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-derived 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
. Leukemia cell inhibitory factor
. Brain-derived neurotropic factor
Section Abbreviations
EGF - epidermal growth factor
FGF - fibroblast growth factor
HGF - hepatocyte growth factor
IGF - insulin-like growth factors
MMPS - matrix metalloproteinases
PDGF - platelet-derived 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 unspecialized cells found in all tissues and have endocrine, paracrine and autocrine effects. Endocrine factors are produced and transported to distant target cells through the bloodstream. Reaching their “goal”, 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 producer cells. Autocrine factors affect 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), can have endocrine effects.
Regulation of neoangiogenesis
The normal functioning of tissues depends on the regular delivery of oxygen by blood vessels. Understanding how blood vessels form has focused much of the research effort in the last decade. Vasculogenesis in embryos is the process by which blood vessels are formed de novo from endothelial cell precursors. Angiogenesis is the process of formation of new blood vessels from a pre-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. Particular interest is focused on tumor growth. It is the formation of a new blood supply that allows the tumor to grow. This process, described as tumor angiogenesis, is also integral in the spread of tumor cells and the growth of metastases. The process of neoangiogenesis is necessary for long-term adaptation of tissues under conditions of damage. In this case, a partial release of growth factors into the blood occurs, which has diagnostic significance.
The following stages of neoangiogenesis are distinguished:
1. increased 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 for regulating 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, endothelial cells 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 in the mechanism of endothelial cell migration great importance plays activation of the expression of endothelial adhesion molecules, for example, E-selectin. In a stable state, endothelial cells do not proliferate and only occasionally (once every 7-10 years) divide. Under the influence of angiogenic growth factors and cytokines, the proliferation of endothelial cells 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 stimulators and inhibitors. At a low ratio of stimulants to inhibitors of vascular formation, neoangiogenesis is blocked or low-intensity; on the contrary, at high ratios, neoangiogenesis is actively triggered.
Stimulators of neoangiogenesis: 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 nonspecific 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 normal, normal vasculature, which matures and stabilizes quickly, tumor blood vessels have structural and functional abnormalities. They do not contain pericytes - cells functionally associated with the vascular endothelium and extremely important for the stabilization and maturation of vascular structures. In addition, vascular1. 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 abnormalities, which are largely due to the 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 that has thereby become active. Although many growth factors are involved in tumor angiogenesis, VEGF has been found to be the most potent and dominant of them. Disruption of the blood supply to the tumor can suppress its subsequent growth. It is assumed that blocking tumor growth is possible by suppressing the formation and activity of growth factors of angiogenesis or by directly affecting newly formed, immature blood vessels. This method of influencing the tumor does not cause eradication, but only limits its growth, transforming the disease into a sluggish chronic process. Anti-VEGF therapy suppresses the growth of new tumor vessels and causes reversal of newly formed vascular beds.
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 VEGF variants are designated 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, whereas VEGF 189 and VEGF 206 are bound to heparin-containing membrane proteoglycans. Unlike other endothelial cell mitogens such as bFGF (the major 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 takes part 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 influence vascular permeability implies the possibility of the involvement of this growth factor in changing the functions of the blood-brain barrier under subnormal and pathological conditions. VEGF-A also causes vasodilation through 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 level in serum is significantly higher. Extremely high levels can be found in the contents of cysts formed in patients with brain tumors or in ascites 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 patients with malignancies may be correlated with elevated platelet counts. 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 increase in 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 include 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 one of the VEGF receptors. As a consequence, interference with VEGF-A function has become a major area of interest for the development of drugs aimed at blocking angiogenesis and metastasis. Currently, more than 110 pharmaceutical companies around the world are involved in the development of such antagonists. Their approaches include antagonists of VEGF-A or its receptors, selective tyrosine kinase inhibitors. Targeting 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 vascular endothelium. Two proteins of this family, VEGF-C and -D, exert a regulatory effect on endothelial cells of 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 gastrointestinal malignancies, where it correlates with invasion, lymph node metastasis and decreased survival.
Vasculoendothelial growth factor D (VEGF-D)
VEGF-D (also known as c-fos-inducible factor, or FIGF) is very similar to VEGF-C. It has 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 the central VEGF receptor-binding homology 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 the formation of a mature, secreted form consisting of monovalent VHD dimers.
Like VEGF-C, VEGF-D binds on the cell surface to tyrosine kinase VEGF 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 pro form of VEGF-D. The expression of the VEGF-D gene in developing embryos, especially in the pulmonary mesenchyme, has been shown. VEGF-D is also localized in tumor cells. In adult tissues, VEGF-D mRNA is expressed in the heart, lungs, skeletal muscle, and small intestine.
VEGF receptors (sVEGFR-1, sVEGFR-2)
Many cytokine receptors exist in a soluble form following proteolytic cleavage and separation from the cell surface. These soluble receptors are able to bind and neutralize cytokines in circulation. There are three receptors for VEGF-A: VEGFR-1 (Flt-1), -2 (KDR) and -3 (Flt-4). All of them contain seven Ig-like repeats in the extracellular domains. VEGFR1-R3 is mainly expressed in proliferating endothelium of the vascular lining and/or infiltrating solid tumors. VEGFR2, however, is more widely represented than VEGFR1 and is expressed in all endothelial cells of vascular origin. VEGFR2 is also present in endothelial and perivascular capillary cells in the lamina 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 binds only VEGF and not PlGF with high affinity.
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 the process of angiogenesis and prevent tumor cell invasion. In vascular endothelial cells, HIV-1 Tat protein-induced angiogenesis is mediated by VEGFR2. Tat specifically binds and activates VEGFR2. Tat-induced angiogenesis is inhibited by agents that can block VEGFR2.
Fibroblast growth factor (FGF)
The FGF family currently includes 19 different proteins. Two forms were initially characterized: acidic (aFGF) and basic (bFGF).
a and bFGF are products of different genes and have 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 range from 16.8 to 25 kDa. No functional differences were found between bFGF forms.
The biological activities of FGF are 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 the 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 a role for 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, leukemia, lymphomas in children and adults and can serve as a prognostic factor for the aggressiveness of the tumor process. bFGF is necessary for the development and maintenance of the vascular system during embryogenesis; it is also the main angiogenic factor in early recovery and cardiovascular diseases.
Epidermal growth factor (EGF)
EGF is a globular protein with m.m. 6.4 kDa, consisting of 53 amino acid residues, which acts as a potent mitogen on various cells of endodermal, ectodermal and mesodermal origin. EGF is found in blood, cerebrospinal fluid, milk, saliva, gastric and pancreatic juices. A growth factor in urine 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 the most important factor mediating the processes of wound healing and angiogenesis. It also acts as an inhibitor of gastric acid secretion. In some biological fluids such as saliva, urine, gastric juice, seminal fluid and milk, high levels of EGF are present.
