Table of contents
1. Regulation of neoangiogenesis
2. Tumor angiogenesis
Vasculoendothelial growth factor
. Vasculoendothelial growth factor C
. Vasculoendothelial growth factor D
. VEGF receptors
. Fibroblast growth factor
. epidermal growth factor
. Transforming growth factor α
. Transforming growth factor β
. platelet growth factor
. placental growth factor
. Hepatocyte growth factor
. Angiogenin
. Angiopoietins-1 and -2
. Pigment factor of epithelial origin
. Nitric oxide
. Matrix metalloproteinases
. Endostatin
. stem cell factor
. Factor that inhibits leukemic cells
. brain neurotropic factor
Section Abbreviations
EGF - epidermal growth factor
FGF - fibroblast growth factor
HGF - hepatocyte growth factor
IGF - insulin-like growth factors
MMRS - matrix metalloproteinases
PDGF - Platelet Growth Factor
PLGF - placental growth factor
TGF - transforming growth factors
TIMP - inhibitors
MMP SCF - stem cell factor
VEGF - Vasculoendothelial Growth Factor
Growth factors are polypeptides with a molecular weight of 5-50 kDa, combined into a group of trophic regulatory substances. Like hormones, these factors have a wide range of biological effects on many cells - they stimulate or inhibit mitogenesis, chemotaxis, and differentiation. Unlike hormones, growth factors are usually produced by non-specialized cells found in all tissues and have endocrine, paracrine, and autocrine effects. Endocrine factors are produced and transported to distant target cells via the bloodstream. Reaching their "target", they interact with specialized high-affinity receptors of target cells. Paracrine factors differ in that they spread by diffusion. Target cell receptors are usually located near the producer cells. Autocrine factors affect the cells that are the direct source of these factors. Most polypeptide growth factors act in a paracrine or autocrine manner. However, certain factors such as insulin-like growth factor (IGF) are able to exert endocrine effects.
Regulation of neoangiogenesis
The normal functioning of tissues depends on the regular delivery of oxygen by the blood vessels. Understanding how blood vessels form has been the focus of much of the research effort in the past decade. Vasculogenesis in embryos is the process by which blood vessels form de novo from endothelial cell precursors. Angiogenesis is the process of formation of new blood vessels from an already existing vascular system. It plays an important role in development, normal tissue growth, wound healing, the reproductive cycle in women (development of the placenta and corpus luteum, ovulation) and also plays a major role in various diseases. Of particular interest is the growth of tumors. It is the formation of a new blood supply system that allows the tumor to grow. This process, described as tumor angiogenesis, is also an integral part of the spread of tumor cells and the growth of metastases. The process of neoangiogenesis is necessary for the long-term adaptation of tissues under conditions of damage. In this case, there is a partial entry of growth factors into the blood, which is of diagnostic value.
The following stages of neoangiogenesis are distinguished:
1. increase in endothelial permeability and destruction of the basement membrane;
2. migration of endothelial cells;
3. proliferation of endothelial cells;
4. "maturation" of endothelial cells and vascular remodeling.
The main mechanism of regulation of neoangiogenesis processes is the release of angiogenic factors, the sources of which can be endothelial and mast cells, macrophages, etc. Under the influence of angiogenic factors, endotheliocytes are activated (mainly in postcapillary venules) and migrate beyond the basement membrane with the formation of branches of the main vessels. It is assumed that the activation of the expression of endothelial adhesion molecules, for example, E-selectin, is of great importance in the mechanism of endotheliocyte migration. In a stable state, endotheliocytes do not proliferate and only occasionally (once every 7-10 years) divide. Under the action of angiogenic growth factors and cytokines, endotheliocyte proliferation is activated, which ends with vessel remodeling, after which the newly formed vessel acquires a stable state.
The growth of new vessels is determined by the balance between its stimulants and inhibitors. At a low value of the ratio of stimulants to inhibitors of vessel formation, neoangiogenesis is blocked or of low intensity, on the contrary, at high values of the ratio, neoangiogenesis is actively triggered.
Neoangiogenesis stimulators: vasculoendothelial growth factor (VEGF), fibroblast growth factor (FGF), angiogenin, epidermal growth factor (EGF), platelet-derived growth factor (PDGF), transforming growth factors α (TGF-α) and β (TGF-β), insulin-like growth factor 1 (IGF-1), NO, interleukin-8 and non-specific factors such as matrix metalloproteinases (MMPs).
Neoangiogenesis inhibitors: endostatin, soluble VEGF receptors (sVEGFR), thrombospondin, angiostatin (plasminogen fragment), vasostatin, restin, MMP inhibitors (TIMP-1, TIMP-2).
Tumor angiogenesis
Unlike the usual, normal vasculature, which quickly matures and stabilizes, tumor blood vessels have structural and functional abnormalities. They do not contain pericytes, cells that are functionally associated with the vascular endothelium and are extremely important for the stabilization and maturation of vascular structures. In addition, vessel1. 2. 3. 4. This tumor network has a chaotic organization, with tortuosity and increased vascular permeability, and its survival and proliferation depend on growth factors. These vascular anomalies, which are largely due to excessive production of growth factors, create conditions favorable for tumor growth.
Cancer cells are characterized by an increase in the level of neoangiogenesis stimulators. In the absence of a blood supply, tumors obtain oxygen and nutrients by diffusion and usually do not grow more than 1–2 mm in diameter. The onset of angiogenesis leads to the formation of a new blood supply and facilitates the rapid growth and metastasis of the tumor, thus becoming active. Although many growth factors are involved in tumor angiogenesis, VEGF has been found to be the most potent and dominant of them. Disturbance of blood supply of a tumor can suppress its subsequent growth. It is assumed that blocking tumor growth is possible by suppressing the formation and activity of angiogenesis growth factors or by direct action on newly formed, immature blood vessels. This method of influencing the tumor does not cause its erradication, but only limits its growth, turning the disease into a sluggish chronic process. Anti-VEGF therapy inhibits the growth of new tumor vessels and induces regression of the newly formed vascular bed.
Vasculoendothelial growth factor (VEGF, VEGF A)
VEGF is a heterodimeric glycoprotein growth factor produced by various cell types. At least 5 variants of VEGF-A have been identified: VEGF 121 , VEGF 165 , VEGF 183 , VEGF 189 , VEGF 206 . Other variants of VEGF are referred to as VEGF-B, -C, -D. VEGF 165 is the predominant form in most tissues. Kaposi's sarcoma expresses VEGF 121 and VEGF 165. VEGF 121 and VEGF 165 are soluble forms, while VEGF 189 and VEGF 206 are bound to heparin-containing membrane proteoglycans. Unlike other endothelial cell mitogens such as bFGF (basic form) and PDGF, VEGF is synthesized as a 226 amino acid precursor.
VEGF is a potential mitogen for vascular epithelial cells. It has a strong effect on vascular permeability, is a powerful angiogenic protein in various experimental systems, and is involved in neovascularization processes in pathological situations. There is a synergistic effect between VEGF and bFGF on the induction of angiogenesis. The ability of VEGF to affect vascular permeability implies the possibility of involving this growth factor in changing the functions of the blood-brain barrier in subnormal and pathological conditions. VEGF-A also causes vasodilation via the NO synthetase pathway in endothelial cells and can activate monocyte migration.
VEGF-A can be detected in the plasma and serum of patients, but its serum level is much higher. Extremely high levels can be found in the contents of cysts formed in patients with brain tumors or in ascitic fluid. Platelets release VEGFA upon aggregation and may be another major source for tumor cells. Various studies have shown that the association of high serum VEGF-A levels with poor prognosis in cancer patients may be correlated with elevated platelets. Tumors can secrete cytokines and growth factors that stimulate the production of megakaryocytes in the bone marrow and increase platelet counts. This, in turn, may lead to another, indirect enhancement of VEGF-A delivery to the tumor. Moreover, VEGF-A is involved in many other pathological processes associated with increased angiogenesis or increased vascular permeability. Examples where VEGF-A plays an important role are psoriasis and rheumatoid arthritis, as well as ovarian hyperstimulation syndrome. Diabetic retinopathy is also associated with high intraocular levels of VEGF-A, and inhibition of VEGFA function can lead to infertility due to blockage of corpus luteum function. The importance of VEGF-A for tumor growth has been clearly demonstrated by using VEGF receptors to block proliferation in vivo, as well as blocking antibodies to VEGF or to one of the VEGF receptors. As a result, interference with VEGF-A function has become a major interest for drug development aimed at blocking angiogenesis and metastasis. Currently, more than 110 pharmaceutical companies worldwide are involved in the development of such antagonists. Their approaches include antagonists of VEGF-A or its receptors, selective inhibitors of tyrosine kinases. Targeting of VEGF signaling may have very important therapeutic implications for many diseases and serve as a basis for the development of future (anti)angiogenic therapies.
Vasculoendothelial growth factor C (VEGF-C)
VEGF-C belongs to the VEGF family. It has been shown to have angiogenic and lymphangiogenic properties. The VEGF family and their receptors are involved in the development and growth of the vascular endothelium. Two proteins of this family, VEGF-C and -D, have a regulatory effect on the endothelial cells of the lymphatic vessels through the VEGFR3 receptor, acting as mitogens.
Expression of VEGF-C is associated with oncohematological diseases. Expression of VEGF-C together with receptors promotes the survival and proliferation of tumor cells. Increased expression of VEGF-C has been shown in malignant tumors of the gastrointestinal tract, where it correlates with invasion, lymph node metastases, and reduced survival.
Vasculoendothelial growth factor D (VEGF-D)
VEGF-D (also known as c-fos-inducible factor, or FIGF) is very close to VEGF-C. It shares structural homology and receptor specificity similar to VEGF-C, so it is believed that VEGF-D and VEGF-C can be classified into the VEGF subfamily. VEGF-D is initially synthesized as a precursor protein containing unique N- and C-terminal propeptides in addition to a central VEGF receptor-binding homologous domain (VHD). N and C-terminal propeptides have not been found in other members of the VEGF family. These propeptides are proteolytically cleaved during biosynthesis resulting in a mature, secreted form consisting of monovalent VHD dimers.
Like VEGF-C, VEGF-D binds on the cell surface to VEGF tyrosine kinase receptor 2 (VEGF R2/Flk-1/KDR) and VEGFR3. These receptors are localized on vascular and lymphatic endothelial cells and are responsible for angiogenesis and lymphogenesis. The mature form of VEGFD binds to these receptors with greater affinity than the original proform of VEGF-D. Expression of the VEGF-D gene has been shown in developing embryos, especially in the lung mesenchyme. VEGF-D is also localized in tumor cells. In adult tissues, VEGF-D mRNA is expressed in the heart, lung, skeletal muscle, and small intestine.
