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 to irradiation.
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(Drug information on 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 platelet aggregation.
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.