EGF plays an important role in carcinogenesis. Under certain conditions, it can cause cell malignancy. EGF induces the proto-oncogenes c-fos and c-myc. 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, the effectiveness of EGF is 50% higher than TGF-α.
Transforming growth factor α (TGF-α)
The main source of TGF-α is carcinomas. Macrophages and keratinocytes (possibly other epithelial cells) also secrete TGF-α. TGF-α stimulates fibroblasts and 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 proteins TGFβ-1, -2, -3 and -4. The main isoform secreted by cells of the immune system is TGF-β1. All TGF-βs consist of 112 amino acid residues. The structure of TGF-β2 has 50% homology with TGF-β1 over the first 20 amino acid residues and 85% for fragment 21-36. No differences in functional activity were found between TGF-β1 and -β2. 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.). TGF-β also targets a variety of cells because expression of its high-affinity receptor is widespread. When TGFβ acts on the immune system, inhibitory effects predominate. The factor suppresses hematopoiesis, the synthesis of inflammatory cytokines, the response of lymphocytes to IL-2, -4 and -7, and the formation of cytotoxic NK and T cells. At the same time, it enhances the synthesis of proteins of the intercellular matrix, promotes wound healing, and has an anabolic effect.
In relation to polymorphonuclear leukocytes, TGF-β acts as an antagonist of inflammatory cytokines. Turning 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 feedback 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. Plasma TGF-β levels positively correlate with tumor vascularization.
Platelet Derived Growth Factor (PDGF)
PDGF is one of the potential mitogenic polypeptides found in human blood. Consists of two chains: A and B, linked in AA-, BB- and AB isoforms. These three isoforms differ in both functional properties and mode of secretion. While the AA and AB forms are rapidly secreted from the producer cell, the BB form remains mainly 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 A or B polypeptide, whereas the β receptor binds only B polypeptide. The entire spectrum of biological effects is due to these three PDGF molecules and two receptors, their differential expression and complex intracellular mechanisms regulating 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 smooth muscle cells of the newborn aorta 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) cells. BB synthesis is associated with macrophages, islet cells of Langerhans, non-angiogenic epithelium and SW (thyroid carcinoma) cell line. Cells that produce both chains (A and B) include 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 may be present, and earlier findings 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 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 PDGF is associated with the development of atherosclerosis, glomerulonephritis, myelofibrosis and keloid formation. Like EGF, PDGF induces the expression of proto-oncogenes such as fos, myc and jun. PDGF is also ubiquitously present in neurons of the CNS, where it is thought to play an important role in cell survival and regeneration, mediating glial cell proliferation and differentiation
Placental growth factor (PlGF)
PlGF - glycoprotein with m.m. 46-50 kDa, belonging to the VEGF family (42% homology with VEGF). PlGF is also homologous, although more distantly, to the PDGF family of growth factors. There are two isoforms of PlGF: -1 and -2, differing in the presence of a heparin-binding domain in PlGF-2. PlGF mediates proliferation of extravillous trophoblast. As the name suggests, PlGF was first identified in normal conditions in the human placenta. It is expressed in other tissues such as capillaries and umbilical vein endothelium, bone marrow, uterus, NK cells and keratinocytes. PlGF is also increased in various pathological conditions, including wound healing and tumor formation. Compared to VEGF, the role of PlGF in neovascularization is less clear. It can increase the lifespan, growth and migration of endothelial cells in vitro, and promote vascular formation in some in vivo models. PlGF activity can occur through direct interaction of the factor with VEGFR1. It has been proposed that VEGFR1 acts as a reservoir for VEGF, and that PlGF, upon binding to the receptor, displaces 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 physiological 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, pancreatic, 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. ANG has 35% sequence identity with pancreatic ribonuclease. It has been shown that at the amino acid level, human angiogenin is 75% identical to mouse ANG and “works” in mouse systems. ANG is expressed by endothelial cells, 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 required for the actions of angiogenin.
Functionally, ANG is most often associated with the process of angiogenesis. It is thought to initially bind to actin, followed by dissociation of the actin-ANG complex followed by activation tissue activator plasminogen. As a result, plasmin is formed, which promotes the degradation of basement membrane components such as laminin and fibronectin. Destruction of the basement membrane is a necessary precondition for endothelial cell migration during neovascularization. Although ANG appears to act primarily extravascularly or perivascularly, circulating ANG has been detected in normal serum at concentrations on the order of ng/mL. In pathological processes, elevated levels of ANG were detected in patients suffering from 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 AK sequences of Ang-1 and -2 are 60% identical. Both Angs interact with the receptor tyrosine kinase-2 (Tie-2), which is present predominantly on endothelial cells. However, there are at least three alternative splicing variants of Ang-1, with two alternative forms failing to activate Tie-2. Thus, they act as endogenous suppressors of the major active form of Ang-1. In addition, Ang-1 and -2 act as competitors for interaction with the Tie-2 receptor, so Ang-2, depending on the cell type, acts as either a suppressor or an activator of the Tie-2 receptor.
Ang-1 and -2 are highly expressed in the embryo during rapid development of vascular tissue. Deletion of the Ang-1 gene leads to lethal consequences in the embryo due to serious defects in the development of the heart and blood vessels. Although Ang-2 does not play as significant a role as Ang-1 in the formation of the vascular system of the embryo, in its absence vascularization is also impaired, which causes early death. In the adult organism, Ang-1 is synthesized predominantly by endothelial cells, megakaryocytes and platelets, and Ang-2 is expressed locally: by the ovaries, uterus, and placenta. Ang-1 regulates the development and remodeling of blood vessels and increases the survival of endothelial cells. The survival of endothelial cells during the interaction of Ang-1 with Tie-2 involves the PI3K/AKT mechanism, and cell migration during the same interaction (ligand/receptor) occurs with the participation of several kinases (PI3K, PAK, FAK). In contrast, 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, as a rule, act as antagonists that jointly regulate 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, it enhances the adhesion and migration of neutrophils and eosinophils, and regulates the permeability of the vascular wall. Ang-1 can also induce the growth and survival of nerve cells and regulate the organization of dendritic cells. Elevated levels of Ang-1 and -2 enhance the angiogenesis of malignancies. High concentrations of circulating Ang-1 are associated with hypertension and cancer pathologies.
Pigment epithelial-derived factor (PEDF)
PEDF (mw 50 kDa, 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 retinal innervation and microvasculature, 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 effects of NO have been widely recognized following its identification as an endothelium-dependent relaxing factor (EDRF), responsible for its potent vasodilatory properties. NO has since been identified as a pleiotropic biological mediator that regulates functions ranging from nervous activity to regulation of the immune system. 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, nitrites (NO 2-) and nitrates (NO 3-), is used for the indirect determination of NO in biological fluids. Examples include altered levels associated with sepsis, reproduction, infections, hypertension, exercise, type 2 diabetes, hypoxia, and cancer.