VEGF receptors (sVEGFR-1, sVEGFR-2)
Many cytokine receptors exist in a soluble form following their proteolytic cleavage and separation from the cell surface. These soluble receptors are able to bind and neutralize cytokines in the circulation. There are three receptors for VEGF-A: VEGFR-1 (Flt-1), -2 (KDR) and -3 (Flt-4). They all contain seven Ig-like repeats in their extracellular domains. VEGFR1-R3 is predominantly expressed in proliferating vascular lining endothelium and/or infiltrating solid tumors. VEGFR2, however, is more widely present than VEGFR1 and is expressed in all endothelial cells of vascular origin. VEGFR2 is also present in endothelial and perivascular capillary cells in the thin lamina of seminiferous tubules, Leydig cells, and Sertoli cells. VEGFR2 binds VEGF-A, -C and -D. Unlike VEGFR1, which binds both PlGF and VEGF with high affinity, VEGFR2 only binds VEGF with high affinity, but not PlGF.
These receptors play an important role in angiogenesis. sVEGFR-1 is an inhibitor of this process. By binding to VEGF, it prevents VEGF from interacting with target cells. Functional inactivation of VEGFR2 by antibodies can disrupt angiogenesis and prevent tumor cell invasion. In vascular endothelial cells, angiogenesis induced by the HIV-1 Tat protein is mediated by VEGFR2. Tat specifically binds and activates VEGFR2. Tat-induced angiogenesis is inhibited by agents capable of blocking VEGFR2.
Fibroblast growth factor (FGF)
The FGF family currently includes 19 different proteins. Initially, two forms were characterized: acidic (aFGF) and basic (bFGF).
a and bFGF are products of different genes and share up to 53% homology. The aFGF molecule is represented by a simple polypeptide chain with m.m. 16.8 kDa. Mm. different forms of bFGF ranges from 16.8 to 25 kDa. Functional differences between bFGF forms were not found.
The biological activity of FGF is diverse. They are mitogens for various cells of neuroectodermal and mesenchymal origin, potential mitogens and stimulators of angiogenesis, support and stimulate the differentiation of cells of various neuronal types in vivo and in vitro. In addition to a and bFGF, the family includes oncoproteins int-2 (FGF-3) and hst (FGF-4), FGF-5, keratinocyte growth factor, and vascular endothelial growth factor. FGF-3 and -4 are closely related to bFGF, which itself is likely to be a potential oncogene. Clinical data support the role of bFGF in tumor neoangiogenesis. Thus, an increase in the level of this factor correlates with the degree of aggressiveness of the process in many solid tumors, leukemias, and lymphomas in children and adults and can serve as a prognostic factor for the aggressiveness of the tumor process. bFGF is essential for the development and maintenance of the vascular system during embryogenesis and is also a major angiogenic factor in early recovery and cardiovascular disease.
Epidermal Growth Factor (EGF)
EGF - globular protein with m.m. 6.4 kDa, consisting of 53 amino acid residues, which acts as a strong mitogen on various cells of endodermal, ectodermal and mesodermal origin. EGF is found in blood, cerebrospinal fluid, milk, saliva, gastric and pancreatic juices. The urinary growth factor known as urogastron is also identical to EGF. The main site of EGF synthesis is the salivary glands. EGF controls and stimulates the proliferation of epidermal and epithelial cells including fibroblasts, renal epithelium, glial cells, ovarian granulosa cells and thyroid cells in vitro. EGF also stimulates the proliferation of embryonic cells and increases the release of calcium from bone tissue. It promotes bone resorption and is a strong chemoattractant for fibroblasts and epithelial cells. EGF alone and in combination with other cytokines is a critical factor mediating wound healing and angiogenesis. It also acts as an inhibitor of gastric acid secretion. Some body fluids, such as saliva, urine, gastric juice, seminal fluid, and milk, contain high levels of EGF.
EGF plays an important role in carcinogenesis. Under certain conditions, it can cause malignancy of cells. EGF induces c-fos and c-myc proto-oncogenes. The biological effects of immunoreactive EGF are similar to those of TGF-α. It is important to note that both factors bind to the same receptors. However, EGF is 50% more effective than TGF-α.
Transforming growth factor α (TGF-α)
The main source of TGF-α are carcinomas. Macrophages and keratinocytes (possibly other epithelial cells) also secrete TGF-α. TGF-α stimulates fibroblasts, endothelial development. It is an angiogenic factor. Like EGF, TGF-α is involved in the regulation of cell proliferation as well as in the regulation of tumor cell growth.
Transforming growth factor β (TGF-β)
The TGF-β family includes a group of homologous heterodimeric TGFβ-1, -2, -3 and -4 proteins. The main isoform secreted by cells of the immune system is TGF-β1. All TGF-β consist of 112 amino acid residues. The structure of TGF-β2 shares 50% homology with TGF-β1 over the first 20 amino acid residues and 85% for fragment 21-36. No differences in functional activity between TGF-β1 and -β2 were found. TGF-β is produced by many types of cells and tissues: activated T-lymphocytes and macrophages, platelets, kidneys, placenta.
The factor is produced in an inactive form containing, along with the main dimer, fragments of additional chains of the precursor molecule. Activation occurs in the form of cleavage of these fragments with the help of proteinases (plasmin, cathepsin, etc.). A variety of cells also serve as targets for TGF-β, since the expression of its high-affinity receptor is widespread. When TGFβ acts on the immune system, inhibitory effects predominate. The factor inhibits hematopoiesis, the synthesis of inflammatory cytokines, the response of lymphocytes to IL-2, -4 and -7, the formation of cytotoxic NK and T cells. At the same time, it enhances the synthesis of proteins in the intercellular matrix, promotes wound healing, and has an anabolic effect.
With regard to polymorphonuclear leukocytes, TGF-β acts as an antagonist of inflammatory cytokines. Switching off the TGF-β gene leads to the development of a fatal generalized inflammatory pathology, which is based on an autoimmune process. Thus, it is an element of the inverse regulation of the immune response and, above all, the inflammatory response. At the same time, TGF-β is also important for the development of the humoral response: it switches the biosynthesis of immunoglobulins to the IgA isotype. Stimulates angiogenesis. The level of TGF-β in blood plasma positively correlates with tumor vascularization.
Platelet Growth Factor (PDGF)
PDGF is one of the potential mitogenic polypeptides found in human blood. It consists of two chains: A and B, connected in AA-, BB- and AB-isoforms. These three isoforms differ both in functional properties and in the mode of secretion. While the AA and AB forms are rapidly secreted from the producer cell, the BB form remains largely associated with the producing cell. Only dimeric forms of PDGF can bind to receptors. Two different types of receptors have been identified. The α receptor binds either the A or B polypeptide, while the β receptor binds only the B polypeptide. The whole range of biological effects is due to these three PDGF molecules and two receptors, their different expression and complex intracellular mechanisms of regulation of their activity. The source of PDGF in serum is platelet α-granules, although macrophages and endothelial cells can also produce this factor. At certain stages, placental cells and neonatal aortic smooth muscle cells also serve as a source of PDGF.
The AA isoform is preferentially secreted by fibroblasts, vascular smooth muscle cells, osteoblasts, astrocytes, COLO (colon carcinoma) and WLM (Wilm's tumor) cell lines. BB synthesis is associated with macrophages, islet cells of Langerhans, non-angiogenic epithelium, and the SW (thyroid carcinoma) cell line. Known among cells producing both chains (A and B) are neurons, kidney mesangial cells, glioma and mesothelioma cell lines, and platelets. Initial data suggested that human platelets contained approximately 70% PDGF-AB and 30% -BB. However, more recent studies have shown that up to 70% PDGF-AA is possible, and earlier data are an artifact. The type of PDGF dimer(s) secreted depends on the mRNA produced and can also be influenced by translation efficiency, secretion and intracellular degradation.
The structural identity of the B chain and the c-sis proto-oncogene suggests that PDGF may play a role in the virus-induced malignant transformation of infected cells. PDGF is involved in the regulation of acute inflammation, wound healing, and scar formation. PDGF released from alveolar macrophages is involved in the development of pulmonary fibrosis. It has also been established that the development of atherosclerosis, glomerulonephritis, myelofibrosis and the formation of keloid is associated with PDGF. Like EGF, PDGF induces the expression of proto-oncogenes such as fos, myc, and jun. PDGF is also ubiquitous in CNS neurons, where it is thought to play an important role in cell survival and regeneration, and in mediating proliferation and differentiation of glial cells.
Placental Growth Factor (PlGF)
PlGF - glycoprotein c m.m. 46-50 kDa, belonging to the VEGF family (42% homology with VEGF). PlGF is also homologous, albeit more distantly, to the PDGF family of growth factors. There are two isoforms of PlGF: -1 and -2, which differ in the presence of a heparin-binding domain in PlGF-2. PlGF ensures the proliferation of extravillous trophoblast. As the name implies, PlGF was first identified under normal conditions in the human placenta. It is also expressed in other tissues such as umbilical vein capillaries and endothelium, bone marrow, uterus, NK cells, and keratinocytes. PlGF is also elevated in various pathological conditions, including wound healing and tumor formation. Compared to VEGF, the role of PlGF in neovascularization is less well understood. It can increase lifespan, growth and migration of endothelial cells in vitro, and promote vascularization in some in vivo models. PlGF activity can be manifested by direct interaction of the factor with VEGFR1. It has been suggested that VEGFR1 acts as a reservoir for VEGF and that PlGF, by binding to the receptor, replaces VEGF, releasing it to activate VEGFR2. PlGF can synergistically enhance VEGF-induced angiogenesis and vascular permeability. The concentration of PlGF increases 4 times from the end of the first to the end of the second trimester of a physiologically proceeding pregnancy.
Hepatocyte growth factor (HGF)
HGF, also called scattering factor (SF), consists of two subunits linked by a disulfide bond: α (69 kDa) and β (34 kDa). HGF is a multifunctional cytokine that acts as a mitogen, which is associated with its function in organogenesis and tissue repair. It has the ability to stimulate blood vessel formation and cell proliferation, suggesting its involvement in malignant growth and metastasis in lung, breast, pancreas, adenocarcinoma, multiple myeloma, and hepatocellular carcinoma. In breast cancer tumor cells, HGF strongly induces bcl-x expression and thus inhibits apoptosis. HGF is continuously produced by bone marrow stromal cells and stimulates hematopoiesis.
