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OPEN 5-Aminolevulinic Acid-Mediated

Sonodynamic Therapy Inhibits
RIPK1/RIPK3-Dependent
received: 13 July 2015
accepted: 29 January 2016 Necroptosis in THP-1-Derived Foam
Cells
Published: 25 February 2016

Fang Tian1, Jianting Yao1, Meng Yan2, Xin Sun1, Wei Wang1, Weiwei Gao1, Zhen Tian2,

Shuyuan Guo1, Zengxiang Dong1, Bicheng Li1, Tielei Gao5, Peng Shan6, Bing Liu6,
Haiyang Wang6, Jiali Cheng1, Qianping Gao1, Zhiguo Zhang4, Wenwu Cao4,7 & Ye Tian1,2,3

Necroptosis, or programmed necrosis, contributes to the formation of necrotic cores in atherosclerotic

plaque in animal models. However, whether inhibition of necroptosis ameliorates atherosclerosis is
largely unknown. In this study, we demonstrated that necroptosis occurred in clinical atherosclerotic
samples, suggesting that it may also play an important role in human atherosclerosis. We established
an in vitro necroptotic model in which necroptosis was induced in THP-1-derived foam cells by serum
deprivation. With this model, we demonstrated that 5-aminolevulinic acid-mediated sonodynamic
therapy (ALA-SDT) inhibited necroptosis while promoting apoptosis. ALA-SDT activated the caspase-3
and caspase-8 pathways in foam cells, which is responsible for the switch from necroptosis to apoptosis.
The inhibition of either caspase-8 or caspase-3 abolished the anti-necroptotic effect of ALA-SDT. In
addition, we found that caspase-3 activation peaked 4 hours after ALA-SDT treatment, 2 hours earlier
than maximal caspase-8activation. Taken together, our data indicate that ALA-SDT mediates the switch
from necroptosis to apoptosis by activating the caspase-3 and caspase-8 pathways and may improve
the prognosis of atherosclerosis.

Foam cells, a hallmark of atherosclerosis, accumulate in the intima of arterial wall and form atherosclerotic
plaques1,2. With the progression of atherosclerosis, some of foam cells undergo necrosis and make up necrotic
core in the centre of atherosclerotic plaque3. Necroptosis, a type of programmed cell death, contributes to necrotic
core formation and enhances inflammation, thereby aggravating atherosclerosis in animal models4. Hence, tar-
geting necroptosis might be a novel methodology for atherosclerosis treatment.
The concept of necroptosis was introduced in 2005 and demonstrated in cultured cells1,2. This type of
cell death occurs when caspase-8 function is compromised upon overwhelming stress and is defined as
caspase-independent programmed cell death3–5. In contrast to apoptosis or necrosis, necroptosis is characterized
by swollen organelles, disintegrated plasma membrane and intact nuclear membrane6,7. The finding of necroptosis
revises the traditional notion of cell death, in which necrosis is described as an unregulated, passive process. This
finding also leads to re-examining the role of cell death in atherosclerosis and other inflammatory diseases8–10.

1
Department of Cardiology, the First Affiliated Hospital, Cardiovascular Institute, Harbin Medical University, Harbin,
150001, P. R. China. 2Department of Pathophysiology and the Key Laboratory of Cardiovascular Pathophysiology,
Harbin Medical University, the Key Laboratory of Cardiovascular Research (Harbin Medical University), Ministry
of Education, Harbin 150081, P. R. China. 3Heilongjiang Academy of Medical Sciences, Harbin 150086, P. R. China.
4
Laboratory of Photo- and Sono-theranostic Technologies and Condensed Matter Science and Technology Institute,
Harbin Institute of Technology, Harbin 150080, P. R. China. 5Department of Forensic Medicine, Harbin Medical
University, Harbin 150001, P. R. China. 6Department of Vascular Surgery, the First Affiliated Hospital of Harbin
Medical University, Harbin 150001, P. R. China. 7Materials Research Institute, The Pennsylvania State University,
University Park 16802, PA, USA. Correspondence and requests for materials should be addressed to Y.T. (email:
[email protected])

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Under the condition of dysfunctional apoptotic signaling, protein kinase RIPK1 (receptor interacting pro-
tein kinase 1) and RIPK3 (receptor interacting protein kinase 3) auto- and transphosphorylate each other, and
form necrosome. The necrosome then phosphorylates the pro-necroptotic protein MLKL (Mixed Lineage Kinase
domain-Like), and phosphorylated MLKL initiates the necroptosis by forming oligomerization and inserting
itself into the membranes of organelles and plasma membrane11–14. Genetic deletion of RIPK1, RIPK3 or MLKL,
inhibits necroptosis in the multiple mouse models of inflammatory diseases8,13,15. In addition, Necrostatin-1, an
inhibitor of RIPK1, is extensively used to prevent necroptosis in preclinical research. Although these methodol-
ogies showed efficacy in slowing down the progression of necroptosis-associated diseases in basic research, their
clinical benefits and safety require further evaluation. So far, the treatment of necroptosis-related diseases16–19
remains hampered by the lack of an ideal therapy16,17,20.
With strong tissue-penetrating and regional focusing characteristics, SDT selectively kills inflammatory or
tumour cells21–23. Its preliminary application in cancer patients showed therapeutic benefits23–26. Previous studies
in our laboratory have demonstrated that ALA selectively accumulates in macrophages and foam cells in ather-
osclerotic plaques27–29. We also showed that ALA-mediated SDT (ALA-SDT) stabilises atherosclerotic plaques
by eliminating foam cells and preventing extracellular matrix degradation, displaying a strong potential for ath-
erosclerosis treatment27. However, the underlying mechanism of ALA-SDT remains elusive. In this study, we
found that ALA-SDT inhibits necroptosis and enhances apoptosis in THP-1-derived foam cells by activating the
caspase-3 and caspase-8 pathways. Switching necroptosis to apoptosis by ALA-SDT may account for its beneficial
effects in atherosclerosis treatment.

