Angiogenesis in canine adrenocortical tumors

Gene expression profiles of Angiopoietin-1 and -2, vascular endothelial growth factor and their receptors.

Miriam Kool, 0051470, 1-10-2008Supervisors: Sara Galac, Hans Kooistra, Jan Mol

Index

Abstract 3Introduction 4

Adrenocortical tumors and Cushing’s syndrome 4Pathogenesis of adrenocortical tumors 10Genes of interest 11Introduction to this study 18

Aims and hypothesis 19Material and methods 20

Tumor and normal adrenal material 20RNA isolation and cDNA synthesis 22Q-PCR for determining expression levels 22Regular PCR for detection of Ang-2443 24Quantification of the full length Ang-2 and Ang-2443 26Quantification of Ang-2 on protein level 27Data analysis 30

Results 32Q-PCR for determining expression levels 32Regular PCR for detection of Ang-2443 35Quantification of the full length Ang-2 and Ang-2443 37

Quantification of Ang-2 on protein level 38Conclusions 41Discussion 43Acknowledgements 51References 52

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Abstract

This paper describes a study concerning the expression of angiogenesis-related genes in cortisol secreting adrenocortical tumors in dogs. The aim of this study was to determine which angiogenesis-related genes are significantly up- or down-regulated in these tumors, when compared to normal adrenal tissue. Six genes, known to be involved in both normal and tumor angiogenesis, were chosen to evaluate: Angiopoietin 1 and 2, their receptor Tie-2, vascular endothelial growth factor (VEGF) and its receptors VEGFR 1 and -2. For quantification of gene expression on messenger RNA level, a quantitative RT-PCR was performed on material of 31 adrenocortical tumors and 9 normal, healthy adrenals. In addition to determining the expression levels of these genes, the presence of Ang-2443, a splice variant of Angiopoietin 2, was also investigated. In dogs this splice variant had not yet been described; therefore the aim was to investigate whether or not this variant was present in dogs, and if so, whether an association with cortisol secreting adrenocortical tumors could be proven. To achieve this, regular PCR and quantitative RT-PCR were performed.To investigate whether the differences in the expression of Ang-2, detected on messenger RNA level, were also present on protein level, a Western blot experiment was performed.

Q-PCR results showed no significant differences in expression levels of Angiopoietin 1, Tie-2 and VEGFR 2, and only minor changes in the expression levels of VEGF and VEGFR 1. However, for Angiopoietin 2, expression analysis showed a significant up-regulation in the tumor group when compared to normal adrenals. This up-regulation was confirmed on the protein level by means of Western blot. The presence of Ang-2443 was demonstrated in both normal adrenals and adrenocortical tumors. As for the full length Ang-2, expression analysis showed that this splice variant was significantly up-regulated in the tumor group. Additionally, a difference in expression levels between adenomas and carcinomas was shown for Ang-2443, with higher levels of up-regulation in the malignant tumors. Western blot results confirmed these findings on protein level.

The results of this study therefore strongly indicate a role of Angiopoietin 2 in the pathogenesis of canine adrenocortical tumors, whereas for the other genes in this study such a role is not implicated by the results. More research is needed to determine the nature of the role of Ang-2 and its splice variant Ang-2443 in the pathogenesis of canine adrenocortical tumors.

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Introduction

Adrenocortical tumors are one of the causes of Cushing’s syndrome; one of the most common endocrine disorders in dogs. In man many studies have been performed regarding the pathogenesis of these tumors, and among these, some studies that identify genes up- or down-regulated in these tumors in comparison to normal adrenal tissue. By means of microarray and quantitative PCR (q-PCR), many differentially expressed genes have been identified in man, among which some genes that are known to play a crucial role in tumor angiogenesis.Because angiogenesis is one of the main factors that facilitate tumor growth, and over-expression of genes involved in angiogenesis has been shown in many tumors in both human and canine origin, we have chosen to direct our attention in this study to the expression profiles of angiogenesis-related genes in canine adrenocortical tumors.

Adrenocortical tumors and Cushing’s syndrome

Tumors of the adrenal cortex are relatively common in dogs. These ATs (ATs) can be divided into groups based on their functionality and their malignancy. ATs may either be functional or non-functional. Most frequently, functional ATs rise from the zona fasciculata and secrete excessive amounts of glucocorticoids. An aldosteronoma, a tumor arising from the glomerular zone, is also possible, but rarely occurs. In a non-functional AT, the tumor cells do not produce any hormones; this kind of AT is called an incidentaloma. The remaining part of this introduction will focus on the functional cortisol secreting AT and the disease it causes in dogs. Apart from the distinction between functional and non-functional ATs, these tumors may also be divided based on their malignancy. In dogs, benign adenomas and malignant carcinomas of the adrenal cortex seem to occur in equal frequencies1,2. Functional ATs give rise to a complex of clinical symptoms called Cushing’s syndrome, which is one of the most common endocrine disorders in dogs. The symptoms in animals are caused by an excess of glucocorticoid secretion by the adrenal cortices.

Regulatory mechanisms of the hypothalamic-pituitary-adrenal axis

For a good understanding of Cushing’s syndrome and of how ATs contribute to this syndrome, a basic understanding of the regulatory mechanisms of the hypothalamic-pituitary-adrenal axis is essential.

Glucocorticoids are synthesized from cholesterol in the adrenal cortex, which consists of three different zones. The middle and inner zones (zona fasciculata and zona reticularis) form one functional unit, and are responsible for the glucocorticoid production. The production of glucocorticoids by the adrenal cortex is stimulated by adrenocorticotropic hormone (ACTH). ACTH is secreted in episodic bursts from the anterior lobe of the pituitary; the frequency and magnitude of these bursts are

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under central nervous system control. Release of ACTH is stimulated by hypothalamic hormones arginine-vasopressin (AVP) and corticotrophin releasing hormone (CRH). Several mechanisms act together to exert control over the amount of glucocorticoids produced. Stress, for example from illness or surgery, induces an increase in the amount of AVP and CRH secreted by the hypothalamus, thus increasing ACTH secretion. Immunological factors can also trigger ACTH release, by producing cytokines that stimulate CRH secretion.The major inhibiting factor in the regulation of glucocorticoid production is the negative feedback mechanism. High levels of glucocorticoids in the blood have an inhibiting influence on the hypothalamus -inhibiting CRH and AVP release- as well as on the pituitary, inhibiting ACTH release. Glucocorticoids also have an inhibitory effect on immune reactions, thus lowering the amount of cytokines produced and creating a second negative feedback loop.

Effects of glucocorticoids on tissue level

Glucocorticoids exert their effects on tissue level by diffusing into the cytoplasm of the target cell, and binding to cytoplasmic glucocorticoid receptors. The glucocorticoid-receptor complexes are then transferred into the nucleus, where they influence mRNA- and protein synthesis. Glucocorticoids increase the synthesis of key enzymes in gluconeogenesis, thus increasing the amount of gluconeogenesis on tissue level, while glucose metabolism and uptake are decreased. Protein synthesis also decreases, leading to larger amounts of free amino acids, which may serve as substrate for gluconeogenesis. Via these mechanisms glucocorticoids cause an elevation of blood glucose concentrations. Glucocorticoids also have a direct stimulatory effect on lipolysis. An indirect effect resulting from the elevated blood glucose levels is an increase in the amounts of insulin released from the endocrine pancreas. The increased insulin levels promote lipogenesis and fat deposition, thus overruling the direct lipolytic effect of glucocorticoids. On the longer term the continued increased insulin levels lead to insulin resistance, which can be the cause of a secondary development of diabetes mellitus.Apart from the metabolic effects, glucocorticoids also affect the immune system in an anti-inflammatory way. Some of these effects are mediated by lipocortin, a glucocorticoid induced protein that inhibits phospholipase A2. This inhibition results in a decrease in the production of leukotrienes, thromboxanes and prostaglandins, which are important inflammatory mediators. Glucocorticoids also have an inhibiting effect on the production of interleukins. The decreased production of interleukins, in turn results in an inhibition of the recruitment of neutrophils, monocytes and macrophages to an area of inflammation.

Types of hypercortisolism

Hypercortisolism, or Cushing’s syndrome typically develops in middle-aged to elderly dogs; small breeds tend to be more frequently affected. In this disease the regulatory mechanisms described above are disturbed, resulting in an over-

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secretion of glucocorticoids. Apart from functional ATs, two other major causes of hypercortisolism can be distinguished: pituitary- or ACTH-dependent hypercortisolism and an iatrogenic form resulting from long-term corticosteroid administration. In about 15% of dogs with spontaneous Cushing’s syndrome, the glucocorticoid excess is caused by a functional AT, producing large amounts of glucocorticoids. This form is also known as ACTH-independent hypercortisolism. As mentioned before, both adenomas and carcinomas occur, in equal frequencies. Because of the negative feedback mechanism, the pituitary release of ACTH is suppressed, resulting in low ACTH concentrations in the peripheral blood. The tumor cells are not sensitive to this feedback mechanism, and keep producing large amounts of glucocorticoids in spite of the low ACTH levels. However, the glucocorticoid-producing cells in the contralateral normal adrenal are normally sensitive to the low concentrations of ACTH, resulting in a decreased glucocorticoid production by these cells, which on the longer term will lead to atrophy of the normal adrenal gland.In the remaining 85% of dogs with non-iatrogenic Cushing’s syndrome, the hyper-secretion of cortisol is caused by a functional tumor in the pituitary gland, which produces large amounts of ACTH, and is much less sensitive to the inhibiting influence of the feedback mechanism. The increased secretion of ACTH results in a continuous stimulation of the adrenal cortices, thus leading to hyper-secretion of glucocorticoids, and on the longer term, adrenal hypertrophy. This form of hypercortisolism is called pituitary-dependent hypercortisolism (PDH). The iatrogenic form of Cushing’s syndrome is caused by long-term use of exogenous corticosteroids in high doses. Because of the feedback mechanism, ACTH secretion will decrease, thus decreasing the level of glucocorticoid production in the adrenal cortices. On the long term this will lead to atrophy of the glucocorticoid producing zones of the adrenal cortices. Because this study concerns the pathogenesis of cortisol secreting ATs, the following discussion of clinical signs, diagnostics and therapeutic options will focus on the ACTH-independent form of hypercortisolism.

Clinical signs and biochemical changes

Clinical and biochemical changes in animals with Cushing’s syndrome are diverse and can be attributed to the actions of glucocorticoids as described above. Common clinical signs associated with hypercortisolism are polyuria and polydipsia. These symptoms are a result of the lowering effect of glucocorticoids on the secretion of AVP by the hypothalamus, combined with an interference of glucocorticoids with the actions of AVP on cellular level in the kidney. The combined gluconeogenic, lipogenic and protein degrading effects result in polyphagia, centripetal obesity with a markedly enlarged abdomen, muscular atrophy and muscle weakness, lethargy and exercise intolerance. The effects of the glucocorticoid excess on the skin and hair coat commonly include alopecia and thinning of the skin. Not every patient will exhibit all of these clinical signs; the range of symptoms exhibited is different for each individual patient.

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Complications that may arise from hypercortisolism can be severe and include secondary diabetes mellitus, systemic hypertension, pulmonary thromboembolism, and congestive heart failure.

The excess of glucocorticoids also causes changes in biochemical and hematological parameters. Biochemically, an increase in alkaline phosphatase (AP) is a very common finding in patients with Cushing’s syndrome. This results from glucocorticoid-induced elevation of a heat stable iso-enzyme of alkaline phosphatase: AP65. Other common biochemical changes include mildly decreased thyroxin (T4) levels, and increased plasma levels of cholesterol, lipids and glucose. Hematological changes in patients may include a decrease in the amounts of lymphocytes and eosinophils, neutrophilic leukocytosis and mild erythrocytosis.

