorescent microscopic imaging system with high speed wave-length switching
was used as described previously (Am J Physiol Cell Physiol. 2013 Mar
1;304(5):C458-66).
Confocal microscopy, Western blot analysis, Laser-sort cytometry and
measurements of cathespin B activities were conducted as we described
currently (Biochim Biophys Acta. 2015 Feb;1853(2):396-408).
METHODS
Figure 2. We demonstrated that Ca2+ plays an important role in the plasma membrane instant resealing, as shown by the Ca2+-dependence of the PI fluorescence rising peaks and its df/dt. In the presence of Ca2+, both LCWE and saponin produced much less membrane injury, as MVECs in Ca2+-free Hank’s solution needed much shorter time to reach maximal df/dt compared to cells in normal Hank’s solution (A, B). The time to peak of PI fluorescence rising and the maximal df/dt were summarized in panel C-F. LCWE or saponin-induced cell membrane injury as shown by maximal df/dt was significantly larger in MVECs bathed in Ca2+-free Hank’s solution compared to the cells in Ca2+ Hank’s solution, as shown by short time to the peak of PI fluorescence rising (C and D) and by increased dt/df (E and F).
Instant cell membrane resealing may importantly protect endothelial cells
(ECs) from injury, and a rapid membrane resealing or repairing during injury
is an important adaptive mechanism, which is essential for the regulation of
cell survival and cell function.
Membrane rafts (MRs) and an important MR sphingolipid, ceramide play a
critical role in cell membrane repair during cell injury, and acid
sphingomyelinase (ASM), the ceramide-producing enzyme that promotes
MR clustering has been reported to participate in the control of cell
membrane resealing.
The present study was designed to test whether the MR- and ceramide-
associated cell membrane resealing is critically implicated in the regulation of
vascular function or related diseases due to endothelial dysfunction and injury.
PI
LC
WE
(1
5
min
)
Co
ntr
ol
F340/F380 PI
Sa
p (
15
min
) C
on
tro
l
F340/F380
Time (min)
0 10 20 30 40
0
5
10
15
0.0
0.1
0.2
0.3
F3
40
/38
0
PI (
df/d
t)
Sap
Fura-2
PI
A
C
E
B
D
F
0 5 10 15 20 25 30
0.0
0.2
0.4
0.6
0
10
20
30
F3
40
/38
0
Time (min)
Fura-2
PI
LCWE PI (
df/d
t)
0 10 20 30 40
0
2
4
6
8
0
5
10
15
F3
40
/38
0
Sap
PI
Fura-2
PI (Δ
)
Time (min) 0 10 20 30
0
10
20
30
0
2
4
6
8
PI (Δ
)
F3
40
/38
0
PI Fura-2
LCWE
Time (min)
FasL and LCWE
Tim
e to
pe
ak (
min
) *
0
5
10
15
20
Vehl MCD
D F
C E
LCWE
*
0
5
10
15
20
Vehl FasL
Tim
e to
pe
ak (
min
)
B
0 5 10 15 20 25 30
0.0
0.4
0.8
1.2
1.6 Vehl+FasL
MCD+FasL
PI (d
f/d
t)
Time (min)
LCWE
0 5 10 15 20 25 30
0.0
0.4
0.8
1.2
1.6 FasL
Vehl
LCWE PI (d
f/d
t)
Time (min)
A
LCWE
*
vehl FasL
PI (d
f/d
t)
0
0.3
0.6
0.9
1.5
PI (d
f/d
t)
FasL and LCWE
*
Vehl MCD 0
0.3
0.6
0.9
1.5
Pro-casp-1
kDa
50
LCWE
Cle-casp-1 17
-
VA64
43 β-actin
+ - +
Vehl
A
B
C
*
#
Nlr
p3
/Ca
sp
-1(P
CC
)
0
0.2
0.4
0.6
0.8
Vehl VA64
Ctrl
LCWE
Nlrp3 / ASC
Nlrp3 / Casp-1
Ctrl LCWE VA64 LCWE-VA64
D
E
Nlr
p3
/AS
C (
PC
C)
0
0.2
0.4
0.6
0.8
Vehl VA64
Ctrl
LCWE *
#
F
IL-1
ß (
pg
/ml) *
#
0
0.6
1.2
1.8
2.4
Ctrl VA64
Vehl
LCWE
Ca
sp
-1 a
ctiva
tio
n *
#
0
0.5
1
1.5
2
2.5
Ctrl VA64
Vehl
LCWE
C E
D F
LCWE
*
0
5
10
15
20
Ca2+ 0 Ca2+ Tim
e t
o p
eak (
min
)
*
0
0.1
0.2
0.3
0.4
Ca2+ 0 Ca2+
LCWE
PI (d
f/dt)
0
5
10
15
Ca2+
Sap
0 Ca2+
*
Tim
e t
o p
eak (
min
)
0
0.2
0.4
0.6
0.8
Ca2+
Sap
0 Ca2+
PI (d
f/dt)
A
B
AO
Scr
AS
M s
i
LC
WE
+ F
asL
V
eh
l L
CW
E +
Fa
sL
V
eh
l
CatB AOI AOI
SUMMARY AND CONCLUSION
BACKGROUND AND AIMS
Figure 4. Next, we demonstrated that membrane injury by LCWE enhanced Nlrp3
inflammasome inflammasome as shown by confocal co-localization of Nlrp3 with ASC or
caspase-1, which was blocked by artificial plasma membrane resealing reagent (VA64) (A, B,
C). By Western blot analysis, cleaved or active caspase-1 was shown increased by LCWE,
which was blocked by VA64 indicating that membrane resealing can block LCWE-induced Nlrp3
inflammasome activation (D). Correspondingly, LCWE-induced caspase-1 activity increase and
IL-1β production, the indicators of inflammasome activation were blocked by VA64, further
confirming the protective role of membrane resealing in the Nlrp3 inflammasome activation.
