PERSPECTIVES OPINION
Possible molecular mechanisms involved in the toxicity of angiogenesis inhibition Henk. M. W. Verheul and Herbert M. Pinedo
Abstract | Contrary to initial expectations, angiogenesis inhibitors can cause toxicities in patients with cancer. The toxicity profiles of these inhibitors reflect the disturbance of growth factor signalling pathways that are important for maintaining homeostasis. Experiences with angiogenesis inhibitors in clinical trials indicate that short-term toxicities are mostly manageable. However, these agents will also be given in prolonged treatment strategies, so we need to anticipate possible longterm toxicities. In addition, understanding the molecular mechanisms involved in the toxicity of angiogenesis inhibition should allow more specific and more potent inhibitors to be developed. Inhibition of angiogenesis, the formation of new vessels from the pre-existing vasculature, was first proposed as a therapeutic strategy against malignancies by Folkman in 1971 (REF. 1). Specific inhibitors of angiogenesis have been identified and developed to block tumour growth and metastasis formation using preclinical in vitro and in vivo models2. In the past decade this strategy has found its way to the clinic. More than 30 years after Folkman’s hypothesis, the first clinical study showed a survival benefit of anti-angiogenic treatment in patients with cancer. In patients with advanced colorectal cancer, the combination of bevacizumab, a humanized monoclonal antibody against vascular endothelial growth factor (VEGF), plus chemotherapy significantly prolonged overall survival by approximately 4.5 months compared with chemotherapy alone3. Bevacizumab in combination with chemotherapy also prolongs the progression-free survival of patients with colorectal cancer, lung cancer and breast cancer4,5. Other anti-angiogenic receptor tyrosine kinase inhibitors (TKIs) targeting the VEGF pathway (primarily VEGF receptor 2 (VEGFR2)), such as sunitinib and sorafenib, have shown clinical efficacy in various cancer types6–9. Target pathways
of these agents also include platelet-derived growth factor receptor-α (PDGFRα) and/or PDGFRβ, fms-related tyrosine kinase 3 (FLT3), the stem-cell factor receptor KIT and the protein product of the RET protooncogene10,11,12. Sorafenib also inhibits the Raf serine/threonine kinase isoforms13. Administration of TKI angiogenesis inhibitors as single agents prolongs the progression-free survival of patients with renal cell cancer and gastrointestinal stromal tumours (GIST) resistant to imatinib, which is a KIT inhibitor8,7,9. The clinical benefit of sunitinib in patients with GIST might be due to the inhibition of KIT, but could also be mediated by the inhibition of VEGFR or PDGFR9. Angiogenesis inhibitors (TABLE 1) primarily target growth factor signalling pathways in proliferating endothelial cells or perivascular cells. Initially, these agents were expected to be active without causing major toxicities or resistance because of the genetic stability of endothelial cells. Under normal physiological circumstances, more than 99% of endothelial cells are quiescent14,15. In healthy adults, angiogenesis is only promoted during wound healing and the menstrual cycle16, and growth factor pathways are not activated17. It was proposed that tumour-stimulated
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endothelial cells have a unique proliferating and migrating phenotype compared with quiescent endothelial cells, and that targeting this phenotype would be so specific that no major side effects could occur, except for during wound healing and the menstrual cycle18. However, recent clinical experiences have changed these expectations19. It now seems that the importance of growth-factorinduced activation of signalling pathways in endothelial cells for maintaining homeostasis in the body was underestimated, and the importance of these pathways in normal physiological processes is drawing increased attention. For example, angiogenesis-related signalling pathways also have an important role in haematopoiesis, myelopoiesis and endothelial cell survival20–22. Toxicities that are being observed emphasize that the generation of new blood vessels is a very complicated multi-factorial biological process in which VEGF has a major role (FIG. 1). Therefore, the possible causes of toxicities induced by angiogenesis inhibitors are plural. As mentioned above, many of these inhibitors target multiple tyrosine kinases of several different pathways, including VEGFR, PDGFR and KIT, and one (ZD6474) also targets the epidermal growth factor receptor (EGFR)23,24. Therefore, toxicities may not only be due to the inhibition of one pathway, but they may also be due to the concomitant inhibition of several pathways. Many of these biological agents are used or will be used as part of combination treatment strategies; for example, in combination with chemotherapy4,25. A few studies have shown increased and alternative toxicity profiles for combination therapies of some angiogenesis inhibitors compared with single agents26,27. This Perspective will discuss the possible mechanisms that underlie toxicities induced by angiogenesis inhibitors, which may help us to better understand normal physiological processes and may lead to the development of more specific and potent angiogenesis inhibitors. Dosing in relation to toxicity The optimal biologically effective dose of angiogenesis inhibitors is difficult to determine. Ideally, this dose should be determined by measuring whether the targeted pathway is sufficiently inhibited,
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PERSPECTIVES Table 1 | Angiogenesis inhibitors in clinical development in solid tumours
Angiogenesis inhibitor
Type of agent
Target
Clinical activity and/or studies
State of clinical development in the US