Establishing a diagnosis

Based on the previously mentioned clinical signs, biochemical and hematologic changes, a presumptive diagnosis of hypercortisolism can be established. If there is no history of corticosteroid administration, the spontaneous forms of Cushing’s syndrome remain as the only options. To confirm this presumptive diagnosis, and to determine whether the hypercortisolism is of adrenal or pituitary origin, some specific tests are indicated. Because of the episodic secretion of ACTH and glucocorticoids in normal individuals, a simple measurement of cortisol in plasma is not sufficient to establish a diagnosis: if measured during an episode of inactivity these values may be within reference ranges in hyperadrenal dogs. Fortunately other tests are available, that can be used apart or in combination to confirm a presumptive diagnosis of Cushing’s syndrome, and to determine the origin. The diagnosis of hypercortisolism can be confirmed by the urinary cortisol/creatinine ratio or the low dose dexamethasone suppression test. For differentiation between the two forms of the disease, the high dose dexamethasone suppression test can be performed and/or the basal plasma ACTH concentration can be measured.

The laboratory test most commonly used in the Netherlands to diagnose hypercortsolism is the urinary cortisol/ creatinine ratio. This test solves the problem of episodic secretion, by measuring the cortisol excretion over a longer period of time, and relating it to the urinary creatinine concentration. That makes this test a reliable parameter of the glucocorticoid production in the adrenal cortices. A second advantage is the possibility to combine this test for baseline glucocorticoid secretion levels, with the high-dose dexamethasone suppression test, to discriminate between pituitary and adrenocortical forms of the disease. The principle behind this, is the fact that most pituitary tumors are less sensitive to the inhibitory influence of glucocorticoids, but not completely insensitive. In other words, the ACTH secretion of a pituitary tumor cannot be suppressed by normal levels of glucocorticoids, but can be suppressed by administering a very high dosage of corticosteroids. In contrast, an adrenal tumor will show no reaction at all to even

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a high dosage of corticosteroids, being completely independent of ACTH stimulation, and thus insensitive to the ACTH mediated feedback mechanism. Thus, by administering a high dose of dexamethasone and measuring the effect on the urine cortisol/creatinine ratio, a distinction can be made between PDH and AT. In dogs with an AT no decrease in cortisol concentrations will occur, whereas in most cases of PDH the cortisol/creatinine ratio will show a decline of 50% or more. In some cases of PDH however, cortisol values do not decline in response to dexamethasone administration, so in patients that show less than 50% decline other tests are needed to discriminate between an AT and an inhibition resistant form of PDH.

The low-dose dexamethasone suppression test is a simple screening test for diagnosing hypercortisolism. This test is based on the resistance of ATs and the relative resistance of PDH to suppression by the negative feedback mechanism, resulting in a marked decline in cortisol concentrations in normal dogs, as opposed to a lack of suppression in the hyperadrenal patient. The last relevant test is the measurement of endogenous ACTH levels in plasma. In dogs with PDH, the ACTH levels will be elevated, since the pituitary tumor causing the disease produces high amounts of ACTH. In dogs with an AT however, the elevated plasma glucocorticoid levels will result in a suppression of pituitary ACTH release through the negative feedback mechanism.

In addition to these laboratory tests, diagnostic imaging can also contribute to the establishment of a diagnosis. Useful techniques in case of an AT include abdominal ultrasonography, thoracic radiography and computed tomography (CT). An AT usually presents as an adrenal mass of variable size and shape on the ultrasound. Because of the negative feedback mechanism, atrophy will have developed in the contralateral adrenal gland, which will therefore be small or undetectable. When performing ultrasonography, a careful examination of the liver for the presence of metastasis and a check for invasion of the tumor into adjacent tissues or blood vessels are other important aspects. Lately, CT has proven to be the most reliable method to visualize adrenals and detect metastasis in the lungs.

Therapeutic options

Once the diagnosis has been established, therapy can be started. In case of an AT the treatment of preference is adrenalectomy. The affected adrenal is completely removed, thus removing the source of the excess glucocorticoid production. If the tumor is located only in one adrenal gland, as is usually the case, adrenalectomy results in a complete cure. If no metastases were present at the time of surgery, the prognosis after adrenalectomy is good; however, dogs with metastases have a poor prognosis. Because of the atrophy of the contralateral adrenal, glucocorticoid supplementation is initially necessary. In the course of a few months, supplementation can be gradually decreased and discontinued when the contralateral adrenal has reached normal functionality. In case of a bilateral tumor,

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bilateral adrenalectomy is an option. After this procedure lifelong supplementation therapy of both glucocorticoids and mineralocorticoids is imperative.If the adrenal tumor cannot be safely removed, for instance if invasion in surrounding tissues and vessels has occurred, or if the condition of the patient does not allow surgery, the only remaining treatment option is the use of Lysodren (o,p`-DDD, Mitotane), a chemical agent which destroys the zones of the adrenal cortex. Lysodren is also the treatment of choice when metastases are present. Although ATs seem to be less sensitive to Mitotane treatment than the hypertrophic adrenals observed in PDH, treatment with Mitotane may still achieve good results. The most important complication of this therapy is the development of iatrogenic hypoadrenocorticism. Therefore, supplementation with glucocorticoids, mineralo-corticoids and salt is needed. In most cases, lifelong therapy with Lysodren, including the supplementation therapy, is necessary.

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Pathogenesis of adrenocortical tumors

In contrast to the extensive knowledge on the pathogenesis of Cushing’s syndrome caused by ATs, little information exists on the pathogenesis of these tumors themselves. Much is still unknown, for example on which molecular and genetic changes precede neoplastic transformation of adrenocortical cells and which factors influence tumor growth rate, cell differentiation, invasion into adjacent tissues or vessels and metastasis.One of the major factors known to be involved in the pathogenesis of many tumors is angiogenesis. For a tumor to grow beyond a certain size, the formation of new blood vessels within the tumor is needed, to provide the tumor cells with the oxygen and energy necessary for growth. Angiogenesis is also involved in facilitating metastasis: the newly formed blood vessels are often less well organized then normal blood vessels, which makes it easier for the tumor cells to gain access to the vascular network, en thus metastasize to distant tissues. Because of the importance of angiogenesis in tumor growth and metastasis, much research has been done to determine how tumor angiogenesis is regulated. Two major groups of genes were identified, which are crucial in both normal and tumor angiogenesis: the Angiopoietin family and the VEGF family. Both families consist of a number of different ligands and receptors; of the Angiopoietin family, Angiopoietin 1 and 2 and receptor Tie-2 are the major players in both normal and tumor angiogenesis. Of the VEGF family, this holds true for VEGF-A (usually just termed VEGF) and VEGF receptor 1 and 2.Much is already known about the role of these genes in regulation of angiogenesis in the normal individual, for instance in embryonic vascular development and vascular remodeling. Furthermore, all of these genes have been implicated in the pathogenesis of several tumor types in both human and canine patients, for instance showing increased expression levels, correlation to increased vessel density, poorer prognosis or less differentiated cells. On their role in the pathogenesis of ATs, however, only little is yet known. Studies using a combination of micro-array and quantitative PCR to determine expression levels of different genes in human ATs, have shown over-expression of Ang-23-5. Also, studies concerning expression of the Angiopoietin and the VEGF family members in mouse adrenal cortex have shown expression of all of these genes in the normal adrenal gland55,56. These data provide some preliminary evidence for the involvement of these genes in adrenocortical pathology. To my best knowledge no information has yet been published on the involvement of the VEGF and Angiopoietin families in the pathogenesis of ATs in dogs. Because of the crucial role of angiogenesis in tumor development, and the known involvement of the Angiopoietin and VEGF families in tumor angiogenesis, these two gene families are the focus of attention of this study. In the following section each of the six genes of interest is introduced, going over its functions and expression levels in the healthy individual and what is known about the functions and expression levels in tumor development. Based on these data, a hypothesis is formulated regarding the expected behavior of each of the genes in the ATs evaluated in this study.

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Genes of interest

Vascular Endothelial Growth Factor

Vascular endothelial growth factor (VEGF) is an angiogenic growth factor, which binds to specific endothelial receptors; VEGFR 1 and 2. It is a crucial factor in the early embryonic development of blood vessels, and plays a role in both vasculogenesis and angiogenesis6,32,34. A null mutation on the VEGF gene results in early embryonic death caused by defects in endothelial cell development, angiogenesis and hematopoiesis, both in homozygous and in heterozygous mice 34,35. In the postnatal period, VEGF continues to play an important role, for instance in organ development and skeletal growth33,35. In the adult individual, VEGF provides an anti-apoptotic signal to endothelial cells, stimulates proliferation and migration of these cells and increases vascular permeability9,12,32,33. VEGF expression has been demonstrated in virtually all vascularized tissues in the healthy adult individual, with the highest levels found in lung alveoli, renal glomeruli and adrenal cortex35,36,43. Interestingly, in mice the expression of VEGF in the adrenal cortex was found to be down-regulated in response to corticosteroid induced hypoadrenocorticism, an effect that in mice could be reversed by supplementing ACTH56. Regulation of VEGF expression levels involves up-regulation by several different factors, including hypoxia, several growth factors, inflammatory cytokines and thyroid hormone31,33,35. VEGF-induced angiogenesis plays a role in the vascular remodeling, which is present in many physiological and pathological processes, for instance in the female reproductive cycle, hair growth, wound healing, ischemia-induced collateral formation, endometriosis, rheumatoid arthritis, psoriasis and tumor growth32. VEGF has been proven to stimulate tumor angiogenesis in vivo and VEGF-negative cells exhibited a strongly impaired ability for tumor growth in nude mice32,34. Apart from its role in stimulating tumor angiogenesis, VEGF may also provide an autocrine survival signal for tumor cells, thus contributing to tumor survival and growth32,50,51. Increased expression and secretion of VEGF by both tumor cells and infiltrating immune cells has been demonstrated in a variety of human tumor types, including mammary carcinoma, hemangiosarcoma, lung cancer and lymphoma. In many of these tumor types, increased VEGF expression is associated with increased vascularity, metastasis, chemo-resistance and a poorer prognosis16,21,31,38. In dogs several studies regarding VEGF expression in diverse tumor types have been performed, showing increased VEGF expression in for instance mammary tumors, seminomas, squamous cell carcinomas and lymphomas13,38-40,42. In addition, increased expression of VEGF was linked to a poorer prognosis in dogs with various spontaneous tumors, receiving radiation therapy. Higher levels of VEGF expression tended to be present in the more aggressive and malignant tumor types, with less differentiated cells and higher vascularity39-41.

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In summary, VEGF is a strong promoting factor of angiogenesis, which plays important roles in both embryonic and adult vasculature. It has also been shown to be an important factor in tumor development, both by stimulating tumor angiogenesis, and by directly stimulating tumor cell survival and growth. In many tumors increased expression has been demonstrated, often in combination with negative prognostic features.Because of its up-regulation in numerous tumor types, and its correlation to negative prognostic features in many of these tumors, in the present study, an up-regulation of VEGF in the ATs was expected. Within the tumor group, the adenomas were expected to show a lesser degree of up-regulation of this gene than the carcinomas.

VEGF receptor 1

VEGF receptor 1 (VEGFR 1), or Fms like tyrosine kinase 1 (Flt-1), is the first of the two endothelial receptors for VEGF. The exact role of this receptor is still subject to discussion. It binds VEGF with a high affinity, but has low tyrosine kinase activity, which gives rise to the theory of it being predominantly a decoy receptor, preventing binding of VEGF to the more active VEGFR 2. In embryonic development VEGFR 1 indeed seems to act as a negative regulator of VEGF activity, as shown by the excessive proliferation of angioblasts and lack of vessel organization, resulting in early embryonic death, in VEGFR 1 null mice44. However, under certain conditions VEGFR 1 signaling does have a stimulatory role, promoting endothelial cell survival, growth and migration, angiogenesis and vascular permeability32,33,44. Possibly, receptor interactions with VEGFR 2 are involved in determining whether VEGFR 1 has a positive or negative regulatory effect under given conditions45. Apart from its roles as both negative and positive regulator in endothelial cells, VEGFR 1 signaling is also involved in hematopoiesis, monocyte and macrophage migration and development of osteoclasts, osteoblasts and the bone marrow cavity33,35,44,46.VEGFR 1 expression in the healthy adult individual was detected in a number of normal cell- and tissue types including endothelial cells, monocytes, macrophages, smooth muscle cells, gastrointestinal epithelium and osteoblasts32,44,47,48. Expression was also shown in the adrenal cortex of the mouse, but was not sensitive to inhibition by iatrogenic hypoadrenocorticism56. VEGFR 1 was shown to respond to hypoxia and macrophage activation with a rise in expression levels33,44. Apart from their roles in the normal physiology, the VEGF receptors have been implicated in many pathological situations, for instance in tumor development, stimulating tumor growth, angiogenesis, metastasis and ascites formation, but also in inflammatory diseases such as arthritis or psoriasis. VEGFR 1 expression was found to be increased in several human tumor types, including colorectal tumors, leukemia’s, renal and mammary carcinomas32,33,37,49. In the study on expression of VEGF and its receptors in canine lymphomas, VEGFR 1 expression was detected in 54% of lymphomas, in variable amounts, with high levels of expression in 23% of the investigated tumors38.

