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Angiogenesis Proangiogenic - Angiogenic growth factors VEGF, FGF, TNF, HGF, IGF,TGF, Proliferin Anti angiogenetic - Thrombospondin -1, 2 Angiostatin Endostatin Platelet factor 4 Angiogenesis Local degradation of basement membrane Directional migration invasion Endothelial cell proliferation Capillary tube morphogenesis Coalescence of capillaries Vascular pruning What Is Metastasis? The ability of cancer cells to: 1. Penetrate into Lymphatic. Blood vessels. 2. Circulate through bloodstream. 4. Invade and grow in normal tissues elsewhere. It is the ability to spread to other tissues and organs that makes cancer a potentially life threatening disease. So what makes metastasis possible for a Cancerous tumor? 1. Cancer cells invade surounding tissues and vessels Blood vessel 2. Cancer cells are transported by the circulatory system to distant sites. 3. Cancer cells reinvade and grow at new location Metastasis Requires Angiogenesis Growth of a new network of blood vessels What Is Tumor Angiogenesis? Tumor angiogenesis Proliferation of a network of blood vessels that penetrates into cancerous growths. Function Supplying nutrients and oxygen and removing waste products. Mechanism Cance cells releas molecules that send signals to surrounding normal host tissue. This signaling activates certain genes in the host tissue that, in turn, make proteins to encourage growth of new blood vessels. Normal Angiogenesis: vasculogenesis Normal Angiogenesis in Adults New blood vessels form in the lining of the uterus during the menstrual cycle. Repair or regeneration of tissue during wound healing. Angiogenesis and Vascular Endothelial Cells The walls of blood vessels are formed by vascular endothelial cells . These cells rarely divide, doing so only about once every 3 years on average. When the situation requires it, angiogenesis can stimulate them to divide. Angiogenesis and Regulatory Proteins High inhibitors Rare cell Low activators= division No angiogenesis Low inhibitors Frequent cell High activators =division Angiogenesis Angiogenesis and Cancer The dilatation theory Before the 1960s Angiogenesis theory Without Angiogenesis, Tumor Growth Stops With Angiogenesis, Tumor Growth Proceeds In another experiment designed to find out whether cancer growth can continue when angiogenesis occurs, researchers compared the behavior of cancer cells in two regions of the same organ. Both locations in the eye had nutrients available, but only one could support angiogenesis. Scientists found that the same starting injection of cancer cells grew to 1-2mm in diameter and then stopped in the region without nearby blood vessels, but grew well beyond 2 mm when placed in the area where angiogenesis was possible. With angiogenesis, tumor growth continued • • What Prompts Angiogenesis? In an experiment designed to find out whether molecules from the cancer cells or from the surrounding host tissues are responsible for starting angiogenesis, scientists implanted cancer cells in a chamber bounded by a membrane with pores too small for the cells to exit. Under these conditions, angiogenesis still began in the region surrounding the implant. Small activator molecules produced by the cancer cells must have passed out of the chamber and signaled angiogenesis in the surrounding tissue • • Activators of Angiogenesis Once researchers knew that cancer cells can release molecules to activate the process of angiogenesis, the challenge became to find and study these angiogenesis-stimulating molecules in animal and human tumors. From such studies more than a dozen different proteins, as well as several smaller molecules, have been meaning ”,angiogenic“ identified as that they are released by tumors as signals for angiogenesis. Among these molecules, two proteins appear to be the most important for sustaining tumor growth: vascular endothelial and basic )VEGF( growth factor VEGF .)bFGF( fibroblast growth factor