NO is formed by the oxidation of L-arginine with the participation of NADPH. Oxidation occurs with the participation of one of 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 suggests, nNOS large quantities expressed by neurons of the CNS and PNS, and also found in cells of other tissues, including skeletal muscle myocytes, lung epithelial cells, and skin mastocytes; eNOS is expressed by endothelium and can also be detected in neurons, skin fibroblasts, keratinocytes, thyroid follicular cells, hepatocytes and smooth muscle cells. iNOS is expressed in a variety of tissues, including chondrocytes, epithelial cells, hepatocytes, glial tissue, and various cell types of the immune system. In general, eNOS and nNOS expression occurs continuously and is regulated by Ca2+-dependent calmodulin, whereas iNOS synthesis is induced by endotoxin and inflammatory cytokines and is relatively insensitive to Ca2+.
Due to the fact that NO is soluble in lipids, it is not stored, but is synthesized de novo and diffuses freely through membranes. The effects of NO on target cells are mediated through various mechanisms. For example, NO-mediated activation of the enzyme guanylyl cyclase (GC) catalyzes the formation of the second messenger 3',5'-cyclic guanosine monophosphate (cGMP). cGMP is involved in a number of biological functions, such as the regulation of smooth muscle contraction, cell lifetime, proliferation, axonal function, synaptic plasticity, inflammation, angiogenesis, and cyclic nucleotide-gated channel activity. NO is also an antitumor and antimicrobial agent through mechanisms of conversion to peroxynitrite (ONOO-), formation of S-nitrosothiols, and reduction of arginine stores. Another putative role of NO is inhibition of mitochondrial respiration through inhibition of cytochrome oxidase. NO can also modify protein activity through post-translational nitrosylation through attachment via the thiol group of cysteine residues.
Matrix metalloproteinases (MMPs)
Human MMPs are a family of matrix-degrading enzymes. MMPs have the ability to degrade almost all components of the extracellular matrix found in connective tissues (collagen, fibronectin, laminin, proteoglycans, etc.). In addition to similarities 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 the formation of MMPs in stromal cells. While promoting invasion of tumor growth and metastasis, MMPs are at the same time powerful stimulators of neoangiogenesis. Endogenous and synthetic MMPs inhibitors are used as potential antitumor agents, the main purpose of which is to suppress neoangiogenesis.
Endostatin
Biologically active C-terminal fragment of collagen VIII with m.m. 20 kDa. Belongs to the family of collagen-like proteins. In order to avoid excessive vascular growth under normal conditions, the processes of formation of new and remodeling of original vessels are controlled by appropriate growth factors. During tumor angiogenesis, penetration of blood 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 undergoing phase 1 clinical trials.
Other diagnostically significant growth factors
Stem Cell Factor (SCF)
Producers of SCF are bone marrow stromal cells, fibroblasts, endothelial cells, and Sertoli cells. Its main target cells are hematopoietic stem cells, early committed precursors of cells 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 precursors. Determination of SCF is of significant interest in the treatment of myelodysplastic syndrome and after bone marrow transplantation.
Leukemia cell inhibitory factor (LIF)
LIF enhances the proliferation of hematopoietic cell precursors. LIF has been shown to cause the development of cachexia syndrome in cancer patients. The LIF receptor component gp130 (CD130) is part of the receptors for IL-6 and -11.
Brain-derived neurotrophic factor (BDNF)
Along with this factor, the family includes nerve growth factor, neurotrophins-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 ENDOTHELIUM GROWTH FACTOR - A CLINICALLY SIGNIFICANT INDICATOR IN MALIGNANT NEOPHOLISM
© E.S. Gershtein, D.N. Kushlinsky, L.V. Adamyan, N.A. Ognerubov
Key words: VEGF; VEGF-R; angiogenesis; tumors; forecast.
The results of our own research 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 cancers, as well as a target of 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 new capillary processes from pre-existing blood vessels. This complex process includes at least four stages: proteolytic destruction of the vascular basement membrane and extracellular matrix, migration and attachment of endothelial cells, their proliferation and, finally, the formation of tubular structures.
Currently, much attention is paid to the problem of neoangiogenesis in malignant tumors, since there is no longer any doubt that a tumor cannot develop and grow without the formation of an extensive network of vessels in it, ensuring the supply of cells with oxygen and nutrients. Interest in this problem arose more than 30 years ago, but until relatively recently, the main characteristic of neoangiogenesis activity in tumors was a microscopic assessment of the density of blood vessels in tumor tissue (microvascular density). And only relatively recently, as a result of studying the molecular mechanisms of angiogenesis, which has been intensively developing in the last 10-15 years, the presence of a number of regulatory angiogenic and antiangiogenic factors, the dynamic balance of which ensures the formation and proliferation of new vessels inside the tumor, was demonstrated.
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 (oFGF and cFGF), epidermal growth factor (EGF), α- and P-transforming growth factors (TGF ), platelet-derived endothelial cell 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
the effect of VEGF on the tumor cells that produce 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 splicing of mRNA, as well as epigenomically during the proteolytic cleavage of synthesized molecules with the participation of the plasminogen activation system. A key regulator of blood vessel growth is VEGF A, while VEGF C primarily regulates lymphangiogenesis. The main soluble forms of VEGF A are molecules of 121 and 165 amino acid residues in size, and they are also the main biologically active forms of VEGF. In tissues, the major isoform of VEGF is believed to be VEGF-165.
On the surface of endothelial cells there are 3 receptors for VEGF, which are typical receptor tyrosine kinases. VEGF receptor type 1 (VEGFR1) is the product of the flt-1 gene, type 2 receptor (VEGFR2) is called KDR and is the human homologue of the mouse flk-1 gene product, and finally type 3 receptor (VEGFR3) is the product of the flt-4 gene . Unlike VEGFR1 and 2, it interacts not with classical VEGF (VEGF A), but with its homologue -VEGF C. All receptors are transmembrane glycoproteins with a mol. weighing 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 most important genes regulated by VEGF in endothelial cells include the c-ets-1 proto-oncogene, which encodes the transcription factor Ets-1. Studies using in situ hybridization have shown that c-ets-1 is expressed in endothelial cells during the early stages of blood formation.