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In summary, VEGFR 1 is a VEGF receptor with both negative and positive regulatory functions, which plays a role in various pathological conditions, including tumor development. Its expression is increased in a number of different tumor types.Because VEGFR 1 has both a negative and a positive regulatory role in angiogenesis, the formulation of a hypothesis regarding its behavior in canine ATs is difficult. However, because of the known up-regulation of this gene in several human tumor types, and the implicated role of this receptor in the pathogenesis of many pathological situations, an up-regulation of this gene in the ATs, seems the most likely outcome of this study.

VEGF receptor 2

VEGF receptor 2 (VEGFR 2), or kinase insert domain receptor (KDR), is the other endothelial receptor for VEGF. Despite its lower affinity for VEGF, this receptor is the primary mediator of VEGF signaling32,33. Binding of VEGF to this receptor promotes endothelial cell survival, proliferation and migration, vessel formation and an increase in vessel permeability33. Mice lacking this receptor show a complete lack of endothelial cell development, a complete absence of vasculogenesis and strongly impaired hematopoiesis, resulting in early embryonic death 8,32,33.In the healthy adult individual, expression of VEGFR 2 has been detected in endothelial cells of almost all vascularized tissue types, osteoblasts, neuronal cells and vascular smooth muscle32,43,44,47. In mice, expression of VEGFR 2 was demonstrated in the adrenal cortex, and VEGFR 2 levels showed a decrease in response to corticosteroid induced hypoadrenocorticism56. Expression levels of VEGFR 2 have been shown to increase in response to hypoxia and high levels of VEGF50,51. Apart from their roles in the normal physiology, the VEGF receptors have been implicated in many pathological situations, for instance in tumor growth. Signaling by VEGFR 2 may contribute to tumor pathology both in a paracrine way, stimulating angiogenesis, and in an autocrine way, stimulating tumor cell survival and growth50,51. In man, increased VEGFR 2 expression has been shown in several tumor types including lymphomas, lung-, renal- and mammary carcinomas, breast- and gastric cancer32,37,52,53. In the previously mentioned study on the expression of VEGF and its receptors in canine lymphoma, only 28% of tumors showed VEGFR 2 expression, however, the levels of expression were markedly higher than those in normal lymph nodes38. Canine mammary tumors were also shown to express VEGFR 2, the levels of expression increasing significantly with malignancy and a less differentiated cell type50,51.

Resuming, VEGFR 2 is the primary mediator of VEGF signaling and binding of VEGF stimulates angiogenesis and vascular permeability. It has been implicated as a factor in the pathogenesis of a variety of tumor types, and increased expression in many tumor types has been demonstrated.Because of its function as the primary mediator of the angiogenesis promoting effect of VEGF, and because of its role in the pathogenesis of various tumor types, an up-regulation of VEGFR 2 in the canine AT’s investigated in this study was expected.

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With regard to the tumor grade, a higher degree of up-regulation in the carcinoma-group was expected.

Angiopoietin 1

Angiopoietin 1 is the primary agonist for the Tie-2 receptor. It plays a crucial role in embryonic vascular development, in which it induces vessel sprouting and remodeling, mediates endothelial cell interactions with, and adherence to, surrounding support cells and is involved in proper development of the cardiac trabeculations6,8. A knockout study showed that Ang-1 negative mice die early in embryonic development, due to defects in angiogenesis, heart development and vessel integrity, whereas over-expression of Ang-1 leads to development of larger amounts of vessels, displaying more branching and a larger diameter8,55. In the adult individual Ang-1 decreases vascular permeability, protecting against vascular leakage, acts as an anti-inflammatory agent, provides a chemotactic signal for endothelial cells and promotes stabilization of vascular networks by inhibiting endothelial cell apoptosis6,7,9,10-12. Expression of Ang-1 has been demonstrated in normal adult vasculature, as well as in a wide range of normal adult tissues including skeletal muscle, ovary, uterus, placenta and prostate. Expression was shown to decrease in response to certain inflammatory mediators, for instance TNF and IL-1α β11,31. In healthy adult mice expression of Ang-1 in the adrenal cortex had been demonstrated. Interestingly, expression levels of Ang-1 were found to be down-regulated in corticosteroid induced hypoadrenocorticism in mice55. In canines a study has been performed determining expression levels of Ang-1 and 2 and VEGF in spontaneous tumors and normal tissues. In this study Ang-1 was found to be expressed in all normal tissues examined, including the adrenal cortex; highest expression levels were found in lung tissue, skeletal muscle and small intestine13.On the expression and role of Ang-1 in tumors, contradicting information exists. Over-expression of Ang-1 has been demonstrated in acute and chronic myeloid leukemia’s, glioblastomas and several breast cancer cell lines. In contrast, artificial over-expression of Ang-1 in a different a human breast cancer cell line, led to a reduction in tumor growth14,29,30. In a number of other studies no significant differences in expression levels were found between normal tissue and tumors. In the previously mentioned study on Ang-1 expression in canine tissues and tumors, over-expression was present in only one of the seven investigated tumors, a mammary spindle cell carcinoma11. To my knowledge no other studies involving Ang-1 expression levels in canine ATs have yet been performed.

In summary, Ang-1 is a stabilizing factor on the vascular endothelium, which acts through its receptor Tie-2 to regulate embryonic angiogenesis, and is expressed in most adult tissue types. Its role in tumor development is still unclear, as studies on this subject showed contradicting results. Because of the contradicting results of studies addressing the expression of Ang-1 in tumors, a hypothesis regarding the expected behavior of this gene in canine ATs was difficult to formulate. However, in most tumors with active angiogenesis a destabilized vascular endothelium is observed, often in combination with increased

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vascular permeability, whereas Ang-1 promotes the opposite effects. Therefore we hypothesize that in the present study, Ang-1 will be down-regulated in canine ATs, when compared to normal adrenocortical tissue. We expect this down-regulation to be present to a greater extend in the malignant tumors.

Angiopoietin 2

Angiopoietin 2 is a competitive antagonist of Ang-1, which binds to the same common receptor, Tie-2, but does not induce receptor phosphorylation. Ang-2 antagonizes the stabilizing effect of Ang-1 on the vascular endothelial cells, leading to a more plastic, unstable state of these vessels. In the presence of VEGF, subsequent angiogenesis is facilitated, as shown by an increase in capillary diameter, proliferation and migration of endothelial cells and sprouting of new vessels. However, in the absence of VEGF, apoptosis and subsequent vessel regression will occur15. During embryogenesis Ang-2 is involved in vascular development as a functional antagonist of Ang-1. Artificial Ang-2 over-expression during this developmental period leads to similar abnormalities as seen in Ang-1 null-mice. Ang-2 may also play a role in embryonic lymphatic system development23.In the healthy individual, Ang-2 is present in high concentrations at sites of vascular remodeling, for instance in areas of wound healing and in the female reproductive tract6,15,55. Ang-2 expression increases in response to hypoxia; high levels of VEGF also result in an increase of its expression31. In a study investigating Ang-1 and Ang-2 expression levels in normal canine tissues and 7 spontaneous canine tumors, it was shown that like Ang-1, Ang-2 is expressed in all normal canine tissues, including the adrenal glands. Highest levels of expression were found in skeletal muscle, lung tissue and small intestine13. Because of its destabilizing effect on the vascular endothelium, which in presence of VEGF leads to angiogenesis, it seems likely that Ang-2 might also play a role in tumor angiogenesis. Over-expression of Ang-2 has indeed been demonstrated in many human tumors, including hepatocellular carcinomas, gastric carcinomas, squamous cell carcinomas, glioblastomas and lung carcinomas16-20,22,30. Furthermore, increased expression of Ang-2 was significantly correlated with increased vascular involvement, more advanced tumor stage and poorer prognosis in several types of human tumors, including hepatocellular carcinomas, gastric carcinomas, acute myeloid leukemia and lung cancer16,17,21,22. In these tumors, over-expression of Ang-2 was often seen in combination with VEGF over-expression17,19,22. Artificial over-expression of Ang-2 led to faster and more aggressive tumor growth, with increased vascular involvement and hemorrhaging in both gastric and hepatocellular carcinomas implanted in mice17,18. Microarray of human ATs has demonstrated an increased expression of Ang-2 in these tumors, which was confirmed and quantified by means of q-PCR3-5. In the previously mentioned study on Ang-1 and 2 expression levels in canine tissues and tumors, increased levels of expression were detected in both of the investigated mammary simple carcinomas, in the splenic hemangiosarcoma and the hepatocellular carcinoma13. To my knowledge no other studies involving Ang-2 expression levels in canine tumors have yet been performed.

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Recently, a splice variant of Angiopoietin 2 has been indentified in man, which is different from the original Ang-2 in missing exon 2. The alternatively spliced isoform is secreted as a 443 amino acid long, glycosylated homodimeric protein and is named Angiopoietin-2443 or Ang-2C. Like Ang-2, it binds to the Tie-2 receptor, but does not induce receptor phosphorylation. Receptor binding of Ang-2443 inhibits binding of Ang-1 and Ang-2 to the Tie-2 receptor, and also inhibits Ang-1 induced receptor phosphorylation61,62. In the study describing the isolation of Ang-2443, mRNA expression of this splice variant was detected alongside of full length Ang-2 expression in endothelial cells, as well as in several non-endothelial tumor cell lines and primary tumors such as hemangioma, gastric carcinoma and breast carcinoma. In the latter case Ang-2443

mRNA expression was tumor specific; in the adjacent normal mammary tissue no Ang-2443 mRNA could be detected61,63. These results suggest that Ang-2443 might function as a competitive antagonist of Ang-1 in the same way as Ang-2, and might play an important role in regulation of angiogenesis, for example in neoplasia and inflammatory processes.

In summary, Ang-2 is the primary antagonist of Ang-1, which has a destabilizing effect on the vascular endothelium, leading to either angiogenesis or vessel regression, depending on the presence or absence of VEGF. It plays an important role in tumor development as shown by its over-expression and correlation to negative features in many tumor types.Based on the existing knowledge on the functions and expression of Ang-2, our hypothesis was that expression levels of Ang-2 would be raised in canine ATs. With regard to the splice variant, our hypothesis is that Ang-2443 will be present in both ATs and normal adrenals, but will show higher levels of expression in the tumors. For both the full length Ang-2 and Ang-2443, a higher expression level in malignant ATs was expected.