Angiogenin (ANG)
ANG is a single chain non-glycosylated polypeptide with m.m. 14 kDa, which belongs to the RISBASE family of ribonucleases (ribonucleases with special biological functions). Molecules of this family exhibit not only ribonuclease activity, but also have special biological effects. The sequence of ANG is 35% identical to pancreatic ribonuclease. Human angiogenin has been shown to be 75% identical to mouse ANG at the amino acid level and “works” in mouse systems. ANG is expressed by endothelial, smooth muscle cells, fibroblasts, columnar intestinal epithelium, lymphocytes, primary adenocarcinoma cells, and some tumor cell lines. The angiogenin receptor is unknown. It is believed that actin, as a receptor or binding molecule, is necessary for the manifestation of the actions of angiogenin.
Functionally, ANG is most commonly associated with the process of angiogenesis. It is believed that initially it binds to actin, and then dissociation of the actin-ANG complex occurs, followed by activation of the tissue plasminogen activator. As a result, plasmin is formed, which promotes the degradation of basement membrane components such as laminin and fibronectin. Basement membrane destruction is a prerequisite for endothelial cell migration during neovascularization. Although ANG appears to act predominantly extravascularly or perivascularly, circulating ANG has been detected in normal serum at concentrations of the order of ng/mL. In pathological processes, elevated levels of ANG were found in patients with pancreatic cancer and arterial occlusion.
Angiopoietins-1 and -2 (Ang)
Ang-1 and -2 are glycoproteins belonging to the family of growth factors that regulate the development of vascular tissue. Ang-1 consists of 498 amino acid residues, Ang-2 - of 467. The AA sequences of Ang-1 and -2 are 60% identical. Both Angs interact with the tyrosine kinase-2 (Tie-2) receptor, which is present predominantly on endothelial cells. However, there are at least three alternative splicing variants of Ang-1, with two alternative forms unable to activate Tie-2. Thus, they act as endogenous suppressors of the main active form of Ang-1. In addition, Ang-1 and -2 act as competitors in interaction with the Tie-2 receptor; therefore, Ang-2, depending on the cell type, acts either as a suppressor or an activator of the Tie-2 receptor.
Ang-1 and -2 are actively expressed in the embryo, with the rapid development of vascular tissue. Deletion of the Ang-1 gene leads to lethal consequences in the embryo due to serious malformations in the development of the heart and blood vessels. Although Ang-2 does not play such a significant role as Ang-1 in the formation of the vascular system of the embryo, but in its absence, vascularization is also impaired, which causes early death. In an adult organism, Ang-1 is synthesized mainly by endothelial cells, megakaryocytes, and platelets, while Ang-2 is expressed locally: by the ovaries, uterus, and placenta. Ang-1 regulates the development and remodeling of blood vessels, increases the survival of endothelial cells. Survival of endothelial cells during the interaction of Ang-1 with Tie-2 includes the PI3K/AKT mechanism, and cell migration during the same interaction (ligand/receptor) occurs with the participation of several kinases (PI3K, PAK, FAK). On the contrary, Ang-2, acting alone, initiates endothelial cell death and vessel regression, although synergistically with VEGF, it can promote the formation of new vessels. If Ang-1 acts synergistically with VEGF, its overproduction leads to increased tissue vascularization. Thus, Ang-1 and -2 tend to act as antagonists co-regulating vascular growth.
The action of angiopoietins is not limited to the vascular endothelium of the bloodstream - they can take part in the formation of vessels of the lymphoid system. Ang-1 has other biological effects, for example, enhances adhesion and migration of neutrophils and eosinophils, regulates the permeability of the vascular wall. Also, Ang-1 can cause the growth and survival of nerve cells, regulates the organization of dendritic cells. Elevated levels of Ang-1 and -2 enhance cancer angiogenesis. High concentrations of circulating Ang-1 are associated with hypertension and cancer.
Pigment factor of epithelial origin (PEDF)
PEDF (50 kDa MW, belongs to the serpin family) was first identified as a factor secreted by retinal epithelial cells and promoting neuronal survival in vitro and in vivo. On the other hand, PEDF has been shown to have the property of inducing apoptosis of capillary endothelial cells, thereby maintaining the avascular nature of the retina. In many ophthalmic diseases characterized by dysregulation of the innervation and microvasculature of the retina, PEDF is an important regulator in ocular diseases. In addition, PEDF has been shown to have multifunctional antitumor activity in experimental neuroblastoma, as PEDF produced by Schwann cells induces a differentiated, less malignant phenotype in neuroblastoma cells, promotes further growth and survival of Schwann cells, and inhibits angiogenesis.
Nitric oxide (NO)
The biological effect of NO has been generally recognized since its identification as an endothelium-dependent relaxing factor (EDRF) responsible for potent vasodilatory properties. Since then, NO has been identified as a pleiotropic biological mediator that regulates functions ranging from nervous activity to immune system regulation. It is a free radical with a short in vivo half-life of about a few seconds. In this regard, the level of more stable NO metabolites, nitrite (NO 2-) and nitrate (NO 3-) is used to indirectly determine NO in biological fluids. Examples include altered levels associated with sepsis, reproduction, infections, hypertension, exercise, type 2 diabetes, hypoxia, and cancer.
NO is formed during the oxidation of L-arginine with the participation of NADPH. Oxidation occurs with the participation of one of the three isoforms of enzymes of the NO-synthase (NOS) family with the formation of citrulline. Members of the NOS family include neuronal (nNOS/NOS1), endothelial (eNOS/NOS3), and inducible (iNOS/NOS2) NO synthases. As the name implies, nNOS is highly expressed in CNS and PNS neurons, and is also found in other tissue cells, including skeletal muscle myocytes, lung epithelial cells, and skin mast cells; eNOS is expressed in the endothelium and can also be detected in neurons, skin fibroblasts, keratinocytes, thyroid follicular cells, hepatocytes, and smooth muscle cells. iNOS is expressed in various tissues, including chondrocytes, epithelial cells, hepatocytes, glial tissue, and in various cell types of the immune system. In general, expression of eNOS and nNOS is continuous and regulated by Ca2+-dependent calmodulin, while iNOS synthesis is induced by endotoxin and inflammatory cytokines and is relatively insensitive to Ca2+ action.
Due to the fact that NO is lipid soluble, it is not stored, but synthesized de novo and freely diffuses through membranes. The effects of NO in target cells are mediated through various mechanisms. For example, NO-mediated activation of the enzyme guanylyl cyclase (GC) catalyses the formation of the second messenger 3',5'-cyclic guanosine monophosphate (cGMP). cGMP is involved in a number of biological functions such as regulation of smooth muscle contraction, cell lifespan, proliferation, axonal function, synaptic plasticity, inflammation, angiogenesis, and cyclic nucleotide-gated channel activity. NO is also an antitumor and antimicrobial agent through the mechanisms of conversion to peroxynitrite (ONOO-), formation of S-nitrosothiols, and depletion of arginine stores. Another proposed role for NO is the inhibition of mitochondrial respiration through the inhibition of cytochrome oxidase. NO can also modify the activity of a protein through post-translational nitrosylation via its addition via the thiol group of cysteine residues.
Matrix metalloproteinases (MMPs)
Human MMPs are a family of matrix-degrading enzymes. MMPs have a degrading ability with respect to almost all components of the extracellular matrix found in connective tissues (collagen, fibronectin, laminin, proteoglycans, etc.). In addition to the similarity at the amino acid sequence level, all MMPs are formed from inactive precursors that are converted into active substrate-degrading proteinases under the influence of extracellular factors. The sources of MMPs formation are fibroblasts, macrophages, smooth muscle cells of the vascular wall, and neutrophils. Any tumor is a powerful inducer of MMPs formation in stromal cells. While promoting invasion of tumor growth and metastasis, MMPs are at the same time powerful stimulators of neoangiogenesis. Endogenous and synthetic inhibitors of MMPs are used as potential antitumor agents, the main purpose of which is the suppression of neoangiogenesis.
Endostatin
Biologically active C-terminal fragment of collagen VIII c m.m. 20 kDa. Belongs to the family of collagen-like proteins. In order to avoid excessive growth of blood vessels under normal conditions, the processes of formation of new and remodeling of the original vessels are under the control of the corresponding growth factors. During tumor angiogenesis, the penetration of vessels into the growing tumor mass is observed. Endostatin specifically inhibits endothelial cell proliferation. Accordingly, it inhibits angiogenesis and tumor growth. Endostatin therapy is currently in Phase I clinical trials.
Other diagnostic growth factors
Stem cell factor (SCF)
SCF producers are bone marrow stromal cells, fibroblasts, endothelial cells, Sertoli cells. Its main target cells are hematopoietic stem cells, early committed progenitors of various hematopoietic lineages, and mast cells. SCF activates the differentiation of multipotent progenitor cells synergistically with IL-3, GM-CSF and IL-7 and erythropoietin. It is involved in maintaining the proliferation of the youngest forms of T-lymphocyte precursors in the thymus. In relation to mast cells, it is a major growth factor and chemotactic agent.
SCF has important clinical significance as an inducer of differentiation of lymphocyte and erythrocyte progenitors. The definition of SCF is of considerable interest in the treatment of myelodysplastic syndrome and after bone marrow transplantation.
Leukemic cell inhibitory factor (LIF)
LIF enhances the proliferation of hematopoietic progenitors. LIF has been shown to cause cachexia syndrome in cancer patients. The LIF receptor component gp130 (CD130) is part of the receptors for IL-6 and -11.
Brain-derived neurotropic factor (BDNF)
Together with this factor, the family includes nerve growth factor, neurotropins-3 and -4. BDNF stimulates the growth of nervous tissue, mainly cholinergic neurons in the brain. BDNF has been shown to affect the growth, metabolism, and internal structure of these cells. The main purpose of neurotropic factors is to protect neurons from apoptosis.UDC 616-006
VASCULAR ENDOTHELIAL GROWTH FACTOR IS A CLINICALLY SIGNIFICANT INDICATOR IN MALIGNANT NEOPLASMS
© E.S. Gershtein, D.N. Kushlinsky, L.V. Adamyan, N.A. Ognerubov
Keywords: VEGF; VEGF-R; angiogenesis; tumors; forecast.
The results of our own studies and the most significant literature data are presented, indicating that the key positive regulator of neoangiogenesis, vascular endothelial growth factor (VEGF), is a clinically significant prognostic factor in various oncological diseases, as well as a target for modern targeted drugs with different mechanisms of action. Its role as a serological marker for diagnosis and monitoring requires further study.
General ideas about the regulation of angiogenesis.