Bevacizumab
Humanized antibody
VEGFA
Colorectal, breast, kidney, lung, prostate, head and neck, oesophageal, pancreatic, gall bladder, cervical and ovarian cancer, GIST, melanoma, mesothelioma, glioma
Approved for colorectal and lung cancer; phase II or III for other cancers
129
Sunitinib
Small-molecule TKI
VEGFR1 and VEGFR2, PDGFR, KIT and FLT3
Kidney, breast, colorectal, prostate, gastric, Approved for kidney cancer; phase II or III for liver, medullary thyroid, head and neck, other cancers ovarian and cervical cancer, STS, GIST, melanoma, NSCLC
47
Sorafenib
Small-molecule TKI
VEGFR2 and VEGFR3, Raf, PDGFR, KIT and RET
Kidney, thyroid, breast, colorectal, ovarian, bladder, pancreatic, prostate and hepatocellular cancer, melanoma, NSCLC, glioblastoma, STS, GIST
Approved for kidney cancer; phase II or III for other cancers
13
GW786034B (pazopanib)
Small-molecule TKI
VEGFR1, VEGFR2, VEGFR3
Renal cell cancer
Phase III
AZD2171
Small-molecule TKI
VEGFR1, VEGFR2, VEGFR3, PDGFRβ, KIT
Phase II or III Breast, kidney, hepatocellular, ovarian, colorectal and head and neck cancer, malignant mesothelioma, malignant melanoma, recurrent small cell lung cancer, glioblastoma, GIST
PTK787/ZK 222584 (vatalanib)
Small-molecule TKI
VEGFR1, VEGFR2, VEGFR3, PDGFRβ, KIT
Colorectal, prostate, renal, breast and pancreatic cancer, glioblastoma, GIST
Phase II or III
VEGF-Trap
Decoy receptor
VEGFA, VEGFB
Ovarian, kidney and breast cancer, glioma, STS
Phase II
133
ZD6474
Small-molecule TKI
VEGFR2, EGFR, FGFR1, RET
NSCLC, glioma, transitional cell carcinoma
Phase II
134
AMG-706
Small-molecule TKI
VEGFR1, VEGFR2, VEGFR3, PDGFR, KIT
NSCLC, breast and colorectal cancer
Phase II
135
AG013736
Small-molecule TKI
VEGFR1, VEGFR2, VEGFR3, PDGFR
Pancreatic cancer
Phase II
136
Refs
130 131
43,132
Source for all data on stage of clinical development was the US National Cancer Institute. EGFR, epidermal growth factor receptor; FGFR1, fibroblast growth factor receptor 1; FLT3, fms-related tyrosine kinase 3; GIST, gastrointestinal stromal tumour; NSCLC, non-small-cell lung cancer; PDGFR, platelet-derived growth factor receptor; STS, soft tissue sarcoma; TKI, tyrosine kinase inhibitor; VEGF, vascular endolthelial growth factor; VEGFR, VEGF receptor.
but it has been difficult to find biomarkers for angiogenesis pathways28. Therefore, the old concept of ‘the more the better’ is applied with the dosing of these agents. In phase I studies, angiogenesis inhibitors were given in increasing doses until severe toxicities became evident. As a direct consequence of dosing until the maximum tolerated dose (MTD), it is possible that some toxicities are not only caused by the disturbance of a specific pathway, but also caused by so-called ‘off target’ effects — small-molecule angiogenesis inhibitors, such as most of the anti-angiogenic TKIs, are chemicals that may have general toxic effects when given at high doses. For bevacizumab the MTD was determined to be 20 mg per kg (body weight), the dose at which about 25% of patients suffered from a grade 3 toxicity (BOX 1). Headache associated with nausea and vomiting was the dose-limiting toxicity, although only one patient stopped treatment at this dose
level for this reason29–31. With increasing doses of anti-angiogenic TKIs, grade 3 and 4 toxicities of oedema, hypertension (increased blood pressure) and fatigue have been observed32–35. Body surface area is the standard method to dose chemotherapeutics in cancer36, and body weight is used to determine the dose of anti-angiogenic antibodies to be given in patients. However, for TKIs, pharmacokinetics, including plasma peak concentrations, are relatively independent of body weight32. Therefore, these agents are prescribed at a fixed dose, independent of body weight or body surface area. Whether the pharmacodynamics of these drugs are independent of these measures remains an open question. Importantly, because the daily oral doses of antiangiogenic TKIs are fixed, toxicity patterns may be increased in patients with low body weight or decreased in patients with high body weight.
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Duration of treatment and toxicity profile At this point, we have not yet had enough experience with anti-angiogenic agents to predict all possible side effects if these agents were to be used in the adjuvant setting for many months, or possibly even years. Despite this, toxicities with different rates of onset may be caused by similar and/or different underlying mechanisms. Short-term toxicities may cause acute life-threatening problems such as gastrointestinal perforations, as have been seen with bevacizumab3. Long-term treatment, for example, may (sometimes in conjunction with previous or concurrent anthracycline chemotherapy) reduce the left ventricular ejection fraction (LVEF), which can also ultimately be life threatening37,38. So far, most studies have not been able to address longterm treatment effects sufficiently, because the longest time that patients have been treated with bevacizumab is 4–6 years, and this was a case study, so generally treatment
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PERSPECTIVES Activation of coagulation cascade Tissue factor ↑ vWF release ↑
Angiogenesis EC proliferation ↑ Migration ↑ Tube formation ↑ Hyperpermeability ↑
Immunomodulation Inhibition of dendritic cell function
Kidney function Protein filtration ↑ Podocyte survival ↑
Blood pressure Baroreceptor response ↓ Vasodilation (NO and PGI2 release) ↑
Vascular homeostasis EC survival ↑ Vascular integrity ↑
Bone marrow function Haematopoiesis and/or myelopoiesis ↑
VEGF
Thyroid function Stimulation of thryroid cells
Figure 1 | The various biological functions of VEGF. EC, endothelial cell; NO, nitric oxide; PGI2, prostacyclin; VEGF, vascular endothelial growth factor; vWF, von Willebrand factor.
