Therapeutic irradiation has the potential to
enhance the destruction of tumor blood vessels and should be
considered when designing clinical trials of antiangiogenic agents.
These agents include biological response modifiers, antibodies,
enzyme inhibitors, therapeutic genes, analogs of bacterial and fungal
products, and cytotoxic drugs. The importance of this approach is
that angiogenesis is an essential component of neoplasia.[1,2]
Blood vessel growth involves several processes, each of which
represents a therapeutic target to combat neoplasia. Endothelial
proliferation is required for the development of a vascular sprout
into the tumor. Endothelial growth can be regulated by a number of
growth factors, including vascular endothelial growth factor,
platelet-derived growth factor, fibroblast growth factor, and
angiopoietins. Endothelial cells migrate into the neoplastic tissue
in a manner that maintains the structure of blood vessels.
Endothelial cell motility is required for movement into the tissue.
The structural and functional integrity of the blood vessel must be
maintained to provide circulation into the tumor. Inhibition of any
of these processes is a strategy to treat most forms of cancer.
The classic antiangiogenic biological response modifier is tumor
necrosis factor-a (TNF). TNF is produced during inflammation and
functions at many levels to prevent angiogenesis, including
inhibition of endothelial cell proliferation and induction of
thrombosis and inflammation. Other more recently identified
biological response modifiers that function as antiangiogenic agents
include angiostatin, endostatin, and antagonists that bind to
angiopoietin receptors.[4,5] Cytokines that are antiangiogenic
include TNF, interferon, platelet factor 4, and interleukin-12.[6-8]
In addition to these naturally occurring proteins, a number of
macromolecules produced by bacteria and fungi have been identified as
antiangiogenic compounds, such as CM101 and TNP470.[9,10] Other
levels of activity of antiangiogenic compounds include antibodies to
integrins or growth factors and cytotoxic agents. Recently, it
has been shown that antiangiogenic agents can be combined with
cytotoxic drugs to enhance tumor control.
Ionizing radiation is also cytotoxic to the vascular
endothelium.[13,14] Growth factors and other biologic response
modifiers can influence radiation-induced cytotoxicity in the
endothelium. For example, elimination of growth factors enhances
endothelial cell killing by ionizing radiation, and administration of
growth factors minimizes endothelial cytotoxicity. In addition to
the direct cytotoxic effects of radiation on endothelial cells,
radiation-induced oxidative injury to the endothelium activates
homeostatic responses. In this regard, platelets are also
activated within irradiated tissues, resulting in platelet
aggregation.[17,18] Ionizing radiation also activates inflammation
through the induction of cell-adhesion molecules and
cytokines.[19-24] These proteins and proteoglycans activate
circulating leukocytes and thereby mediate the inflammatory response
As described below, each of these radiation-induced processes can be
enhanced within the tumor vasculature to achieve obliteration of
blood vessels. The advantage of ionizing radiation over other
cytotoxic and vasculitic agents is that it can be localized precisely
to tumors, thus sparing surrounding normal tissues. In this regard,
enhancement of vasculitic properties of ionizing radiation directed
to the tumor vascular endothelium may provide a therapeutic advantage
in the treatment of cancer.
Radiation-Induced Cell-Adhesion Molecule Expression in Irradiated Endothelium
Ionizing radiation induces oxidative injury in the endothelium. The
endothelium responds to maintain homeostasis by preserving the
barrier function in blood vessels. This is accomplished by activation
of inflammation and platelet aggregation, which are transient
responses and can resolve if endothelial repair is sufficient. The
mechanisms by which radiation activates these homeostatic responses
is, in part, through the induction of cell-adhesion molecules. This
requires the activation of the nuclear transcription factor (NFkB),
which regulates transcription of the intracellular adhesion molecule
(ICAM)-1 and E-selectin genes.[22,25]
ICAM-1, E-selectin, and P-selectin are induced by irradiation of the
endothelium and bind to receptors on circulating leukocytes to
initiate inflammation.[21,26] Prior to adhesion to cellular-adhesion
molecules (CAM), leukocytes are relatively quiescent but mediate the
inflammation in response to endothelial injury. For example,
E-selectin and P-selectin bind to counter-receptors on monocytes to
increase the procoagulative state through the release of cytokines
and tissue factor.[28,29] Radiation, therefore, initiates the
inflammatory cascade through the activation of the endothelium.
P-selectin (GNP 140, CD62P) contributes to the inflammatory response
following translocation from the vascular endothelial cytoplasm to
the luminal surface of irradiated blood vessels. P-selectin is
translocated to the blood-tissue interface of the endothelium and is
not released from storage reservoirs, but remains tethered to the
endothelial cell membrane. This proteoglycan is stored in
Weibel-Palade bodies within the cytoplasm of endothelial cells and in
a-granules of platelets.
P-selectin is rapidly translocated to the vascular lumen after tissue
injury to initiate the adhesion and activation of platelets and
leukocytes. We studied the histologic pattern of P-selectin
expression in irradiated tumor blood vessels, and observed that P-selectin
was localized within the endothelium of tumor vessels prior to
treatment. At 1 to 6 hours following irradiation, P-selectin was
localized to the lumen of blood vessels.
To determine whether radiation-induced vascular lumen localization of
P-selectin was tumor type-specific or species-specific, we studied
tumors in rats, C3H mice, C57BL6 mice, and nude mice. P-selectin
localization to the vascular lumen was present in all tumors and all
species studied. Irradiated intracranial gliomas showed P-selectin
localization to the vascular lumen within 1 hour, whereas blood
vessels in normal brain showed no P-selectin staining in the
endothelium and no localization to the irradiated vascular lumen.