Angiogenesis is the process of branching of new capillary processes from pre-existing blood vessels. This complex process includes at least four stages: proteolytic destruction of the basement membrane of vessels and extracellular matrix, migration and attachment of endothelial cells, their proliferation, and, finally, the formation of tubular structures.
At present, much attention is paid to the problem of neoangiogenesis in malignant tumors, since there is no doubt that a tumor cannot develop and grow without the formation of an extensive network of vessels in it, which provide cells with oxygen and nutrients. Interest in this problem arose more than 30 years ago, but until relatively recently, the main characteristic of the activity of neoangiogenesis in tumors was the microscopic assessment of the density of vessels in the tumor tissue (microvascular density). And only relatively recently, as a result of studying the molecular mechanisms of angiogenesis, which has been intensively developing over the past 10–15 years, the presence of a number of regulatory angiogenic and antiangiogenic factors has been demonstrated, the dynamic balance of which ensures the formation and spread of new vessels inside the tumor.
Many known growth factors and cytokines are involved in the regulation of angiogenesis in one way or another, such as basic and acidic fibroblast growth factors (bFGF and cFGF), epidermal growth factor (EGF), a- and P-transforming growth factors (TGF ), platelet endothelial growth factor/thymidine phosphorylase, tumor necrosis factor, interleukins, etc. However, the most important positive regulator of angiogenesis is undoubtedly vascular endothelial growth factor (VEGF), also called vascular permeability factor. The uniqueness of this protein lies in the fact that, unlike all other growth factors, it is mitogenic only in relation to endothelial cells, although recent data indicate that autocrine is also possible.
the effect of VEGF on tumor cells producing it.
VEGF is a homodimeric, highly glycosylated protein with a mol. weighing 46-48 ^a, existing in at least five isoforms with similar biological activity, but significantly different in bioavailability. The bioavailability of VEGF is largely determined by the size of the molecule and is regulated at the genetic level during alternative mRNA splicing, as well as epigenomically during proteolytic cleavage of the synthesized molecules with the participation of the plasminogen activation system. The key regulator of blood vessel growth is VEGF A, while VEGF C regulates predominantly lymphangiogenesis. The main soluble forms of VEGF A are molecules of 121 and 165 amino acid residues in size, they are also the main biologically active forms of VEGF. It is believed that in tissues the main isoform of VEGF is VEGF-165.
On the surface of endothelial cells, there are 3 receptors for VEGF, which are typical receptor tyrosine kinases. The type 1 VEGF receptor (VEGFR1) is a product of the flt-1 gene, the type 2 receptor (VEGFR2) is named KDR and is the human homologue of the mouse flk-1 gene product, and finally the type 3 receptor (VEGFR3) is a product of the flt-4 gene. . Unlike VEGFR1 and 2, it does not interact with classical VEGF (VEGF A), but with its homologue -VEGF C. All receptors are transmembrane glycoproteins with a mol. mass 170235 ^a. Effective binding of VEGF to receptors requires its interaction with heparin-like components of the extracellular matrix.
In addition to the mitogen-activated protein kinase cascade common to most receptor kinases, which regulates the expression of genes associated with proliferation, the c-ets-1 proto-oncogene, encoding the transcription factor Ets-1, is one of the most important genes regulated by VEGF in endothelial cells. Studies using in situ hybridization have shown that c-ets-1 is expressed in endothelial cells at early stages of blood formation.
venous vessels. Its product Ets-1 contributes to the manifestation of the angiogenic phenotype of these cells, activating gene transcription and subsequent synthesis of proteins of the most important proteases that cleave the extracellular matrix (ECM), such as the urokinase-type plasminogen activator, stromelysin, collagenase 1, MMP-1, 3 and 9, as well as p2-integrin. These effects reach a maximum 2 hours after the addition of VEGF (as well as other angiogenic factors - cFGF, bFGF and EGF) and are inhibited by antisense oligonucleotides to ets-1. Activation of proteases has three important consequences for the stimulation of angiogenesis: it facilitates the disintegration of endothelial cells and their invasion into the basal layer of vessels, generates ECM degradation products that promote endothelial cell chemotaxis, and also activates and mobilizes growth factors located in the ECM.
The role of VEGF in the regulation of angiogenesis in breast cancer. The first evidence of the relationship between VEGF expression and angiogenesis activity in breast tumors was obtained on clinical material and published in 1994-1995. a group of Japanese researchers. In the first immunohistochemical study, which included 103 patients with breast cancer, they showed that the density of microvessels and its increase, determined by immunochemical staining for factor VIII antigen, was significantly higher in tumors with intense staining for VEGF than in tumors with weak staining. VEGF is localized predominantly in the cytoplasm of tumor cells. Subsequently, they expanded the examined group of patients to 328 people and, confirming the above regularities, also showed that VEGF expression correlates with the expression of another angiogenic factor, platelet endothelial cell growth factor. Later, these authors carried out a quantitative immunoenzyme analysis of the content of VEGF in the tissues of primary breast cancer and showed that the concentration of VEGF in highly vascularized tumors was significantly higher than in poorly vascularized ones. At the same time, no relationship was found between tissue levels of VEGF and two other potentially angiogenic factors - bFGF and hepatocyte growth factor. The concentration of these two factors also did not correlate with microvessel density.
Interesting data were also obtained. Using an immunohistochemical method, they compared the expression of VEGF, its flt-1 receptor, as well as bFGF and a- and P-TGF in breast cancer and surrounding intact breast tissue. It turned out that of all the parameters studied, only VEGF expression was significantly increased in tumor cells compared to normal ones. An increase in VEGF expression in breast cancer tissue compared to non-tumor breast tissue was also demonstrated by RNA hybridization methods. All these studies provided the first evidence for the important role of VEGF in neoangiogenesis in breast cancer and its importance for tumor growth. To more directly prove this hypothesis, experimental studies were required to confirm the effect of VEGF produced by breast cancer cells on angiogenesis. One of the first such proofs can be considered the work
H. Zhang et al. in which the VEGF-121 gene was transfected into estrogen-
dependent cell line of breast cancer MCF-7. Expression and secretion of VEGF by transfected cells (V12) was confirmed by three independent methods: competitive radioreceptor assay,
stimulation of growth of human endothelial cells in vitro and activation of angiogenesis in the rabbit cornea. When transplanted into athymic mice, clone V12 cells gave rise to more vascularized tumors with a more heterogeneous vascular distribution than the original MCF-7 cells. The growth rate of tumors that originated from V12 cells was higher than that of tumors from the original cell line, while the hormone dependence of the cells and their sensitivity to tamoxifen were preserved. Thus, it was shown that breast cancer cells, constantly producing VEGF, have certain growth advantages.
Another evidence of the effect of VEGF on the growth and metastasis of breast cancer are experiments with antibodies to this factor. So, in experiments on mice with spontaneous breast cancer, characterized by a high frequency of metastasis to the lungs, it was shown that polyclonal antibodies to VEGF inhibit tumor growth by 44% and reduce the number and size of lung metastases by 73 and 84%, respectively.
An interesting model for testing the angiogenic potential of various breast tissues in vivo was developed by H. Lichtenbeld et al. . They placed pieces of tumor and normal breast tissue in a chamber formed by the dorsal skin fold in nude mice and evaluated the induction of angiogenesis. It was found that all breast cancer samples, as well as breast tissues with hyperplasia and apocrine metaplasia, significantly activate angiogenesis. Histologically unchanged areas of breast tissue in patients with breast cancer stimulated angiogenesis in 66% of cases, while healthy gland tissues obtained during cosmetic surgery did not affect angiogenesis. In all cases, the induction of angiogenesis occurred in parallel with the production of VEGF by tumor or mammary gland cells.
The classical model of angiogenesis regulation in breast cancer (just like in any other tumor) provides for the presence of a paracrine system in which growth factor (VEGF) is produced by tumor cells, and its signal-perceiving receptors are located on vascular endothelial cells. The existence of such a paracrine system in breast cancer is well illustrated by the data of L. Brown et al. who studied tissue samples of 68 patients with breast cancer by in situ RNA hybridization and showed that in cells of invasive, metastatic and intraductal breast carcinoma there is a pronounced expression of VEGF, and in cells of the vascular endothelium penetrating these tumors, a pronounced expression of VEGFR1 and VEGFR2. Similar data were obtained and
A. Kranz et al. however, these authors also found VEGFR2 on ductal epithelial cells of the mammary gland. There is also other evidence that VEGF receptors are located on breast cancer cells, and the level of expression of VEGF and VEGFR2 correlates with the tumor cell proliferation index, determined by the expression of the Ki-67 antigen. It has been shown that both tumor and stromal cells isolated from primary human breast carcinomas produce VEGF in vitro, and its level
production is significantly higher than that of the corresponding cells isolated from the normal mammary gland. At the same time, PCR analysis showed that VEGFR2 predominates in tumor cells, while only VEGFR1 is expressed in stromal cells. Thus, in addition to its direct function of stimulating neoangiogenesis, VEGF in breast cancer can also play the role of an auto/paracrine regulator of tumor and/or stromal cell proliferation.
It is assumed that VEGF can play another role in breast cancer: through the flt-1 receptors, it stimulates the migration of macrophages into the tumor tissue, which, in turn, are aniogenesis stimulators, since they synthesize various angiogenic factors, incl. and VEGF itself. In particular, R. Leek et al. , having examined tissue samples from 96 patients with breast cancer, demonstrated a positive correlation between the index of tumor tissue infiltration by macrophages and the level of VEGF expression.
Secretion of VEGF by breast cancer cells is induced by various external and internal factors. P. Scott et al. , studying the effect of hypoxia, hypoglycemia, acidity, female sex steroid hormones and vitamin D on the expression of the 4 main VEGF isoforms by cultured breast cancer cells with different biological phenotypes, showed that these cells differ significantly both in the basal expression of VEGF mRNA and in its sensitivity to various stimuli. At the same time, hypoxia turned out to be the most powerful VEGF-inducing stimulus for all cell types, and steroid hormones had practically no effect on VEGF expression. R. Bos et al. showed that HIF-1, a transcription factor induced under hypoxia conditions, plays an important role in the stimulation of neoangiogenesis under the action of hypoxia, the high level of which in breast cancer tissue correlates with a high proliferation index, increased expression of VEGF and estrogen receptors (ER). The expression of HIF-1 and VEGF in breast cancer cells is not associated with the level of expression of the apoptosis inducer p53. At the same time, the apoptosis inhibitor bcl-2 enhances the stimulatory effect of hypoxia on the synthesis of VEGF in breast cancer cells. Hybridization analysis showed that MCF-7 cell clones overexpressing bcl-2 and having an increased metastatic potential and resistance to adriamycin have a higher level of mRNA expression of the most angiogenic isoforms of VEGF - VEGF-121 and VEGF-165 - than the original clone MCF- 7. In experiments in vivo bcl-2-transfected cells formed tumors with a greater degree of vascularization and greater expression of VEGF than the original cells.