times are shorter39. For anti-angiogenic TKIs, the maximal treatment time has been approximately 2–2.5 years40. Therefore, the long-term side effects of these agents are difficult to adequately review at this point. Furthermore, we foresee that in adjuvant studies, treatment with an anti-angiogenic agent may be given for at least 1 year, and it is unknown at the moment whether continued treatment will lead to long-term toxicities or whether toxicities might emerge in the following years after patients have stopped treatment. Drug resistance in relation to toxicity Tumours eventually become resistant to angiogenesis inhibitors in almost all treated patients, but the underlying mechanism of resistance against angiogenesis inhibitors has not yet been clarified. Preclinical in vivo studies indicated that tumours may activate alternative pathways to stimulate the angiogenic process in order to escape from VEGFR inhibition. For example, higher quantities of basic fibroblast growth factor (bFGF) were detected when the VEGF pathway was blocked in mice41. Classical resistance mechanisms, such as increased drug metabolism or an increased number of drug efflux pumps located on the cell membrane might also have a significant role42. A reciprocal increase in expression of the growth factor or its receptor (that is, VEGF or VEGFR) might be induced by treatment, and could also provoke toxicity of these agents. For example, in clinical trials with anti-angiogenic TKIs, increased plasma VEGF levels have been observed6,43. Increased levels of the growth factor or its receptor might lead to more problems once treatment with the agent is
halted owing to increased VEGFR activation after removing the receptor inhibitor in the presence of increased VEGF levels. In the following sections, we will discuss the toxicities of angiogenesis inhibitors that have reached phase II or III trials or have been approved and target at least the VEGF pathway (TABLE 1). The most important
and specific angiogenesis inhibitor-related toxicities are summarized according to their possible underlying cause (TABLE 2). Bleeding, wound healing and perforations Bleeding complications, gastrointestinal perforations and disturbed wound and ulcer healing can all occur as a result of antiangiogenesis therapy, and are most probably caused by disturbance of the tight endothelial cell–platelet interaction that maintains vascular integrity. Minor subungual splinter bleedings up to fatal lung bleedings in patients with centrally located lung cancer have been reported in clinical trials with both bevacizumab and anti-angiogenic TKIs44,45. Overall, in up to 44% of patients treated with bevacizumab, grade 1–2 (BOX 1) bleeding complications, such as epistaxis (nosebleeds), have been reported46. Low-grade bleeding complications have occurred during treatment with antiangiogenic TKIs as well, occurring in up to 26% of patients treated with sunitinib and up to 60% of patients treated with sorafenib44,47. Angiogenesis is essential for wound healing. A slightly increased complication rate of wound healing following surgery during angiogenesis inhibitor treatment has been
Glossary Acral erythema
Leukopenia and lymphopenia
Redness of the most distal extremities caused by capillary congestion (a general sign of inflammation).
A low leukocyte or lymphocyte count in the circulating blood, both of which increase the risk of infections.
Baroreceptors Located in the carotid arteries in the neck, these receptors are stretched by high blood pressure, reducing the activation of the vasomotor centre. They also activate the vasomotor centre in response to low blood pressure.
Megakaryocytes The precursor cells of platelets, located in the bone marrow.
Perivascular cells Dendritic cells Immune cells that process antigens and present them to other immune cells.
Cells that surround vessels, including pericytes, myofibroblasts and smooth muscle cells.
Podocytes Encephalopathy Alteration in brain function and/or structure. Common symptoms include progressive loss of memory and cognitive ability, subtle personality changes, inability to concentrate, lethargy and progressive loss of consciousness.
Gastrointestinal perforations Can occur in the wall of the stomach, small intestine or large bowel, resulting in intestinal contents flowing into the abdominal cavity.
Cells that form the visceral epithelium in the kidney and are involved in the glomerular filtration barrier.
Reversible posterior leukoencephalopathy syndrome A rapidly evolving neurological syndrome. The underlying mechanism seems to be related to an increased permeability and reactivity of brain vasculature.
Subungual splinter bleeding Glomerulus A capillary bed surrounded by the Bowman’s capsule in the kidney, which regulates blood filtration and urine generation.
A small amount of bleeding that occurs under a finger or toe nail.
Thrombocytopenia Ischaemia
A low platelet count in the circulating blood.
An inadequate blood supply to an organ.
Vascular resistance Left ventricular ejection fraction The fraction of blood pumped out of the left ventricle with each heart beat.
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The resistance to flow that must be overcome to push blood though a vessel; determined by diameter, stiffness and length of the vessel.
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PERSPECTIVES Box 1 | Toxicity grading system: common toxicity criteria (CTC) The CTC-system is used to determine the severity of the toxicity induced by an anticancer agent. There are five levels of toxicity: grade 1–grade 5. For each different type of toxicity the severity is described in five different levels. Grade 1 is the mildest toxicity, grade 4 is the most severe lifethreatening toxicity and grade 5 is defined as a fatal toxicity. Often grade 3 toxicity is defined as the dose-limiting toxicity for a certain agent. However, some grade 3 toxicities are well manageable. For example, grade 3 neutropenia (decreased number of white blood cells) is acceptable as long as it is not accompanied by infections.