Results
Necroptosis occurs in human atherosclerosis.  Foam cell necroptosis occurs in atherosclerotic lesions
and contributes to advanced atherosclerosis in animal models8. However, to date, evidence showing that necrop-
tosis exists in human atherosclerotic plaques is missing. To investigate whether this type of cell death takes place
in human atherosclerotic plaques, we collected human atherosclerotic tissues from 12 autopsies and 6 patients
undergoing carotid endarterectomy (CEA) (Supplementary Figure e). Compared with normal arterial wall in the
same section, arterial wall with atherosclerotic lesions was grossly thickened, exhibiting discernible lipid-contain-
ing plaque. Based the observation with haematoxylin-eosin (HE) staining, atherosclerotic plaques were separated
into two types, with or without a necrotic core (Fig. 1a). For the plaques with necrotic cores, necrosis primarily
occurred at the centre of atheroma (Fig. 1a). To determine the cell composition of atherosclerotic plaque, we
relied on transmission electron microscopy (TEM). In the plaques without necrotic cores, the main cellular com-
ponents were smooth muscle cells, which migrate and proliferate from media to the intima responding to arterial
wall damage. In contrast, plaques with necrotic cores were full of lipid-rich foam cells, and some of the foam cells
around the necrotic core displayed typical necroptotic morphology6, including disrupted membrane, translucent
cytoplasm, swollen organelles and intact nucleus (Fig. 1a). The protein levels of RIPK1/3 are positively correlated
with the extent of necroptosis30. Therefore, we examined the expression levels of RIPK1 and RIPK3 in atheroscle-
rotic lesions. Western blotting analysis showed that the levels of these two proteins were significantly increased
in atherosclerotic tissues with necrotic cores compared with plaques without necrotic cores (Fig. 1b). Consistent
with the western blotting results, the levels of the RIPK1-RIPK3 complex were dramatically elevated (Fig. 1c) in
atherosclerotic tissues with necrotic cores. Taken together, these results indicate that necroptosis indeed occurs in
human atherosclerotic lesions and is correlated with necrotic core formation.

RIPK1/RIPK3-dependent necroptosis in THP-1-derived foam cells is induced by serum starva-

tion.  The insufficient supply of nutrients results in foam cell death in atherosclerotic plaques31. To mimic
the pathological process of foam cell death, we treated THP-1-derived foam cells with serum-free medium. To
discern which type of cell death occurred in foam cells after serum starvation, we used TEM to observe cellular
morphology. Twenty-four hours after serum deprivation, all types of cell death, including apoptosis, necroptosis,
and necrosis, were observed in foam cells (Fig. 2). The morphological changes of necroptotic cells are distinct
from that of apoptotic or necrotic cells. In contrast to apoptotic cells (Fig. 2b), membrane leakiness and organelle
swelling were observed to occur early in necroptotic cells (Fig. 2c); but apoptotic bodies were not formed in these
cells. Campared with cells undergoing uncontrolled necrosis (Fig. 2d), necroptotic cells showed swollen nuclear,
increased cell volume with translucent cytoplasm (Fig. 2c); however, these cells did not display a ruptured nuclear
membrane. After serum starvation, the intracellular ATP levels and the release of lactate dehydrogenase (LDH)
were measured at different time points. As shown in Fig. 3a, the intracellular ATP levels significantly decreased
with the extension of serum deprivation (Fig. 3a). At the same time, lactate dehydrogenase (LDH) was drastically
released into culture medium (Fig. 3b). These results suggested that THP-1-derived foam cells underwent cell
death in response to serum starvation. Consistent with these observations, flow cytometry analysis also showed
an increased rate of cell death after serum starvation, as indicated by positive PI staining (Fig. 3d).
In addition to the increased rate of cell death, the incubation of foam cells with serum-free medium for 24
and 48 hours led to a significant elevation of RIPK1 and RIPK3 protein levels (Fig. 3c,d). More important, the
level of the RIPK1-RIPK3 complex, a representative marker of necroptosis, was significantly increased over time
after serum starvation (Fig. 3e). In addition, the levels of MLKL oligomerization were also significantly elevated
after serum starvation (Fig. 3f). Summarily, the above results indicate that necroptosis was successfully induced
in foam cells by serum deprivation.
Since PI staining cannot distinguish necroptosis from necrosis, we next determined the percentage of necrop-
totic cells among the dead cells after serum starvation. To this end, we used a necroptotic-specific inhibitor,
Necrostatin-1 (Nec-1), to block serum deprivation-induced necroptosis. Using Annexin V and propidium
iodide (PI) staining, we determined the rates of necrosis (PI-positive) and apoptosis (PI-negative and Annexin
V-positive) by flow cytometry. As shown in Fig. 4a, Nec-1 treatment dramatically reduced foam cell death from

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Figure 1.  Morphological and biochemical features of necroptosis in human atherosclerosis.