and bFGF are produced by many kinds of cancer cells and by certain .types of normal cells, too • • • The Angiogenesis Signaling Cascade VEGF and bFGF are first synthesized inside tumor cells and then secreted into the surrounding tissue. When they encounter endothelial cells, they bind to specific proteins, called receptors, sitting on the outer surface of the cells. The binding of either VEGF or bFGF to its appropriate receptor activates a series of relay proteins that transmits a signal into the nucleus of the endothelial cells. The nuclear signal ultimately prompts a group of genes to make products needed for new .endothelial cell growth • • Endothelial Cell Activation The activation of endothelial cells by VEGF or bFGF sets in motion a series of steps toward the creation of new blood vessels. First, the activated endothelial cells produce matrix a special ,)metalloproteinases (MMPs class of degradative enzymes. These enzymes are then released from the endothelial cells into the surrounding tissue. The MMPs break down the extracellular matrix—support material that fills the spaces between cells and is made of proteins and polysaccharides. Breakdown of this matrix permits the migration of endothelial cells. As they migrate into the surrounding tissues, activated endothelial cells begin to divide. Soon they organize into hollow tubes that evolve gradually into a mature network of blood vessels • • Inhibitors of Angiogenesis Although many tumors produce angiogenic molecules such as VEGF and bFGF, their presence is not enough to begin blood vessel growth. For angiogenesis to begin, these activator molecules must overcome a variety of angiogenesis inhibitors that normally restrain blood vessel growth. Almost a dozen naturally occurring proteins can inhibit angiogenesis. Among this group ,angiostatin of molecules, proteins called thrombospondin appear to and ,endostatin be especially important. A finely tuned balance, between the concentration of angiogenesis inhibitors and of activators such as VEGF and bFGF, determines whether a tumor can induce the growth of new blood vessels. To trigger angiogenesis, the production of activators must increase as the production of inhibitors decreases • • • Angiogenesis Inhibitors and Metastasis The discovery that angiogenesis inhibitors such as endostatin can restrain the growth of primary tumors raises the possibility that such inhibitors might also be able to slow tumor metastasis. In one striking study, researchers injected several kinds of mouse cancer cells beneath the animals' skin and allowed the cells to grow for about two weeks. The primary tumors were then removed, and the animals checked for several weeks. Typically, mice developed about 50 visible tumors from individual cancer cells that had spread to the lungs prior to removal of the primary tumor. But mice treated with angiostatin developed an average of only 2-3 tumors in their lungs. Inhibition of angiogenesis by angiostatin had reduced the rate of spread (metastasis) by about 20-fold • • • Angiogenesis and Tumor Dormancy It has been known for many years that cancer cells originating in a primary tumor can spread to another organ and form tiny, microscopic tumor masses (metastases) that can remain dormant for years. A likely explanation for this tumor dormancy is that no angiogenesis occurred, so the small tumor lacked the new blood vessels needed for continued growth. One possible reason for tumor dormancy may be that some primary tumors secrete the inhibitor angiostatin into the bloodstream, which then circulates throughout the body and inhibits blood vessel growth at other sites. This could prevent microscopic metastases from growing into visible tumors • • • Cancer in Angiogenesis-Deficient Mice Additional support for the idea that interfering with the process of angiogenesis can restrain tumor growth has come from genetic studies of mice. Scientists have recently created strains of mice that lack two