Tie-2 receptor

Tyrosine kinase receptor Tie-2 (or TEK) is the common receptor for the Angiopoietin family, and binds both Ang-1 and Ang-2. Tie-2 regulated pathways play a crucial role in embryonic development of blood vessels, regulating vessel sprouting, remodeling and stabilization and promoting endothelial attachment to the underlying supportive tissues7. Mice lacking Tie-2 die in an early embryonic stage, showing incomplete cardiac development, widespread hemorrhage, decreased numbers of endothelial cells, fewer and simpler vessels, and decreased amounts of, and adhesion to, vessel support tissue7,23-25. Apart from its role in embryonic angiogenesis, Tie-2 signaling also seems to be involved in embryonic hematopoiesis, as hinted by the presence of this receptor on several hemopoietic cell types23. Tie-2 deficient embryo’s have a pale and anemic appearance and lack the ability to develop definitive hemopoietic cells24,26.In the healthy adult individual, Tie-2 is present in endothelial cells and several hemopoietic cells, and is up-regulated at sites of angiogenesis7,23,28,29. Expression levels of Tie-2 rise in response to hypoxia and certain inflammatory cytokines, such

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as TNF and interleukin-1α β31. In the adult individual, Tie-2 is involved in regulation of both vascular stabilization and vascular remodeling. Persistent expression and -Ang-1 induced- phosphorylation of Tie-2 provides a survival signal for endothelial cells, preventing apoptosis, increasing vascular stability and decreasing vessel permeability7,23. On the other hand, Ang-2 binding to the Tie-2 receptor promotes detachment of the endothelial cells from surrounding support tissue. In the presence of VEGF this detachment facilitates vessel sprouting, leading to new vessel formation and vascular remodeling. However, in absence of VEGF this detachment precedes apoptosis and vessel regression. In mice, expression of Tie-2 in the adrenal cortex has been demonstrated, which, like Ang-1, was down-regulated in response to corticosteroid induced hypoadrenocorticism. Apart from its functions in regulation of vascular integrity, Tie-2 signaling may also play a role in adult hematopoiesis28. The up-regulation of Tie-2 in sites of angiogenesis, and the increase in its expression levels in response to hypoxia and inflammatory mediators, suggest that Tie-2 might also show increased expression in tumors, in which angiogenesis, hypoxia and inflammatory mediators are frequently present. Indeed, Tie-2 mediated pathways appear to play a role in tumor pathogenesis. In man, an increased expression of the Tie-2 receptor has been demonstrated in a variety of tumors, including acute and chronic myeloid leukemia’s, glioblastomas, mammary carcinomas, and gastric and hepatocellular carcinomas14,17,27-30. High expression levels of Tie-2 were often correlated with negative features, like less differentiated cells, increased tumor size and cellularity and a poorer prognosis21,27. To my knowledge no studies have yet been performed on the expression of Tie-2 in canine tumors.

Resuming, Tie-2 is the common receptor for Ang-1 and 2, and plays important roles in both vascular stabilization and remodeling. Because of its actions, a role in tumor angiogenesis seems plausible, and over-expression of this gene and a correlation to poor prognosis and negative features have indeed been shown in many human tumor types.Based on the functions and expression data of Tie-2, a raised expression level of Tie-2 in the canine ATs included in this study was expected. Furthermore, a higher expression in the carcinomas was expected, when compared to adenomas.

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Introduction to this study

From the previous descriptions of Ang-1 and 2, their receptor Tie-2 and VEGF and its receptors, can be concluded that all of these genes have important functions in both physiological processes involving angiogenesis and pathological situations. In particular, a role in tumor pathogenesis has been implicated for all of them. A better understanding of the functions, mechanisms of action and expression profiles of these genes, in both healthy and diseased tissue, thus may contribute to a better understanding of tumor pathogenesis. Furthermore, research into the role of these genes in tumor pathogenesis may lead to the development of new diagnostic and prognostic markers and ultimately, new therapeutic strategies.Already, research into these genes in human medicine has led to the discovery of the use of some of them as prognostic markers. Also, experimental therapeutic strategies, for example blocking the VEGF signaling pathway, have shown promising results. In veterinary medicine however, much less is known about the function of these genes in tumor pathogenesis or their use in the development of prognostic and diagnostic markers, whereas I could find no information at all on their use in new therapeutic strategies.

So, in spite of the large amount of knowledge already present on these genes and their functions, much also still remains to be discovered, especially in the veterinary field. With this study we hope to gain more knowledge on the role of the Angiopoietin and VEGF families in the pathogenesis of canine ATs.

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Aims and hypotheses

The aim of this study was to determine which genes involved in angiogenesis, are significantly up- or down regulated in ATs, as compared to normal adrenal tissue. A comparison between adenomas and carcinomas is also made, to determine whether there is a significant difference in expression levels between these two groups of tumors.In addition to determining the expression levels of these genes, we were interested to know whether the splice variant of Angiopoietin 2, which has recently been identified in humans, is also present in dogs. If this splice variant could be detected in dogs, we were also interested in its expression levels, and in differences in expression profiles between the ATs and normal adrenal tissue.

To determine the expression levels of the genes of interest on messenger RNA level, a quantitative RT-PCR was performed on samples from 31 ATs and 9 normal adrenal cortices. Six genes that play a prominent role in both normal and tumor angiogenesis were investigated: Angiopoietin 1 and 2, their receptor Tie-2, VEGF and its receptors VEGF receptor 1 and 2. For detecting the presence of the splice variant of Angiopoietin 2, a regular PCR was performed on all of the samples, which was designed to discriminate between the full length Ang-2 and the splice variant. For quantification of the expression levels of both the full length Ang-2 and its splice variant, a quantitative RT-PCR was performed, using primers specific for these two isoforms. Additionally, a Western blot experiment was performed to determine the amounts of Angiopoietin 2 protein present in the samples.

Based on present knowledge about the functions of the target genes, and what is known about their role in tumor pathogenesis, a hypothesis was formulated regarding the expected behavior of these genes in canine ATs. For Ang-2, Tie-2, VEGF, VEGFR 1 and 2 an up-regulation of gene expression was expected in the tumor group. Additionally, for Ang-2 we expect to confirm the presence of Ang-2443, and to find higher expression of this splice variant in the tumors. For Ang-1 a down-regulation in the tumor-group was expected.

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Material and methods

Tumor and normal adrenal material

In this study 31 ATs (table 1) were examined, along with 9 samples of normal adrenal tissue. Both adenomas and carcinomas were included. All ATs used in this study were derived from patients presented to the university clinic for companion animals at the faculty of veterinary medicine in Utrecht between 2001 and 2008. After establishing a diagnosis, ATs were removed surgically and the tumor material was stored at -70°C. After surgical removal, all tumors were subjected to histological evaluation, to confirm the diagnosis and to determine whether an individual tumor was benign or malignant. All of the tumors were recently evaluated by one pathologist. A list of the tumors used in this study is depicted in table 1.

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Number Owner name Tumorclassification

Patient nr. Pathology nr.

   1 Baljet A 501795 P05033042 Gercke, van C 307649 P03095513 Brouwer A 221016 P02077714 Does C 311316 P03129165 Doring C 228380 P03009926 Reek, van de C 412393 P0501181 7 Frederiks C 219680 P02072618 Fischer C 600914 P9539 Geest, van A 306624 P030805310 Groenedijk A 216001 P020193211 Hazekamp C? 601184 P231912 Keizer C? 602408 P253413 Mooyman A 304360 P030781014 Rooy A 225696 P021336515 Struyk C 216027 P020169316 Veen, van der C 202841 P030431017 Verberk C H01.0223B P010132318 Vermeulen C 410807 P041268019 Verweel C 228018 P021447720 Waldeck C 600227 P173421 Wilmes A 400056 P040161022 Jellema  A 508028  23 Jong, de  A 603713 P070199124 Ribbens A 609558 P07003125 Ottens  C 700948 P070220926 Laar, van  A H01.3118.Y  27 Harms  A H01.5333.C P0200347 28 Neumayer C 506709 P050662529 Askeland C 608328 P060962530 Vente  A 702869  31 Dijkstra  A 704792  

Table 1: This table lists the ATs used in this study, including their histological qualification, patient- and pathology-numbers. In the “tumor classification” column, A stands for adenoma and C for carcinoma. For four of the tumors, pathology numbers were not available, and only patient numbers are listed.

Normal, healthy adrenals were derived from dogs belonging to the General animal laboratory of Utrecht University. These dogs were euthanized for reasons other than Cushing’s syndrome and their adrenal glands were removed after euthanasia. These normal adrenal glands were also stored at -70°C, and were used in this study as a control group.

RNA isolation and cDNA synthesis

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RNA was isolated from the tissue samples using the RNeasy® mini kit, according to the manufacturer’s protocols. An optional extra DNAse step was performed to eliminate genomic DNA as much as possible. After RNA isolation, the total concentration of RNA was measured using Nanodrop® technology. Samples containing less than 100 ng/ l were excluded from the study. Subsequently cDNAμ synthesis was performed using the iSCRIPT® cDNA synthesis kit, according to manufacturer’s protocols. All RNA and cDNA samples were stored ad -20 °C.

Q-PCR for determining expression levels

To quantify the expression levels of the genes of interest on mRNA level, a quantitative polymerase chain reaction (q-PCR) was performed on cDNA of all samples. Primers for q-PCR were developed using the DNA-star® Primer Select program and the M-fold® program. The following criteria were used to select appropriate primer pairs:

- a product length between 100 and 200 base pairs- no alternate bindings sites for the primers- primers should span at least one intron, of preferably 3000 base pairs or

more- preferably, primers should end on either C or G- small difference in Tm of upper and lower primer- product loop free temperature below optimal annealing temperature

For validation of the primers, the temperature range and optimal annealing temperature were determined and PCR products were sequenced to determine whether the correct product had been formed. To determine the temperature range and the optimal annealing temperature, q-PCR gradients were run for each of the primer pairs, with annealing temperatures ranging from 55 to 65°C. One sample of normal adrenal tissue (M) was used for running all of the gradients.

After completing the PCR gradient reactions, results were analyzed to find the proper temperature range and the optimal annealing temperature for each of the primer pairs. Criteria for the temperature range within which the primer pairs work appropriately, were a reaction efficiency between 95 and 105% and a slope around -3,321. Within this range, the optimal annealing temperature was defined as the temperature, at which the efficiency was closest to 100%,

To ascertain that the correct products had been formed, PCR products were amplified in the Big Dye Cycle, and subsequently purified and sequenced. The protocols for the Big Dye Cycle, purification and sequencing are added to this report as appendices. For 5 out of 6 primer pairs, specific and correct products had been formed. However, the primer pair for Ang-2 produced a second - aspecific - product, therefore new q-PCR primers were developed using OligoExplorer®, a different program for primer design. The same primer criteria were used as described for

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primer design using the Primer Select program. However, no intron-spanning primer pairs were detected by the program, so a non intron-spanning primer pair was chosen. The procedure for validation of this new primer pair was identical to the process for validation of the other primer pairs, as described above. After sequencing, this primer pair was shown to deliver only the correct product, therefore it was suitable for use in q-PCR.

For all of the primer pairs the sequences, appropriate temperature range, optimal annealing temperature, positioning on the gene and product length are given (table 2).

Q-PCR primers (Position)Primer sequence

Optimal annealing temperature

(Range)

Product length

Angiopoietin 1 (926 -1087) 62 °C (61-63 °C) 162 bpUpper primer AAT AAT ATG CCA GAA CCC AAA AAGLower primer CCC CAG CCA ATA TTC ACC AGA G

Angiopoietin 2 (1002-1171) 57 °C (57-63 °C) 170 bpUpper primer CGG CGT GAA GAT GGC AGT GTT GLower primer GCC TCG TTT CCC TCC CAG TCC

Ang-2 2nd pair (96-182) 64 °C (61,4-64,5 °C) 87 bpUpper primer AGA AGC ATG GAC AGC ATC GGLower primer GTT GTC TGT TTC TGG CAG GAG G

Tie 2 (25-138) 58 °C (57-64 °C) 104 bpUpper primer CAG CTT ACC AGG TGG ACA TTT TTGLower primer GTC CGC TGG TGC TTG AGA TTT AG

VEGF (6-107) 58 °C 102 bpUpper primer CTT TCT GCT CTC CTG GGT GCLower primer GGT TTG TGC TCT CCT CCT GC

VEGF R 1 (Flt 1) (189-378) 63 °C (56-64 °C) 190 bpUpper primer GGC TCA GGC AAA CCA CACLower primer CCG GCA GGG GAT GAC GAT

VEGF R 2 (KDR) (3606-3785) 64 °C 181 bpUpper primer GGA AGA GGA AGT GTG TGA CCC CLower primer GAC CAT ACC ACT GTC CGT CTG G

Table 2: This table lists the primer pairs used for the target genes in this study, giving the sequences, appropriate temperature ranges, optimal annealing temperatures, positioning on the gene and product lengths.