(a) Representative images showing human atherosclerotic plaques by H&E staining and TEM (scale
bars =  200 μ m). Magnified insets represent the atherosclerotic plaque without a necrotic core (NC, in red
frame), and the atherosclerotic plaque with a necrotic area (in black frame). An “X” indicates a necrotic area
in the atherosclerotic plaques (scale bars =  450 μ m). The right panel represents TEM images of atherosclerotic
plaques with or without a necrotic core (scale bars =  2 μ m). TEM image in red frame shows multiple sheets
of smooth muscle cells (SMC) in atherosclerotic plaque without a necrotic core. TEM image in black frame
indicates cellular composition of atherosclerotic plaque with a necrotic core, and shows the typical necroptotic
morphology of foam cells (FC), including a disrupted membrane, translucent cytoplasm, swollen organelles and
intact nucleus. (b) Western blotting analysis of RIPK1 and RIPK3 levels in atherosclerotic plaque tissues with
or without a necrotic core. Protein expression was quantified with reference to GAPDH. (n ≥  6. *P <  0.05 and
**P <  0.01 compared to the group without a necrotic core). (c) Immunoprecipitation assay indicates the increase
of RIPK1-RIPK3 complex in atherosclerotic plaques with necrotic core. The data represent means ±  S.E.M.

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Figure 2.  Electron microscopic characteristics of different types of cell death in foam cells. (a) A viable
foam cell with large lipid droplets, normal cell organelles, as well as intact plasma and nuclear membranes.
(b) Apoptotic cell showing nuclear shrinkage, chromatin condensation (triangle), and apoptotic body formation
(arrow). (c) Necroptotic cell showing swollen nucleus (arrow), increased cell volume, disrupted cell membrane
integrity, and swollen organelles (triangle). (d) Necrotic cell showing signs of a ruptured nucleus and plasma
membrane, a loss of intracellular contents (arrow), and ruptured organelles (triangle). scale bar is 2 μ m.

41.94 ±  5.69% to 21.20 ±  2.90%; but had little effect on the apoptotic rate. This result suggested that around half
of dead cells were attributed to necroptosis after the foam cells were deprived of serum. TEM analysis of around
200 cells also showed a similar result. As shown in Fig. 4b, Nec-1 treatment significantly decreased necroptotic
cells. Furthermore, both immunofluorescence and immunoprecipitation analysis showed that Nec-1 attenuated
the formation of the RIPK1-RIPK3 complex (Fig. 4c,d). In addition, Nec-1 treatment dramatically reduced the
levels of MLKL oligomerization in foam cells after serum starvation (Fig. 4e).
To further dissect the functional roles of RIPK1, RIPK3 and MLKL in serum deprivation-induced necrop-
tosis, we utilised siRNAs to knockdown RIPK1, RIPK3 and MLKL, respectively. The knockdown efficiency of
different siRNAs was checked with western blot analysis (Supplementary Figure f–k). According to the results of
western blot, RIPK1-sh#1, RIPK3-sh#1 and MLKL-sh#2 were chosen for further experiments. As illustrated in
Fig. 5a,e,b,f, knockdown of RIPK1 or RIPK3 resulted in the significant decrease of LDH release and MLKL oli-
gomerization in serum starved foam cells. Furthermore, the formation of RIPK1-RIPK3 necrosome was blocked
by knocking down either RIPK1 or RIPK3, indicated by both immunoprecipitation and immunofluorescence
assays (Fig. 5c,g,d,h). These results suggest that RIPK1 and RIPK3 are required for necroptosis occurred in foam
cells undergoing serum deprivation. As expected, MLKL downregulation by specific siRNA also significantly
abolished LDH release and MLKL oligomerization induced by serum starvation (Fig. 5I,j); whereas did not affect
the formation RIPK1-RIPK3 complex (Fig. 5k,l). These data confirmed that MLKL functions as a downstream of

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Figure 3.  Activation of necroptosis and formation of the RIPK1-RIPK3 complex are increased after
serum-free starvation in foam cells. (a) The reduction of intracellular ATP in THP-1 foam cells after serum
deprivation. (n =  5; *P <  0.05 and ***P <  0.001 compared to the 0-h group). (b) The increase of LDH release
from THP-1 foam cells after serum deprivation. (n =  6; *P <  0.05 and ***P <  0.001 compared to the 0-h group).
(c) Western blotting analysis shows the elevation of RIPK1 and RIPK3 levels in foam cells undergoing serum
starvation for 24 or 48 hours. In right panel, protein expression was quantified relative to GAPDH. (n =  6;
*P <  0.05, **P <  0.01 and ***P <  0.001 compared to 0-h group, #P <  0.05 compared to 24-h group) (d) Flow
cytometry analysis shows the percentage of necrotic and necroptotic cells (PI-positive), after serum starvation
for 24 or 48 hours. The quantitative analysis is shown in bar graphs. (n =  6; **P <  0.01 and ***P <  0.001
compared to the 0-h group; ##P <  0.01 compared to the 24-h group). (e) The increase of RIPK1-RIPK3 complex
in foam cells after serum starvation, indicated by immunoprecipitation. (f) The lysates were incubated with
or without EGS (ethylene glycol bis(succinimidyl succinate)) and subjected to SDS-PAGE separation and
immunoblotting. MLKL oligomerization was detected using a MLKL phosphospecific antibody. GAPDH is
shown as a loading control. The data represent means ±  S.E.M.

RIPK1 and RIPK3. Summarily, it is convincing that serum starvation induces RIPK1/RIPK3-dependent necrop-
tosis in THP-1-derived foam cells.

ALA-mediated SDT inhibits RIPK1/RIPK3-dependent necroptosis.  The above observations indi-

cate the successful establishment of an in vitro necroptotic model, in which necroptosis is induced in foam cells
by serum deprivation. Using this model, we next tested whether ALA-SDT affects necroptosis. Twelve hours
after serum starvation, foam cells were treated with ALA-SDT. TEM analysis revealed that ALA-SDT signifi-
cantly suppressed necroptosis induced by serum starvation; in contrast, dramatically enhanced apoptotic cells
(Fig. 6a,b). This result was further supported by flow cytometry analysis. The percentage of cell death (PI-positive
cells) decreased from 50.08 ±  4.22% to 31.73 ±  9.24% after ALA-SDT treatment. Meanwhile, the apoptotic rate
(PI-negative and Annexin V-positive) increased from 1.02 ±  0.77% to 5.75 ±  1.31% (Fig. 6c). Although ALA-SDT
treatment did not alter the protein levels of RIPK1 and RIPK3 individually, it disrupted necrosome formation.
ALA-SDT remarkably attenuated a number of cells with RIPK1-RIPK3 colocalisation (Fig. 6d), and levels of
the RIPK1-RIPK3 complex (Fig. 6e) and MLKL oligomerization in foam cells (Fig. 6f). These results suggest
that ALA-SDT switchs the cell death mechanism in foam cells from necroptosis to apoptosis by disrupting the
RIPK1-RIPK3 complex.