On the other hand, it has been shown that VEGF, which is a survival factor for endothelial cells, not only stimulates their proliferation, but also suppresses apoptosis by inducing bcl-2 expression. Interestingly, VEGF had a similar effect on breast cancer cells, i.e., it was an anti-apoptotic factor not only for endothelial cells, but also for tumor cells proper.
Various growth factors and signaling systems are involved in the regulation of VEGF expression in breast cancer cells. Several studies in particular have demonstrated the important role of the erbB family of receptor tyrosine kinases and some of their ligands. Thus, L. Yen et al. by examining a panel of cell lines
Breast cancer with stable overexpression of the ligand-free "receptor-dispatcher" erbB-2 showed that heregulin-P1, interacting with the erbB-3 and erbB-4 receptors, induces VEGF secretion in most of the studied breast cancer cell lines, but not in cells of normal mammary glands. Basal VEGF secretion was increased in cells with elevated levels of erbB-2, and in T47D cells with functionally inactivated erbB-2, not only basal VEGF secretion was reduced, but also its induction under the action of heregulin. Subsequently, it was shown that the effect of heregulin on VEGF synthesis includes one of the classical signaling pathways involving phosphatidylinositol 3-kinase and protein kinase B (Akt) with subsequent induction of the transcription factor HIF-1, which stimulates the expression of the VEGF gene.
Apparently, some growth factors of the TGF-β family also act as regulators of VEGF expression in breast cancer cells. The concentrations of TGF-P1 and VEGF in tumors and blood serum of BC patients positively correlated with each other, and in in vitro experiments TGF-R1 induced VEGF production by cultured MDA-MB-231 cells. Another study showed that the simultaneous high expression of TGF-β2 and its receptors is characteristic of tumors with a high density of microvessels.
Until now, the issue of hormonal regulation of VEGF synthesis in breast cancer cells by sex steroids, especially estrogens, remains controversial. Although estrogen induction of VEGF-mediated angiogenesis in the endometrium is virtually certain, the existence of a similar mechanism in breast cancer has not been clearly proven. J. Rujhola et al. on cell culture MCF-7 showed that 17P-estradiol (E2) causes a two-phase increase in the synthesis of VEGF mRNA, accompanied by the accumulation of the corresponding protein in the culture medium. This effect was blocked by pure antiestrogen ICI 182.780, which suggests that RE was involved in its implementation. At the same time, such classical antiestrogens as tamoxifen and toremifene, which have a partial estrogenic effect, not only did not inhibit the VEGF-inducing effect of E2, but also induced VEGF synthesis by themselves. The participation of ER in the regulation of VEGF synthesis in breast cancer cells is also confirmed by molecular biological studies by S. Hyder et al. who demonstrated that the VEGF gene contains two sequences that are homologous to classical estrogen-sensitive elements and specifically bind both forms of ER - ER-a and ER-r.
However, the nature of the action of estrogens and antiestrogens on VEGF synthesis seems to depend on the type of breast cancer cells. For example, J. Kurebayashi et al. described the human breast cancer cell line KPL-1, whose growth was stimulated by ICI 182.780 and suppressed by E2 in vivo. At the same time, E2-propionate suppressed angiogenesis and stimulated apoptosis in tumors formed by KPL-1 cells. Under in vitro conditions, E2 did not affect either the synthesis of VEGF or the rate of cell proliferation. Interestingly, VEGF expression in KPL-1 cells was induced by medroxyprogesterone acetate.
The inducing effect of progestins on VEGF synthesis by breast cancer cells was also noted by S. Hyder et al. . Examining the T47-D cell line, they found
showed that progesterone dose-dependently increased the level of VEGF in the culture medium by 3-4 times with a maximum effect at a concentration of 10 nM. At the same time, other steroid hormones (estrogens, androgens and glucocorticoids) did not affect the production of VEGF, and the effect of progestins was not manifested in other BC cell lines - hormone-dependent MCF-7, ZR-75 and hormone-independent MDA-MB^L Effect of progesterone on T47-cells D was blocked by the antiprogestin RU-486, which suggests the involvement of the classical receptor mechanism. Interestingly, according to K. Heer et al. , the level of VEGF in the blood serum of women is significantly reduced in the luteal phase of the menstrual cycle and is inversely related to the level of progesterone in the blood serum. Serum obtained during this period stimulated the production of VEGF by MCF-7 cells to a lesser extent than serum obtained in the first phase of the menstrual cycle.
Interesting patterns regarding the hormonal regulation of angiogenesis in the mammary gland were demonstrated by R. Greb et al. . Having studied the expression of the main VEGF-A isoforms in tumors and surrounding unaltered mammary gland tissues of 19 patients with breast cancer by PCR analysis, they found that the levels of VEGF expression in the unaltered gland were significantly higher in premenopausal patients than in postmenopausal patients, and significantly decrease with increasing age of patients. At the same time, VEGF expression in tumor tissue did not depend on the age and menopausal status of patients. The authors believe that angiogenesis is under hormonal control in the normal mammary gland, while this control is lost in malignant transformation.
In addition to the most well-known and widespread angiogenic factor VEGF-A, described above, there are several other members of the VEGF family - VEGF-B, C and D. The function of VEGF-C has been most clearly defined to date: it is believed that it stimulates lymphangiogenesis by interacting with receptors VEGF type 3 (flt-4) located on lymphatic endothelial cells. In experimental studies on nude mice using a new marker of lymphatic endothelium LYVE-1, it was shown that overexpression of VEGF-C in breast cancer cells significantly enhances intratumoral lymphangiogenesis and stimulates the formation of metastases in regional lymph nodes and lungs. Previously, J. Kurebayashi et al. PCR analysis showed that, unlike VEGF-A and B, which are present in breast cancer tissue regardless of its stage, VEGF-C is detected only in tumors that have metastasized to the lymph nodes, and VEGF-D is found only in inflammatory breast cancer. On the other hand, according to R. Valtola et al. , VEGF-C receptor expression is indeed increased in invasive and intraductal breast carcinomas compared to normal breast and fibroadenomas, but increased expression of type 3 VEGF receptors was observed on endothelial cells of blood vessels, and not lymphatics. In this regard, the authors believe that VEGF-C, like VEGF-A, is an angiogenic factor mainly for blood vessels, although they do not exclude its participation in the regulation of lymphangiogenesis.
In general, the role of lymphangiogenesis and the ligand-receptor systems that regulate it in the processes of metastasis of solid tumors and, in particular, breast cancer, has recently received increasing attention.
Thus, VEGF plays an important and diverse role in breast cancer, stimulating tumor growth and spread through complex paracrine and autocrine effects both directly on the endothelium of blood vessels, and on tumor cells and tumor stroma, tumor-infiltrating macrophages, and lymphatic vessel cells. All this allows us to consider VEGF as a very promising biological marker for the prognosis of breast cancer and one of the main targets of antiangiogenic antitumor therapy.
Clinical significance of VEGF determination in breast cancer. Above, we have already cited a number of works in which clinical material using various methods (immunohistochemical, enzyme immunoassay, hybridization) demonstrated an increased expression of VEGF in breast cancer tissue and its relationship with traditional indicators characterizing the activity of neoangiogenesis in tumor tissue. In total, according to the results of the analysis of the Medline database, the study of the clinical significance of the tissue level of VEGF in breast cancer was carried out by 14 groups of researchers. It should be noted again that almost all researchers who made such comparisons, regardless of the methods used, noted an increase in VEGF expression in breast cancer tissue compared to the surrounding histologically unchanged breast tissue, as well as benign tumors. There are no contradictions in the question of the direct correlation of the level of VEGF expression with the activity of neoangiogenesis in the tumor tissue.
For the first time, an unfavorable prognostic value of high VEGF expression in breast cancer was noted by M. Toi et al. . After retrospectively analyzing the results of observation of 328 patients in whose tumors VEGF expression was assessed by immunohistochemical method, they showed that, in univariate analysis, the prognosis of relapse-free survival in patients with VEGF-positive tumors was significantly worse than in patients with VEGF-negative tumors. The value of VEGF for the prognosis of disease-free survival was also demonstrated by M. Relf et al. , which determined the expression of the corresponding RNA in the tumor tissues of 64 patients with breast cancer. At the same time, according to A. Obermair et al. , the level of VEGF, measured by enzyme immunoassay, did not have a significant impact on the prognosis of relapse-free survival of 89 patients with breast cancer examined by them.
The most interesting should be recognized as studies in which the prognostic value of VEGF was evaluated in various clinical groups of patients with breast cancer, taking into account the ongoing treatment. The results of such a detailed analysis are published by two groups G. Gasparini et al. and B. Linderholm et al. . In a 1997 paper, G. Gasparini et al. presented the results of quantitative ELISA determination of the concentration of VEGF in the cytosols of tumors of 260 patients with breast cancer without metastases to the lymph nodes. Patients were followed up for a mean of 66 months. At the same time, VEGF in a wide range
concentration zone (from 5 to 6523 pg/mg of protein) was found in 95% of tumors. Its level did not correlate with known prognostic factors: age and menopausal status of patients, histological type, size and receptor status of the tumor, however, it turned out to be a statistically significant predictor of relapse-free and overall survival according to the results of both single-factor and multivariate analysis. Thus, the cytosolic level of VEGF is an indicator of prognosis in patients with early stages of breast cancer, which makes it possible to form a group with an increased risk of recurrence and metastasis.
In a subsequent publication by this group of authors, a comparative assessment of the prognostic value of VEGF and another angiogenic factor thymidine phosphorylase (TF - platelet endothelial cell growth factor) was carried out in patients with breast cancer with metastases to the lymph nodes who received chemotherapy according to the CMP scheme (137 patients) or hormone therapy with tamoxifen (164 patients). Cytosolic concentrations of VEGF were similar in both groups. In the group of patients treated with tamoxifen, the level of VEGF positively correlated with the age of the patients and was inversely related to the level of steroid hormone receptors. In this group, the level of VEGF, along with the number of affected lymph nodes and the concentrations of ER and RP, was a significant independent prognostic factor according to the results of univariate and multivariate analysis. The best results from tamoxifen treatment should be expected in patients with low levels of VEGF in the tumor and the involvement of less than three lymph nodes in the tumor process. The low level of VEGF turned out to be an independent factor of a favorable prognosis in the group of patients receiving chemotherapy. In this group, TF is also a significant prognostic factor, while the prognosis is favorable with a high level of this protein.