To allow for normalization of gene expression, 4 reference genes were chosen, that are known to be stably expressed throughout different tissue types, both in normal

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and in tumor tissue. These genes were RPL8 (ribosomal protein L8), RPS5 (ribosomal protein S5), RPS19 (ribosomal protein S19) and HPRT (hypoxanthine phosphoribosyltransferase). The expression levels of these reference genes were also measured by q-PCR in all of the samples (Table 3).

Reference gene primers q-PCR

Sequence Optimal annealing temperature

RPL8 55 °Cupper CCA TGA ATC CTG TGG AGClower GTA GAG GGT TTG CCG ATG

RPS5 62,5 °Cupper TCA CTG GTG AGA ACC CCC Tlower CCT GAT TCA CAC GGC GTA G

RPS19 61 °Cupper CCT TCC TCA AAA AGT CTG GGlower GTT CTC ATC GTA GGG AGC AAG

HPRT 56 °Cupper AGC TTG CTG GTG AAA AGG AClower TTA TAG TCA AGG GCA TAT CC

   

Table 3: This table lists the primer pairs used for the reference genes, giving the sequences and optimal annealing temperatures.

After validation of the primer pairs, q-PCR was performed for all of the genes of interest and the reference genes, on both tumor- and normal samples. All reactions were run at the optimal annealing temperature and according to the same protocol.

Regular PCR for detection of Ang-2443

To detect the presence of Ang-2443, PCR primers were developed to distinguish between the two isoforms. These primers were developed using the DNA-star®

Primer-select program; primer conditions were the same as for development of the q-PCR primers, except for the product length. The primers were designed to anneal on either sides of exon 2, so that the full length Ang-2 will produce a long replication product of 553 basepairs, whereas Ang-2443 should result in a PCR product that is 155 basepairs shorter, as a result of the deletion of exon 2. Primer locations and expected PCR products are shown in a schematic diagram (figure 1). Primer characteristics are listed in table 4.

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Fig. 1: In this schematic diagram the primer locations (black arrows) are shown in both the full length Angiopoietin 2 and the splice variant. The replication product is indicated by the thick red line.

Regular PCR primers

(Position)Primer sequence

Optimal annealing

temperature

Product length

Ang-2 (106-658) 54 °C

Upper ACA GCA TCG GGA GAA GGC AGT ATC553 bp

(full length)

Lower TCT TCT TTT ATT GAC CGT AGT TGA398 bp

(splice variant) 

Table 4: This table shows the characteristics of the primer pair used for detection of Ang-2 443, giving the primer sequences, appropriate temperature range, optimal annealing temperature, positioning on the gene and product lengths.

To validate the primer set, a PCR was run on one of the normal samples (M), at the optimal annealing temperature as given by the primer select program (54 °C). Running the PCR at lower temperatures resulted in aspecific binding of the primers. Because no PCR products were formed when using ordinary Taq polymerase, platinum Taq was used. The resulting PCR products were separated by gel electrophoresis, using a ladder to determine the length of the fragments. Two bands were detected after gel electrophoresis of the test sample, with an approximate length of 550 and 400 basepairs. Bands were carefully cut from the gel, and the DNA was isolated from the gel using the QIAquick® gel extraction kit, and following the protocol provided by the manufacturer. After gel extraction, DNA concentrations of the extracted products were measured using Nanodrop® technology. To determine the identity of the PCR products, sequencing was performed, and the resulting sequence was blasted against the canine genome. Sequencing results showed that the primers were working properly, and the correct products had been formed.The next step was to determine whether Ang-2443 was present in all of the samples of both ATs and normal adrenals. To achieve this, a regular PCR was performed on all of the samples, using the primers described above. PCR-products were

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subsequently separated by gel electrophoresis, and the bands on the gel were evaluated.

Quantification of the full length Angiopoietin 2 and Ang-2443

Evaluation of the bands after regular PCR showed the presence of both full length Ang-2 and Ang-2443 mRNA in all of the samples, with varying band intensity. These results were of course very promising; however, because no quantitative information can be derived from this evaluation, a way to quantify the relative expression levels of the full length Ang-2 and Ang-2443 was needed. To achieve this, specific q-PCR primers were developed that would replicate only the full length Ang-2 and the splice variant, respectively. These primers were developed using the DNA-star® Primer Select program and the M-fold® program, using the same criteria as mentioned previously. Because the program could not detect any primers specific for the splice variant, that met all of these criteria, criteria for product length were loosened to a maximum of 250 basepairs, after which a suitable primer pair could be detected. To specifically replicate the full length Angiopoietin 2, a primer pair was chosen of which the upper primer annealed on exon 2, which is not present in the splice variant. For replication of the Ang-2443, a primer pair was selected, of which the upper primer annealed to the transition of exon 1 to exon 3, a transition which is not present in the full length Angiopoietin 2. Primer locations and expected PCR products are shown in a schematic diagram (figure 2). Primer characteristics are listed in table 5.

Fig. 2: the primer locations for both primer pairs (black arrows). The thick red line indicates the replication products in the full length and the splice variant, respectively.

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Q-PCR primers (Primer location)Primer sequence

Optimal annealing temperature

(Range)

Product length

 Ang-2 full length (376-485) 65 °C (61,4 - 65°C) 110 bp

Upper AGA ACC AGA CTG CCG TGA TLower TGT TGT CTG ATT TAA TAC TTG TGC

Ang-2443 (293-518) 64,5 °C 226 bpUpper TACGCA GTG GCT AAT TAA GGT ATTLower CTG GAG CTG ATC TTT CTC TTC TTT

 

Table 5: This table shows the characteristics of the q-PCR primer pairs used for quantification of the full length Ang-2 and Ang-2443, giving the primer sequences, appropriate temperature ranges, optimal annealing temperatures, positioning on the gene and product lengths.

For validation of these primer pairs, a temperature gradient was run at temperatures ranging from 55 °C to 65 °C. Results were analyzed to determine the optimal annealing temperature and the temperature range within which the PCR runs properly. PCR products were sequenced and blasted against the canine genome to determine whether the correct products had been formed. Sequence analysis showed that the correct products had been formed in both primer pairs.

Quantification of Ang-2 on protein level

To confirm whether the up-regulation of Angiopoietin 2 on mRNA level, as seen in the q-PCR experiments, was also present on protein level, a Western blot experiment was performed. In this way, the levels of Angiopoietin 2 protein were quantified in all of the tumors and normal adrenals, in order to make a comparison between the levels of Ang-2 protein in normal adrenal glands and ATs.As a first step, protein was isolated from all of the ATs and normal adrenal glands, following the protocol for isolation of protein from tissues. After isolation, protein levels were measured by spectrophotometry using the DC protein assay (BioRad®). A standard dilution series was made using Bovine Serum Albumin (BSA) in concentrations ranging from zero to 1.50 mg/ml. Absorbance was measured in all of the samples and in the standards, using triplicates. From the absorbance in the dilution series, a standard line was calculated, from which the protein concentrations in the samples could be deduced. Because the protein concentrations in the samples were much higher than in the dilution series, all samples were diluted 25 times in PBS before measurement. Protein concentrations in all of the samples were sufficient for further analysis by Western Blotting.

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Protein separation was achieved by performing gel electrophoresis. Because the molecular weights of the full length Ang-2 and Ang-2443 are 68 and 61 kD (glycosylated) or 57 and 51 kD (deglycosylated) respectively, for good separation of both isotypes, the gel was chosen with the highest discriminatory rate between 50 and 75 kD. The gel that fitted these demands best was a 7.5% polyacrylamide gel. Gels were cast according to the protocol provided by BioRad®, after which gel electrophoresis was performed. To visualize the progression of electrophoresis and to allow for accurate estimation of protein size, in each gel a ladder was included, consisting of dual color Precision plus Protein Standard (BioRad®). After gel electrophoresis, proteins were transferred onto Hybond® ECL nitrocellulose membrane. To prevent aspecific binding of the antibodies to the membrane, blocking of the membrane was performed using 4% ECL in TBST 0,1%. The primary antibody consisted of goat polyclonal Ang-2 (C-19): sc-7015, purchased from Santa Cruz biotechnologies, in a working dilution of 1: 200. Blots were incubated overnight with the primary antibody, at a temperature of 4 °C. Initially, the secondary antibody consisted of chicken anti-goat HRP conjugated IgG: sc-2961, purchased from Santa Cruz biotechnologies. Dilutions of 1: 5000 and 1: 40.000 were used. Blots were incubated with the secondary antibody for one hour at room temperature. However, no valid results were achieved using this secondary antibody. Regardless of the antibody concentration and the method of blocking, an extremely high amount of background staining occurred, making the blots unsuitable for analysis. Because no solution could be found for this problem, an alternative secondary antibody was used, consisting of donkey anti-goat HRP conjugated IgG: sc-2020, also purchased from Santa Cruz biotechnologies. This secondary antibody was used in a dilution of 1 : 20.000; good results were achieved.Antibody staining and detection were performed using the ECL advanced Western blotting detection kit (Amersham RPN2135). Protein detection was performed by chemiluminescence. For detection, blots were incubated for 2 minutes with the detection reagent. After incubation, detection was performed using the ChemiDoc®

machine. All blots were first developed for 10 seconds, after which a density measurement was performed on the bands, to calculate the optimal exposure time. Blots were then exposed for this period of time to achieve optimal results. Protocols for protein isolation, gel casting, gel electrophoresis, blotting and antibody staining and detection are added to this report as an appendix.To check that the lanes in the gel had been evenly loaded with protein, and to correct for differences in protein amounts, a loading control was performed, using Tubulin (Tubulin antibody TUB-1A2, ab11325, AbCam®) of mouse origin. The antibody was used in a 1: 2000 dilution and the expected band size for this protein was 55 kD. To perform the loading control experiment, blots were stripped to remove all antibodies, and subsequently blocked and incubated with Tubulin antibody for one hour at room temperature. A goat anti-mouse secondary antibody was used. To confirm the specificity of the Angiopoietin 2 staining, a blocking peptide for Ang-2 was used (Ang-2 blocking peptide, sc-7015P, Santa Cruz biotechnologies). By incubating the primary antibody solution with an excess of blocking peptide, all

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antibody is adsorbed to blocking peptide, and thus inactivated, leaving no active antibody in the solution. Therefore, no staining of the blots should occur. To achieve this, the primary antibody was dissolved in TBST 0,1% with 4% BSA at a dilution of 1: 200. Blocking peptide was then added in a concentration of 5 times the primary antibody concentration, and the resulting solution was left to incubate for 2 hours at room temperature. Blots were stripped to remove all antibodies, and subsequently blocked and incubated overnight with the pretreated Ang-2 antibody. Protocols for antibody staining and detection were the same as for the previous Western blot experiments.

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Data analysis

Q-PCR data were prepared for analysis using IQ5® software. This software analyses the raw data produced by the q-PCR, and calculates the Ct value for each sample. The Ct value is the number of cycles needed for the amount of fluorescence emitted by a sample to cross a set threshold. This threshold was set to 20 for all of the plates, to ensure that Ct values were comparable between different plates. A low Ct value stands for early crossing of the threshold, and thus high expression of the gene in question. From the Ct values of the standard dilution series, a standard line is calculated. In this standard line, the Ct values and the log starting quantity are set out against each other, resulting in a linear curve; the standard curve. After an ideal q-PCR, the slope of this curve is -3,321, which gives an efficiency of 100%. To see whether a PCR reaction had run properly, for each reaction the standard curve was evaluated. Efficiencies between 95% and 105% were considered acceptable for the reference genes, whereas for the target genes, efficiencies between 90% and 110% were considered acceptable. If necessary, deviating Ct values were left out in the calculation of the standard line to achieve these efficiencies, on the condition that this change did not cause samples to fall outside the reach of the standard line.

