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

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min

)

0

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Ca2+

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PI (d

f/dt)

A

B

AO

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AS

M s

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WE

+ F

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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