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2011 Issue 4, November: Progress in our understanding of the pathobiology of hypoxia and angiogenesis
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2011 Issue 3, August: The molecular pathology of sarcomas
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2011 Issue 2, April: Recent advances in the molecular pathology of micro-RNAs
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2011 Issue 1, February: Neuropathology: Advances in technology and biology
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2010 Issue 4, December: Recent advances in the pathobiology of lymphoma
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2010 Issue 3, September: Recent advances in renal pathology
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2010 Issue 2, June: Recent advances in breast cancer
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2010 Issue 1, April: p53: Recent advances in our understanding of this key tumour suppressor
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The Journal of Pathology 2011 Virtual Issue Number 4, November

Progress in our understanding of the pathobiology of hypoxia and angiogenesis

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Compiled and annotated by Adrian M Jubb
Department of Pathology, Genetech Inc., 1 DNA Way, South San Francisco, CA, 94080, USA

Introduction

Cancer progression is accompanied by changes in the microenvironment in which cancer cells reside (reviewed in [1]). These changes may be either permissive to malignant growth, secondary to manipulation of the stroma by the tumour itself, or they may be a reaction of the body to try and limit malignant growth. Hypoxia and angiogenesis are pervasive and inter-related features of the tumour microenvironment. Therapeutic modulation of angiogenesis has proven clinical benefit in certain types of cancer, and hypoxia has roles in resistance to both chemotherapy and radiotherapy. In this virtual issue we have compiled a collection of articles that report recent developments in research to better understand the effects of hypoxia and angiogenesis in cancer, and how they can be therapeutically manipulated.

In addition, manipulating the physiological responses to hypoxia, which include angiogenesis, may be therapeutically relevant to several other disease processes, including acute hypoxic brain injury, chronic obstructive pulmonary disease and retinal hypoxia/neovascularization. Work to understand the effects of hypoxia and the mechanisms of angiogenesis in cancer has also informed research into these diseases, and several articles that have advanced our understanding of the broader implications of hypoxia and angiogenesis are highlighted.

1	Jekyll and Hyde: the role of the microenvironment on the progression of cancer Michael Allen, J Louise Jones The Journal of Pathology 2011; 223: 163-177. (Invited review)

The hypoxia/angiogenesis axis in renal cell carcinoma

Clear cell renal carcinomas are highly vascular, secondary to the upregulation of vascular endothelial growth factor (VEGF)-A as a consequence of inactivation of the Von Hippel Lindau (VHL) tumour suppressor gene. Under normoxic conditions, VHL binds hypoxia inducible factor (HIF)-1alpha (the rate limiting component of the HIF-1 dimer), resulting in its ubiquitination and rapid turnover. In hypoxia, the binding of VHL to HIF-1alpha is blocked by biochemical modification of the HIF-1alpha protein. As a consequence, HIF-1alpha accumulates and a transcriptional program is activated, which includes the upregulation of VEGF-A expression. VEGF-A is the primary proangiogenic ligand in renal cell carcinomas and results in prolific angiogenesis and a high microvessel density. Baldewijns and colleagues [2] review advances in this field, including the emerging hypothesis that activation of a related hypoxia inducible complex, HIF-2, is responsible for the majority of the oncogenic effects of VHL loss in renal cell carcinoma. Indeed, HIF-1 and HIF-2 are reported to have partially antagonistic roles and the relative balance of the two complexes may affect tumourigenesis and tumour progression. Subtyping renal cancer in this way may permit more effective personalized therapies that block pathways downstream of the specific HIF complexes.

The HIF-1 dependent transcriptional program is broad, and has recently been shown to include microRNAs such as miR210. MiR210 is highly expressed in renal cell carcinoma, yet in vitro data from Nakada et al. [3] suggest that miR210 overexpression causes an accumulation of cells at the G2/M checkpoint by downregulating E2F3 among other genes, which is counterintuitive to a role in oncogenesis. However, accumulation of cells at the G2/M checkpoint resulted in centrosome amplification and aneuploidy, thereby promoting chromosomal instability. Thus HIF-1 may have oncogenic effects beyond its conventional role in driving angiogenesis.