Another focus in preparing the q-PCR data for analysis was the melting curve. This curve shows the melting temperature of the resulting PCR product, which is product-specific. If one specific product is formed, the melting curve should show one single peak, at the temperature expected for that product. For the initial primer set of Angiopoietin 2, the melting curve consistently showed an abnormality. For the majority of the samples the melting curve had its peek at the expected temperature; however some of the samples showed a peek with a different melting temperature. Some samples also showed a double peek, with both the normal and the abnormal temperatures. To determine the cause of this abnormality, gel electrophoresis was run on 6 of the PCR products; two with normal peeks, two with abnormal peeks and two with double peeks. A double band on the gel was seen in only one of the samples, in which the abnormal product was shorter than the normal product. To indentify this abnormal product, sequencing was performed, and the resulting sequence was blasted against the canine genome. Blast results showed that the second product was not Angiopoietin related; the sequence matched part of the sequence of the canine mitochondrion.These results show that the initial primer pair for Ang-2 has an alternate binding site, and is therefore not suitable for use in expression analysis of this gene. New q-PCR primers were developed, and after validation of these primers, q-PCR for Angiopoietin 2 was repeated. This time a single peak at the expected temperature was seen in the melt curve of all of the samples, and data were valid and suitable for further analysis. The other five target genes also showed qualitatively good data, which were suitable for expression analysis.

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To confirm that the reference genes were stably expressed and suitable as reference genes, the relative expression data of these genes were analyzed using GeNorm®. This program calculates the stability of gene expression and gives an index which indicates whether or not a gene is stable enough to serve as a reference gene. An index below or equal to 1,5 is accepted for a reference gene, whereas a higher index indicates that the gene is not stable enough to serve as a reference gene. All of the reference genes used in this study had an index below 1,5 and were thus considered suitable for use as reference genes.

After preparation, CT values were exported to excel and analyzed using REST-excel, a program designed for comparing and analyzing q-PCR expression data. This program operates by comparing two groups, for instance normal controls versus tumors. The CT values of both target- and reference genes for both groups are entered into the program. From these values, the program calculates the normalized expression and then compares this normalized expression between the two groups. The results of this comparison are given in a fold change; a number that says how many times the gene is up- or down-regulated in the second group, as compared to the first. Apart from the fold change, the p-value is calculated, which represents the chance that the calculated difference between the two groups is based on coincidence. A p-value below 0.05 was considered significant.

The following groups were compared to each other:- Normal adrenals versus all tumors (N vs. T)- Normal adrenals versus adenomas (N vs. A)- Normal adrenals versus carcinomas (N vs. C)- Adenomas versus carcinomas (A vs. C)

The version of REST-excel used in this study does not support the use of multiple reference genes. Therefore, all of the comparisons were calculated separately for each of the reference genes. Fold changes given in the “Results” section are mean fold changes, calculated from the REST-excel results for each of the reference genes in one comparison.

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Results

Q-PCR for determining expression levels

After q-PCR had been performed on all of the target genes and reference genes, data analysis was performed using REST-excel. The first step was to check whether all data were valid and suitable for further analysis. In REST-excel calculations, the expression levels of one of the reference genes - RPL8 - showed significant down-regulation in the tumor-group as compared to the normal controls. The expression levels of RPL8 showed significant changes in all comparisons, except adenomas versus carcinomas, with fold changes around 1,5 (Fig. 3).

Fig. 3: In this figure, the changes in expression levels for RPL8 are depicted per category; significant results are marked with an asterisk. The categories stand for the different comparisons: normal versus all tumors (N vs. T), normal versus adenomas (N vs. A), adenomas versus carcinomas (A vs. C) and normal versus carcinomas (N vs. C). On the Y-axis, the fold change is set out; a fold change of 1 or minus 1 indicates no change in expression levels.

For this reason, RPL8 was excluded from this study as a reference gene, even though its expression was stable enough in GeNorm. The other three reference genes did not show any significant changes in expression levels between the tumor- and the control group, and were therefore suitable for use as reference genes.

As expected, expression of all genes was demonstrated in all of the samples, of both normal adrenals and adrenal tumors. In the following section the results of expression analysis will be discussed for each of the genes separately.Expression analysis of VEGF showed non-significant down-regulation in the whole tumor group, as well as in the carcinomas, whereas non-significant up-regulation was seen in the adenomas. When comparing adenomas to carcinomas, VEGF was

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shown to be significantly down-regulated in the carcinomas, with a fold change of approximately 8. VEGFR 1 showed a down-regulation in all of the comparisons, which only reached significance in the comparison of normal adrenals versus carcinomas. In this comparison, VEGFR 1 was shown to be down-regulated with a fold change around 2,5. Expression analysis for the last member of the VEGF family, VEGFR 2, did not show any significant changes in expression levels in any of the comparisons. The expression changes in the VEGF family are summarized in figure 4.

Fig. 4: This figure summarizes the expression changes in the VEGF family; significant results are marked with an asterisk. The results are shown per gene, and per category; categories represent the same comparisons as described for RPL8.

For the Angiopoietin family, expression analysis showed no significant changes in expression levels of Ang-1 and receptor Tie-2.In contrast, a clear up-regulation in the tumor-group was seen for Angiopoietin 2, which was significant in most of the REST-excel calculations for the following comparisons: normal adrenals versus all tumors (N vs. T), normal adrenals versus adenomas (N vs. A) and normal adrenals versus carcinomas (N vs. C). Only in the calculation using RPS5 as a reference gene, significance was not reached, but p-values were near-significant. Fold changes were comparable for all three reference genes and for all three comparisons, and ranged between 2 and 2,5. No significant difference in expression levels was seen between adenomas and carcinomas. The expression changes in the Angiopoietin family are summarized in figure 5.

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Fig. 5: This figure summarizes the expression changes in the VEGF family; significant results are marked with an asterisk. The results are shown per gene, and per category; categories represent the same comparisons as described for RPL8.

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Regular PCR for detection of Ang-2443

After development of the primers, the first step was to determine whether or not the splice variant was present in canine adrenal tissue. To achieve this, PCR was performed on a sample of normal adrenal tissue, and gel electrophoresis was performed on the resulting PCR products. The PCR of this sample resulted in the presence of two bands on the gel, with product lengths of around 550 and 400 basepairs respectively (Fig. 6).

Fig. 6: This photograph shows the results of electrophoresis; the ladder is located on the left side, and the three columns on the right represent the sample of normal adrenal tissue. On the gel, two bands are seen, with product lengths around 550 and 400 basepairs respectively.

To confirm that the bands on the gel represented the full length Angiopoietin 2 and Ang-2443, all bands were carefully cut from the gel. After DNA extraction from the gel, sequencing was performed, and the resulting sequences were blasted against the canine genome. The 550 bp product corresponded with the full-length sequence of Angiopoietin 2 (Fig. 7). The 400 bp product corresponded with two sequences in the Angiopoietin 2 gene, the first stretch terminating at basepair 311, the second starting at basepair 468 (Fig. 7). These positions correspond exactly with the expected sequence of the splice variant, where exon 2 (bp 312-467) should be missing. These results prove that the splice variant that had previously been described in man - Ang-2443 - is also present in canine adrenal tissue, and that the primers designed for detecting this splice variant function properly.

Fig 7: This schematic diagram shows the BLAST results of the 550- and the 400-basepair products, aligned to the Angiopoietin 2 gene.

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The next step was to determine whether Ang-2443 was present in all of the samples used in this study. To achieve this, the same PCR was performed on all of the samples of adrenal tumors and normal adrenals. PCR products were separated by gel electrophoresis, and presence of the 550 and 400-basepair bands was evaluated.Both bands were present in all of the samples, indicating the presence of both the full length and the alternatively spliced Angiopoietin 2 in all of the tumor and normal samples (Fig. 8). Band intensities varied considerably among the different samples. Remarkably, the most intensely stained bands corresponded mostly with malignant ATs. The intensity of the upper and lower bands, when compared to each other, also showed variation among the different samples, although the upper band was always the more prominent of the two.

Fig. 8: This photograph shows the results of electrophoresis ater performing regular PCR for detection of Ang-2443 on all of the samples. On the photograph, the ladder is located on the upper left side and on the lower right en left sides, samples 1 through 20 on the upper lane and samples 21 through 40 on the lower lane.

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Quantification of the full length Angiopoietin 2 and Ang-2443

After validation of both primer sets, and running of the q-PCR, data were analyzed in the same way as for the previous q-PCR experiments. Preparation of the data for REST-excel analysis included analysis of the standard curve and melting curve and a confirmation of the stability of the reference genes, using GeNorm® and REST-excel®. For all of the reference genes and for the full length Angiopoietin 2, the standard curve efficiencies were within the accepted limits (95-105 % for the reference genes and 90-110 % for the targets). However, for Ang-2443 the standard curve did not reach an efficiency below 125 %. Because REST-excel corrects for the efficiency, and better results could not be achieved with these primers, the results were accepted for further calculation in spite of the high efficiency. GeNorm and REST-excel calculations showed that all three reference genes were stable throughout the groups and thus suitable for use as reference genes.After preparation, CT-values for the reference genes and target genes were exported to excel, and expression analysis was performed using REST-excel. For comparison, the same groups were used as for the previous q-PCR experiments.REST-excel analysis showed a significant up-regulation of both the full length Angiopoietin 2 and Ang-2443 in the comparisons of normal adrenals versus all tumors (N vs. T), normal adrenals versus adenomas (N vs. A) and normal adrenals versus carcinomas (N vs. C). Fold changes were consistently around 2 times higher in the splice variant, indicating that in ATs, Ang-2443 is more strongly up-regulated than its full length counterpart. Expression levels for both genes tended to be higher in carcinomas than in adenomas. However, this difference in expression levels was only significant for Ang-2443. The expression changes for Ang-2 and Ang-2443 are summarized in figure 9.

Fig. 9: This figure summarizes the expression changes for the full length Angiopoietin 2 and Ang-2 443. Significant differences are marked with an asterisk. The results are shown per gene, and per category; categories represent the same comparisons as described for RPL8.

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Quantification of Angiopoietin 2 on protein level

After antibody staining and detection, a prominent band at 68 kD was detected in all of the samples and a faint 61 kD band in most of the samples. Band positions correspond with the expected protein sizes for the full length Ang-2 and Ang-2443

respectively. Band intensities vary considerably between samples for both isotypes (Fig. 10).

Fig. 10: This photograph shows the detection result of one of the blots, with the ladder located on the right side, and 4 samples located in the columns to the left.

After use of the blocking peptide, both the 68 kD band and the 61 kD band were no longer visible. Absence of these two bands when blocking peptide is used, shows that the bands indeed represent Angiopoietin 2; the upper band corresponding with the full length variant, and the lower band corresponding with Ang-2443.In part of the samples, a number of aspecific bands were detected in addition to the specific Ang-2 bands. This might be explained by the fact that a polyclonal primary antibody was used in this experiment, which often results in more aspecific binding. After treatment with blocking peptide, these additional bands were no longer visible, indicating that these bands most likely consist of degrading products of Ang-2. The absence of these bands after use of blocking peptide further confirms the specificity of the antibody. The loading control experiment using Tubulin antibodies showed presence of the expected band at 55 kD for all of the samples. Unfortunately, band intensities differed considerably between different samples, indicating uneven amounts of protein loaded. Therefore a correction of Ang-2 band intensities was necessary. To achieve an accurate estimation of Ang-2 expression in each of the samples, the relative quantities for both Ang-2 and Tubulin, as given by the Quantity One®

program on the ChemiDoc, were normalized to the exposure period in seconds.