ALA-mediated SDT inhibits RIPK1-RIPK3 necrosome through caspase-8 activation.  To further

explore the mechanism underlying the anti-necroptotic effect of ALA-SDT, we checked the status of caspase-8,
whose activation can block the initiation of necroptosis by inhibiting the formation of RIPK1-RIPK3 com-
plex and MLKL oligomerization. We found that ALA-SDT increased both cleaved caspase-8 levels and activity
(Supplementary Figure c,d, Fig. 7a,b). To investigate whether the enhanced caspase-8 activity accounted for the
anti-necroptotic effect of ALA-SDT, we pre-treated foam cells with a caspase-8-specific inhibitor, z-IETD-fmk
(ZIETD), and then subjected the cells to ALA-SDT. As expected, pre-incubation with ZIETD inhibited the
elevation of cleaved caspase-8 levels and caspase-8 activity in ALA-SDT treated foam cells (Fig. 7a,b). Further
experiments showed that pre-treatment with ZIETD abolished the anti-necroptotic effect of ALA-SDT through
stabilizing the RIPK1-RIPK3 complex (Fig. 7c,d) and reducing MLKL oligomerization (Fig. 7e). Together, our

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Figure 4.  Effects of necrostatin-1 on foam cell necroptosis induced by serum starvation. (a) Flow
cytometry analysis (left penal) shows pre-treament with Nec-1 block cell death induced by serum deprivation.
The right panel shows the quantification data of flow cytometry analysis (n =  6; ***P <  0.001 compared to
the control group, NS =  no significant difference). (b) TEM photomicrograph of foam cells with or without
Nec-1 treatment (upper panel). In total 200 cells counted for each sample, the numbers of necroptotic cells
was recorded and is shown in the lower panel. (c) Immunoprecipitation assay indicates that Nec-1 treatment
inhibits the formation of RIPK1-RIPK3 complex in foam cells after serum starvation. (d) Confocal microscopy
images showing the distribution and colocalisation of RIPK1 and RIPK3 in serum-starved foam cells (scale
bars =  8 μ m). (e) The lysates were incubated with or without EGS (ethylene glycol bis(succinimidyl succinate)),
subjected to SDS-PAGE separation, and analysed by immunoblotting with MLKL phosphospecific antibody.
The data represent means ±  S.E.M.

data suggest that ALA-SDT leads to the activation of caspase-8, inhibits the formation of the RIPK1-RIPK3
necrosome, decreases MLKL oligomerization, and consequently protects foam cells from necroptosis.

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Figure 5.  Effects of RIPK1, RIPK3 and MLKL on foam cell necroptosis induced by serum starvation.
(a,e) and (i) THP-1-derived foam cells were transfected with siRNA targeting MLKL, RIPK1 and RIPK3,
as indicated. The foam cell death rate was measured using an LDH assay, separately. (n =  6; *P <  0.05 and
***P <  0.001 compared to the siRNA-negative group). (b,f) and (j) MLKL phosphorylation and oligomerization
was detected using an MLKL phosphospecific antibody. GAPDH was used as a loading control (bottom
panel). (c,g) and (k) The lysates from cells transfected with or without siRNA were immunoprecipitated with
an antibody against RIPK1, followed by western blotting analysis with an antibody against RIPK3. The input
whole-cell lysates were probed with RIPK1 and RIPK3 antibodies. (d,h) and (l) Confocal microscopy images
showing the distribution and colocalisation of RIPK1 and RIPK3 in serum-starved foam cells transfected or not
with siRNA (scale bars =  8 μ m). The data represent means ±  S.E.M.

Caspase-3 is an upstream regulator of caspase-8 activation in ALA-SDT-treated cells.  We next

asked how caspase-8 is activated after ALA-SDT treatment. To this end, we monitored the alteration of caspase-3
and caspase-8 after ALA-SDT treatment. The levels of the active form of both caspase-3 and caspase-8 were
significantly elevated at 2, 4 and 6 hours after treatment (Fig. 8a,b). However, the dynamics of their enzymatic
activity were quite different. Caspase-3 activation peaked 4 hours after ALA-SDT treatment, 2 hours earlier than
the maximal caspase-8 activation. This result suggested that caspase-3 may be an upstream enzyme responsible
for caspase-8 activation. Indeed, pre-treatment with a caspase-3 specific inhibitor, z-DEVD-fmk (ZDEVD), abol-
ished the caspase-8 activation in ALA-SDT-treated cells (Fig. 8c,d). Since ALA-SDT prevented foam cells from
necroptosis by activating caspase-8, we explored whether ZDEVD could reverse the anti-necroptotic effect of
ALA-SDT. As expected, pre-incubation with ZDEVD markedly inhibited the anti-cell death effect of ALA-SDT
(Fig. 8e). Meanwhile, the enhanced apoptosis following ALA-SDT was also suppressed by this inhibitor (Fig. 8e).
Immunoprecipitation assay showed that pre-incubation with ZDEVD stabilised the RIPK1-RIPK3 complex
(Fig. 8f) and MLKL oligomerization (Fig. 8g). In summary, ALA-SDT exerted its anti-necroptotic effect through
activating the caspase-3 and caspase-8 pathways.

