Abstract Macrophage apoptosis: a double edge sword

1
Abstract Macrophage apoptosis a double edge
sword?
Apoptosis, a form of genetically programmed cell
death, plays an essential role in different
physiologic and pathologic processes including
atherosclerosis, in which it affects all cell
types including endothelial cells, vascular
smooth muscle cells (VSMCs), and macrophages.
Over the course of the plaque progression, pro-
and anti-apoptotic signals abound. In other organ
systems, apoptosis limits the number of a
particular cell type that accumulates in the
lesion. The issue in atherosclerosis, however, is
clearly more complex. The loss of VSMCs can be
detrimental for plaque stability since most of
the fibrous cap collagen required for the tensile
strength of the cap is produced by VSMCs.
Apoptosis of macrophages, on the other hand,
could be beneficial for plaque stability if
apoptotic bodies were removed. Several
investigators have reported, however, that
apoptotic bodies in the advanced atherosclerotic
plaque are often not scavenged, can activate the
coagulation cascade, potentially leading to
plaque rupture and luminal thrombosis. Many of
the apoptotic bodies are of macrophage origin.
Moreover, interventions like statin therapy have
shown that beneficial effects on the plaque,
namely shrinkage of the lipid core, decrease of
the inflammatory burden and thickening of the
fibrous cap, are accompanied by a decrease in
apoptotic activity. It is therefore not
surprising that most investigators believe that
apoptosis is detrimental to plaque stability.
2
Abstract (cont)Macrophage apoptosis a double
edge sword?
Our group has long been interested in the thermal
heterogeneity of the atherosclerotic plaque and
on the effect of plaque heating on the processes
of inflammation and apoptosis. In a recent study
by Dr. Birendra Lal in Dr. Yong-Jian Gengs
laboratory at the University of Texas Houston,
eleven freshly living human carotid
endarterectomy specimens were heated in DMEM
medium at 42?C for 15 minutes followed by
incubation at 37?C for 6 hours. In unheated
controls, 4 of the VSMCs and 8 of macrophages
were TUNEL positive. In the specimens with the
short term heating, 46 of the macrophages and
10 of the SMCs were TUNEL positive.
Immunostaining for tumor necrosis factor-?
(TNF-?) and interleukin-6 (IL-6) demonstrated
lower levels of both cytokines in the heated
group. Moreover, thermal stimulation also
inactivated NF-?B (a transcription factor
involved in cytokine expression, cell
proliferation, etc) in macrophages derived from
THP-1 cells by phorbol esters as demonstrated by
gel shift assays.
3
Abstract (cont)Macrophage apoptosis a double
edge sword?
In another set of experiments performed by Dr.
Mitra Rajabi in Dr. Yong-Jian Gengs laboratory
at the University of Texas Houston, mouse VSMCs
were divided in two groups, half heated at 42?C
for 15 minutes before returning to 37?C. Two
hours after heating, both heated and non-heated
dishes were divided in 3 groups a) TNF-?
10ng/ml, b) TNF-? 10ng/ml and IFN-? 10ng/ml, and
c) no cytokines. After 12, 36 and 48 hours, the
nitrite production, a marker of iNOS expression,
was statistically significant lower in the heated
as compared to the non-heated groups.
4
Abstract (cont)Macrophage apoptosis a double
edge sword?
We therefore believe that specific therapies like
local gentle heating have a potential therapeutic
effect by decreasing markers of inflammation
coupled to their pro-apoptotic effects on
macrophages. In addition, the operator in the
catheterization laboratory could add adjuvant
therapy like balloon dilation, stenting and
anticoagulation, thereby preventing the potential
complications of plaque rupture and thrombosis
from happening in vivo. In summary, although
large body of evidence considers apoptosis in the
plaque to be risky and detrimental, we believe
that under certain controlled conditions, gentle
heating could decrease the plaque vulnerability.
5
APOPTOSIS ATHEROSCLEROSIS