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After normalization, Ang-2 band intensities were corrected for Tubulin band intensities in the corresponding samples.For the comparison of expression levels, two methods were used separately. The first method was a semi-quantitative method, in which band intensities for Ang-2, Ang-2443 and Tubulin were scored. Possible scores ranged from – (no band visible), ± (band visible, but with difficulty) to +++ (very thick band with high intensity). After scoring of the bands, results were compared between the following groups: normal controls (N), adenomas (A), carcinomas (C) and the whole tumor group (T). Results of this method of comparison showed that expression levels of Ang-2 were consistently much higher than those of Ang-2443. Both variants were present in higher amounts in the tumor groups, and for Ang-2443 a difference between adenomas and carcinomas seemed to be present.Because of the inaccuracy of this semi-quantitative assessment, a second method of comparison was also applied, in which the relative quantities as measured by the Quantity One® program were used for comparing the expression levels. For this method, the relative quantities as given by the Quantity One® program and corrected for Tubulin staining as described above, were grouped and means were calculated for each group. Groups were the same as for the first method of comparison. Results are shown in figures 11 and 12.

Fig. 11: This figure shows the expression changes on protein level for the full length Ang-2. The normalized and corrected band intensities are depicted on the Y axis and represent the expression level of the protein.

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Fig. 12: This figure shows the expression changes on protein level for Ang-2443. The normalized and corrected band intensities are depicted on the Y axis and represent the expression level of the protein.

The expression levels of the full length Ang-2 protein are considerably higher in the tumor group, in comparison to the normal controls. No difference is seen between adenomas and carcinomas (Fig. 11). Expression levels of Ang-2443 protein are generally much lower than those of the full length variant. However, like the full length Ang-2, Ang-2443 shows markedly higher expression levels in the tumor groups as compared to normal controls. Furthermore, for Ang-2443, a clear difference in expression levels is present between adenomas and carcinomas, with higher expression levels in the malignant tumors (Fig. 12). These results are in correspondence with the results of the semi-quantitative scoring method.

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Conclusions

From the results of this study, a number of conclusions can be drawn, with regard to the expression of the target genes in ATs. Of the six target genes, Angiopoietin 2 showed the most promising results. Expression analysis of the q-PCR results showed significant up-regulation of Ang-2 mRNA in the ATs, when compared to normal adrenal tissue. Additionally, by means of regular PCR and sequencing, the presence of Ang-2443, a splice variant of Ang-2, which misses exon 2, has been shown in both ATs and normal adrenal glands. This splice variant had previously been described in man, and has been linked to tumorigenesis in some human studies. To my best knowledge, this is the first study in which this splice variant has been described in the canine species. Expression analyses of the full length Ang-2 and Ang-2443 have been performed, to see whether the splice variant is differentially expressed between normal adrenal tissue and ATs. Results of this analysis show that both the full length Ang-2 and Ang-2443 are significantly up-regulated on mRNA level in the ATs. Fold changes were consistently around two times higher in the splice variant than in the full length, indicating that the splice variant is more strongly up-regulated in these tumors than the full length Ang-2. Another interesting outcome was that unlike the full-length Ang-2, Ang-2443 showed a clear up-regulation in carcinomas as compared to adenomas.To confirm the q-PCR findings, a Western blot experiment was performed, to quantify and compare the expression levels of both the full length Ang-2 and its splice variant, on protein level. Results of this experiment show a clear up-regulation on protein level of both the full length Ang-2 and Ang-2443 in the tumor groups, when compared to normal controls. For the full length Ang-2, no difference in expression levels between adenomas and carcinomas was seen, whereas for Ang-2443 markedly higher expression levels were seen in the carcinomas. The Western blot results thus confirm the q-PCR findings regarding the expression of Ang-2 and Ang-2443 in ATs. Both q-PCR and Western blot results thus strongly indicate a role of Ang-2 in the pathogenesis of ATs in dogs. Because both the full length Ang-2 and Ang-2443 are over-expressed in ATs, it seems likely that both isotypes are involved in tumor pathogenesis. The stronger up-regulation of Ang-2443 and its differential expression between adenomas and carcinomas might indicate a specific role of this splice variant in the pathogenesis of ATs.

For the other genes, the results of expression analysis were less prominent. No significant changes in mRNA expression levels were detected for Ang-1, Tie-2 and VEGFR 2. These results indicate that Ang-1, Tie-2 and VEGFR 2 are most probably not involved in ATigenesis in dogs. Expression analysis of VEGF and VEGFR 1 showed no significant changes in most of the comparisons. However, VEGF was significantly down-regulated in the comparison of carcinomas versus adenomas (C vs. A) and VEGFR 1 was significantly down-regulated in the comparison of normal adrenals versus adenomas (N vs. A). The roles of VEGFR 1 and VEGF are in canine

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AT pathogenesis are therefore still unclear, as down-regulation was in both cases only present in one of the comparisons and no changes were detected in the other comparisons. Furthermore, in the case of VEGF, down-regulation is not the expected result in case of a contribution to tumor pathogenesis of this gene. Therefore, more research is needed in order to draw valid conclusions about a possible role of these genes in canine AT pathogenesis.

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Discussion

Before discussing the actual results of expression analysis, some general remarks are in place about the materials en methods used in this study.

The first point of discussion concerns the method of tumor classification. As previously described, for comparison a distinction was made between normal adrenal glands, the whole tumor group, adenomas and carcinomas. The expression data within these groups were compared to each other, to detect differences in expression levels between the groups. The distinction between adenomas and carcinomas was based upon histological evaluation of the tumors and upon clinical data regarding metastasis. However, the classification of canine ATs raises some difficulties. A differentiation between benign and malignant tumors is often difficult, when no obvious metastases or invasive growth can be detected. As a result, different pathologists may come to different classifications on histological evaluation.To overcome this problem in the present study all the tumors were evaluated by one single pathologist. The tumor was regarded malignant when at least one or more of the following criteria were met: the presence of metastasis, vascular ingrowth and capsular invasion by the tumor cells. Tumors that lacked these three criteria were characterized as adenomas. This classification will most likely be reliable for most of the tumors, but the reliability would be greatly enhanced by using and combining more criteria. Therefore, caution is needed in the interpretation of expression analysis based on this tumor classification. In a recent publication on the indicators of malignancy in canine ATs, a large number of different criteria were assessed for their potential in AT classification66. This assessment resulted in a list of criteria, with a significant association with either adenomas or carcinomas. Significant indicators of malignancy in ATs were: a diameter over 2 cm, peripheral fibrosis, capsular invasion, trabecular growth pattern, hemorrhage, necrosis, and single-cell necrosis. Significant indicators of benign ATs were: hematopoiesis, fibrin thrombi, and cytoplasmic vacuolation. Apart from these morphologic characteristics, the potential of the immunohistochemical proliferation marker KI-67 in distinguishing benign and malignant ATs was also evaluated. This evaluation showed, that a high KI-67 proliferation index was significantly correlated to malignancy, and a threshold value of 2,4 would correctly classify 96% of all ATs. Thus, for AT classification, a combined evaluation of these morphological criteria and a KI-67 staining would produce the most reliable results. Re-assessing the ATs in that way would thus be a valuable recommendation for improving the reliability of the results of expression analysis.

A second point of discussion is the amount of angiogenesis itself. As explained in the introduction, angiogenesis plays an important role in the pathogenesis of many tumors. For this reason, genes that are involved in the regulation of angiogenesis were chosen as target genes for this study. In the hypothesis that genes involved in angiogenesis would be up-regulated, the presumption is made that angiogenesis is

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also more active in these ATs: if no increase in angiogenesis would be present, no up-regulation of any of these genes would be expected.The results of this gene expression study, especially the up-regulation of Ang-2, indicate that angiogenesis may indeed be increased. However, this cannot be concluded from the results of expression analysis alone. To confirm the involvement of increased angiogenesis in the pathogenesis of these tumors, an evaluation of the vascularity of these tumors should be performed. This would also strengthen the conclusions of this study. A difference in expression levels of some genes involved in angiogenesis is obviously present and the involvement of these genes in AT pathogenesis is therefore likely; however, without an evaluation of the vascularity of the tumors, no definitive conclusions can be drawn concerning the connection between the expression differences and actual angiogenesis. If an evaluation of the vascularity of the tumors were available, the correlation between increased vascularity and increased gene expression could be calculated, and a conclusion could be drawn concerning the relation between those two variables. Such an evaluation would thus be a valuable recommendation for future research.

Another point of discussion is the number of tumor- and normal samples used in this study. As described in material and methods, 31 ATs were evaluated, along with 9 normal adrenals. In an ideal situation, these groups should be of approximately the same size, so that a better comparison can be made. In this study, the negative effect of unequal group sizes could be seen in some of the comparisons. In these comparisons, a trend is visible in the expression levels, which does not reach significance, but might do so if the number of normal controls were doubled. Therefore, repeating the q-PCR analyses using more normal controls, so that equal group sizes are achieved, would considerably strengthen the results of this study.

To gain more insight in the influence of ACTH in the regulation of target gene expression, an ACTH-stimulation experiment was performed. However, in this experiment no differences between the stimulated group and the control samples could be detected, in either expression levels of the target genes, or cortisol concentrations. The absence of a rise in cortisol levels in the ACTH treated group shows that no ACTH stimulation has occurred in the tissue fragments. Therefore, from this experiment no conclusions could be drawn about the regulating effect of ACTH stimulation on the target genes, and the results of this experiment were thus left out of this report.However, as previously mentioned, ACTH has been reported to influence most of the target genes investigated in this study and ACTH levels are known to be extremely low in dogs with functional ATs. These low ACTH levels might well influence the target gene expression in these dogs, and thus the results of this study. Therefore, it may be a valuable addition to repeat this stimulation experiment, using multiple samples of both normal adrenal tissue and ATs and looking carefully at the conditions for this experiment. In this way, the influence of ACTH on the expression levels of all of the genes of interest in the canine adrenal gland can be made clear.

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For the Western blot experiment, a note of discussion concerns the amounts of protein loaded to the gel for electrophoresis. As described in the “Results” section, the loading control experiment did not result in equal amounts of Tubulin staining for each of the samples. This indicates that uneven amounts of protein have been loaded to the gel. The explanation for this uneven loading may lie in a number of different problems, for example inaccuracies in the protein measurement, pipetting errors or inadequate mixing of the dilutions for protein measurement. Another possible explanation lies in the homogeneity of the protein samples. In some of the samples, the protein lysate does not appear to be entirely homogeneous and although all samples were mixed before pipetting, this may still have led to differences in the amount of protein loaded for each sample. To minimize the negative effects of the unequal protein loading, two methods of comparison were applied, as described in the “Results” section; a semi-quantitative method involving scoring of the band intensities, and a method using the relative quantities as calculated by the ChemiDoc® software. For the last method, all Western blot results were normalized for the exposure time and subsequently corrected for Tubulin quantities. In this way the relative quantities could still be compared to each other in spite of the differences in the amounts of protein loaded. Both methods gave roughly the same results, which increases the reliability of these results. However, better and more reliable results would be achieved by repeating the experiment, and making sure that the amount of protein loaded is the same for each sample. If further research were to be done on this subject, repeating this experiment that way might thus be a valuable recommendation.

The results of the q-PCR, regular PCR and Western blot experiments performed in this study revealed some interesting, and sometimes unexpected, outcomes. In the next paragraphs a discussion of these results will follow for each of the target genes separately, taking into account the expectations, based on literature research, and examining potential explanations for unexpected results.

VEGFBased on the literature, an up-regulation of VEGF in the tumor group was expected, as VEGF is known to be up-regulated in many different types of tumors, both in man and in dogs. Additionally, in many studies, a correlation was shown between VEGF-levels and malignancy, in which malignant tumors tended to have higher levels of VEGF expression16,21,31,38,13,38,40,42. However, the results of expression analysis do not show an up-regulation of VEGF in the ATs investigated in this study. Instead, a non-significant down-regulation is seen in most comparisons, and even a significant 8-fold down-regulation when comparing carcinomas to adenomas. Another interesting result is that although VEGF is either not significantly changed or even down-regulated, Angiopoietin 2 is significantly up-regulated. In the literature, these factors often show a significant correlation; both are up-regulated together17,19,22. Also, VEGF is an important stimulatory factor of Ang-2 expression; high levels of VEGF induce an increase in Ang-2 expression31. Therefore the strong up-regulation of Ang-2, while VEGF remains unchanged or is even down-regulated, is a remarkable outcome.