found. Surgery on patients with colorectal cancer during bevacizumab plus chemotherapy treatment versus chemotherapy alone increased wound healing problems from 3.4 to 13%48, and post-operative treatment also increased wound healing problems from 0.5 to 1.3% in patients treated with bevacizumab plus chemotherapy compared with chemotherapy alone48. These differences did not reach statistical significance, probably because of the relatively low incidence, but seem to be consistent with other clinical reports. Wound healing problems have not been seen in patients treated with anti-angiogenic TKIs, but this might be explained by the fact that wound healing has not been adequately studied with these agents in the clinic. Furthermore, the effects of anti-angiogenic TKIs on wound healing is variable in preclinical studies, either causing delayed wound healing or no effect at all49–51. Healing of gastrointestinal ulcers also depends on angiogenesis, and a relationship between gastrointestinal perforations and anti-angiogenic antibody therapy has been found52. In patients with
colorectal cancer, giving bevacizumab in combination with chemotherapy resulted in a higher incidence of gastrointestinal perforations compared with chemotherapy alone (1.5% versus 0%, respectively3). This difference did not reach statistical significance between the groups in this study, most probably because of the low incidence in relation to the number of patients per group. In addition, an analysis of a surveillance registry on the use of bevacizumab in combination with chemotherapy in 1,953 patients with metastatic colorectal cancer found that gastrointestinal perforations were observed in 2.3% of patients older than 65 and in 1.2% of patients younger than 65 (REF. 53). This complication has not been reported before with chemotherapy regimens in colorectal cancer alone, and therefore seems to be due to the addition of bevacizumab. Importantly, gastrointestinal perforations due to bevacizumab treatment have only been observed clinically in colorectal and ovarian cancer3,54. Most of these perforations occurred in the tumour area, but some
Table 2 | Possible molecular mechanisms in the toxicity of angiogenesis inhibition
Toxicity
Possible underlying mechanism
Bleeding, disturbed wound healing
Platelet dysfunction; decreased expression of endothelial TF
Thrombotic events
Endothelial cell apoptosis; lack of endothelial cell renewal leading to exposure of the ECM to the circulating blood (results in platelet activation); increased TF expression; reduced TM and NO; direct platelet activation
Hypertension
Decreased NO and/or PGI2 production; inappropriate density of vessels (arterioles and capillaries); vascular stiffness; disturbed endothelin function
Hypothyroidism
Disturbed thyroid cell function; reduced vascularity of thyroid
Fatigue
Hypothyroidism
Proteinuria and oedema
Podocyte dysfunction owing to VEGF blockade; hypertension
Leukopenia, lymphopenia and immunomodulation
Inhibition of haematopoiesis and/or myelopoiesis; impaired dendritic cell function
Dizziness, nausea, vomiting and diarrhoea
Mucosa disturbance
Skin toxicity including rash Epidermal cell apoptosis and hand-foot syndrome ECM, extracellular matrix; TF, tissue factor; TM, thrombomodulin; NO, nitric oxide; PGI2 , prostacyclin; VEGF, vascular endothelial growth factor.
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occurred at sites of intestinal ulceration and ischaemic inflammation, and radiation therapy seems to be a provoking factor55. There are limited data on gastrointestinal perforations in patients treated with antiangiogenic TKIs. In a dose-finding study with sunitinib in 28 patients, tumour cavity formation occurred in six patients, and in two of those patients subsequent fistula formation occurred, reflecting perturbation of physiological barriers such as the skin and intestines32. One source of survival factors, including VEGF56, for quiescent endothelial cells is the platelet compartment57,58 (BOX 2). Platelets are small cell fragments, derived from megakaryocytes that circulate in the blood, and are involved in blood clot formation. Platelets have been shown to secrete VEGF in wound-healing areas, and the inhibition of platelet activation results in a significant decrease of VEGF in wounds59. Furthermore, the angiogenesis-promoting activity of platelets has been demonstrated in in vitro and in vivo assays60–62. An overview of normal homeostasis of endothelial–platelet interactions and the possible interference of angiogenesis inhibitors in this interaction is shown in FIG. 2 and FIG. 3. Recently, we showed that platelets take up bevacizumab, thereby blocking their angiogenic activity (H.V., M. Lolkema, D. Qian, Y. Hilkes, E. Liapi, J-.W. Akkerman, R. Pili and E. Voest, unpublished observations). During wound healing platelets release their contents, which are primarily growth factors. We propose that the uptake of bevacizumab by platelets neutralizes VEGF, and is related to bleeding, wound healing and gastrointestinal complications in patients. Whether TKIs that inhibit angiogenesis also influence platelet release of VEGF or primarily affect endothelial cells remains to be determined. Interestingly, platelets express VEGF receptors, and platelet activation is enhanced by VEGF, suggesting that anti-angiogenic TKIs might inhibit platelet activation63. In addition, anti-angiogenic TKIs can induce thrombocytopenia in patients32,38, which can disturb platelet–endothelial cell interactions, and the side effects of angiogenesis inhibitors may also be potentiated by chemotherapyinduced thrombocytopenia26. The perivascular cells that surround endothelial cells are another source of VEGF for endothelial cells64. The inhibition of vascular signalling pathways in endothelial and perivascular cells also disturbs normal endothelial cell homeostasis22,65.
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PERSPECTIVES Box 2 | Vascular integrity and platelets In the 1960s, it was shown that isolated organs could be kept alive for a few days when perfused with platelet rich but not platelet poor plasma126. These experiments suggested that plateletderived growth factors are essential for the homeostasis of the endothelial cell lining. Serum, which is used in vitro to grow cells, including endothelial cells, contains most of these plateletderived angiogenic growth factors. In patients, platelets have also been recognized to maintain the vascular integrity127,128. Very low platelet counts are associated with oedema and the extravasation of blood plasma and cells.
As a consequence, proper wound healing is inhibited49,51,66. Therefore, spontaneous bleedings and gastrointestinal perforations may be caused by the disturbance of endothelial cell homeostasis.
In addition, angiogenesis and coagulation are closely related biological processes67. VEGF has a role in the coagulation cascade by inducing tissue factor (TF) expression on endothelial cells60,68–70. TF is the main regulator
of the coagulation cascade, inducing thrombin formation from prothrombin, which in turn activates platelets and converts fibrinogen into fibrin to cause clot formation71 (FIG. 3). The inhibition of angiogenesis might be partly mediated by the downregulation of TF expression by endothelial cells. Endothelial-cellinduced coagulation promotes wound healing and presumably angiogenesis. Therefore, inhibition of the TF pathway might be responsible for inadequate wound healing. Treatment with SU5416 (a small-molecule TKI that primarily inhibits VEGFR2 that is no longer in clinical development) reduced the VEGF-induced expression of TF in endothelial cells in vitro72.