Radiation-induced P-selectin localization to the vascular lumen
increased in a time-dependent manner until 24 hours after irradiation (Figure
1). Since P-selectin in platelets may account for this
time-dependent increase in staining, we employed immunohistochemistry
for platelet antigen GP-IIIa to differentiate between endothelial and
platelet localization of P-selectin.
We found that GP-IIIa staining was not present 1 hour after
irradiation, but increased at 6 hours and 24 hours. P-selectin
localization to the vascular lumen at 6 to 24 hours was, in part,
associated with platelet aggregation. These findings indicate that
radiation-induced P-selectin staining in the vascular lumen of
neoplasms is associated with platelet aggregation. Radiation-induced
localization of P-selectin to the vascular lumen is specific to the
microvasculature of malignant gliomas and is not present in the blood
vessels of the irradiated normal brain.
The presence of P-selectin in membranous organelles, such as
a-granules of platelets and Weibel-Palade bodies in endothelial
cells, enables it to be readily available for hemostasis. P-selectin
initiates the slowing of circulating platelets, implicating it in
hemostasis and thrombosis.[32-34] Platelets roll on stimulated
endothelium by interacting with endothelial P-selectin. Platelet
activation is inhibited by antiP-selectin antibodies.
P-selectindeficient mice have prolonged bleeding times compared
to those of wild-type mice. Moreover, the slowing of platelet
flow through blood vessels is markedly impaired in
P-selectindeficient mice. We observed that localization of
P-selectin to the vascular lumen was associated with platelet
aggregation and P-selectin staining on platelets. P-selectin
exocytosis, therefore, contributes to the proinflammatory and
prothrombotic response of the endothelium to radiation.
Radiation Induces a Procoagulative State in the Endothelium
Weibel-Palade bodies contain several proteins and proteoglycans that
initiate thrombosis and inflammation. These include P-selectin, von
Willebrand factor, interleukin-8, and CD63. To determine whether
radiation produces exocytosis of Weibel-Palade bodies, we irradiated
the vasculature within the mouse thorax and performed
immunohistochemistry for P-selectin. This experiment demonstrated
that rapid exocytosis of Weibel-Palade bodies occurs within 30
minutes of irradiation. We used human umbilical vein endothelial
cells to study the mechanisms of radiation-mediated Weibel-Palade
bodies exocytosis in vitro. We found that exocytosis was most
efficient at 2 to 5 Gy, whereas higher radiation doses cause
apoptosis in endothelial cells, which interferes with exocytosis.
The Shwartzman reaction shows the close interrelation between the
inflammatory and hemostatic systems. During this reaction, a
hemorrhagic vasculitis is provoked by local injection of
lipopolysaccharide, followed by injection of TNF-a
into the same site. The predominant feature of this vasculitis is
platelet and neutrophil sequestration along the vasculature
endothelium. Microthrombi composed of platelets, neutrophils, and
fibrin occlude the capillaries and venules. The thrombotic component
of this vasculitis is markedly attenuated in mice that are deficient
in the P-selectin gene. Depletion of neutrophils or platelets
attenuates this vasculitis during the Shwartzman reaction.
The mechanism of the increased procoagulative state is
transcriptional induction of tissue factor, which is a transmembrane
glycoprotein on monocytes. Tissue factor is a high-affinity receptor
for coagulation factors VII and VIIa. The resulting tissue
factor-VIIa complex rapidly catalyzes the conversion of factor X to
factor Xa and factor IX to factor IXa, leading to the formation of
thrombin. Monocyte-derived tissue factor can activate both the
intrinsic and extrinsic coagulation cascades.
Irradiation of blood vessels produces a procoagulative state. The
mechanisms are related to the release of von Willebrand factor[40,41]
and interaction with leukocytes. E-selectin and P-selectin bind
to leukocytes and thereby activate expression of a number of genes
including tissue factor and TNF.[28,29] Tissue factor expression
promotes thrombosis.[38,39] TNF has also been shown to increase
monocyte tissue factor generation.[38,39] Therefore, there are
several potential mechanisms by which radiation creates a
procoagulative state in the vasculature.
GP-IIIa is a platelet antigen that is not found in the vascular
endothelium. We utilized antiGP-IIIa antibodies to determine
whether the time-dependent increase in P-selectin staining is due to
platelet aggregation. Lewis lung carcinoma tumors in C57BL6 mice were
irradiated and stained with antiGP-IIIa antibody. Little
GP-IIIa staining was observed in blood vessels at 1 hour following
irradiation. However, GP-IIIa staining increased at 6 and 24 hours
following irradiation (Figure 2).
These findings indicate that the increased P-selectin staining
within the vascular lumen of irradiated tumors was partially due to
To verify that platelet aggregation was present in these irradiated
blood vessels, tissue sections were stained with antiGP-IIIa
antibodies that stained the platelets. We found no P-selectin or
GP-IIIa staining in the brain or kidney, but both P-selectin and
GP-IIIa staining were present in the irradiated lung, intestine, and
tumor vessels. The P-selectin knockout mouse was used to study the
correlation between platelet aggregation (ie, GP-IIIa accumulation)
and P-selectin staining in the vascular endothelium. The GP-IIIa
staining was not localized to the lumen of irradiated blood vessels
in the knockout mouse, but extravasated into the irradiated lung,
intestine, and tumors. Red blood cells also extravasated from
irradiated tissues. Therefore, P-selectin accumulated in irradiated
blood vessels correlated with maintenance of the barrier function of
the endothelium. Knockout of the P-selectin gene leads to
extravasation of platelets and red blood cells.
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