2	VHL and HIF signalling in renal cell carcinogenesis Marcella M Baldewijns, Iris JH van Vlodrop, Peter B Vermeulen, Patricia MMB Soetekouw, Manon van Engeland, Adriaan P de Bruïne The Journal of Pathology 2010; 221: 125-138. (Review)
3	Overexpression of miR-210, a downstream target of HIF1α, causes centrosome amplification in renal carcinoma cells Chisato Nakada, Yoshiyuki Tsukamoto, Keiko Matsuura, Tung Lam Nguyen, Naoki Hijiya, Tomohisa Uchida, Fuminori Sato, Hiromitsu Mimata, Masao Seto, Masatsugu Moriyama The Journal of Pathology 2011: 224; 280-288. (Original paper)

Glioma as a Model of VEGF Dependent Angiogenesis

The tumour initiating/stem cell hypothesis is contentious, and relies upon preclinical model data that suggest sorted fractions of tumour cells show different abilities to re-form tumours in vivo. CD133 is one such marker of putative tumour stem cells. In gliomas, CD133 expression is seen adjacent regions of microvascular proliferation, suggesting that perivascular regions represent a tumour stem cell niche. Ping et al. reported that CD133+ glioma cells were more likely to express the chemokine receptor CXCR4 [4]. Stimulation of CXCR4 with its ligand, CXCL12, induced VEGF-A expression and angiogenesis. These data further confirm the interaction between putative CD133+ tumour stem cells and the vascular microenvironment, and provide an additional avenue for anti-angiogenic therapy.

Anti-VEGF-A therapy is currently under evaluation in glioblastoma, where it has demonstrated changes in response and symptomatic improvement. However, disease progression is still inevitable. Several preclinical carcinoma models suggest that anti-VEGF drugs are most effective against immature vessels, but dual targeting of VEGF and PDGF signaling is effective against both immature and mature vasculature. Nevertheless, Navis et al. [5] have shown that PDGFRbeta blockade has little additive efficacy to anti-VEGF therapy in a diffuse orthotopic glioma model. Their data shows the value of using relevant models to make clinical decisions.

4	The chemokine CXCL12 and its receptor CXCR4 promote glioma stem cell-mediated VEGF production and tumour angiogenesis via PI3K/AKT signalling Yi-fang Ping, Xiao-hong Yao, Jian-yong Jiang, Lin-tao Zhao, Shi-cang Yu, Tao Jiang, Marie CM Lin, Jian-hong Chen, Bin Wang, Rong Zhang, You-hong Cui, Cheng Qian, Ji Ming Wang, Xiu-wu Bian The Journal of Pathology 2011; 224: 344-354. (Original paper)
5	Effects of targeting the VEGF and PDGF pathways in diffuse orthotopic glioma models Anna C Navis, Bob C Hamans, An Claes, Arend Heerschap, Judith WM Jeuken, Pieter Wesseling, William PJ Leenders The Journal of Pathology 2011; 223: 626-634. (Original paper)

Not all VEGFs are made equal

The VEGF signaling pathways, with their numerous ligands receptors and co-receptors, are further complicated by the existence of ligand splice variants with distinct biochemical and physiological properties. All splice variant lengths of VEGF-A may also exist in two additional forms, “a” and “b”, which differ at their C-terminal end. The “b” sequence is a minor variant that has been shown to act as a competitive inhibitor to the pro-angiogenic “a” sequence. Peiris-Pages et al. [6] demonstrated that VEGF-A₁₆₅b is expressed in less aggressive neuroblastoma cell lines. Moreover, the authors showed that the “b” isoform was able to suppress the growth and angiogenesis of a neuroblastoma xenograft model. This study has numerous potential implications for anti-VEGF therapy, including the development of specific antibodies that target the “a” but not “b” isoforms, and the use of VEGF-A₁₆₅b as a drug itself.

6	Balance of pro- versus anti-angiogenic splice isoforms of vascular endothelial growth factor as a regulator of neuroblastoma growth Maria Peiris-Pagès, Steven J Harper, David O Bates, Pramila Ramani The Journal of Pathology 2010; 222: 138-147. (Original paper)

Vascular Integrins

Angiogenesis may occur through the proliferation and migration of endothelial cells in preexisting vessels or the seeding, proliferation and migration of endothelial progenitor cells mobilized from the bone marrow. Genetic and pharmacological manipulation of members of the integrin family of cell surface adhesion molecules has been shown to modulate pathological angiogenesis. Moreover, mice deficient in beta3-integrin are reported to show increased expression of endothelial VEGF receptor 2 and increased pathological angiogenesis. To clarify the role of beta3-integrin in bone marrow endothelial progenitors, as opposed to in endothelium from pre-existing vessels, Watson et al. [7] transplanted beta3-integrin null bone marrow into several mouse tumour models. Their data revealed enhanced mobilization of endothelial progenitor cells in response to VEGF-A, and an increase in nonfunctional blood vessels. Authors from the same laboratory [8] also observed downregulation of alpha6-integrin on blood vessels supporting breast carcinoma. To test the hypothesis that alpha6-integrin was functionally involved in tumour-associated angiogenesis, the authors created an alpha6-integrin^flox/flox.Tie1-Cre mouse to knockout alpha6-integrin in endothelial cells. Genetic ablation of endothelial alpha6-integrin resulted in enhanced growth and angiogenesis of several preclinical tumour models, secondary in part to increased VEGF receptor 2 expression. These studies exemplify the complex roles of integrins, both in endothelial progenitors and preexisting vasculature, and their interplay with the VEGF signaling pathway in regulating tumour angiogenesis.