Endothelial cell survival and growth factors (VEGF, PDGF and others)
Platelet
Blood flow
Blood vessel Endothelial cell Subendothelial matrix: collagen and vWf
Perivascular cell Thrombin FVIIIa
FXa Thrombin
Prothrombin
FVa FXa PrC
FVIIa
FVIIa
↑AT-III activity
Thrombin APC
Plasminogen
Plasmin
TFPI
GAGs
tPA
NO
PGI2
TM TF Ecto-ADPase
NO eNOS
Endothelial cell
PGI2
PGH2 PS
Nucleus
Figure 2 | Platelet–endothelial cell interactions and the anti-coagulatory activity of the quiescent endothelium. During anti-angiogenic therapy, the ability of the endothelium to maintain homeostasis and to heal and prevent blood loss might be disturbed. To maintain vascular integrity, quiescent endothelial cells prevent the activation of the coagulation cascade. Incidentally, some platelets become activated and release growth factors in low quantities that will prevent the apoptosis of endothelial cells. The factors that are involved in this anti-coagulatory activity are: nitric oxide (NO), which prevents platelet activation and is synthesized by endothelial NO-synthetase (eNOS); prostacyclin (PGI2), which is an inhibitor of platelet activation and which, along with NO, is released by endothelial cells; endothelial membrane-associated ecto-ADPase, which prevents platelet activation by breaking down ADP (a weak platelet activator); glycosaminoglycans (GAGs), such
as heparan sulphate, which create a negatively charged cell surface and might increase antithrombin-III (AT-III) activity; tissue factor pathway inhibitor (TFPI), thrombomodulin (TM), AT-III and protein C (PrC), which inhibit thrombin activity directly or through the TF-mediated activation of prothrombin into thrombin; and finally, tissue type plasminogen activator (tPA), which converts plasminogen into plasmin to break down any fibrin formed despite endothelial anti-thrombotic activity75. The inhibition of angiogenesis disturbs platelet–endothelial homeostasis, and may cause endothelial cell apoptosis owing to a lack of sufficient survival factors (such as vascular endothelial growth factor (VEGF)). FVa, coagulation factor V; FVIIa, coagulation factor VII; FVIIIa, coagulation factor VIII; FXa, coagulation factor X; PDGF, platelet-derived growth factor; PGH 2 , prostaglandin H 2 ; PS, prostacyclin synthase; vWF, von Willebrand factor.
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PERSPECTIVES
Perivascular cell Endothelial cell Fibrin
tPA
Plasmin
Thrombin
Fibrinolysis
Plasminogen PAI
Activated platelets
Blood vessel Fibrinogen TF ↓ TM
FVIIa
Thrombin Coagulation cascade
Growth factors vWF
Collagen
Subendothelial matrix
Figure 3 | Angiogenic growth factor stimulation of the endothelium causes concomitant activation of the coagulation cascade and angiogenesis. In wound healing, platelets become activated in large amounts by the pro-coagulatory activity of the endothelium itself or by the presentation of the subendothelial matrix owing to endothelial cell retraction. Subsequently, endothelial cells will become activated to migrate, proliferate and form new vessels. In addition, the anti-thrombotic homeostasis of endothelial cells can also be disturbed when endothelial cells undergo apoptosis, as cells that become apoptotic first increase the expression of tissue factor (TF) and become pro-thrombotic76. TF and coagulation factor VII (FVIIa) activate the coagulation cascade, resulting in thrombin formation. Thrombin, in turn, induces platelet aggregation and activation, and the conversion of fibrinogen into fibrin. Furthermore, the fibrinolytic cascade, which breaks down fibrin, is inhibited by decreasing tissue type plasminogen activator (tPA) activity and increasing plasminogen activator inhibitor (PAI) expression. The activation of the coagulation cascade and clot formation is essential for wound healing, but may also cause pathological thrombus formation and obstruction of blood flow (thrombosis), and has an important role in angiogenesis. Owing to platelet and perivascular cell–endothelial cell interactions, inhibitors of angiogenesis may cause delayed wound healing or bleeding by blocking growth factors released from platelets. In addition, inhibitors of angiogenesis may cause endothelial cell apoptosis with subsequent thrombus formation. TM, thrombomodulin; vWF, von Willebrand factor.
Thrombotic events Thrombosis is defined as a pathological thrombus formation in a vessel that causes an obstruction to the flow of blood. It has been shown that the coagulation cascade, mainly regulated by TF, is involved in deep venous thrombosis (DVT). Platelets have an important role in arterial thrombosis. Patients with cancer are at an increased risk of a thrombotic event, because tumour cells release pro-thrombotic factors or stimulate endothelial cells to become pro-thrombotic73. As mentioned above, increased endothelial TF expression in relation to VEGF stimulation has been demonstrated and therefore VEGF-stimulated endothelial cells have a thrombotic phenotype60,68–70. Interestingly, thrombotic events, which can be fatal, have been observed in patients treated with angiogenesis inhibitors, especially when these agents are given in combination with chemotherapy3,26,74. Bevacizumab primarily increased the risk for arterial thrombosis46. The incidence of drug-related thrombotic events seems low with antiangiogenic TKIs as monotherapy. However, thrombotic
events did occur clinically with chemotherapy plus SU5416 (REF. 26), an antiangiogenic TKI no longer in clinical development, but most antiangiogenic TKIs have not been tested in combination treatment strategies with similar chemotherapy regimens. Under normal physiological circumstances, endothelial cells play a major role in preventing blood cells from adhering to the vasculature and subsequent coagulation (FIG. 2). To do this, endothelial cells produce and secrete many factors to prevent the activation and propagation of the coagulation cascade. These include endotheliumderived nitric oxide (NO) synthesized by endothelial cell nitric oxide synthetase (eNOS), endothelial membrane-associated ecto-ADPase, thrombomodulin, prostacyclin (PGI2), glycosaminoglycans, TF pathway inhibitor (TFPI) and tissue type plasminogen activator (tPA)75. NO, ecto-ADPase and PGI2 prevent platelet aggregation and activation. Thrombomodulin and TFPI inhibit the TF-mediated activation of prothrombin into thrombin, whereas tPA converts plasminogen into plasmin for the immediate breakdown
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of fibrin that may be formed despite endothelial anti-thrombotic activity. In addition, endothelial cells bind anti-thrombotic proteins such as thrombomodulin, TFPI and protein C. Heparan sulphates contribute to the negatively charged endothelial cell surface and promote the adhesion of anti-thrombinIII, an anti-coagulant that inhibits thrombin activity. When the anti-thrombotic homeostasis of endothelial cells is disturbed, the cells undergo a programmed phenotypic change and become pro-thrombotic by increasing the expression of TF76. As mentioned above, VEGF stimulation is also able to induce this pro-thrombotic change60,68–70. Therefore, one may expect that an inhibitor of VEGF would reduce the pro-thrombotic state. However, this effect of VEGF seems dose dependent. At high concentrations, VEGF stimulates coagulation by inducing TF activity, vascular permeability and endothelial cell proliferation and migration, but at low concentrations, VEGF is a survival and maintenance factor for the endothelial cell lining. Blockade of the VEGF pathway might induce the apoptosis of these quiescent endothelial cells in vivo rather than inhibiting the angiogenic activity of VEGF77. It has been shown that not