Figure 6 shows the effects of ASM siRNA transfection on
FasL/LCWE-induced cathepsin B activation. It was shown
that FasL/LCWE increased lysosome membrane
permeability, as shown by decreased orange fluorescence
intensity for acridine orange staining of MVECs. IN
addition, cathepsin B activation as shown by increased red
fluorescene intensity of CatB staining. These findings
confirm that cathepsin B is involved in FasL/LCWE-
induced Nlrp3 inflammasome activation in MVECs, which
is associated with impaired membrane resealing.
A Ca2+-dependent membrane resealing was shown in
MVECs in response to LCWE or sapanin, which was
markedly attenuated by FasL, a death receptor ligand that
stimulates membrane raft (MR) clustering in ECs.
FasL-induced impairment of membrane resealing and
consequent enhancement of plasma membrane injury
were almost completely blocked by a MR disruptor,
methyl-β-cyclodextrin (MCD).
The impaired plasma membrane resealing by FasL led to
the enhanced formation and activation of Nlrp3
inflammasome in MVECs.
FasL-enhanced inflammasome activation was blocked
by an artificial membrane resealer VA64 and ASM
inhibition, and this enhanced inflammasome activation is
due to increased lysosomal permeability and cathepsin B
release.
These results suggest that a Ca2+-dependent and MR-
associated instant cell membrane resealing occurs
instantly in ECs in response to injurious factors, which
prevents Nlrp3 inflammasome activation by stabilization of
lysosomes.
Figure 3. We further tested whether MRs clustering alters membrane resealing during LCWE-induce plasma membrane injury in MVECs by measuring the peak of the flow velocity of PI and df/dt. The peak response was more sensitive after pretreatment of FasL for 30 min. The lipid raft disruptor, MCD could attenuate FasL-induced effects on this peak response (A, B). The time to peak response was much shorter in FasL-treated cells than its vehicle. However, after pretreatment of cells with MCD with FasL together, the time of peak response was restored the level of vehicle treated cells (C, D). In contrast, the df/dt was increased significantly by FasL indicating weak or no resealing. However. MCD markedly reduced FasL-induced increases in df/dt (E, F). These results suggest that FasL-induced MRs clustering impair cell membrane resealing, which can be blocked by disruption of the MRs.
Figure 5. Similarly, silencing of ASM gene, a MR key lipid-ceramide producing enzyme markedly
inhibited FasL enhancement of LCWE-induced Nlrp3 inflammasome formation and activation in
MVECs, as shown by confocal microscopic co-localization of Nlrp3/ with ASC or caspase-1 (A-
C) for Nlrp3 inflammasome formation, cleaved or active capase-1 level and caspase-1 activity
(D, E).
0
5
10
15
20
scr siASM
Vehl LCWE+FasL
Ca
sp
-1 a
ctivity
*
#
E
kDa
50
17
43 β-actin
Cle-casp-1
pro-casp-1
scr siASM
D
LCWE - + - +
A
0
0.1
0.2
0.3
0.4
scr siASM
Vehl LCWL+FasL
*
#
Nlr
p3
/AS
C (
PC
C)
B C
0
0.15
0.3
0.45
0.6
scr siASM
Vehl
LCWE+FasL
*
#
Nlr
p3
/Ca
sp
1 (
PC
C)
Nlrp3/ASC
scr
Vehl LCWE + FasL Vehl
siASM
Nlrp3/Casp1
LCWE + FasL