Discussion
The present studies demonstrate that necroptosis occurs in human atherosclerotic lesions and may contribute
to the progression of atherosclerosis. The necroptotic markers, RIPK1, RIPK3 and the RIPK1-RIPK3 com-
plex, are significantly increased in human atherosclerotic tissues with necrotic core. More important, we found
that ALA-SDT inhibits necroptosis, while promotes apoptosis by activating caspase-3 and caspase-8 (Fig. 8h).
This necroptosis to apoptosis switch suggests that ALA-SDT might be an efficient regimen for the treatment of
atherosclerosis.
Necroptosis is a newly identified form of programmed cell death and displays unique morphological features
that are distinct from apoptosis and necrosis6. Necroptosis is initiated by formation of the necrosome, of which
the major component is the RIPK1-RIPK3 complex12,13. Activated RIPK3 then phosphorylates the pseudo-kinase
MLKL, inducing its oligomerization and insertion into the plasma membrane and leading to necroptosis4,32.
Necroptosis not only facilitates the formation of the necrotic core in atherosclerotic lesions but also drives the
infiltration of inflammatory cells12,33. High rates of necroptosis in advanced atherosclerotic plaques contribute to
the destabilisation of atherosclerosis and increase the risk of plaque rupture and thrombosis8. Nonetheless, the

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Figure 6.  Effects of ALA-SDT on RIPK1/RIPK3-dependent necroptosis in foam cells. (a) ALA-SDT inhibits
necroptosis in foam cells. TEM photomicrographs show the representative morphological characteristics of
untreated foam cells and ALA-SDT treated foam cells (scale bars =  10 μ m). The white arrow shows apoptotic
cells, and necroptotic foam cells are indicated with a black arrow. (b) Among the 200 cells counted, the numbers
of apoptotic and necroptotic cells were recorded separately for each sample. (c) Flow cytometry analysis (left
penal) shows that ALA-SDT inhibits necroptosis, while enhaces apoptosis in foam cells. Right panel: the mean
percentages of PI-positive cells and Annexin V-positive/PI-negative cells were analysed by flow cytometry.
(n =  6; **P <  0.01 and ***P <  0.001 compared to the control group). (d) Confocal microscopy images shows that
ALA-SDT inhibits the colocalisation of RIPK1 and RIPK3 in serum-starved foam cells. (scale bars =  8 μ m).
(e) ALA-SDT blocks the formation of RIPK1-RIPK3 complex in serum-starved foam cells. (f) ALA-SDT blocks
the levels of pMLKL oligomerization in serum-starved foam cells. The data represent means ±  S.E.M.

involvement of necroptosis in human atherosclerosis has never been experimentally evaluated or verified. In this
study, to our knowledge, it is the first time to confirm necroptosis in clinical atherosclerotic lesions. First, some
foam cells in human atherosclerotic plaques displayed necroptotic morphological features, including enhanced
cytoplasmic translucency, swollen organelle, increased cell volume, disrupted cell membrane and intact nucleus.
Second, the expression of RIPK1 and RIPK3 was higher in human atherosclerotic samples with necrotic cores
compared with tissues from the same vessel without necrotic cores. Third, levels of the RIPK1-RIPK3 necrosome
were increased in human atherosclerotic lesions harbouring necrotic cores. In summary, the above evidence

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Figure 7.  The effects of ZIETD on caspase-8 activation and RIPK1/RIPK3-dependent necroptosis in ALA-
SDT-treated foam cells. (a) Z-IETD-fmk (ZIETD, a caspase-8 specific inhibitor, 20 μ M), inhibits the increased
protein levels of cleaved caspase-8 in ALA-SDT treated foam cells (upper panel). Protein expression was
quantified in the lower panel relative to GAPDH. (n =  4; ***P <  0.001 compared to the control group,
##
P <  0.01 compared to the SDT group). (b) ZIETD inhibits the increased protein levels of cleaved caspase-8
in ALA-SDT treated foam cells (n =  5; ***P <  0.001 compared to the control group, ###P <  0.001 compared
to the SDT group) (c) ZIETD stabilizes the RIPK1-RIPK3 complex in ALA-SDT treated foam cells.
(d) ZIETD reverses the anti-cell death effect of ALA-SDT, analysed with flow cytometry (left panel). Right
panel shows the quantification data of flow cytometry analysis. (n =  6; **P <  0.01 and ***P <  0.001 compared to
the control group, ###P <  0.001 compared to the SDT group) (e) ZIETD blocks the decreased levels of pMLKL
oligomerization in ALA-SDT treated foam cells. The data represent means ±  S.E.M.