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One possible explanation for the absence of the expected up-regulation of VEGF is that there is a difference in the behavior of VEGF between different tumor types and/or different species. Although raised VEGF levels have been shown in various tumors, both in man and in dogs, no studies have been performed concerning VEGF levels in ATs in either species. Possibly, VEGF behaves differently in these tumors; a difference between canine and human patients is also a possibility. Another possible explanation may be found in the type of tumor used in this study, and the regulation of VEGF expression. In this study, only functional ATs are investigated; in other words, tumors from dogs suffering from Cushing’s syndrome . In these dogs, the negative feedback mechanism results in a strong decrease in ACTH levels. This decrease might influence VEGF levels, as ACTH is one of the factors known to up-regulate VEGF expression, and a study in mice showed that its depletion in iatrogenic hypercortisolism led to a decrease in VEGF levels, which could be reversed by ACTH supplementation56.However, these theories still do not provide an explanation for the discrepancy in the behavior of VEGF and Angiopoietin 2. In order to find possible explanations, a literature search was performed, looking for factors that positively affect Ang-2 expression, but have no influence – or a negative influence – on VEGF expression. In the figure 13, a schematic representation is given of the regulatory mechanisms of the expression of both genes.

Fig. 13: In this diagram, a schematic representation is given of the regulatory mechanisms involved in the expression of VEGF and Ang-2. Factors that elevate Ang-2 expression, but have no influence (or a negative influence) on VEGF expression, are depicted in green.

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An interesting candidate to explain the discrepancy in behavior of both factors is glucose. High glucose levels induce Ang-2 expression, while VEGF expression is down-regulated in vivo in response to hyperglycemia64,65 (Fig. 13). As explained in the introduction, the high levels of glucocorticoids produced in dogs with Cushing’s syndrome , cause an increase in gluconeogenesis, and consequently an increase in blood glucose levels. That makes glucose an interesting candidate to explain the difference in behavior between VEGF and Ang-2. However, it needs to be mentioned than none of the dogs had suffered from diabetes mellitus.Apart from glucose, some other factors can be identified from the regulation diagram that up-regulate Ang-2, but have no effect on VEGF expression. These factors include for instance PI 3, HER 2 and basic fibroblast growth factor and are depicted in green in the diagram. To identify which factor actually causes the discrepancy, more research is needed. Recommendations for further research might include q-PCR for the factors that positively influence Ang-2, but not VEGF, and analysis of the plasma glucose levels in the patients from which the tumors were excised, to see if a correlation between glucose levels at the time of surgery and the expression levels of both genes can be found.The strong down-regulation of VEGF in adrenocortical carcinomas is more difficult to explain, as in the literature in many cases a positive correlation was shown between VEGF levels and malignancy, and to my best knowledge, no studies have yet reported a down-regulation of VEGF in malignant tumors. Further research is therefore needed to ascertain that VEGF is indeed down-regulated in canine adrenocortical carcinomas, and if confirmed, to determine possible mechanisms and causes of this down-regulation.

VEGFR 1 Expression analysis shows a down-regulation of VEGFR 1 mRNA, which reaches significance only in the comparison between normal adrenals and adenomas. These results contradict our hypothesis that VEGFR 1 would be up-regulated in ATs. However, as was discussed in the introduction to this gene, the exact role of this receptor is still subject to discussion, and although VEGFR 1 has been shown to be up-regulated in some studies32,33,37,49, this up-regulation was not consistently present in different kinds of tumors. A tumor-type dependent behavior of the expression levels of this gene is therefore likely. On the expression levels in ATs, to my knowledge no information was available in either man or dog. Furthermore, the predominant theory on the function of VEGFR 1 is that it acts as a decoy receptor, preventing the binding of VEGF to VEGFR 2. In this theory, VEGFR 1 would act as a negative regulatory receptor for angiogenesis. Down-regulation of this receptor would therefore be in line with our hypothesis that angiogenesis is increased in ATs, and genes stimulating angiogenesis should be up-regulated, whereas genes inhibiting angiogenesis should be down-regulated.

VEGFR 2Based on the role of VEGFR 2 as the primary mediator of VEGF’s stimulatory action on angiogenesis, and on its up-regulation in some tumors in the literature, our hypothesis was that this gene would be up-regulated in the tumors. However, the

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results of this study do not correspond with the hypothesis, as VEGFR 2 does not show any difference in expression levels in any of the comparisons made in REST excel.As for VEGF, there are a few possible explanations for these unexpected results. First, an increase in VEGFR 2 expression levels in tumors has only been shown in some studies, whereas in other studies no changes in VEGFR 2 levels were seen. Therefore, the behavior of VEGFR 2 may differ, depending on the tumor type and species, like the behavior of VEGFR 1. Unfortunately, no information on VEGFR 2 expression levels in ATs was available.Another possible explanation lies in the regulatory mechanism of VEGFR 2. As for VEGF, expression of this receptor was shown to decrease in response to low levels of ACTH, as was shown in mice with iatrogenic hypercortisolism56. As ACTH decreases to undetectable levels in dogs with Cushing’s syndrome as a result of the negative feedback mechanism, a lack of ACTH stimulation might explain the low levels of VEGFR 2.Finally, as VEGFR 2 is the primary mediator of VEGF action, it may not be surprising that both behave in the same way. The combination of normal to low VEGF levels and unchanged VEGFR 2 levels may simply indicate that the VEGF pathway is not active in these tumors. Further research is needed to determine whether the VEGF pathway is involved in canine AT pathogenesis, and if so, in what way it is involved.

Angiopoietin 1As described in the introduction to this gene, the formulation of a hypothesis regarding the behavior of Ang-1 in canine ATs was difficult, as contradicting information exists on its expression in tumors14,29,30. Therefore our hypothesis was based on the knowledge of the function of this gene: Ang-1 acts as a stabilizing factor on the vascular endothelium, whereas in most tumors with active angiogenesis a destabilized vascular endothelium is observed. Also, in many tumors this destabilization is observed in combination with increased vascular permeability, whereas Ang-1 functions in decreasing vascular permeability and protecting against vascular leakage6,7,9,10-12. For these reasons, we expected to find down-regulation of Angiopoietin 1 in canine ATs.However, the results of this study contradict our hypothesis, as no significant differences in expression levels were seen in any of the comparisons for Angiopoietin 1. A possible explanation for this unexpected result may be that as Ang-1 and Ang-2 function as competitive antagonists, the amount of angiogenesis and the stabilization or destabilization of the vascular network is a result of the balance between these two genes. A raise in Ang-2 levels might thus be sufficient in changing the balance towards destabilization and angiogenesis, without a decrease in Ang-1 being present. To test this hypothesis and to determine whether or not Ang-1 is involved in canine AT angiogenesis, further research is necessary.

Angiopoietin 2 As described in the introduction to this gene, Angiopoietin 2 is a regulatory protein in angiogenesis, which causes a destabilization of the vascular endothelium. In the

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presence of VEGF, this destabilization precedes proliferation and migration of endothelial cells and vessel sprouting, resulting in increased angiogenesis15. Because of these features, a role of Ang-2 in tumor pathogenesis in general and in tumor angiogenesis in particular was deemed likely. Indeed, numerous studies in both man and dog, have shown elevation of Ang-2 expression in different kinds of tumors, including human ATs3-5,16-20,22,30. In many cases this elevation was shown to correlate to increased vascularity, and often a correlation with tumor grade was also shown16,17,21,22. Based on these findings, the hypothesis was formulated that Ang-2 would also be involved in the pathogenesis of the canine ATs investigated in this study, and levels of Ang-2 expression in these tumors would be higher than those in normal adrenal glands. In accordance with this hypothesis, expression analysis performed on the ATs in this study, showed a clear up-regulation of Ang-2 on mRNA level, which was significant in almost all comparisons. To determine whether this up-regulation was also present on protein level, a Western blot study was performed. Results of this study showed a clear up-regulation of Ang-2 protein in the ATs, thereby confirming the q-PCR results.As already discussed, the presence of VEGF is needed for Ang-2 to play a role in stimulation of tumor angiogenesis. Although VEGF was not over-expressed in the ATs used in this study, it was present in all of the samples, thus enabling the angiogenesis-promoting role of Ang-2. These results therefore support the hypothesis that increased expression of Ang-2 contributes to tumor angiogenesis in ATs in dogs, and in that way plays a role in the pathogenesis of these tumors. To further strengthen this theory, it would be worthwhile to perform histological examination of all of the tumors, in which the vascularity is assessed. A correlation between elevated levels of Ang-2 and increased vascularity would provide further evidence that Ang-2 is indeed involved in AT pathogenesis by influencing tumor angiogenesis. Another useful recommendation for further research on this subject would be to perform immunohistochemistry, in order to indentify the exact localization of Ang-2 production within ATs. Another interesting outcome of this study was the existence of Ang-2443, a splice variant of Ang-2, which differs from the full length variant by missing exon 2. As discussed in the introduction, this splice variant had previously been identified in man and some studies have indicated a role in human tumor pathogenesis for this variant61,62. Because to our best knowledge, no information was available on the presence, or the role in tumor pathogenesis, of Ang-2443 in dogs, this study aimed to investigate the presence of this splice variant and if present, to quantify its expression. As discussed in the “Results” and “Conclusions” sections, in this study the presence of Ang-2443 in canine ATs and normal adrenal glands was confirmed by means of regular PCR, and its expression quantified by means of q-PCR and Western blot. Expression analysis showed an increased expression of both the full length Ang-2 and Ang-2443 in the investigated tumors on both mRNA level and protein level. These results are in line with our hypothesis, and with the up-regulation of Ang-2 as seen in the previous q-PCR experiments. An interesting outcome was that the fold changes were consistently around twice as high in Ang-2443 than in the full length

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variant; Ang-2443 was thus up-regulated to a higher extend than the full length Ang-2. Another interesting outcome was that unlike the full length Ang-2, Ang-2443

showed a clear difference in expression levels between adenomas and carcinomas, both on mRNA and on protein level. These results might indicate a specific role of Ang-2443 in the pathogenesis of these tumors, and are consistent with the specific up-regulation of this splice variant in some tumors in man. However, which specific role Ang-2443 might play in tumor angiogenesis is still unclear, as studies in humans showed that Ang-2 and Ang-2443 most likely function in the same way, as a competitive antagonist of Ang-1. Further research is needed to confirm a specific role in tumor angiogenesis of Ang-2443, and to determine the nature of this role.

Tie-2Based on the functions of Tie-2 and its raised expression in a variety of human tumor types, an up-regulation of this gene in the ATs was expected. However, in this study, no change in Tie-2 expression was detected in any of the comparisons.A possible explanation for this discrepancy lies in the regulation of Tie-2 expression. As for VEGF, VEGFR 2 and Ang-1, regulation of Tie-2 expression is ACTH dependent. A study showed that Tie-2 expression decreased in mice with iatrogenic hypercortisolism, which could be reversed by supplementing ACTH55. Because of the negative feedback mechanism, ACTH concentrations in dogs with corticosteroid producing ATs are extremely low, which may explain why no rise in Tie-2 levels was seen in these tumors. Another possibility is that the behavior of Tie-2 depends on the tumor type or species, and that over-expression of this gene is simply not involved in canine AT pathogenesis. To determine which of these theories is the most likely, more research is needed.

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Acknowledgements

Coming to the end of this paper, I would like to thank all the persons who have contributed to this study. Many thanks I owe to my supervisors Jan Mol, Hans Kooistra and Sara Galac, for their continuing guidance, support, input and suggestions, and for making it possible for me to stay just a few months longer and thus enabling me to really finish this project, instead of just handing it over to the next student. I would also like to thank Elpetra Sprang-Timmermans, Monique van Wolferen and Adri Slob for their great help and guidance in the laboratory work and their continuing input, without which I could never have done any of this. The other research analysts also deserve a thank you, for their help in the lab and for always answering my many questions. The same goes for the PhD students, especially Ana and Gaya for their help and suggestions, both during the weekly meetings and in the lab. And last but not least all of the other students and employees at the department, for their companionship and support, and for making the past nine months not only an extremely interesting research period, but also al lot of fun.

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