In one of the latest studies by O. Oa8ragipi and al. natural inhibitors of angiogenesis, thrombospondins 1 and 2, were also included in the multivariate prognosis model in patients with early stages of breast cancer; however, their contribution to the prediction of disease-free and overall survival was not statistically significant.
Thus, according to the data of this research group, summarized in several review articles, VEGF is the most promising predictor of angiogenesis activity in breast cancer. Its high level indicates
about poor prognosis for both early and advanced breast cancer. Among other regulators of angiogenesis, only TF makes a certain contribution to the prognosis, and its significance is manifested only during chemotherapy of advanced breast cancer.
The prognostic value of VEGF in non-advanced breast cancer has also been studied and confirmed by B. buchiergoit et al. . They determined by enzyme immunoassay the content of VEGF in tumor cytosols of 525 patients without metastases in the lymph nodes (T1.2K0M0), 500 of whom did not receive any postoperative treatment. The median follow-up was 46 months. In contrast to the previously cited researchers, they found a direct correlation between the level of VEGF and tumor size, as well as its degree of malignancy, and an inverse correlation.
regulation of VEGF and RE levels. Survival of patients with cytosolic VEGF levels below the median level (2.4 pg/μg DNA) was significantly higher than in patients with lower levels of VEGF. On multivariate analysis, VEGF levels were found to be the most significant independent prognostic factor, outperforming other known measures. A significant decrease in survival with a high level of VEGF in the tumor was also found in the prognostically favorable group of EC-positive patients.
According to the same authors, a high level of VEGF also has an unfavorable prognostic value when patients with early stages of breast cancer undergo locoreginal radiation therapy. 302 patients were examined with a median follow-up of 56 months. VEGF turned out to be the only independent predictor of overall survival (relative risk 3.6) in the entire group, as well as disease-free survival in the most prognostically favorable groups of patients with small tumors (T1) and with EC-positive tumors. The authors believe that a high intratumoral level of VEGF may correspond to a radioresistant phenotype and indicate the need for additional systemic treatment.
B. Linderholm et al. also examined a group of 362 patients with breast cancer with lymph node metastases, 250 of whom received adjuvant hormone therapy and 112 - adjuvant chemotherapy. In a univariate analysis, VEGF proved to be a significant prognostic factor for relapse-free and overall survival in the entire patient population, as well as in the endocrine therapy group. In the group of patients receiving chemotherapy, the level of VEGF had an impact only on overall survival. In multivariate analysis, VEGF retained its significance only for overall survival.
Thus, this group of researchers also demonstrated the predictive value of VEGF for various clinical groups of patients with breast cancer, which was summarized in a 2000 publication that included data on 833 patients with various stages of breast cancer. This work also demonstrated the prognostic value of the simultaneous study of VEGF and mutant p53. The relative risk of death increased by 2.7 times in the group with a high content of VEGF and p53 positive and only in
1.7 in groups with one of these adverse factors.
In a collaborative study that included a total of 495 patients from two different clinics, based on data from univariate and multivariate analysis, which included along with traditional indicators also angiogenin, bFGF and plasminogen activators, it was shown that VEGF is the most important prognostic parameter for breast cancer patients without metastases in The lymph nodes. And recently, another group of researchers confirmed that the intratumoral level of VEGF makes an additional contribution to the so-called Nottingham prognostic index, which is used to form high-risk groups among patients with early stages of breast cancer.
A special place is occupied by the study of J. Foekens et al. [b1], who determined the concentration of VEGF in the preserved extracts by the enzyme immunoassay.
max 845 patients with advanced breast cancer with developed recurrence of the disease. Of these patients, 618 received adjuvant postoperative hormonal therapy with tamoxifen, and 227 patients received postoperative chemotherapy. It turned out that the cytosolic concentration of VEGF in the tumors of patients who relapsed during the first year of observation was significantly higher than in patients with a longer relapse-free period. It was also noted that the level of VEGF in the primary tumor is higher in patients with primary metastasis to the internal organs than in patients with metastasis to the bones and soft tissues. A high level of VEGF, according to single and multivariate analysis, was an independent indicator of low sensitivity to both tamoxifen and chemotherapy.
In general, 13 out of 14 studies published by 8 independent groups of researchers demonstrated that high VEGF levels are an independent factor in the poor prognosis of breast cancer in the early stages and / or its low sensitivity to traditional types of hormonal or chemotherapy in a widespread process. In this regard, it was proposed to consider the possibility of including various antiangiogenic drugs in the adjuvant therapy regimens for patients with high intratumoral concentrations of VEGF. It should be noted that unified methodological approaches and criteria for identifying patients with high VEGF levels have not yet been developed, and further collaborative studies will be required to create them.
In parallel with the study of the clinical significance of the tissue level of VEGF in breast cancer, the question of whether the increased expression of VEGF in the tumor is reflected in the level of this protein in the serum/plasma of the blood and whether the concentration of circulating VEGF is an adequate characteristic of its content and activity of angiogenesis in the tumor is being studied. In 1996-1997 The first studies were published in which an increase in the level of VEGF in the blood of cancer patients was demonstrated. Thus, Y. Yamamoto et al. [b2], having examined a large group of patients and donors, including 137 patients with breast cancer, found that the level of VEGF in the blood serum of 8.8% of patients with breast cancer exceeds the threshold level set by them of 180 pg/ml. In this case, the serum level of VEGF correlated with the prevalence of the process and with the level of VEGF expression in the tumor tissue, and the main isoform of VEGF in serum was VEGF-γ5.
L. Dirix et al. [b3] examined a group of 132 patients with metastatic cancer with different primary diagnoses. They considered the level of VEGF to be elevated, exceeding the 95% confidence interval of the control group and amounting to 500 pg/ml. VEGF was elevated in 57% of patients with untreated metastatic cancer, regardless of its location. During treatment, the level of VEGF increased in 2/3 patients with disease progression and in less than 10% of patients with positive dynamics.
P. Salven et al. [64] also showed that in various types of tumors (including breast cancer), the serum level of VEGF in disseminated cancer (171711 pg/ml; median - 214 pg/ml) is significantly higher than in healthy donors (1-177 pg / ml; median - 17 pg / ml) and in patients with a localized process (8-664 pg / ml; median - 158 pg / ml). In 74% of untreated patients
with disseminated cancer, the level of VEGF in the blood serum exceeded 200 pg/ml, and decreased after successful treatment. Similar patterns were noted by A. Kraft et al. [b5]: according to their data, an elevated level of VEGF is observed in the serum of 0-2G% of patients with a localized tumor process and 11-65% of patients with a metastatic process. However, it should be noted that in this study, VEGF levels in healthy donors (30–1752 pg/mL; median, 294 pg/mL; upper 95% confidence interval, 883 pg/mL) were significantly higher than those reported by other authors. In this study, as well as in the work of B. Zebrowski et al. [bb], demonstrated a significant increase in the concentration of VEGF in ascitic fluids in cancer patients compared with ascites of non-tumor origin.
In a later work, P. Salven et al. [b7] presents the results of determining the concentration of VEGF in the blood serum of 105 patients with benign and malignant breast tumors. It was shown that the levels of VEGF in the blood serum of patients with metastatic breast cancer (7-1347 pg/ml; median - 186 pg/ml) were significantly increased compared with patients with benign tumors (2-328 pg/ml; median -57 pg/ml). VEGF levels in patients with locally advanced breast cancer (11-539 pg/ml; median - 104 pg/ml) are also higher than in patients with benign tumors, but the difference is not statistically significant. In patients with metastatic cancer receiving specific treatment, the level of VEGF was significantly lower than in patients receiving only symptomatic treatment. It is also interesting that with a locally advanced process, the level of VEGF in the blood serum of patients with invasive ductal carcinoma (median - 107 pg / ml) was significantly higher than in patients with invasive lobular cancer (median - 44 pg / ml), and in the latter it was even lower than in benign tumors. This observation is in good agreement with the data of A. Lee et al. [b8], who showed that the content of VEGF mRNA and protein in the tissue of ductal breast cancer was significantly higher than in the tissue of lobular cancer. At the same time, ductal and lobular carcinoma did not differ in microvessel density, and only in ductal carcinoma was there a direct correlation between the level of VEGF mRNA and protein and vascular density indices. It can be assumed that VEGF is a regulator of angiogenesis mainly in ductal breast cancer.
Of interest are comparative studies in which the content/expression of VEGF was simultaneously determined in tumor tissue and blood serum. G. Callagy et al. [b9], who determined the tissue expression of VEGF by immunohistochemical method, came to the conclusion that this indicator, but not the serum concentration of VEGF, correlates with the density of microvessels and the stage of breast cancer and is therefore a more reliable prognostic factor than the level of VEGF in blood serum. They also found no relationship between serum VEGF levels and its expression in the tumor.
The most representative comparative study was conducted by J. Adams et al. . They determined the content of VEGF in serum and blood plasma and the expression of VEGF in the tumor (immunohistochemically) of 201 patients with localized and advanced breast cancer, benign breast tumors.
Leza and healthy women. In metastatic breast cancer, there was a significant increase in the level of VEGF both in plasma and in blood serum compared to the norm. The content of VEGF in blood plasma in patients with metastatic breast cancer was also significantly increased in comparison with patients with benign tumors and localized breast cancer. In localized breast cancer, only an increase in the level of VEGF in blood plasma was observed compared with the control. The authors believe that the measurement of VEGF in blood plasma reflects its production by the tumor to a greater extent, since serum VEGF is predominantly of platelet origin. Paradoxically, the highest serum and plasma levels of VEGF were found in breast cancer patients in remission on tamoxifen treatment. The level of circulating VEGF did not correlate with any of the known clinicopathological factors, including microvascular density and tissue expression of VRPP.
Thus, the possibility of using indicators of VEGF levels in the blood (both in serum and plasma) as an adequate replacement for tissue expression of this protein in assessing the activity of angiogenesis in breast cancer and predicting the outcome of the disease and the effectiveness of therapy has not yet been proven.
UECE-dependent angiogenesis as a target of anticancer therapy in breast cancer. Given the critical role of angiogenesis in supporting the growth and spread of breast cancer and the key role of VEGF in this process, many authors have long concluded that targeted suppression of VEGF expression and/or its effects may be a promising approach to the development of new regimens for adjuvant therapy of this disease. It was proposed to use as antiangiogenic agents substances that relatively non-specifically block the interaction of various growth factors with tyrosine kinase receptors, in particular, less toxic analogs of suramin. It was also assumed that antiangiogenic agents could be especially effective when combined with drugs activated under hypoxic conditions, since the suppression of angiogenesis would create conditions favorable for the activation of these drugs. The fact that purely anti-angiogenic therapy is most likely to lead not to tumor regression, but to stop its further growth, was especially emphasized. In this regard, the need to develop clinical and biochemical criteria for evaluating the effectiveness of antiangiogenic drugs was pointed out.