genes, called Id1 and Id3, whose absence hinders angiogenesis. When mouse breast cancer cells are injected into such angiogenesis-deficient mutant mice, there is a small period of tumor growth, but the tumors regress completely after a few weeks, and the mice remain healthy with no signs of cancer. In contrast, normal mice injected with the same breast cancer cells die of cancer within a few weeks. When lung cancer cells are injected into the same strain of angiogenesis-deficient mutant mice, the results are slightly different. The lung cancer cells do develop into tumors in the mutant, but the tumors grow more slowly than in normal mice and fail to spread (metastasize) to other organs. As a result, the mutant mice live much longer than normal mice injected with the same kinds of lung .cancer cells • • • Angiogenesis Inhibitors in the • Treatment of Human Cancer Researchers are now asking if • inhibiting angiogenesis can slow down or prevent the growth and spread of cancer cells in humans. To answer this question, almost • two dozen angiogenesis inhibitors are currently being tested in cancer patients. The inhibitors being tested fall into several different categories, depending on their mechanism of action. Some inhibit endothelial cells directly, while others inhibit the angiogenesis signaling cascade or block the ability of endothelial cells to break down the extracellular matrix Drugs That Inhibit Angiogenesis Directly One class of angiogenesis inhibitors being tested in cancer patients are molecules that directly inhibit the growth of endothelial cells. Included in this category is endostatin, the naturally occurring protein known to inhibit tumor growth in animals. Another drug, combretastatin A4, causes growing endothelial cells to commit suicide (apoptosis). Other drugs, which interact with a molecule called integrin, also can promote the destruction of proliferating endothelial cells • • Old Drug With a New • Use Another interesting drug • is thalidomide, a sedative used in the 1950s that was subsequently taken off the market because it caused birth defects when taken by pregnant women. Although this drug clearly would not be suitable for pregnant women, its ability to prevent endothelial cells from forming new blood vessels might make it useful in treating non.pregnant cancer patients Drugs That Block the Angiogenesis Signaling Cascade A second group of angiogenesis inhibitors being tested in human clinical trials are molecules that interfere with steps in the angiogenesis signaling cascade. Included in this category are antiVEGF antibodies that block the VEGF receptor from binding growth factor. Another agent, interferon-alpha, is a naturally occurring protein that inhibits the production of bFGF and VEGF, preventing these growth factors from starting the signaling cascade. •Also, several synthetic drugs capable of interfering with endothelial cell receptors are being tested in cancer patients . On to Clinical Trials Researchers have answered many questions about angiogenesis, but many questions still remain. Scientists do not know whether using angiogenesis inhibitors to treat cancer will trigger unknown side effects, how long treatment will need to last, or whether tumor cells will find alternative routes to vascularization. To answer such questions, human clinical trials are currently under way. For an updated list of ongoing and currently planned clinical trials involving angiogenesis inhibitors, including phone numbers for obtaining additional information, refer to the National Cancer Institute CancerTrial ™ Web site, which has a section Angiogenesis Inhibitors in devoted to .Clinical Trials • • • Tumor angiogenesis Tumorgrowth Hypoxia Growth factors Vessels sprouting Tumor growth Rheumatoid Arthritis Blindness Stroke Cancer AIDS complications EXCESSIVE Psoriasis INSUFFICIENT Infertility Heart Disease Scleroderma Ulcers Tumorangiogenesis Stimulators vs. Inhibitors „ANGIOGENIC SWITCH“ Prevascular