•        While apoptosis is a key negative
  regulator of the cell density in oncogenesis,
  organ development, and immune response, the role
  of apoptosis in atherosclerosis is more complex.
•         Variation in the rate of apoptosis of
  different cell types promotes differences in
  growth rates, structure and stability of the
  plaques.
•         Several cytokines known to be
  pro-apoptotic, such as tumor necrosis-a (TNF-a),
  interleukin-1ß, and interferon ? (IFN- ?) and
  products of genes involved in the cell cycle
  regulation (Fas/Fas ligand, caspase, p53 and
  c-Myc) have been found in vascular cells and
  atherosclerotic plaques.
•   Apoptotic rate is higher in advanced
  plaques

6
APOPTOSIS OF DIFFERENT CELL TYPES IN PLAQUE
TISSUE
ENDOTHELIAL CELLS

•        Lesion-prone regions show increased
  endothelial cell (EC) turnover ratio.
•        ECs undergo apoptosis when coming in
  contact with circulating or local factors like
  angiotensin II, oxidized LDL, reactive oxygen
  species (ROS) and inflammatory cytokines.
•         Apoptotic ECs assume pro-coagulant
  characteristics due to increased exposure to
  phosphatidylserine and loss of normal
  anticoagulant membrane properties.
•         Apoptotic ECs increase migration of
  monocytes and T-lymphocytes.

7
APOPTOSIS OF DIFFERENT CELL TYPES IN PLAQUE
TISSUE

VASCULAR SMOOTH MUSCLE CELLS

• Apoptosis of vascular smooth muscle cells
  (VSMC) reduces the rate of plaque growth. At the
  same time, since VSMCs are the source of
  interstitial collagen fibers type I, plaque
  stability might be affected.
•  Migration of macrophages to areas of VSMC
  apoptosis has been described.
•  Overall effects of VSMCs apoptosis are complex
  and difficult to predict but generally felt to be
  deleterious for plaque stability.

8
APOPTOSIS OF DIFFERENT CELL TYPES IN PLAQUE
TISSUE

MACROPHAGES  

•  

•   Macrophages may activate several matrix
  metalloproteinases which degrade interstitial
  collagen, thus weakening the fibrous cap.
•  Macrophages produce cytokines that may induce
  VSMCs apoptosis.
•  Loss of macrophages results in decreased
  scavenging products of cell degradation, leading
  to accumulation of necrotic debris and
  coagulation activation.
•  Therefore, apoptosis of macrophages, may have
  both pro- and anti-destabilizing effects.

9
APOPTOSIS OF DIFFERENT CELL TYPES IN PLAQUE
TISSUE

T-LYMPHOCYTES

•        Lymphocytes produce molecules with
  important regulatory functions on the plaque cell
  death (cytokines, perforin, Fas).
•        Apoptosis of lymphocytes is not well
  understood in the context of atherosclerosis.

10
Effect of heat on apoptosis of macrophages and
smooth muscle cells

At 37 C, the proportion of apoptotic SMC and
macrophage were 4 and 8 respectively. At 42
C, these proportions increased to 10 and 46
respectively.
11
Effect of heat on macrophage apoptosis

TUNEL and HAM-56 double staining. There is
significant increase in the number of TUNEL
positive macrophages after heating (8 to 46)
12
Effect of heat on SMC apoptosis

TUNEL and ? actin double staining. There is
insignificant increase in the number of TUNEL
positive SMCs after heating (4 to 10).
13
Effect of heat on macrophage ultrastructure

Human carotid atherectomy specimen. A Normal
macrophage in unheated plaque. B Two apoptotic
macrophages inheated plaque condensed chromatin
is same in both cells.
14
Effect of heat on macrophage ultrastructure
(cont)

C Enlarged view of B. D Foam cell at the end
stage of apoptotic process. Extra cellular debris
is also present.
15
Effect of heat on TNF -? immunoreactivity

TNF- ? immunoreactivity decreases markedly with
heating
16
Conclusion

• Gentle short-term thermal treatment induces
  apoptosis in human atherosclerotic lesions,
  reduces expression of pro-inflammatory cytokines
  TNF? and IL-6, and inactivates NF?B (as
  demonstrated by electrophoretic mobility shift
  assay, data not shown).
• These data suggest that thermal treatment may
  have potential for treating advanced
  atherosclerotic lesions by reducing inflammation
  and triggering apoptosis in macrophages.