venous vessels. Its product Ets-1 promotes the manifestation of the angiogenic phenotype of these cells, activating gene transcription and subsequent protein synthesis of the most important proteases that break down the extracellular matrix (ECM), - 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, oFGF and EGF) and are inhibited by antisense oligonucleotides to ets-1. Activation of proteases has three important consequences for stimulating angiogenesis: it facilitates the disintegration of endothelial cells and their invasion into the basal layer of blood vessels, generates ECM degradation products that promote the chemotaxis of endothelial cells, 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 from clinical material and published in 1994-1995. a group of Japanese researchers. In the first study, conducted by immunohistochemistry and including 103 patients with breast cancer, they showed that microvessel density and its growth, determined by immunochemical staining for factor VIII antigen, were 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, having confirmed the above patterns, also showed that the expression of VEGF correlates with the expression of another angiogenic factor - platelet-derived endothelial cell growth factor. Later, these authors conducted a quantitative enzyme-linked immunosorbent analysis of VEGF content in primary breast cancer tissues and showed that the concentration of VEGF in highly vascularized tumors was significantly higher than in weakly vascularized ones. However, no relationship was found between tissue levels of VEGF and two other potentially angiogenic factors - oFGF and hepatocyte growth factor. The concentrations of these two factors also did not correlate with microvessel density parameters.
Interesting data were also obtained. Using an immunohistochemical method, they compared the expression of VEGF, its receptor flt-1, also oFGF and α- and P-TGF in breast cancer and surrounding normal breast tissue. It turned out that of all the parameters studied, only the expression of VEGF 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 has also been demonstrated by RNA hybridization methods. All of 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 breast cancer cell line MCF-7. The expression and secretion of VEGF by transfected cells (V12) was confirmed by three independent methods: a competitive radioreceptor assay,
stimulation of the growth of human endothelial cells in vitro and activation of angiogenesis in the rabbit cornea. When transplanted into nude mice, clone V12 cells produced more vascularized tumors with a more heterogeneous distribution of blood vessels than the parental MCF-7 cells. The growth rate of tumors arising 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 has been shown that breast cancer cells that constantly produce VEGF have certain growth advantages.
Another proof of the influence of VEGF on the growth and metastasis of breast cancer is experiments with antibodies to this factor. Thus, in experiments on mice with spontaneous breast cancer, characterized by a high frequency of metastases to the lungs, it was shown that polyclonal antibodies to VEGF inhibit tumor growth by 44% and reduce the number and size of pulmonary 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 mammary tissue into a chamber formed by the dorsal skin fold of nude mice and assessed the induction of angiogenesis. All breast cancer samples, as well as breast tissue with hyperplasia and apocrine metaplasia, were found to significantly activate angiogenesis. Histologically unchanged areas of breast tissue from breast cancer patients stimulated angiogenesis in 66% of cases, while healthy breast tissue obtained through cosmetic surgery did not affect angiogenesis. In all cases, the induction of angiogenesis occurred in parallel with the production of VEGF by tumor or breast cells.
The classical model of regulation of angiogenesis in breast cancer (as in any other tumor) provides for the presence of a paracrine system in which growth factor (VEGF) is produced by tumor cells, and its receptors that perceive the signal 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 from 68 breast cancer patients using RNA in situ hybridization and showed that in the cells of invasive, metastatic and intraductal breast carcinoma there is a pronounced expression of VEGF, and in the vascular endothelial cells penetrating these tumors there is a pronounced expression of VEGFR1 and VEGFR2. Similar data were obtained
A. Kranz et al. , however, these authors also found VEGFR2 on mammary ductal epithelial cells. There is other evidence that breast cancer cells contain VEGF receptors, 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 levels
production is significantly higher than that of corresponding cells isolated from normal mammary glands. In this case, PCR analysis showed that VEGFR2 predominates in tumor cells, and only VEGFR1 is expressed in stromal cells. Thus, in addition to its direct function - stimulation of non-angiogenesis, 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 stimulators of aniogenesis, because they synthesize various angiogenic factors, incl. and VEGF itself. In particular, R. Leek et al. , having examined tissue samples from 96 breast cancer patients, demonstrated a positive correlation between the index of tumor tissue infiltration by macrophages and the level of VEGF expression.
The secretion of VEGF by breast cancer cells is induced by various external and internal factors. P. Scott et al. , studying the influence 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 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 virtually no effect on VEGF expression. R. Bos et al. showed that in the stimulation of neoangiogenesis under the influence of hypoxia, an important role is played by HIF-1, a transcription factor induced under hypoxia conditions, 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 stimulating 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 increased metastatic potential and resistance to adriamycin have a higher level of mRNA expression of the most angiogenic VEGF isoforms - VEGF-121 and VEGF-165 - than the original MCF- clone 7. In in vivo experiments, bcl-2-transfected cells formed tumors with a greater degree of vascularization and higher expression of VEGF than parental 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 the expression of bcl-2. 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 themselves.
Various growth factors and signaling systems are involved in the regulation of VEGF expression in breast cancer cells. In particular, several studies 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 “manager receptor” 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 normal breast cells glands. Basal VEGF secretion was increased in cells with increased levels of erbB-2, and in T47D cells with functionally inactivated erbB-2, not only basal VEGF secretion was reduced, but also its induction by heregulin. It was subsequently shown that the effect of heregulin on the synthesis of VEGF involves one of the classical signaling pathways involving phosphatidylinositol 3-kinase and protein kinase B (Akt), followed by the induction of the transcription factor HIF-1, which stimulates the expression of the VEGF gene.
Regulators of VEGF expression in breast cancer cells are apparently also some growth factors of the TGF-β family. The concentrations of TGF-P1 and VEGF in tumors and blood serum of breast cancer patients positively correlated with each other, and in in vitro experiments, TGF-P1 induced the production of VEGF by cultured MDA-MB-231 cells. Another study showed that 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 the induction of VEGF-mediated angiogenesis in the endometrium by estrogen is virtually certain, the existence of a similar mechanism in breast cancer has not been clearly demonstrated. J. Rujhola et al. using MCF-7 cell culture showed that 17P-estradiol (E2) causes a biphasic increase in the synthesis of VEGF mRNA, accompanied by the accumulation of the corresponding protein in the culture medium. This effect was blocked by the pure antiestrogen ICI 182.780, which suggests the participation of ER in its implementation. At the same time, classical antiestrogens such as tamoxifen and toremifene, which have a partial estrogenic effect, not only did not inhibit the VEGF-inducing effect of E2, but also themselves induced VEGF synthesis. The participation of RE 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 appears to depend on the type of breast cancer cells. Thus, J. Kurebayashi et al. described the human breast cancer cell line KPL-1, the growth of which 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 the synthesis of VEGF by breast cancer cells was also noted by S. Hyder et al. . Studying the T47-D cell line, they discovered
It was found 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 breast cancer 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, suggesting the involvement of a 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 VEGF production 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 unchanged breast tissue of 19 patients with breast cancer using PCR analysis, they found that the levels of VEGF expression in the unchanged gland were significantly higher in premenopausal patients than in postmenopausal patients, and significantly decrease with increasing age of patients. At the same time, the expression of VEGF in tumor tissue did not depend on the age and menopausal status of patients. The authors believe that in normal mammary glands angiogenesis is under hormonal control, but during malignant transformation this control is lost.