phase Vascular phase Proliferation - Progression - Metastases - Survival Mast cells Macrophages Recruitment activation Tumor Lytic enzymes Extra cellular matrix Blood vessel Angiogenic factors Hypoxia in Angiogenesis Carmeliet, Nature, 2000 Vascular endothelial growth factor Four isoforms: 121, 165, 189, 206 VEGF 121: diffusible VEGF 165: cell surface Two receptors: Flt-1, KDR Endothelial cell proliferation and migration Extracellular matrix degeneration Vascular permeability (VPF) • • • • • Vascular Morphogenesis Ligands and EC selective RTKs Vascular Endothelial Growth Factor VEGF-R2 (Flk 1/KDR): angioblast/EC proliferation VEGF-R1 (Flt 1): capillary tube formation Angiopoietin-1 / Angiopoietin-2 (-) Tie2: vessel maturation, periendothelial recruitment Tie1: intercapillary hemodynamic balance Distinct phenotype – complimentary function Pathological Vascular Growth Carmeliet, Nat Med, 2000 Cascade of Vessel Formation Vasodilatation Vascular permeability Extravasation of plasma proteins EC detachement and migration EC proliferation and sprouting Lumen formation Endothelial survival and differentiation Remodeling of endothelial network Cascade of Vessels Formation ANGIOPLAST Regression Extracellular matrix Physical barrier Production of angiogenic factors Reservoir of inactive growth factos (bFGF) Macrophages: secrete GFs Mediation of EC binding (integrins) EC migration, signalling, apoptosis • • • • • VEGF/bFGF - MMP – matrix degradation Interactions in Tumorangiogenesis Jones, BJUI, 1999 Cancer Cells & Tumor Vessels • Cancer cells occupy 4% of vascular surface • Tumor intra-vasation in 2 days • Shedding of 106 cells / day / gram Angiogenesis in Bladder cancer MVD = prognostic indicator in TCC MVD + p53 = aggressive subset identified bFGF higher in metastatic pts. VEGF, TGF- higher in TCC Urinary VEGF, aFGF higher in TCC pts. VEGF mRNA ~ recurrence + progression • • • • • • Tumorangiogenesis and LN-Metastases T2-4 bladder cancer, n=41 Cx, pelvic LN dissection MVD (FVIII-RA), 200x neg. LN (n=27): 56.2 MV (SD 29.5) pos. LN (n=14): 138.1 MV (SD 37.9) p<0.0001 Angiogenesis in Prostate cancer MVD higher in PCa MVD ~ stage (superior to grade, preop. PSA) MVD predictor for metastasis after Px TURP-MVD ~ failure of radiotherapy Biopsy-MVD (+ GS, PSA): extraprostatic ext. Urine-bFGF: BPH > PCa > control Serum-bFGF: PCa > BPH VEGF: ? PSA = antiangiogenic • • • • • • • • • Microscopic Tumor Extension Capsular penetration Seminal vesicle extension Pelvic lymph node metastases Pathologic upstaging: 11%-60% Sensitive markers: serum, urine, imaging? Tumorangiogenesis and PCa Microvessel density ~ Pathologic stage Organ confined tumor vs. non organ confined tumor n=31, retropubic Px, CD31, 200x, 0.74 mm2 Stage Mean Range Organ confined (n=23) 49.7 23-97 Positive margins (n=5) 81.6 54-104 N1 (n=3) 87.2 65-111 MVD in Prostatic Carcinoma CD31, 200x Urine VEGF and T-Stage in PCa VEGF-Elisa (R&D Systems), TNM n=14 (64.6, ± 7.5 yrs.) T2: n=6, Urine VEGF = 191 pg/ml (± 75) T3: n=8, Urine VEGF = 309 pg/ml (± 163) p=0.04 T2a/T2b and T3a/T3b: n.s. Angiogenic Topography in PCa PCa, rad Px, n=60 (64 ± 6 yrs) MVD endothelial antigen CD34 VEGF165 monoclonal Ab 7,9 mm2 area, serial sections Distribution and topography CD34+VEGF165 Topography divided in 4 categories: Identical, intersecting, adjacent, no contact • • • • • • Angiogenic Topography in PCa CD34 VEGF Angiogenic Topography in PCa MVD between four groups n.s. • VEGF165-expression and highest MVD • - identical: 19 (31.6%) - intersecting: 18 (30%) - adjacent: 11 (18.3%) - no contact: 12 (20%) Close topographical relation in 80% • Angiogenesis and NE PCa with high NE differentiation: poor • prognosis NE and Neovascularisation? • PCa, rad Px, n=102 (65.2 ± 6.6 yrs) • Chromogranin A, CD34, 200x, morphometry • NE: scattered cells • small clusters (<10 cells) large clusters (>10 cells) Angiogenesis and NE Scattered cells 14.6 ± 2.2 Small cluster 82.8 ± 16.3 Chromogranin A, 200x