only proliferating endothelial cells, but also apoptotic endothelial cells, become pro-coagulatory78. This might then lead to thrombotic events in areas prone to thrombosis, such as areas of blood stasis or narrow vessels79,80. Another related explanation for the increased risk of thrombosis is that by inhibiting the VEGF pathway, the renewal capacity of endothelial cells in response to trauma is disturbed. The inability of endothelial cells to proliferate may cause an increased exposure of the underlying extracellular matrix81, allowing the extracellular matrix proteins collagen and von Willebrand factor to become available and activate platelets. In our opinion, the fact that bevacizumab-induced thrombosis occurs predominantly in arteries might be due to disturbed platelet function in these patients. Platelets are known to have an important role in arterial thrombosis80. As previously described, platelets take up bevacizumab (H.V., M. Lolkema, D. Qian, Y. Hilkes, E. Liapi, J-.W. Akkerman, R. Pili and E. Voest, unpublished observations). This uptake reduces the stimulatory activity of platelets on endothelial cells, as within 8 hours after treatment more than 97% of platelet VEGF is neutralized (H.V., M. Lolkema, D. Qian, Y. Hilkes, E. Liapi, J-.W. Akkerman, R. Pili and E. Voest, unpublished observations). Consequently, platelets can no longer provide the endothelial cells with VEGF,
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PERSPECTIVES thereby disturbing platelet–endothelial cell homeostasis. Recently, platelet activation induced by the incubation of platelets with a mixture of bevacizumab, heparin and VEGF has been observed, and this activation might have a role in bevacizumab-associated thrombosis82. Another factor that could explain a difference in venous versus arterial thrombosis is that there might be a difference in cell signalling in the endothelial cells that line veins and arteries83. Hypertension and decreased LVEF The treatment of cancer patients with angiogenesis inhibitors is associated with hypertension and a reduced LVEF32,33,84. Although regular anti-hypertensive agents are quite effective in reducing this blood pressure increase, bevacizumab or anti-angiogenic TKI-induced hypertension might be life threatening (so-called malignant hypertension) and cause damage to the eyes, brain, kidneys and/or lungs. When VEGF is blocked by bevacizumab, up to 20–30% of patients experience increases in blood pressure, primarily systolic29,85. When anti-angiogenic TKIs are administered, 15–60% of patients develop hypertension86. A reduced LVEF has been observed during treatment with sunitinib in up to 5–11% of patients, and in up to 5% of patients treated with bevacizumab6,37,38. Some patients treated with sunitinib had a decline in LVEF of more than 20%, but no clinical signs or symptoms of congestive heart failure were observed. For bevacizumab, congestive heart failure has been observed in up to 2% of patients with breast cancer, but this is probably related to previous chemotherapy (anthracyclines)87. Blood pressure is regulated by cardiac output, blood volume regulation through the kidney, baroreceptors and the vasculature itself through the release of hormones such as endothelin 1. FIG. 4 shows the most important factors in normal blood pressure regulation at the vascular level and possible interfering effects of angiogenesis inhibitors. The first indications that VEGF was important for blood pressure regulation came from preclinical studies in which animals were infused with VEGF. In these in vivo experiments a clear drop in blood pressure after VEGF administration was observed88,89. A study by Li et al. showed that VEGF regulation of blood pressure is primarily mediated by VEGFR2. In a clinical trial in which VEGF was used to treat ischaemia, a drop of systolic blood pressure of 22% was observed90. The diastolic
blood pressure was also decreased but to a lesser extent90. The underlying mechanism of VEGF-mediated regulation of blood pressure has been extensively studied. Endothelial cells promote vasodilation by secreting NO and PGI2 (REF. 91). VEGF is known to induce the release of these factors by endothelial cells. Downstream of the VEGF receptor on endothelial cells, the PI3K (phosphatidylinositol 3-kinase) and MAPK (mitogen-activated protein kinase) signalling cascades are responsible for eNOS induction and NO production92. Blocking VEGFR signalling will therefore decrease the production of these vasodilators, leading to vascular resistance and increased blood pressure. Inhibition of the MAPK and Akt pathways by angiogenesis inhibitors, leading
to the downregulation of the release of PGI2 and NO from vascular or perivascular cells, may also be directly involved in treatmentinduced hypertension93. Independent of vasodilator release, VEGF can induce hypotension through an endothelial baroreceptor response94. Upon infusion with VEGF, the baroreceptor signalling cascade was decreased in rats, thereby decreasing blood pressure. In the case of VEGF inhibition, one might expect the opposite disturbance — increased signalling with increased blood pressure. The exact interaction of VEGF and the baroreceptors is unknown. Hypertension in response to angiogenesis inhibitors might not only be induced by the lack of vasodilatory effects, but may also be due to a decrease in the number of small
Smooth muscle layer Perivascular cell Endothelial cell Blood flow
Subendothelial matrix Decrease blood pressure: relaxation of smooth muscle cells
Increase blood pressure: contraction of smooth muscle cells
NO/PGI2 release by ECs/platelets ↑
Possible effects of angiogenesis inhibitors on blood pressure Reduced NO/PGI2
Baroreceptor response ↓
Baroreceptor response ↑
Baroreceptor response disturbance
Sympathicus ↓
Sympathicus ↑
Unknown
ATP/shear stress ↓
ATP/shear stress ↑
Unknown
Blood volume ↓
Blood volume ↑
Unknown
Low activity of endothelin
Increased activity of endothelin
Increased activity of endothelin Inappropriate density of vessels/vascular stiffness
Figure 4 | Blood pressure regulation by the vascular system. The normal blood pressure regulation determined by the interplay of vascular cells, smooth muscle cells and baroreceptor regulation is shown. On the right are the possible underlying mechanisms for how angiogenesis inhibitors disturb normal blood pressure homeostasis. Endothelial cells (ECs) promote vasodilation through the secretion of nitic oxide (NO) and prostacyclin (PGI2)91. Vascular endothelial growth factor (VEGF) is known to induce the release of these factors by endothelial cells, and therefore the inhibition of VEGF may lead to hypertension. Baroreceptors are located in the carotid arteries in the neck and send signals to the brain and subsequently to the vasomotor centre. If these receptors are stretched too much by high blood pressure they reduce their activating signals to the vasomotor centre, whereas in the case of low blood pressure they activate the vasomotor centre. Hypertension in response to angiogenesis inhibitors might not only be induced by the lack of vasodilatory effects, but may also be due to an inappropriate reduction in the density of capillaries and arterioles, which may induce peripheral vascular resistance resulting in hypertension95. In addition, hypertension might be caused by an increase in vascular stiffness84. Blockade of VEGF signalling may also disturb the balance between VEGF and endothelin. Endothelin is a potent endogenous vasoconstrictor, and its expression is correlated with that of VEGF. The exact role of their interaction is unknown, but one may expect that they function to keep the blood pressure tightly in balance64,96.