Currently, only in the United States, more than 20 drugs are undergoing clinical trials, mainly phase I, that affect angiogenesis in one way or another. Among them are such rather specific preparations as monoclonal antibodies in VEOP (GepeIeeI) and its receptors (IMC-1C11), inhibitors of the internal tyrosine kinase of the VEGF receptor (for example, 7B6474), blockers of VEGF mitogenic signal transmission (SIII668 and 8III416), natural (Neoya8-M ) and synthetic (Magita8Ia1;, Pgnita8Ia1;, BMB-275291, COb-3) inhibitors of matrix proteinases, natural (angiostatin and endostatin) and synthetic (TYP-470) inhibitors of proliferation or inducers of apoptosis (Combre8Ia1m) of endothelial cells and
a number of drugs with a different or unclear mechanism of action.
I Royctap divides anti-angiogenic drugs into direct and indirect inhibitors of angiogenesis. He refers to direct inhibitors substances that directly affect endothelial cells. These are the already mentioned angiostatin, endostatin, TYP-470, Cob-NaNn, as well as such natural inhibitors of angiogenesis as thrombospondins, pigment epithelium factor. A characteristic feature of such drugs is that they generally do not cause resistance in endothelial cells and can therefore be used for a long time. Indirect inhibitors include drugs that affect the production of angiogenic factors by tumor cells or block the action of these factors at one stage or another. These are monoclonal antibodies or antisense nucleotides to VEGF and its receptors. Since the action of indirect inhibitors is directly related to the tumor cell and its ability to produce angiogenic factors, the likelihood of resistance to these drugs is about the same as for traditional antitumor agents.
Interest in antiangiogenic anticancer therapy is currently so great that the number of publications devoted to preclinical and clinical studies in this area is measured in hundreds, so in this review we will focus only on works directly related to breast cancer.
The most advanced clinically is a drug with an unknown mechanism of anti-angiogenic action - thalidomide. In the 1970s it was used as a sedative and was banned due to its teratogenic side effect, which was due to its anti-angiogenic properties. Currently, attempts are being made to use the anti-angiogenic potential of thalidomide in the treatment of malignant tumors, and it is already undergoing phase II clinical trials. Among the 66 patients included in this study who received 100 mg of thalidomide every night, there were 12 patients with breast cancer. There was no objective response to the drug in breast cancer patients, although a partial response or stabilization was observed in 6 of 18 kidney cancer patients included in the same study.
At the same time, in experiments on the induction of angiogenesis in the cornea of a rabbit under the action of a UEOP-producing clone of MCP-7 cells, it was shown that the thalidomide analog linomide at a dose of 100 mg/kg of body weight effectively inhibits this process.
M. A8apo e1 a1. showed that the anti-VEGF monoclonal antibody MU833 inhibited the growth of human breast cancer xenografts in nude mice. However, the inhibitory effect of MU833 did not correlate either with the amount of VEGF secreted by the tumor or with the expression of the VEGF receptor. In other experiments, transplantation into the subcutaneous dorsal chamber of athymic mice of spheroids formed by breast cancer cells of the MCP-7, 7R-75 and BK-VR-3 lines showed that the monoclonal antibody to VEGF A.4.6.1 at a daily dose of 200 μg significantly inhibits the angiogenic activity of these cell lines and enhances the antitumor activity of doxorubicin in these models.
Significant antitumor activity on breast cancer xenografts was also noted in the low molecular weight specific inhibitor of vEGF receptor tyrosine kinase (both types 1 and 2) - 7B4190. A single oral administration of this drug at doses that did not have a direct antiproliferative effect on tumor cells significantly inhibited the growth of formed tumors with a size of about 0.5 cm3. Antiangiogenic activity against breast cancer xenografts has another tyrosine-kinase inhibitor - a fairly well-known and already undergoing clinical trials drug 7B1839 (Ire88a), which is a selective inhibitor of EGF receptor tyrosine kinase. It is assumed that this drug does not act directly on VEGF receptors, but suppresses the induction of VEGF synthesis under the action of EGF receptor ligands. Apparently, another blocker of the EGF-dependent pathway of mitogenic signal transmission, Herceptin, a humanized monoclonal antibody to ErbB2/neu, also has a similar indirect effect on angiogenesis.
Effective inhibitors of angiogenesis are also drugs that disrupt the function of microtubules. So, as early as 1997, Klauber N. ei a1. showed that 2-methoxyestradiol and taxol, which have similar properties, inhibit VEGF-induced angiogenesis by 54% and 37%, respectively. At the same time, 2-methoxyestradiol suppressed the growth of human breast cancer implanted in nude mice by 60%. The antiangiogenic properties of taxol have also been demonstrated by B. bau et al. in mice with a well-vascularized transgenic mammary tumor MeL. The effect appeared at non-cytotoxic doses of taxol (3-6 mg/kg/day) and was associated with suppression of VEGF secretion.
This property of taxol was proposed to be used to evaluate the effectiveness of the treatment of patients with metastatic breast cancer. Their study included 14 patients who received taxol monotherapy at a dose of 175 mg/m2 IV for three courses of 21 days each. In all patients, the serum level of VEGF was determined by enzyme immunoassay before the start of treatment and after each of the 21-day courses. In 3 patients, a partial response to treatment was noted, in 6 - stabilization and in 5 - progression of the disease. Serum VEGF levels before treatment were significantly elevated in 8 of 14 patients. The mean VEGF level decreased after treatment in patients with partial response and stabilization and did not change significantly in patients with progression. Moreover, the percentage of normalization of the level of VEGF or its decrease by more than 50% was significantly higher in patients with a partial effect (5/9) than in patients with progression (0/5). The authors believe that the stabilizing effect of taxol in patients with advanced breast cancer may be associated with the suppression of VEGF secretion and, accordingly, with the inhibition of angiogenesis.
Published experiments on immunodeficient mice demonstrated the possibility of using gene therapy to suppress angiogenesis and inhibit the growth of breast cancer. Mice with mature Mca-4 tumors were injected twice with an interval of 7 days into the tumor with a plasmid containing the gene for the natural angiogenesis inhibitor endostatin. 14 days after the first injection,
reduction in the weight of tumors in experimental mice by 51% compared with the control group. At the same time, an increase in the distance between tumor cells and the nearest vessels, a decrease in the total density of vessels, and an increase in apoptosis in tumors containing and expressing the endostatin gene were observed.
Another genetic engineering approach to anti-angiogenic therapy of breast cancer is the use of anti-sense cDNA to VEGF. S.A. Im et al. human breast cancer cells of the MDA231-MB line were transfected with an adenoviral vector containing such cDNA to VEGF-165 (Ad5CMV-alphaVEGF). In an in vitro system, this transfection resulted in a decrease in VEGF secretion without significant effect on cell growth. In vivo injection of Ad5CMV-alphaVEGF into tumors formed by MDA231-MB cells in nude mice resulted in the suppression of their growth, a decrease in VEGF protein expression in tumor tissue, and a decrease in microvascular density compared with the group that was injected with a vector that did not contain anti-VEGF cDNA .
Thus, the possibility of using various types of direct and indirect antiangiogenic therapy in the treatment of patients with breast cancer has been experimentally proven. Unfortunately, none of these methods has yet been proven effective in the clinic. Moreover, most authors are inclined to believe that antiangiogenic therapy (especially direct), which is predominantly cytostatic rather than cytotoxic in large tumors, should be used not as an independent method of treatment, but as an important addition to standard therapy regimens.
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nocTynma b pegaKunro 16 Hoa6pa 2013 r.
Gershtein E.S., Kushlinskiy D.N., Adamyan L.V., Ognerubov N.A. VASCULAR ENDOTHELIAL GROWTH FACTOR-CLINICALLY VALUABLE MARKER IN MALIGNANT NEOPLASMS
Results of the authors’ investigations and the most representative literature data indicating that the key positive neoangiogene-sis regulator - vascular endothelial growth factor (VEGF) - is a
clinically significant prognostic factor in various oncologic diseases, and molecular target for several modern drugs with different mechanisms of action are demonstrated. Its role as a serological factor for diagnostics and monitoring needs further investigation.
Key words: VEGF; VEGF-R; angiogenesis; tumors; forecast.
Gershtein Elena Sergeevna, Russian Cancer Research Center. N.N. Blokhin RAMS, Moscow, Russian Federation, Doctor of Biological Sciences, Professor, Leading Researcher, Laboratory of Clinical Biochemistry, Research Institute of Clinical Oncology, e-mail: [email protected]
Gerstein Elena Sergeyevna, N.N. Blokhin Russian Oncologic Scientific Center RAMS, Moscow, Russian Federation, Doctor of Biology, Professor, Leading Research Worker of Clinical Bio-chemistry of SRI of Clinical Oncology Laboratory, e-mail: [email protected]
Kushlinsky Dmitry Nikolaevich, Russian Scientific Center for Obstetrics, Gynecology and Perinatology named after A.I. IN AND. Kulakova, Moscow, Russian Federation, oncogynecologist, e-mail: [email protected]
Kushlinskiy Dmitriy Nikolayevich, Research Center for Obstetrics, Gynecology and Perinatology named after V.I. Kulakov, Moscow, Russian Federation, Gynaecological Oncologist, e-mail: [email protected]
Adamyan Leyla Vladimirovna, Russian Scientific Center for Obstetrics, Gynecology and Perinatology named after A.I.
IN AND. Kulakov, Moscow, Russian Federation, Doctor of Medical Sciences, Professor, Academician of the Russian Academy of Medical Sciences, Deputy. director, email: [email protected]
Adamyan Leyla Vladimirovna, Research Center for Obstetrics, Gynecology and Perinatology named after V.I. Kulakov, Moscow, Russian Federation, Doctor of Medicine, Professor, Academician of RAMS, Vice Director, e-mail: [email protected]
Ognerubov Nikolay Alekseevich, Tambov State University. G.R. Derzhavin, Tambov, Russian Federation, Doctor of Medical Sciences, Professor, Head. Department of Oncology, Operative Surgery and Topographic Anatomy, e-mail: [email protected]
Ognerubov Nikolay Alekseyevich, Tambov State University named after G.R. Derzhavin, Tambov, Russian Federation, Doctor of Medicine, Professor, Head of Oncology, Operative Surgery and Topographical Anatomy Department, e-mail: [email protected]
For 30 years it has been suggested that angiogenesis - the process of formation of new blood vessels - could become an important target for antitumor therapy. And only recently this opportunity has been realized. Clinical data have shown that a humanized monoclonal antibody, bevacizumab, that targets a key pro-angiogenic molecule, vascular endothelial growth factor (VEGF), can increase life expectancy in patients with metastatic colorectal cancer when given as first-line therapy in combination with chemotherapy drugs. Here, we will discuss the functions and significance of VECF to show that VEGF is a valid point of action for anticancer therapy.