In addition to the most well-known and widespread angiogenic factor VEGF-A, described above, there are several more members of the VEGF family - VEGF-B, C and D. The function of VEGF-C is most clearly defined to date: it is believed that it stimulates lymphangiogenesis by interacting with receptors VEGF type 3 (flt-4), located on endothelial cells of lymphatic vessels. Experimental studies on nude mice using a new lymphatic endothelial marker LYVE-1 showed that overexpression of VEGF-C in breast cancer cells significantly enhances intratumor lymphangiogenesis and stimulates the formation of metastases in regional lymph nodes and lungs. Previously, J. Kurebayashi et al. using PCR analysis demonstrated 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 detected 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 with normal breast and fibroadenomas, but increased expression of type 3 VEGF receptors was observed on endothelial cells of blood vessels, not lymphatic vessels. In this regard, the authors believe that VEGF-C, like VEGF-A, is an angiogenic factor primarily 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 regulating 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, using clinical material using various methods (immunohistochemical, enzyme-linked immunosorbent, hybridization), increased expression of VEGF in breast cancer tissue and its relationship with traditional indicators characterizing the activity of neoangiogenesis in tumor tissue was demonstrated. In total, according to the results of an analysis of the Medline database, the study of the clinical significance of tissue VEGF levels in breast cancer was carried out by 14 groups of researchers. It should be noted again that almost all researchers who have made such comparisons, regardless of the methods used, note an increase in VEGF expression in breast cancer tissue compared to the surrounding histologically unchanged breast tissue, as well as with benign tumors. There is also no controversy regarding the direct correlation of the level of VEGF expression with the activity of neoangiogenesis in tumor tissue.
For the first time, the unfavorable prognostic value of high VEGF expression in breast cancer was noted by M. Toi et al. . Having retrospectively analyzed the results of observation of 328 patients in whose tumors VEGF expression was assessed by immunohistochemical method, they showed that in a 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 importance of VEGF for predicting disease-free survival was also demonstrated by M. Relf et al. , which determined the expression of the corresponding RNA in 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 effect on the prognosis of relapse-free survival of the 89 patients with breast cancer they examined.
The most interesting studies should be those in which the prognostic value of VEGF was assessed in various clinical groups of breast cancer patients, taking into account the treatment. The results of such a detailed analysis were 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 a quantitative enzyme-linked immunosorbent 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 for an average of 66 months. At the same time, VEGF in a wide range
concentration zone (from 5 to 6523 pg/mg 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, but it turned out to be a statistically significant predictor of relapse-free and overall survival according to the results of both univariate and multivariate analysis. Thus, the cytosolic level of VEGF is an indicator of prognosis in patients with early stages of breast cancer, allowing the formation of a group increased risk 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-derived endothelial cell growth factor) was carried out in patients with breast cancer with metastases to the lymph nodes who received chemotherapy according to the SMR regimen (137 patients) or hormone therapy with tamoxifen (164 sick). Cytosolic VEGF concentrations were similar in both groups. In the group of patients receiving tamoxifen, the level of VEGF was 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 in the results of univariate and multivariate analysis. Best results Treatment with tamoxifen should be expected in patients with low levels of VEGF in the tumor and involvement of less than three lymph nodes in the tumor process. A low level of VEGF turned out to be an independent factor of favorable prognosis in the group of patients receiving chemotherapy. In this group, TF is also a significant prognostic factor, and the prognosis is favorable with high levels of this protein.
In one of the latest studies of O. Oa8ragipii a1. The multifactorial prognosis model for patients with early stages of breast cancer also included natural inhibitors of angiogenesis - thrombospondins 1 and 2, but their contribution to the prediction of relapse-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 prognostic indicator of angiogenesis activity in breast cancer. Its high level indicates
about the unfavorable 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 for advanced breast cancer.
The prognostic value of VEGF in non-advanced breast cancer has also been studied and confirmed by B. biliary et al. . They determined by enzyme immunoassay the content of VEGF in the cytosols of tumors of 525 patients without metastases in the lymph nodes (T1.2K0M0), 500 of whom did not receive any postoperative treatment. Median follow-up was 46 months. Unlike the previously cited researchers, they found a direct correlation between the level of VEGF and the size of the tumor, as well as the degree of its malignancy and an inverse correlation.
lation of VEGF and RE levels. The survival rate of patients with cytosolic VEGF concentrations below the median level (2.4 pg/µg DNA) was significantly higher than in patients with lower VEGF levels. In multivariate analysis, VEGF level was the most significant independent prognostic factor, superior to other known indicators. A significant decrease in survival with a high level of VEGF in the tumor was also found in the prognostically favorable group of ER-positive patients.
According to the same authors, a high level of VEGF 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 was the only independent predictor of overall survival (relative risk 3.6) in the entire group, as well as relapse-free survival in the most prognostically favorable groups of patients with small tumors (T1) and ER-positive tumors. The authors suggest that high intratumoral VEGF levels may correspond to a radioresistant phenotype and indicate the need for additional systemic treatment.
B. Linderholm et al. also examined a group of 362 breast cancer patients with metastases to the lymph nodes, 250 of whom received adjuvant hormone therapy and 112 - adjuvant chemotherapy. In a univariate analysis, VEGF turned out to be a significant predictor of disease-free and overall survival in the entire patient population, as well as in the group receiving endocrine therapy. In the group of patients receiving chemotherapy, the level of VEGF had an effect only on overall survival. In multivariate analysis, VEGF remained significant only for overall survival.
Thus, this group of researchers also demonstrated the prognostic significance of VEGF for various clinical groups of breast cancer patients, which was summarized in a 2000 publication that included data on 83 patients with various stages of breast cancer. This work also demonstrated the prognostic significance of simultaneous study of VEGF and mutant p53. The relative risk of death increased by 2.7 times in the group with high VEGF content and positive p53 and only in
1.7 in groups with one of these unfavorable factors.
In a cooperative study, which included a total of 495 patients from two various clinics, based on data from univariate and multivariate analysis, which included, along with traditional indicators, also angiogenin, oFGF and plasminogen activators, it was shown that VEGF is the most important prognostic parameter for patients with breast cancer without metastases to 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, used to form high-risk groups among patients with early stages of breast cancer.