indicates that necroptosis occurs in clinical atherosclerosis, suggesting that it may play an important role in
human atherosclerotic progression.
Diverse stimuli, such as TNF-α , ox-LDL and lipopolysaccharides, in combination with caspase inhibitors,
are able to trigger necroptosis in different macrophage cell lines, for instance, J774A.1, RAW264.7 and mouse
peritoneal macrophages9,32,34. However, these stimulations cannot efficiently induce THP-1 macrophage necrop-
tosis (data not shown). This result is in agreement with Mocarski’s observation35. Low energy/ATP in the centre
of atherosclerotic plaques due to insufficient nutrients results into foam cell death and the formation of necrotic
core36. According to this knowledge, we treated THP-1-derived foam cells with serum-free medium to mimic
the microenvironment in atherosclerotic plaques. Serum deprivation efficiently induced necroptosis in THP-
1-derived foam cells, confirmed by morphological observation and molecular characterisation. Necroptosis
accounts for approximately half of cell-death events induced by serum starvation; the other half could be caused
by apoptosis or unregulated necrosis mediated by unknown mechanisms. More important, Nec-1, a RIPK1 inhib-
itor, as well as siRNA knockdown of RIPK1, RIPK3 and MLKL, significantly blocked cell death, further confirm-
ing that necroptosis is occurred in THP-1-derived foam cells under the condition of serum deprivation. Since
energy is essential for cell survival, to what extent energy deficiency drives foam cells to go to necroptosis, necro-
sis or apoptosis remains to be determined in the future. The induction of necroptosis by modulating ATP levels
should be further optimized and would be a good model for the basic and translational studies of necroptosis.
The RIPK1-RIPK3 necrosome is a critical initiator of necroptosis and a main target for blocking necropto-
sis32,37. Activated caspase-8 prevents necroptosis by blocking formation of the RIPK1-RIPK3 complex and, sub-
sequently, MLKL oligomerization38. In the present study, caspase-8 activation, disruption of the RIPK1-RIPK3
complex, and reduced MLKL oligomerization were observed in ALA-SDT-treated cells. In addition, a caspase-
8-specific inhibitor (ZIETD) reversed the anti-necroptotic effect of ALA-SDT, suggesting that the activa-
tion of caspase-8 plays a critical role in the anti-necroptotic effects of ALA-SDT. More important, ALA-SDT
enhanced apoptosis while inhibiting necroptosis. In contrast to necroptosis and necrosis, apoptotic cells release
anti-inflammatory factors, exhibit reduced inflammation reactions and result in little damage to surrounding
tissues. Preventing necroptosis by initiating apoptosis may explain, at least in part, the benefit of ALA-SDT in
atherosclerosis treatment.
Apoptosis and necroptosis share several common molecular pathways, implying that these two types of cell
death may be two sides of the same coin and could be switched from one to the other under certain conditions14.

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Figure 8.  The effects of ZDEVD on caspase-8 activation and the anti-necroptotic effect of ALA-SDT in
foam cells. (a) The changes of protein level of cleaved caspase-3/8 in foam cells at the different time point after
ALA-SDT. (b) The dynamics of activated caspase-3/8 activity after ALA-SDT. (n =  4; *P <  0.05 and ***P <  0.001
compared to the control group). (c) Pretreatment with a caspase-3-specific inhibitor, z-DEVD-fmk (ZDEVD,
20 μ M) inhibits the increased protein levels of cleaved caspase-8 in ALA-SDT treated foam cells. (d) ZDEVD
blocks the increased activity of caspase-8 in ALA-SDT treated foam cells. (n =  5; ***P <  0.001 compared to the
control group, ###P <  0.001 compared to the SDT group). (e) ZDEVD reverses the anti-cell death effect of ALA-
SDT, analyzed with flow cytometry (left panel). The right panel shows the quantification data of flow cytometry
analysis. (n =  3; *P <  0.05 and **P <  0.01 compared to the control group, #P <  0.05 compared to the SDT
group). (f) ZDEVD stabilizes the RIPK1-RIPK3 complex in ALA-SDT treated foam cells. (g) ZDEVD blocks
the decreased pMLKL oligomerization levels in ALA-SDT treated foam cells. (h) A proposed model of the ALA-
SDT induced anti-necroptotic effect. The data represent means ±  S.E.M.

Necroptosis occurs when apoptotic pathway is impaired. For instance, caspase-3 specific and pan-caspase inhib-
itors induce cell necroptosis39,40. The relationship between apoptosis and necroptosis is still fragmentary and
elusive. It has been proposed that apoptosis has an antagonistic effect on necroptosis. This hypothesis is supported
by a genetic study, in which RIPK3 deficiency rescues embryonic lethality induced by caspase-8 or FADD knock-
out41,42. Our studies also corroborate this hypothesis. ALA-SDT activated the caspase-3 and caspase-8 pathways
in foam cells, initiated apoptosis, and thereby inhibited necroptosis. Additionally, caspase-3 and caspase-8 inhib-
itors reversed the anti-necroptotic effect of ALA-SDT, suggesting that caspase-3 and caspase-8 may mediate the
switch between apoptosis and necroptosis.
Verification of necroptosis in human atherosclerotic plaques suggests its potential role in the pathogenesis and
development of atherosclerosis. The inhibition of necroptosis mitigates pathological changes in multiple disease
models34,43. For instance, the RIPK1 inhibitor, Nec-1 or RIPK3-deficiency tremendously benefits inflammatory
diseases in vivo, such as myocardial infarction and atherosclerosis34,43. The RIPK3 inhibitors GSK’843 and GSK’872
are effective in alleviating liver injury44. ALA-SDT, in our study, is another methodology to inhibit necroptosis
while to enhance apoptosis, implying its translational potential for the treatment of necroptosis-related diseases,
including atherosclerosis. Next, it would be necessary to confirm the anti-necroptotic effect of ALA-SDT in ath-
erosclerotic animal model.

Methods
Reagents.  5-Aminolevulinic acid (ALA) and phorbol 12-myristate 13-acetate (PMA) were purchased from
Sigma-Aldrich (St Louis, MO, USA). Necrostatin-1 (Nec-1), z-DEVD-fmk (ZDEVD) and z-IETD-fmk (ZIETD)
were from Santa Cruz Biotechnology (CA). Cu-oxidised LDL (ox-LDL) and red fluorescent marked DiI-ox-
LDL were both purchased from Peking Union-Biology Co. Ltd (Beijing, China). Hoechst-33342, DAPI, the ATP
assay kit, the lactate dehydrogenase (LDH) release assay, and the caspase-3 and caspase-8 activity assay kit were
obtained from Beyotime Institute of Biotechnology (Jiangsu, China). The ApoAlert Annexin V-FITC kit was
purchased from BD Biosciences (Franklin Lakes, NJ, USA). Other drugs and chemicals used in this study were

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obtained from Sigma-Aldrich. EGS (ethylene glycol bis(succinimidyl succinate)) was obtained from Thermo
Scientific.