7	Deficiency of bone marrow β3-integrin enhances non-functional neovascularization Alan R Watson, Simon C Pitchford, Louise E Reynolds, Natalie Direkze, Mairi Brittan, Malcolm R Alison, Sara Rankin, Nicholas A Wright, Kairbaan M Hodivala-Dilke The Journal of Pathology 2010; 220: 435-445. (Original paper)
8	Genetic ablation of the alpha 6-integrin subunit in Tie1Cre mice enhances tumour angiogenesis Mitchel Germain, Adèle De Arcangelis, Stephen D Robinson, Marianne Baker, Bernardo Tavora, Gabriela D'Amico, Rita Silva, Vassiliki Kostourou, Louise E Reynolds, Alan Watson, J Louise Jones, Elisabeth Georges-Labouesse, Kairbaan Hodivala-Dilke The Journal of Pathology 2010; 220: 370-381. (Original paper)

Lymphangiogenesis, an irrelevance or a permissive route for metastasis?

Lymphatic angiogenesis is governed by distinct signaling pathways mediated by specific VEGF family members, such as VEGF-C and VEGF-D, and VEGF receptors, such as VEGF receptor 3 and neuropilin 2. However, in malignancy many of these receptors are expressed on vascular endothelium, blurring the distinction between angiogenic blood vessels and angiogenic lymphatics. There is also debate in the literature as to whether lymphatic metastasis occurs through preexisting lymphatic channels, which have been invaded by malignant cells, or newly formed lymphatic networks. In an attempt to clarify this quagmire, Koch et al. [9] examined the role of VEGF-D in developmental and tumourigenic lymphangiogenesis. The authors observed that VEGF-D^-/- mice bearing an orthotopic pancreatic cancer showed a reduction in lymph vessel density and lymph node metastasis. By contrast, VEGF-D had no influence on lymphangiogenesis during embryonic development of skin wound healing, suggesting that it has a context specific role in certain malignancies.

To assess the relevance of lymphatic versus blood vessel invasion in lymph node negative human breast cancer, Mohammed and colleagues [10] used immunohistochemistry to identify lymphatic and non-lymphatic blood vessels. They observed that lymphatic invasion accounted for 97% of cases with vascular invasion and was a multivariate prognostic factor. Taken together, these articles suggest that blocking lymphangiogenesis may be a valid therapeutic strategy in early malignancy.

9	VEGF-D deficiency in mice does not affect embryonic or postnatal lymphangiogenesis but reduces lymphatic metastasis Marta Koch, Daniela Dettori, An Van Nuffelen, Joris Souffreau, Lucia Marconcini, Goedele Wallays, Lieve Moons, Françoise Bruyère, Salvatore Oliviero, Agnes Noel, Jean-Michel Foidart, Peter Carmeliet, Mieke Dewerchin The Journal of Pathology 2009; 219: 356-364. (Original paper)
10	Objective assessment of lymphatic and blood vascular invasion in lymph node-negative breast carcinoma: findings from a large case series with long-term follow-up Rabab AA Mohammed, Stewart G Martin, Ali M Mahmmod, R. Douglas Macmillan, Andrew R Green, Emma C Paish, Ian O Ellis The Journal of Pathology 2011; 223: 358-365. (Invited commentary)

Pathophysiology of acute and chronic hypoxia

Research to understand the role of hypoxia in cancer has also informed the pathogenesis and treatment of several non-malignant conditions, including cerebral palsy, chronic obstructive pulmonary disease (COPD) and pathological retinal angiogenesis.

An acute hypoxic insult to the developing brain is a common cause of cerebral palsy and neurodevelopmental impairment. The brain responds to such an insult by stabilizing hypoxia inducible proteins, such as the alpha subunit of the HIF-1 complex. HIF-1 transcriptionally upregulates a number of genes to limit hypoxic damage, including erythropoietin (EPO), which has antiapoptotic effects. Yamada et al. exploited this mechanism, by treating a preclinical model of hypoxia with recombinant EPO, demonstrating a reduction in apoptosis [11]. Given that recombinant human EPO is available as a drug, these data have important preclinical implications for the design of clinical trials to evaluate the use of EPO in hypoxic brain injury.