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PERSPECTIVES arteries and arterioles as a consequence of inhibiting new vessel formation. It is known that inappropriate reduction in the density of capillaries and arterioles may induce peripheral vascular resistance, resulting in hypertension95. Recently, Veronese and co-workers found that hypertension induced by sorafenib is primarily due to an increase in vascular stiffness, whereas no relation with humoral factors or volume expansion was detected84. In this study, 16% of the patients treated with sorafenib suffered from an increased systolic blood pressure of more than 20 mmHg. All humoral factors that are involved in blood pressure regulation, such as catecholamines and aldosterone, failed to show a relationship between their deregulation and hypertension during treatment with sorafenib84. These results further support the idea that the effect of angiogenesis inhibitors on blood pressure is determined at the level of the vasculature itself. Blockade of VEGF signalling may also disturb the balance between VEGF and endothelin. Endothelin is a potent endogenous vasoconstrictor and its expression correlates with that of VEGF. The exact role of their interaction is unknown, but one might expect that they function to keep the blood pressure tightly in balance64,96. Increased peripheral resistance of the vasculature, similar to blood volume overload, may be responsible for both hypertension and a reduced LVEF, and both may precede congestive heart failure97. Therefore, these two toxicities are closely related. The relationship between LVEF and angiogenesis was established by Yau and co-workers98. They studied the effect of angiogenic gene therapy and found that the transplantation of VEGF- and insulin-like growth factor 1 (IGF1)-expressing bone-marrow-derived cells was able to improve left ventricular cardiac function due to increased cardiac angiogenesis. This study, together with the clinical observations that angiogenesis inhibitors reduce the LVEF, underscore the need for measures of LVEF to be determined routinely in patients treated with angiogenesis inhibitors. Hypertension and a reduced LVEF during treatment may complicate the opportunity to use these agents in adjuvant or life-long treatment strategies86. Another side effect that could be related to hypertension is encephalopathy99. Two case reports of bevacizumab and one case report of sorafenib-associated reversible posterior leukoencephalopathy syndrome (RPLS) have been published100–102. The most frequent cause of RPLS is thought to be hypertension, and it might be related to endothelial dysfunction.
Hypothyroidism Anti-angiogenic TKIs can affect thyroid homeostasis. After treatment with TKIs (for example, sunitinib) an increase in thyroid stimulating hormone (TSH) and a decrease in the levels of the circulating thyroid hormones (T3 and T4) indicative of hypothyroidism were observed in up to 36% of patients103. Furthermore, anti-angiogenic TKIs can induce severe symptoms of fatigue32,35, which in some patients might be related to a disturbed thyroid function. VEGF expression is low in hypofunctional thyroid glands104. In addition, in vitro experiments showed that VEGF reduces TSH-induced iodine uptake by thyroid cells, whereas the inhibition of VEGF restores iodine uptake105. The precise role of VEGF in the thyroid signalling cascade is unknown, reflected by the fact that bevacizumab treatment, unlike treatment with anti-angiogenic TKIs, has not been associated with disturbed thyroid homeostasis30,106,107. For this reason, other factors such as PDGF or KIT may have a role in thyroid homeostasis, but so far no data have been published on the role of these factors in thyroid function. VEGF also has an important role in endocrine glands other than the thyroid. For example, in pancreatic islets VEGF has a role in the formation of fenestrations (small openings) of the islet capillaries. The fine tuning of blood glucose regulation was disturbed in mice with VEGF-deficient islets or after treatment with an angiogenesis inhibitor108. Interestingly, in mice treated with a VEGFR inhibitor, a significant reduction of vessel density was observed in several organs, including the thyroid gland, adrenal cortex, pituitary, choroid plexus and small intestinal villi108. However, we are unaware of any clinical data on the effects of VEGF on these other glands. Proteinuria and oedema Proteinuria, the presence of an excess of proteins in the urine, may reflect renal (kidney) damage. Renal function is partly regulated by VEGF109, and the inhibition of VEGF causes mild proteinuria110,111. As proteins are normally reabsorbed from urine, proteinuria is due to a decreased reabsorption or increased filtration. Up to 30% of patients treated with bevacizumab developed proteinuria87,112, but this proteinuria was mostly asymptomatic. Interestingly, anti-angiogenic TKIs are significantly less associated with proteinuria in patients, indicating that the exact role of VEGF in proteinuria is more complicated, and that the effect of bevacizumab is probably due to
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more than just the inhibition of the VEGF signalling cascade. The importance of VEGF for the kidney is reflected by its expression in the normal renal cortex as well as in kidney tumours113–115, and VEGF is highly expressed in the glomeruli and tubules, and is involved in kidney repair116. However, the underlying mechanism of increased proteinuria owing to treatment with angiogenesis inhibitors is unknown. A preclinical study in rats showed that VEGF has a role in the function of podocytes and renal endothelial cells to prevent proteinuria111. In an elegant study, Guan et al. showed that VEGF can act as a survival factor for podocytes and thereby prevent glomerulonephritis117. Alternatively, or in parallel, the endothelial cell lining might be disturbed in patients treated with angiogenesis inhibitors, causing the extravasation of plasma proteins