Cell cultures.  THP-1 monocytes are a human monocyte line (American Type Culture Collection,
Manassas, VA, USA). The cells were cultured in RPMI 1640 medium (HyClone, Logan, UT, USA) containing
10% foetal bovine serum (FBS) (HyClone, Logan, UT, USA), 20 μ g/mL penicillin and 20 μ g/mL streptomycin
(Sigma-Aldrich, St Louis, MO, USA), and maintained at 37 °C in a humidified atmosphere containing 5% CO2.
To differentiate THP-1 monocytes into macrophages, they were seeded onto cover slips in 35-mm Petri dishes
at a density of 0.5 ×  106 cells/mL and treated with 100 ng/mL PMA for 72 hours. The macrophages were incu-
bated with 100 μ g/mL ox-LDL for 48 hours in serum-free RPMI 1640 to form foam cells. The inhibitors ZIETD,
ZDEVD and Nec-1 were used at appropriate concentrations (ZDEVD, 20 μ M; ZIETD, 20 μ M; Nec-1, 20 μ M).

RNA interference.  Two different siRNAs against RIPK1, RIPK3 and MLKL,respectively, and a negative con-
trol siRNA were designed by GenePharma Co.,Ltd. (Shanghai, China), according to a previously described pro-
tocol. ThesiRNA sequences were as follows: RIPK1-sh#145: 5′ -GCACAAATACGAACTTCAA-3′ , RIPK1-sh#246:
5′-CCACUAGUCUGACGGAUAA-3′, RIPK3-sh#112: 5′-UAACUUGACGCACGACAUCAGGCUGUU-3′,
RIPK3-sh#212: 5′-GCAGUUGUAUAUGUUAACGAGCGGUCG-3′. MLKL-sh#147: 5′-GCGTATATTTGGGA
TTTGCAT-3′, and MLKL-sh#246: 5′ -CAAACUUCCUGGUAACUCA-3′. The siRNAknockdowns were per-
formed using X-tremeGENE siRNA Transfection Reagent (Roche) according to the manufacturer’s protocol.
Briefly, 1 ×  106 cells wereseeded in 35-mm Petri dishes to obtain approximately 80–90% confluence. Two hundred
microlitres of a mixture containing siRNA (2 μ g for each) and transfection reagents (12 μ L for each) was added
to the cells for 6 h at 37 °C, and then replaced with fresh culture medium. The levels of RIPK1, RIPK3 and MLKL
were determined using western blotting analysis.

Sonodynamic therapy treatment.  The SDT equipment used in this study, including an ultrasonic gener-
ator, transducer and power amplifier, was assembled by the Harbin Institute of Technology (Harbin, China). The
homemade ultrasonic transducer (diameter: 3.5 cm; resonance frequency: 1.0 MHz; duty factor: 10%; repetition
frequency: 100 Hz) was placed in a water bath 30 cm below the cells. The ultrasonic intensity used was 0.4 W/
cm2, as measured by a hydrophone (Onda Corp, Sunnyvale, CA, USA). THP-1-derived foam cells (0.5 ×  106 cells/
mL) were seeded in 35-mm Petri dishes, consistent with the size of the therapeutic instrument probe, before SDT
treatment. The cells were incubated with 1 mM ALA (Sigma-Aldrich, St Louis, MO, USA) in the dark for 6 hours
prior to treatment with the SDT equipment.

Human sample collection.  We performed an autopsy of 12 individuals aged 36 to 58 years from the
Department of Forensic Medicine of Harbin Medical University. In addition, 6 human tissue samples were col-
lected from atherosclerotic patients of 40–60 years of age by carotid endarterectomy (CEA) at the First Affiliated
Hospital of Harbin Medical University. Informed written consent was obtained from all patients. The study pro-
tocol conformed to the Ethical Guidelines of the 1975 Declaration of Helsinki, and was approved by the First
Affiliated Hospital of Harbin Medical University (People’s Republic of China) Human Ethics Committee.

HE staining.  All tissues were sliced at 5 μ m, mounted on glass slides coated with poly L-lysine, and subjected
to haematoxylin and eosin staining according to routine histopathological methods. Histopathological changes
were observed under a light microscope.

Transmission electron microscopy.  The cells were rinsed three times with PBS and then fixed with 2.5%
glutaraldehyde in 0.1 M phosphate buffer saline (PBS, pH 7.3) for 1 hour at room temperature. Next, the prepared
samples were determined by transmission electron microscopy (TEM) (JEM-1220, Japan) according to conven-
tional methods. For quantitative analysis of necrotic foam cells. Total 200 cells were counted, and the number of
necrotic foam cells was recorded.

Western blotting analysis.  Proteins were extracted with RIPA buffer from foam cells 4 hours after SDT
treatment. The proteins were separated on 7.5%, 10%, 12% or 15% SDS-PAGE gels and transferred to a nitro-
cellulose membrane. The membranes were incubated with primary antibodies, including those against cleaved
caspase-3, cleaved caspase-8, RIPK1, RIPK3, MLKL, pMLKL and GAPDH (Cell Signaling Technology, Inc.,
USA). Antibody labelling was detected using a ChemiDoc MP imaging system (Universal Hood III, Bio-Rad
Laboratories, Inc., USA) with BeyoECL Plus (Beyotime, Jiangsu, China), according to the manufacturer’s instruc-
tions. The labelling was analysed with Image Lab software (version 4.1, Bio-Rad).