Large cluster 291.3 ± 40.1 Angiogenesis and NE G Ia / Ib / IIa, Gleason 2-6, n=36 • G IIb / IIIa / IIIb, Gleason 7-10, n=66 • Low grade PCa: 33.8 ± 6.7 NE cells • High grade PCa: 90.5 ± 16.4 NE cells • (p=0.028) Angiogenesis and NE pT2 n=36 1p<0.05 pT3 n=58 pT4 n=6 49.6 NE tumor ± 10.63 cells 73.3 ± 15.23 201.8 ± 86.61,2 2.56 MVD ± 0.183 2.79 ± 0.163 1.33 ± 0.411,2 vs. pT2 tumors, 2p<0.05 vs. pT3 tumors, 3p<0.05 vs. pT4 tumors Angiogenesis and NE Low grade PCa, high NE • CD34: 2.4% ± 0.26 High grade PCa, high NE • CD34: 3.3% ± 0.26 (p=0.026) Angiogenesis, NE and Proliferation S-VEGF: high angiogenic potential • Ki-67: proliferation • Neuroendocrine differentiation (NE) • Correlation of S-VEGF and Ki-67 in • neuroendocrine differentiated PCa? Angiogenesis, NE and Proliferation PCa, rad Px, T2b-3a, n=9, ( 65.8 yrs) S-VEGF (quantitative ELISA) Chromogranin A Ki-67 (MIB-1-Ab) • • • • Expression, topography • Angiogenesis, NE and Proliferation S-VEGF correlates with Ki-67 in areas of • PCa with high NE differentiation. (p=0.0228, r=0.67) S-VEGF does not correlate with Ki-67 in • areas with little NE differentiation. (p=0.2110) Vascular Morphogenesis Microvessel Maturity Newly formed immature vessels vs. Established and mature vessels Coating of periendothelial cells: Pericytes, smooth muscle cells, myocardial cells Anti-angiogenic therapy: MVD , change of ratio immature/mature vessels Vulnerability of uncoated ECs SMC-EC Interaction ECs initiate angiogenesis • Periendothelial cells for vascular maturation • Vascular myogenesis / mural cells Vessel stability Inhibition of EC proliferation Production of extracellular matrix Viscoelastic and vasomotor properties Specialized functions of ECs • • • • • Angioarchitecture HMW-MAA (MAb 225.28S)• Staining restricted to pericytes• Staining „clusters of cellular processes“• Pericytes aquire HMW-MAA• HMW-MAA pos. pericytes = proliferation?• HMW-MAA in PCa?• Antibody supplied by S. Ferrone, Roswell Park Cancer Insitute, USA Angioarchitecture HMW-MAA MAb 225.28S, 40x Human Plasminogen - Kringle 5 K5 K1-4=Angiostatin™ Kringle 5 in Urine of Cancer Patients Breast cancer Lung cancer Rheumatoid arthritis Prostate cancer • • • • Band reacts only with K5 Ab, not K1-3 or K4 Abs K5 has a molecular mass of 10459 kDa HMVEC bFGF/VEGF Stimulated Migration Control (15 ng/ml bFGF/VEGF) 400 pM K5 (15 ng/ml bFGF/VEGF) 36 Cells Migrated 2 Cells Migrated 40x magnification Migration Inhibition of HMVEC Cells by K5 with Various Activators None K5 (400 pM) Endothelial Cell Migration 120 100 80 60 40 20 0 None bFGF aFGF VEGF PDGF TGF- IL-8 HGF/SF Pharmacokinetic MRI-Model Gd Gd k21 Gd Gd Gd Gd extracellular space Amplitude A Pharmacokinetic MRI-Model k21 Amplitude A - Signal intensity - Vessel density k21 - Exchangerate - Vessel density / Vessel permeability Pharmacokinetic MRI-Model MR-Spectroscopy in PCa 200 100 O2 0 min 0 1,5 3 4,5 6 0 1,5 3 4,5 6 min 200 100 0 Change of signal intensity [%] MR-Spectroscopy in PCa PCa, before inhalation PCa, after inhalation MR-Spectroscopy in PCa Change of signal intensity in ROI Angiogenic Surroundings Tumorproliferation / Apoptosis Microvessel sprouting Recruitment of periendothelial cells Angioarchitecture Neuroendocrine differentiation Hormonal influence (Androgens) • • • • • • Paracrine pathway of angiogenic stimulation? Tumor microenvironment? Antiangiogenesis? VEGF and Androgens in PCa Androgens induce VEGF in hormone-sensitive tissues VEGF induces EC proliferation Hormonal ablation in androgen-sensitive tumors Withdrawal of tumor cell VEGF: Apoptosis of ECs AND tumor cells surrounding ECs Regression of tumor mass Problem: Recurrence due to hormone refractory variants Antiangiogenic Trials Natl Cancer Inst Database, 8/99 Tumorangiogenesis and Cancer: Relevance? Diagnostic: Screening (Serum, Urine) Staging (Expression of GF, MVD, Imaging) Prognosis Therapeutic: Antiangiogenesis (30 cancer drugs on trial NCI) Market: $ 3 billion by 2005 (FT Pharmaceuticals)