into the extracellular matrix or urine. However, whether proteinuria in patients treated with angiogenesis inhibitors is of major importance is currently unknown. Proteinuria is, in general, reversible after halting the anti-angiogenic agent. In addition, a clear correlation between the induction of hypertension and the prevalence of proteinuria is known. In most clinical trials, patients with pre-existing proteinuria were excluded from study entry, and therefore studies to determine the safety of angiogenesis inhibitors in this category of patients is of major interest. Oedema (fluid extravasation), can also occur as a result of angiogenesis inhibition, and may be a direct consequence of proteinuria. When large amounts of proteins are lost in the urine, the balance of the osmotic pressure between the blood and the interstitium is disturbed, causing the extravasation of fluid reflected by oedema (nephrotic syndrome). This complication has only occurred in a few patients during anti-angiogenic therapy, but for sunitinib it is one of the dose-limiting toxicities32. Haematopoiesis and immunomodulation VEGF receptors are expressed by almost all haematopoietic cells and endothelial precursors118,119. In addition, circulating monocytes and platelets also express VEGFR1 and/or VEGFR2 (REF. 120). Leukopenia and lymphopenia, as well as thrombocytopenia, are probably casued by the inhibition of haematopoiesis and/or myelopoiesis during angiogenesis inhibition. Bone marrow cells, such as megakaryocytes, also express VEGFRs121. In addition, VEGF is known to inhibit the function of dendritic cells122. Whether the inhibition of VEGFinduced immunomodulation is important
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PERSPECTIVES in the clinic is unknown, but an effect on the immune system may become clear during longer term therapy. Skin toxicity and hair discoloration The inhibition of angiogenesis, especially by anti-angiogenic TKIs, can cause severe skin toxicities. Growth factor signalling pathways are involved in the homeostasis of the skin. For example, hair depigmentation, hair loss and acral erythema (a severe and painful side effect) are common during treatment with sunitinib or sorafenib as recently reviewed by Robert et al.123. One of these skin toxicities, also named hand-foot syndrome, has clear distinct features from classical chemotherapy-induced hand-foot syndromes. Analysis of skin biopsies of these areas in patients indicate that epidermal cells are swollen, capillaries are dilated and apoptotic endothelial cells are present32. For sorafenib, similar skin toxicities have been observed in up to 42% of patients124. In this study it was also explored whether skin toxicity of sorafenib would predict tumour response, but this hypothesis could not be confirmed. The observation that endothelial cells are affected in the skin suggests that the skin toxicity is a direct consequence of the biological activity of these agents. This toxicity might be mediated by the interaction of stroma and endothelial cells. Hair discoloration is also a common side effect of sunitinib and closely follows the treatment schedule of 4 weeks of treatment followed by a 2 week rest period32. It seems that these effects may be caused by the inhibition of KIT rather than VEGF, as KIT has been shown to have a role in the skin toxicities of imatinib125, and these side effects are not usually observed with bevacizumab therapy. Further studies are needed to explore the biological mechanisms of this toxicity, and should be relatively easy to perform because of the availability of skin tissue for repeated biopsies. Although these side effects can range from mild to severe, in general they are well tolerated by patients and are seldom a reason to lower the dose of anti-angiogenic TKIs.
regulation (FIG. 1). At present, angiogenesis inhibitors have been shown to prolong progression-free survival but only have a small effect on overall survival in patients with cancer. We expect that the development of new, more potent agents and more active combination strategies will lead to an improved anti-tumour effect in patients. However, more potent agents could possibly also be more toxic, and therefore understanding the underlying biological mechanisms of these toxicities may provide the knowledge to optimize this treatment strategy. Henk M. W. Verheul is at the University Medical Center Utrecht, Heidelberglaan 100, 3584 CX, Utrecht, The Netherlands. Herbert M. Pinedo is at the VU Medical Center Amsterdam, De Boelelaan 1015, 1081 HV, Amsterdam, The Netherlands. Correspondence to H.M.P. e-mail:
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Conclusion The exciting development of angiogenesis inhibition as a cancer treatment has generated a new drug-related toxicity profile, which also gives more insight into the biological process of angiogenesis. The toxicities emphasize that the generation of new blood vessels is a very complicated multi-factorial biological process that involves various pathways in the body such as the coagulation cascade, the immune system and blood flow
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Acknowledgements H.V. is a recipient of the American Society of Clinical Oncology (ASCO) Young Investigator’s award 2006 and of a Drug Development fellowship at the Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins Medical Institution. This work was supported in part by The Adriana van Coevorden Society (H.V.) and for a major part by the Spinoza award (H.M.P.).
Competing interests statement The authors declare no competing financial interests.
DATABASES The following terms in this article are linked online to: Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query. fcgi?db=gene bFGF | EGFR | eNOS | FLT3 | IGF1 | KIT | PDGFRα | PDGFRβ | PI3K | RET | TFPI | VEGF
FURTHER INFORMATION US National Cancer Institute: www.cancer.gov/clinicaltrials Access to this links box is available online.
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