A special place is occupied by the study by J. Foekens et al. [b1], who determined the concentration of VEGF in preserved extracts using the enzyme immunoassay method.
total of 845 patients with advanced breast cancer who developed a relapse of the disease. 618 of these patients received adjuvant postoperative hormonal therapy with tamoxifen, and 227 patients received postoperative chemotherapy. It turned out that the cytosolic concentration of VEGF in tumors of patients in whom relapse occurred 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 internal organs than in patients with metastasis to bones and soft tissues. High VEGF levels, according to univariate and multivariate analysis, turned out to be an independent indicator of low sensitivity to both tamoxifen and chemotherapy.
In general, 13 of 14 studies published by 8 independent groups of researchers demonstrated that a high level of VEGF is an independent factor in the poor prognosis of breast cancer in the early stages and/or its low sensitivity to traditional types of hormone or chemotherapy in advanced breast cancer. In this regard, it was proposed to consider the possibility of including various antiangiogenic drugs in adjuvant treatment regimens for patients with high intratumor VEGF concentrations. It should be noted that unified methodological approaches and criteria for identifying patients with high levels of VEGF have not yet been developed, and further cooperative 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 increased expression of VEGF in the tumor is reflected in the level of this protein in serum/plasma and whether the concentration of circulating VEGF is an adequate characteristic of its content and angiogenesis activity in the tumor is being studied. In 1996-1997 The first studies were published that demonstrated an increase in the level of VEGF in the blood of cancer patients. 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 exceeded their established threshold level of 180 pg/ml. The serum level of VEGF correlated with the extent of the process and with the level of VEGF expression in tumor tissue, and the main isoform of VEGF in serum was VEGF-^5.
L. Dirix et al. [b3] examined a group of 132 patients with metastatic cancer with different primary diagnoses. They considered the VEGF level to be elevated if it exceeded the 95% confidence interval of the control group and amounted to 500 pg/ml. VEGF was elevated in 57% of patients with untreated metastatic cancer, regardless of site. During treatment, VEGF levels increased in 2/3 patients with disease progression and in less than 10% of patients with positive dynamics.
P. Salven et al. [b4] also showed that in various types of tumors (including breast cancer), the serum level of VEGF in disseminated cancer (171,711 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
In patients with disseminated cancer, the level of VEGF in the blood serum exceeded 200 pg/ml, and with successful treatment it decreased. Similar patterns were noted by A. Kraft et al. [b5]: according to their data, an increased 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 in the work of B. Zebrowski et al. [bb], a significant increase in the concentration of VEGF in ascitic fluids in cancer patients was demonstrated compared with ascites of non-tumor origin.
In more recent 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 has been shown that VEGF levels in the blood serum of patients with metastatic breast cancer (7-1347 pg/ml; median - 186 pg/ml) are significantly increased compared to 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 cancer (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 for benign tumors. This observation is in good agreement with the data of A. Lee et al. [b8], who showed that in the tissue of ductal breast cancer the content of VEGF mRNA and protein is significantly higher than in the tissue of lobular cancer. At the same time, ductal and lobular cancer did not differ in microvessel density, and only in ductal cancer was there a direct correlation between the levels of VEGF mRNA and protein and indicators of vessel density. It can be assumed that VEGF is a regulator of angiogenesis primarily in ductal breast cancer.
Of interest are comparative studies in which the content/expression of VEGF in tumor tissue and blood serum was simultaneously determined. G. Callagy et al. [b9], who determined the tissue expression of VEGF using an immunohistochemical method, came to the conclusion that it is this indicator, but not the serum concentration of VEGF, that 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 the blood serum. They also did not find a relationship between serum VEGF levels and its expression in the tumor.
The most representative comparative study was carried out by J. Adams et al. . They determined the content of VEGF in serum and plasma and the expression of VEGF in tumors (immunohistochemically) of 201 patients with localized and advanced breast cancer, benign breast tumors
forests and healthy women. In metastatic breast cancer, a significant increase in the level of VEGF was noted in both plasma and serum compared to normal. The content of VEGF in blood plasma in patients with metastatic breast cancer was also significantly increased compared with patients with benign tumors and localized breast cancer. In localized breast cancer, only an increase in the level of VEGF in the blood plasma was observed compared to the control. The authors believe that the measurement of VEGF in blood plasma largely reflects its production by the tumor, since serum VEGF is predominantly of platelet origin. Paradoxically, the highest levels of VEGF in serum and plasma were found in patients with breast cancer who were in remission during treatment with tamoxifen. Circulating VEGF levels did not correlate with any of the known clinicopathological factors, including microvascular density and tissue expression of UROR.
Thus, the possibility of using VEGF levels in the blood (both serum and plasma) as an adequate replacement for the tissue expression of this protein when 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.
ECEC-dependent angiogenesis as a target for antitumor therapy in breast cancer. Given the critical role of angiogenesis in maintaining 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 adjuvant treatment regimens for this disease. It has been proposed to use substances that relatively nonspecifically block the interaction of various growth factors with tyrosine kinase receptors, in particular, less toxic analogues of suramin, as antiangiogenic agents. It was also assumed that antiangiogenic agents could be especially effective when combined with drugs activated under hypoxia conditions, since suppression of angiogenesis would create conditions favorable for the activation of these drugs. Particular emphasis was placed on the fact that purely antiangiogenic therapy will most likely lead not to regression of the tumor, but to stopping its further growth. In this regard, the need to develop clinical and biochemical criteria for assessing the effectiveness of antiangiogenic drugs was pointed out.
Currently, in the United States alone, more than 20 drugs that affect angiogenesis in one way or another are undergoing clinical trials, mainly phase I. Among them are such quite specific drugs as monoclonal antibodies in VEOP (Genereil) 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 SV416), natural (Leova8-M ) and synthetic (Magita8la1, Propita8a1, VMB-275291, COb-3) inhibitors of matrix proteinases, natural (angiostatin and endostatin) and synthetic (TYP-470) inhibitors of proliferation or inducers of apoptosis (Combre8la1m) of endothelial cells and
There are also a number of drugs with different or unclear mechanisms of action.
I Roictap divides antiangiogenic drugs into direct and indirect angiogenesis inhibitors. He refers to direct inhibitors as substances that directly affect endothelial cells. These are the already mentioned angiostatin, endostatin, TYP-470, CotbinlaPn, as well as natural angiogenesis inhibitors such as thrombospondins and pigment epithelial factor. A characteristic feature of such drugs is that they, as a rule, do not induce 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 occurring is approximately the same as for traditional antitumor agents.
The interest in antiangiogenic antitumor therapy is currently so great that the number of publications devoted to preclinical and clinical studies in this area is measured in the 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 antiangiogenic action - thalidomide. In the 1970s it was used as a sedative and was banned due to its teratogenic side effects, which were due to its antiangiogenic properties. Attempts are currently being made to exploit the antiangiogenic 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 thalidomide every night, there were 12 patients with breast cancer. There was no objective response to the drug in patients with breast cancer, although a partial response or stabilization was observed in 6 of 18 patients with kidney cancer included in the same study.