By contrast, the HIF-1 complex is chronically upregulated in the large airways of patients with chronic obstructive pulmonary disease (COPD). In this setting Polosukhin et al. report that HIF-1 has deleterious effects, inducing goblet cell hyperplasia and driving the production of viscous mucus that can impair ventilation [12]. Thus, in contrast to the acute setting, blockade of the HIF-1 pathway in the context of COPD may be beneficial.

11	Erythropoietin protects against apoptosis and increases expression of non-neuronal cell markers in the hypoxia-injured developing brain Miko Yamada, Christopher Burke, Paul Colditz, David W Johnson, Glenda C Gobe The Journal of Pathology 2011; 224: 101-109. (Original paper)
12	Hypoxia-inducible factor-1 signalling promotes goblet cell hyperplasia in airway epithelium Vasiliy V Polosukhin, Justin M Cates, William E Lawson, Aaron P Milstone, Anton G Matafonov, Pierre P Massion, Jae Woo Lee, Scott H Randell, Timothy S Blackwell The Journal of Pathology 2011; 224: 203-211. (Original paper)

Modulating the angiogenic response in the retina

Retinal neovascularization in response to hypoxia or another proangiogenic stimulus is a frequent event in age-related macular degeneration, diabetic retinopathy and retinopathy of prematurity. Treatment with anti-VEGF-A results in significant improvements in visual acuity in many patients with these conditions. However, VEGF-A may be expressed by many cell types in the retina, and their relative contributions to neovascularization are poorly understood. To better understand the mechanisms behind retinal neovascularization secondary to VEGF, Bai et al. [13] performed a conditional VEGF-A knockout in Muller cells, which are one potential source of VEGF-A. They observed that, under normal conditions, knockout of VEGF-A in Muller cells had no phenotype. However, under conditions of ischaemia, the knockout mice showed significant reductions in neovascularization and vascular leakage. This study confirms the importance of VEGF-A, and provides a cellular context to better understand the mechanisms of VEGF-A driven pathological neovascularization.

In addition to the neovascular response, hypoxia is directly toxic to retinal ganglion cells. Sivakumar and colleagues demonstrated that this cell death is mediated in part by TNFalpha and IL-1beta from microglial cells [14]. Blockade of these signaling pathways with antibodies resulted in reduced apoptosis of retinal ganglion cells. This provides a preclinical rationale to support anti-VEGF therapy with anti-TNFalpha/IL-1beta antibodies in eye diseases caused by retinal hypoxia.

13	Müller cell-derived VEGF is a significant contributor to retinal neovascularization Yanyan Bai, Jian-xing Ma, Junjing Guo, Juanjuan Wang, Meili Zhu, Ying Chen, Yun-Zheng Le The Journal of Pathology 2009; 219: 446-454. (Original Paper)
14	Retinal ganglion cell death is induced by microglia derived pro-inflammatory cytokines in the hypoxic neonatal retina Viswanathan Sivakumar, Wallace S Foulds, Chi D Luu, Eng-Ang Ling, Charanjit Kaur The Journal of Pathology 2011; 224: 245-260. (Original paper)

Conclusion

The physiological and pathological responses to hypoxia and mechanisms of angiogenesis are complex and context specific. Nevertheless, the tools now exist to appropriately model these systems preclinically and to therapeutically manipulate specific aspects of these pathways. Clearly dissecting out the relative contributions of specific pathway members by genetic and pharmacological means in the relevant preclinical models is crucial to the success of any clinical trials of drugs that manipulate these biological phenomena.

Questions

The following questions can be answered by reading and reflecting upon the above annotation and the papers that are cited within it. Within the Royal College of Pathologists Continuing Professional Development (CPD) scheme, CPD points may be earned by writing reflective notes on the papers in this Virtual Issue and the questions are designed to act as a focus for this activity. To do this, you may wish to use the Royal College of Pathologists' reflective notes form.

Question 1	How would you develop a drug that disrupted HIF-2 signaling in renal cell carcinoma? What would you want to see preclinically to demonstrate activity and what patient population would you target to best identify clinical efficacy?
Question 2	Given the central role for VEGF-A signaling in highly angiogenic renal cell carcinomas and glioblastomas, why have anti-VEGF-A drugs not shown greater activity in these indications?
Question 3	A proportion of patients with wet age-related macular oedema do not respond to anti-VEGF-A therapy with ranibizumab/bevacizumab. What other pathways could be targeted to improve the anti-angiogenic effects of these agents?
Question 4	What are the potential toxic effects of anti-angiogenic therapy and for which patients would it not be suitable?
Question 5	Inflammation and angiogenesis are inter-related in malignant and non-malignant disease. How do inflammatory cells trigger the angiogenic switch?

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