What is VEGF?
VEGF is one member of a family of structurally related proteins that are ligands for the VEGF receptor family. VEGF influences the development of new blood vessels (angiogenesis) and the survival of immature blood vessels (vascular support) by binding to and activating two closely related membrane tyrosine kinase receptors (VEGF receptor-1 and VEGF receptor-2). These receptors are expressed by the endothelial cells of the blood vessel wall (Table 1). Binding of VEGF to these receptors triggers a signaling cascade that ultimately stimulates vascular endothelial cell growth, survival, and proliferation. Endothelial cells are involved in such diverse processes as vasoconstriction and vasodilation, antigen presentation, and also serve as very important elements of all blood vessels - both capillaries and veins or arteries. Thus, by stimulating endothelial cells, VEGF plays a central role in the process of angiogenesis.
Why is it important to do vascular endothelial growth factor (VEGF human)?
VEGF is extremely important for the formation of an adequate functioning vascular system during embryogenesis and in the early postnatal period, but its physiological activity is limited in adults. Experiments on mice showed the following:
- Targeted damage to one or two alleles of the VEGF gene leads to the death of the embryo
- VEGF inactivation during early postnatal development also leads to death
- Damage to VEGF in adult mice is not associated with any overt abnormalities, as its role is limited to follicular development, wound healing, and the reproductive cycle in females.
The limited value of angiogenesis in adults means that the suppression of VEGF activity is a feasible therapeutic goal.
Already in July, the first Russian gene therapy drug for the treatment of ischemia of the leg vessels may appear on the market. Last September, neovasculgen (as it is called) was registered with Roszdravnadzor. It is possible that soon it will be offered for public procurement. The human stem cell institute, a biotechnology company that created the drug and is developing and trying to market drugs and services “based on cellular, gene and post-genomic technologies,” is talking about the new product as a breakthrough in science. However, many experts evaluate the new drug differently, arguing that it is actually about "confusing patients."
In his speech on June 3, Medical Director of the Human Stem Cell Institute (HSCI) Roman Deev noted that currently only three gene therapy drugs are registered in the world, one of which is neovasculgen, and in Europe this is the first gene therapy drug in general. “Out of 1,500 clinical trials in the field of gene therapy, about 20 are in the direction of treating patients with vascular pathology, and neovasculgen has already shown its effectiveness, while some drugs have gone the distance,” Deev emphasized. It seems that domestic drug manufacturers have something to be proud of! But is the new drug really effective and safe, and how much will its use cost patients?
The Society of Evidence-Based Medicine Specialists draws attention to the fact that the Human Stem Cell Institute is not a scientific institution, but a commercial organization.The drug, created by geneticists, was tested in the clinics of Yaroslavl, Ryazan, Moscow, prescribing for inoperable forms of chronic ischemia of the legs to patients over 40 years old. Did two injections. Doctors have data that after the injection of the drug, the patient could walk without pain no longer 100 meters, as before the injection, but up to 800 meters.
The cost of two injections is about 100 thousand rubles. “The mechanism of action of neovasculgen is based on the principle of therapeutic angiogenesis,” explained Artur Isaev, director of the HSCI. – The drug is a circular DNA molecule that contains a site responsible for the synthesis of vascular endothelial growth factor. Local administration of the drug stimulates the growth and development of new vessels. The researchers are confident that for many patients, the drug could be an alternative to amputation. The percentage of "success" of therapy, according to Professor R.E. Kalinin (Ryazan State Medical University), amounted to 93.6%.
In Russia, the system of angioplasty and vascular treatment of vessels has not been debugged. What is considered "high-tech care" to prevent amputations has been a daily practice in most countries for many years.
The situation in Russia is also bad with medicines. Senior Research Fellow, Institute of Surgery. Vishnevsky Leonid Blatun says that in the presence of perfect ointments and medicines, patients in the clinics of the Russian Federation "really have access to only the most outdated drugs", since modern drugs are not included in the standards of treatment.
How safe is neovasculgen? It should be emphasized that when a new gene is introduced into a human cell, the patient may experience oncological risks. That is why drugs with this mode of action have not been approved before. “The theory that a researcher can act on a cell growth factor, stimulate it by introducing an autogen that will produce protein growth, is generally correct,” says Academician Valentin Vlasov, director of the Institute of Chemical Biology and Fundamental Medicine. - That is, with the help of gene technology, a virus is taken, and it delivers the desired gene into the cell.
On this topic
Law enforcement agencies did not initiate a criminal case against Elena Bogolyubova, a resident of Moscow, who ordered a drug not registered in Russia by mail for her terminally ill son.
“I am familiar with the Stem Cell Institute project and the neovasculgen drug,” says Valentin Vlasov. - In this case, there is no question of a virus vector. I do not exclude that in some very short time after the injection, protein synthesis occurs with the help of this product, and it seems that it does not bring anything bad to the patient, but whether it brings something good, in order to assert this, a very serious evidence base is needed ".
The expert noted that it is rather difficult to draw such a conclusion based on the images provided: “How to look at them, with what resolution the X-ray images were taken, how they were developed - this is all on the conscience of the researchers. It seems to be a branching of small vessels. The report on the drug was pompous, but I can say that if there is such an effect, then it is very short in time, it can last only a few days. And there is no reason to expect a miraculous effect from the drug. According to academician Vlasov, scientists need to achieve the process of long-term protein production, and this can only be achieved by “inserting” the desired gene into the cell, but researchers have not yet been able to do this safely for the patient.
Even the journal that published the results of the Neovasculgen study looks like it belongs to the same company. According to experts, the questions cause haste in conducting clinical trials, the lack of randomization in them (a special algorithm for conducting, excluding interest in the results). The place of injection of the drug was called into question, its description is “plasmid construction”.
As a result, the experts came to the conclusion that we can talk about "confusing the consumer", since large vessels in which there is no blood flow are never restored. The researchers promise benefits to patients for two years, but the actual trial lasted only six months. The lack of reported side effects of such a drug is also suspicious. The desire of scientists to find new possibilities for therapy is not disputed. But all this requires many years of research and strong evidence before application.
Patients with critical ischemia of the lower extremities in 20–50% of cases survive the so-called primary amputations, but only slightly more than half of those operated on retain both legs a year later. Every fifth person dies, and in every fourth case, a “big amputation” is already performed. It is obvious that many patients will literally stand in line for a miracle cure. There will be a huge number of diabetics among them.
In Russia, the number of patients with diabetes mellitus complicated by diabetic foot syndrome is about 4 million people. Such a complication in half of the cases is the main indicator for amputation. In almost half of patients, the treatment of this complication begins late. At the same time, compared with European countries, very few low-traumatic endovascular operations on the vessels of the legs are performed in Russia. According to the Russian State Medical University. N.I. Pirogov, in the EU countries, 8% of complications of peripheral vessels of the legs end in amputation, while in Russia this figure is much higher and reaches more than 50% in diabetes mellitus. According to the President of the Russian Academy of Medical Sciences, Director of the Endocrinological Research Center of the Ministry of Health and Social Development Ivan Dedov, about 8-10% of diabetic patients are affected by diabetic foot syndrome, and up to 50% of them can be classified as at risk. After amputations, the mortality of patients doubles, but if such patients are not operated on, then within two years they will die of gangrene.
In surgical interventions in patients with type 2 diabetes
In type 2 diabetes, angiogenesis is out of balance. DM is characterized by hyperglycemia and various metabolic disorders. They disrupt the balance between pro-angiogenic and anti-angiogenic regulators and lead to inadequate neovascularization in diabetes mellitus (DM). In turn, disorders of angiogenesis and vasculogenesis are important mechanisms in the development of vascular complications of DM. Thus, the development of macrovascular complications is accompanied by suppression of the intensity of angiogenesis and vasculogenesis.
in poorly controlled diabetes mellitus (DM), the healing process of soft tissues slows down. At the same time, one of the factors is a decrease in the level of local growth factors, which limits the possibility of building soft tissues of the gums as part of implant surgeries. It has also been proven that in patients with diabetes, the amount of collagen produced by fibroblasts decreases, which leads to a slowdown in wound contraction. Violation of carbohydrate metabolism entails an increase in matrix metalloproteases (MMPs) and a decrease in nitric oxide (NO), transforming growth factor beta-1 (TGFβ1), which slows down the processes of ECM formation. Clinical studies show that in diabetes mellitus, angiogenesis imbalance can be achieved using both angiogenesis inhibitors and stimulants. Stimulation of angiogenesis and vasculogenesis using stem cells and growth factors is a promising direction in the treatment of angiogenesis deficiency in diabetes mellitus, which affects the reduction in the healing process of soft tissues and the formation of macroagniopathy.
Given the above, in the postoperative period in patients with DM, it seems promising to stimulate the process of angiogenesis due to cycotins and vascular endothelial growth factor.
It is known that vascular endothelial growth factor and cycotins stimulate angiogenesis, and thus increase tissue oxygen saturation (pO2), which is one of the soft tissue repair factors. A decrease in the level of this growth factor leads to a slowdown in the process of epithelialization. The research results show that growth factors and cytokines have a decisive influence on the speed and quality of reparative processes in diabetic patients.
So in dentistry, when building up gum tissue, implant operations, you can use collagen membranes saturated with vascular endothelial growth factor or carry out the Plasmodent procedure based on the introduction of platelet-rich plasma taken from the patient's blood. Such plasma contains growth factors and is a stimulator of the angiogenesis process. Currently, implant surgeries are performed in patients with DM only when the level of glycated hemoglobin is less than 6.0. This indicator is achieved due to the temporary transfer of the patient for the period of the operation and the postoperative period for insulin injections. However, in type 2 diabetes, the patient has hyperinsulinemia due to insulin resistance. It is possible that the use of vascular endothelial growth factor to stimulate the process of soft tissue repair will allow shifting the glycated hemoglobin index to higher values, compensating for violations of angiogenesis from hyperglycemia with vascular endothelial growth factor. It seems that the procedure for the introduction of platelet-rich plasma can be used in any surgical intervention in patients with diabetes.