At the same time, earlier experiments on the induction of angiogenesis in the rabbit cornea under the influence of the VEOP-producing clone of MCP-7 cells showed that the thalidomide analogue linomide at a dose of 100 mg/kg body weight effectively inhibits this process.
M. A8apo e1 a1. , showed that the anti-VEGF monoclonal antibody MY833 suppresses the growth of human breast cancer xenografts in nude mice. However, the inhibitory effect of MY833 did not correlate with either the amount of VEGF secreted by the tumor or the expression of the VEGF receptor. In other experiments, when transplanting spheroids formed by breast cancer cells of the MCP-7, 7YA-75 and BK-VYA-3 lines into the subcutaneous dorsal chamber of athymic mice, it was shown that the monoclonal antibody to VEGF A.4.6.1 at a daily dose of 200 μg significantly suppresses 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 observed 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 in doses that did not have a direct antiproliferative effect on tumor cells significantly suppressed the growth of established tumors measuring about 0.5 cm3. Another tyrosine kinase inhibitor has antiangiogenic activity against breast cancer xenografts - the well-known drug 7B1839 (Ige88a), which is a selective inhibitor of EGF receptor tyrosine kinase and is already undergoing clinical trials. It is assumed that this drug does not directly act on VEGF receptors, but suppresses the induction of VEGF synthesis under the influence of EGF receptor ligands. Apparently, another blocker of the EGF-dependent pathway of mitogenic signal transmission, Herceptin, a humanized monoclonal antibody to Herb2/neu, has a similar indirect effect on angiogenesis.
Drugs that disrupt microtubule function are also effective inhibitors of angiogenesis. So, back in 1997, Klauber N. eii 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 into athymic mice by 60%. The antiangiogenic properties of taxol have also been demonstrated by B. bau e1 a1. on mice with a well-vascularized transgenic breast tumor MeH. The effect was observed 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 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 using the enzyme-linked immunosorbent method before the start of treatment and after each of the 21-day courses. In 3 patients there was a partial response to treatment, in 6 - stabilization and in 5 - progression of the disease. Serum VEGF levels before treatment were significantly elevated in 8 of 14 patients. The average VEGF level decreased after treatment in patients with partial effect and stabilization and did not change significantly in patients with progression. Moreover, the percentage of normalization of VEGF levels or its reduction by more than 50% was significantly higher in patients with partial response (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 suppression of VEGF secretion and, accordingly, 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 administration it was detected
reduction in the weight of tumors in experimental mice by 51% compared to the control group. At the same time, an increase in the distance between tumor cells and the nearest vessels, a decrease in the overall density of vessels, and an increase in apoptosis were observed in tumors containing and expressing the endostatin gene.
Another genetic engineering approach to antiangiogenic therapy for breast cancer is the use of anti-sense cDNA to VEGF. S.A. Im et al. transfected human breast cancer cells of the MDA231-MB line with an adenoviral vector containing the same cDNA for VEGF-165 (Ad5CMV-alphaVEGF). In an in vitro system, this transfection resulted in decreased VEGF secretion without significantly affecting cell growth. Injection of Ad5CMV-alphaVEGF in vivo into tumors formed by MDA231-MB cells in nude mice resulted in suppression of tumor growth, decreased expression of VEGF protein in tumor tissue, and decreased microvascular density compared to the group injected with a vector containing no anti-VEGF cDNA .
Thus, the possibility of using various types direct and indirect antiangiogenic therapy in the treatment of patients with breast cancer. Unfortunately, none of these methods have yet proven their effectiveness in the clinic. Moreover, most authors are inclined to believe that antiangiogenic therapy (especially direct), which is predominantly cytostatic rather than cytotoxic for large tumors, should not be used 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
Gershtein E.S., Kushlinskiy D.N., Adamyan L.V., Ognerubov N.A. VASCULAR ENDOTHELIAL GROWTH FACTOR-CLINICALLY VALUABLE MARKER IN MALIGNANT NEOPLASMS
Results of 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 targets 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; prognosis.
Gershtein Elena Sergeevna, Russian Oncology Research Center named after. N.N. Blokhin RAMS, Moscow, Russian Federation, Doctor of Biological Sciences, Professor, Leading Researcher at the Laboratory of Clinical Biochemistry of the 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 of Obstetrics, Gynecology and Perinatology named after. IN AND. Kulakova, Moscow, Russian Federation, gynecological oncologist, e-mail: [email protected]
Kushlinskiy Dmitriy Nikolayevich, Research Center for Obstetrics, Gynecology and Perinatology named after V.I. Kulakov, Moscow, Russian Federation, Gynecological Oncologist, e-mail: [email protected]
Adamyan Leila Vladimirovna, Russian Scientific Center of Obstetrics, Gynecology and Perinatology named after.
IN AND. Kulakova, Moscow, Russian Federation, Doctor of Medical Sciences, Professor, Academician of the Russian Academy of Medical Sciences, Deputy. director, e-mail: [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, Tambovsky State University them. G.R. Derzhavina, 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 anticancer therapy. And only recently this opportunity was realized. Clinical data have demonstrated that the humanized monoclonal antibody drug bevacizumab, which targets a key proangiogenic molecule, vascular endothelial growth factor (VEGF), can prolong the life of patients with metastatic colorectal cancer when administered as first-line therapy in combination with chemotherapy drugs. Here we discuss the functions and significance of VECF to demonstrate that VEGF is a reasonable target for anticancer therapy.
What is VEGF?
VEGF is one of the members 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 endothelial cells in the wall of blood vessels (Table 1). The binding of VEGF to these receptors initiates 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 adequately functioning vascular system during embryogenesis and in the early postnatal period, but in adults its physiological activity is limited. Experiments on mice showed the following:
- Targeted damage to one or two alleles of the VEGF gene leads to the death of the embryo
- Inactivation of VEGF during early postnatal development is also fatal
- Damage to VEGF in adult mice is not accompanied by any obvious abnormalities because its role is limited to follicular development, wound healing, and the reproductive cycle in females.
The limited importance of angiogenesis in adults means that inhibition of VEGF activity represents a feasible therapeutic goal.
Already in July, the first Russian gene therapy drug for the treatment of vascular ischemia in the legs may appear on the market. Last September, neovasculgen (as it is called) was registered with Roszdravnadzor. It is possible that it will soon be offered for government procurement. The biotech company that created the drug, the Human Stem Cell Institute, which develops and tries to promote drugs and services “based on cellular, gene and post-genomic technologies,” speaks of the new product as a breakthrough in science. However, many experts view the new drug differently, arguing that it is actually about “patient confusion.”