MTT assay.  Cell viability was determined with MTT assay (Sigma-Aldrich, St Louis, MO, USA). Cells
(1 ×  105 cells/mL) were seeded in 96-well culture plates. Ox-LDL was added to the treatment group for 4 hours
at concentration of 50–200 μ g/mL. The control group was incubated with RPMI 1640 medium without ox-LDL.
Cytotoxicity was determined by adding 10 μ L MTT solution (5 mg/ml in PBS) to each well, and the mixture was
incubated for 4 hours at 37 °C in a CO2 incubator. The formed formazan crystals were dissolved in a solution of
100 μ l 10% sodium dodecyl sulphate (SDS) and 0.01 M HCl. The absorbance was measured at 570 nm using a
Model 680 Microplate Reader. The macrophage viability of the treated samples was then analysed in comparison
to the control.

ATP assay.  For the ATP assay, a luminescence-based commercial kit was obtained from Beyotime Institute
of Biotechnology (Jiangsu, China). Intracellular ATP levels were measured at suitable time points in serum-free

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RPMI 1640 according to the manufacturer. Luminescence was measured using a Glomax 20/20 luminometer
(Promega). The ATP level was determined as a multiple of that observed in the untreated control group.

LDH release assay.  Foam cell necrosis was assessed using LDH assay kit. Cells were seeded in 35-mm Petri
dishes and measured at suitable time points in serum-free RPMI 1640 according to the manufacturer. The absorb-
ance was recorded at 490 nm using a micro-plate reader. The basal level of LDH release was determined with
50 μ L supernatant from cells cultured with medium. The calculation of LDH release was as follows: (LDH release
from treated group minus basal release) divided by (maximal LDH release minus basal release).

Flow cytometry analysis.  We stained cells using Annexin V-FITC and PI Detection Kit (BD Pharmingen,
USA) prior to the analysis with flow cytometry. Briefly, cells were seeded in 35-mm Petri dishes at a density of
1 ×  106 cells per well. The cells were cultured in 2 mL phenol red-free medium containing 10% FBS, and necrop-
tosis was then induced as described above. Then, the foam cells were measured at suitable time points according
to the manufacturer. The results were analysed using BD FACSDiva Software v7.0 (Becton-Dickinson, USA). For
proper statistical analysis, more than 10000 cells per group were counted, and each assessment was repeated three
times.

Immunofluorescence.  The cells were fixed with 4% paraformaldehyde for 10 min at room temperature and
then permeabilized with 0.2% Triton X-100 for 10 min at room temperature. The cells were blocked with 3% BSA
(Sigma-Aldrich, St Louis, MO, USA) at room temperature for 30 minutes. Next, the cells were incubated with
anti-RIPK1 (Cell Signaling Technology, Inc., USA) and anti-RIPK3 (Santa Cruz Biotechnology, CA) antibod-
ies for 2 hours at 37 °C. Then, the cells were rinsed with PBS and incubated with Dylight 594 and DyLight 488
AffiniPure secondary antibodies (EarthOx, San Francisco, CA, USA) for 1 hour. Next, the cells were counter-
stained with DAPI for 15 minutes at 37 °C in the dark. After being washed with PBS, the cells were visualised by
confocal microscopy (Carl Zeiss LSM700).

Caspase activity analysis.  The cells were collected by trypsinisation, rinsed twice with PBS, re-suspended
in lysis buffer supplied by the caspase-3 and/or caspase-8 activity assay kit and incubated on ice for 15 minutes.
After centrifugation, the supernatant was measured for protein concentration using the Bradford method. Total
protein (0.1 mg) was used for the caspase activity assay, with Ac-DEVD-pNA or Ac-IETD-pNA as the substrates
of caspase-3 and caspase-8, respectively. Absorbance at 405 nm resulting from the production of pNA was contin-
uously recorded using a Model 680 Microplate Reader after incubation for 2 hours at 37 °C.

Statistical analysis.  All the experiments were repeated at least 3 times independently. The data repre-
sent means ±  S.E.M. SAS 9.1 (SAS Institute Inc., Cary, NC) were used for statistical analysis. The differences
between groups were determined by one-way analysis of variance (ANOVA) followed by the Bonferroni post
hoc test, when appropriate. For analysis of statistical difference between two groups, a Student’s two-tailed t-test
was applied. Differences among groups were determined by variance (ANOVA), and a Bonferroni post hoc test.
P-values of 0.05 or less were considered statistically significant.

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Acknowledgements
The authors express their sincere gratitude to Professor Min Li for his help in text revision. This study was
supported by the National Natural Science Foundation of China (NSFC) (81371709, 81171483, 81400250,
81341051, 81400339 and 81301343), the State Key Program of NSFC (81530052) and the Foundation for
Innovative Research Groups of NSFC (81121003).

Author Contributions
F.T., M.Y., W.W., W.W.G., Z.X.D., S.Y.G. performed the experiments. F.T. analysed the data. F.T. designed the
research. F.T., J.T.Y. and M.Y. wrote the manuscript. Z.T., J.T.Y. and X.S. helped the text revision. Y.T. supervised
the work. T.L.G., B.C.L., P.S., J.L.C., Q.P.G., B.L. and H.Y.W. assembled human specimens. Z.G.Z. and W.W.C.
provided sonodynamic equipment.

Additional Information
Supplementary information accompanies this paper at http://www.nature.com/srep
Competing financial interests: The authors declare no competing financial interests.
How to cite this article: Tian, F. et al. 5-Aminolevulinic Acid-Mediated Sonodynamic Therapy Inhibits RIPK1/
RIPK3-Dependent Necroptosis in THP-1-Derived Foam Cells. Sci. Rep. 6, 21992; doi: 10.1038/srep21992
(2016).
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Scientific Reports | 6:21992 | DOI: 10.1038/srep21992 13