In his speech on June 3, the 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 generally the first gene therapy drug. “Out of 1,500 clinical trials in the field of gene therapy, about 20 are aimed at treating patients with vascular pathology, and neovasculgen has already shown its effectiveness, while some drugs have fallen out of the running,” Deev emphasized. It seems that domestic drug manufacturers have something to be proud of! But is the new medicine really effective and safe, and how much will its use cost patients?
In the Society of Specialists evidence-based medicine draw 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 clinics in Yaroslavl, Ryazan, and Moscow, prescribed for inoperable forms of chronic leg ischemia to patients over 40 years of age. Two injections were given. Doctors have evidence that after administering the medicine, the patient could walk without pain not 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 HSCI director Artur Isaev. – The drug is a circular DNA molecule that contains a region responsible for the synthesis of vascular endothelial growth factor. Local administration of the drug stimulates the growth and development of new blood vessels.” Researchers are confident that for many patients the drug can become an alternative to amputation. The percentage of “success” of therapy, according to Professor R.E. Kalinin (Ryazan Medical State University), amounted to 93.6%.
In Russia, the system of angioplasty and vascular treatment of blood vessels has not been established. What is considered “high-tech care” to prevent amputations has become routine practice in most countries many years ago.
Things are bad in Russia with medicines too. Senior Researcher at the Institute of Surgery named after. Vishnevsky Leonid Blatun says that despite the availability of advanced ointments and medicines, patients in clinics of the Russian Federation “really have access to only the most outdated means,” since modern means are not included in the standards of treatment.
How safe is neovasculgen? It must be emphasized that when a new gene is introduced into a human cell, the patient may experience cancer risks. This is why drugs with this mode of action have not previously received approval. “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 Valentin Vlasov, director of the Institute of Chemical Biology and Fundamental Medicine, Academician of the Russian Academy of Sciences. - That is, with the help gene technology a virus is taken and it delivers the required gene into the cell.
On this topic
Law enforcement agencies did not initiate a criminal case against Moscow resident Elena Bogolyubova, who ordered a drug not registered in Russia by mail for her terminally ill son.
“I am familiar with the project of the Stem Cell Institute and the drug neovasculgen,” says Valentin Vlasov. – In this case, there is no question of a virus vector. I do not rule out that in a very short time after the injection, protein synthesis occurs with the help of this product, and it does not seem to bring anything bad to the patient, but whether it brings anything good, in order to assert this, a very serious evidence base is needed "
The expert noted that it is quite difficult to draw such a conclusion from the photographs provided: “How to look at them, with what resolution the X-rays were taken, how they were developed - this is all on the conscience of the researchers. It seems that small vessels are branching. The report about the drug was pompous, but I can say that if such an effect exists, it is very short in time, it can only last a few days. And there is no reason to expect a miraculous effect from the drug.” According to Academician Vlasov, scientists need to achieve long-term protein production, and this can only be achieved by “inserting” the desired gene into a cell, but researchers have not yet been able to do this safely for the patient.
Even the journal in which the results of the study of the drug neovasculgen were published looks like it belongs to the same company. According to experts, questions arise from the haste in conducting clinical trials and the lack of randomization in them (a special algorithm for conducting them that excludes interest in the results). The place of administration of the drug and its description – “plasmid construct” – raised doubts.
As a result, experts came to the conclusion that this may be a case of “consumer confusion,” since large vessels in which there is no blood flow are not restored. The researchers promised benefits for patients for two years, but the trial actually lasted only six months. The absence of declared side effects from such a drug is also suspicious. The desire of scientists to find new treatment options is not disputed. But all this requires for long years searches and significant evidence before use.
Patients with critical ischemia of the lower extremities in 20–50% of cases experience so-called primary amputations, but only slightly more than half of those operated on retain both legs after a year. Every fifth person dies, and in every fourth case a “major amputation” is performed. Obviously, many patients will literally stand in line for a miracle cure. Among them there will be a huge number of diabetics.
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, treatment for this complication begins late. At the same time, in comparison 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 EU countries 8% of complications of peripheral vessels of the legs end in amputation, while in Russia this figure is significantly higher and in diabetes mellitus reaches more than 50%. 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 patients with diabetes are affected by diabetic foot syndrome, and up to 50% of them can be classified as at risk. After amputations, the mortality rate of patients doubles, but if such patients are not operated on, they will die of gangrene within two years.
During surgical interventions in patients with type 2 diabetes
In type 2 diabetes there is an imbalance in angiogenesis. Diabetes is characterized by hyperglycemia and various metabolic disorders. They disrupt the balance between proangiogenic and antiangiogenic regulators and lead to inadequate formation of new vessels in diabetes mellitus (DM). In turn, disorders of angiogenesis and vasculogenesis are important mechanisms in the development vascular complications SD. 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 is slowed down. In this case, one of the factors is a decrease in the level of local growth factors, which limits the possibility of building up soft gum tissue as part of implant surgery. It has also been proven that in patients with diabetes, the amount of collagen produced by fibroblasts is reduced, which leads to a slowdown in wound contraction. Impaired carbohydrate metabolism entails an increase in matrix metalloproteases (MMP) and a decrease in nitric oxide (NO), transforming growth factor beta 1 (TGFβ1), which causes a slowdown in the formation of ECM. Clinical studies show that in diabetes mellitus, angiogenesis imbalance can be achieved using both angiogenesis inhibitors and its stimulators. Stimulation of angiogenesis and vasculogenesis with the help of stem cells and growth factors is a promising direction for the treatment of insufficiency of agnogenesis in diabetes mellitus, which affects the reduction in the healing process of soft tissues and the formation of macroagniopathy.
Taking into account the above, in postoperative period In patients with diabetes, it seems promising to stimulate the process of angiogenesis using cycotins and vascular endothelial growth factor.
It is known that vascular endothelial growth factor and cycotin stimulate angiogenesis, and thus increase tissue oxygen saturation (pO2), which is one of the factors for soft tissue repair. A decrease in the level of this growth factor leads to a slowdown in the epithelization process. Research results show that growth factors and cytokines have a decisive influence on the speed and quality of reparative processes in patients with diabetes mellitus.
So in dentistry, when growing gum tissue, implantological 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, implantological operations are performed in patients with diabetes only when the level of glycated hemoglobin is less than 6.0. This indicator is achieved by temporarily transferring the patient to insulin injections during the operation and postoperative period. 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 the glycated hemoglobin indicator to shift to higher values, compensating for the disturbances of agnogenesis from hyperglycemia with vascular endothelial growth factor. It appears that the procedure for administering platelet-rich plasma can be used in any surgical procedure for patients with diabetes.
