Abbreviations

VEGF — vascular endothelial growth factor

ANG — angiopoietin

ENG — endoglin

MMP — matrix metalloproteinase

SNP — single nucleotide polymorphism

TGF — transforming growth factor

PDGF — platelet-derived growth factor

ALK — activin receptor-like kinase

HIF — hypoxia-inducible factor

TGF — transforming growth factor

TIE — endothelial cell — specific tyrosine kinase

Arteriovenous malformations (AVM) are annually observed in 1.1 per 100 thousand adults. This disease is not the most common cerebrovascular disease [1, 2]. AVMs occur sporadically and as part of genetic syndromes. Hereditary hemorrhagic telangiectasia (Osler—Weber—Rendu syndrome) is a syndrome primarily associated with AVM. Genetic, molecular and cellular factors of development, growth and rupture of AVMs have been described over the past two decades [3—8]. Discovery of new data on the main mechanisms of AVM development can be an incentive to identify biomarkers of risk and prognosis and new methods of treatment [7].

Today, it is known that over 860 genes are involved in development of cerebral AVMs with overexpression of 300 genes and inhibition of 560 genes. Single nucleotide polymorphisms (SNP) of angiogenic factors as sequence of DNA variations were first associated with development and risk of rupture of cerebral AVMs [3,5,7].

There are 2 stages of development of cerebrovascular system. The first one (vasculogenesis) is formation of circulatory vascular system in early embryogenesis, when mesoderm cells differentiate into hemangioblasts (common precursors of hematopoietic cells and endothelial cells (angioblasts)). Vasculogenesis takes place outside the neural tube and results formation of primary vascular plexus. All further transformations of the vascular network occur during the second stage (angiogenesis). This stage is accompanied by development of new vessels from previous vascular structures [8, 9]. Primary vascular plexus turns into a highly organized vasculature due to growth and branching of capillaries. Angiogenesis begins from local destruction of the wall of previously existed vessel, activation of proliferation and migration of endothelial cells. Endothelial cells merge into tubular structures. Vascular walls are subsequently formed around these structures. Analysis of the processes regulating biological activity of two main types of cells (endothelial cells and pericytes) is essential to understand the mechanisms of angiogenesis [5, 8, 9, 10]. Endothelial cell activation is ensured by growth factors and components of extracellular matrix. Growth factors are released by endothelial cells themselves. Cessation of these factors returns endothelial cells to a rest state. Growth factors is a group of protein molecules inducing DNA synthesis in a cell. Each type of growth factor binds with its own specific receptor and triggers the processes of growth regulation, differentiation, expression of genes and apoptosis. Growth factors are divided into inducers and inhibitors. The last ones normally prevail over inducers. As a result, angiogenesis is suppressed in the adult body and only 0.01% of endothelial cells are capable for mitoses. Reduced release of inhibitors or increased secretion of inducers stimulate angiogenesis [9].

AVM is a conglomeration of arteries and veins. These vessels form a network of direct arteriovenous shunts with high blood flow velocity. According to the generally accepted histopathological concept, there is no true capillary system in AVM. Nevertheless, there is a group of dilated capillaries near AVM (the so-called “giant bad capillaries”). Their diameter is 10—25 times greater than diameter of normal vessels. These enlarged capillaries are connected with AVM structures and normal capillaries through arterioles and venules. This abnormal vascular system impairs hemodynamics and causes brain ischemia. It is possible that this abnormal capillary network is a morphological reserve for AVM growth and recurrence. Cells cultured from surgical samples of brain AVMs are characterized by 1.8—6.4-fold higher proliferation rate than normal resting cerebrovascular endothelial cells. These values are similar to growth rate of endothelial cells in progressive tumors [11].

The main regulator of physiological and pathological angiogenesis is vascular endothelial growth factor (VEGF). This factor stimulates growth and division of only endothelial cells. Vascular endothelial growth factor activates two closely related membrane tyrosine kinase VEGF receptors localized on the surface of endothelial cells (VEGFR1 and VEGFR2). VEGF expression is maximal in embryonic period. However, this growth factor is normally suppressed in adult cerebral vasculature. Expression of embryological substrates in AVM implies that development of malformations is an active process beginning in antenatal period and continuing after birth.

Equal reaction of all endothelial cells against VEGF angiogenic stimulus would result partial destruction of vasculature and impaired blood supply of tissues in this area. To prevent this event, there is a mechanism for initiation of only some endothelial cells inside the vessel (the so-called "tip cells") for angiogenic proliferation. These cells occupy a leading position in growth of new vessels, respond to VEGF gradient determining direction of migration and lead the advancement of growing capillary [12—14]. Phenotype of tip cells is cardinally changed under the influence of angiogenic stimulus of VEGF. These cells acquire invasiveness and ability to move and activate surface proteases (MMPs) which destroy adjacent basal membrane. These processes change interactions between tip cells and surrounding endothelial cells. Selection of tip cells is controlled by Notch receptors and their transmembrane ligands Dll4 (Delta like ligand 4) [13]. VEGF activates expression of Dll4 and its Notch receptors in endothelial cells. In response, tip cells eject filopodia towards tissue VEGF gradient. However, VEGF does not result proliferation of tip cells themselves. Reduced expression of Dll4 or blockade of Notch signaling pathways enhance formation of tip cells. This process is followed by excessive increase of local angiogenesis and disruption of proper development of new vessels. Overexpression of VEGF in antenatal period causes excessive development of abnormal vessels and AVM.

Selection and advancement of tip cells are followed by development of new capillaries though proliferation and migration of other endothelial cells (stalk cells). These cells are also stimulated by VEGF. Thus, VEGF independently controls migration of two different subpopulations of endothelial cells: tip cells and stalk cells. Murphy P.A. (2014) et al. reported expression of proteins involved in Notch signaling pathways in cerebral AVMs samples [13,15]. The processes of angiogenesis are regulated by multiple genes. However, significant congenital vascular defects and death of embryos arise only in DNA deletion of one allele of VEGF and Dll4 genes [16, 17]. Endothelial overexpression of Notch-4 and its ligands (Jagged 1 and 2-ligands of stalk cells and Dll 1,3,4-ligands of tip cells) is accompanied by development of cerebral AVMs in mice [18]. Increased expression of Notch ligands (Jagged 1 and 2) alters synthesis of arterial and venous endothelial markers (Ephrin B2 and Eph B4) in the vessels of experimental AVMs. This results violation of specificity of endothelial cells (arterial and venous) during formation of AVMs [16, 17]. Thus, Notch and its ligands are involved in pathogenesis of cerebral AVM through several pathways: (1) enhancement of angiogenesis; (2) deterioration of vascular wall structure; (3) changes in arterial and venous specificity of endothelial cells [19].

The main attention of researchers was drawn to regulation of endothelial cells after discovery of angiogenic factors. However, cells of the vascular walls are also functionally important, since impaired development of the walls causes increased vascular permeability. Initial stage of vessel maturation is fusion of the newly formed capillary with other capillaries. This process is associated with behavioral changes of tip cells. These cells come into contact with other tip cells or existing capillaries and stop moving. Occurrence of highly adhesive intercellular interactions in this area results formation of vascular lumen. Subsequent blood flow stabilizes the vessel de novo while oxygen delivery reduces local expression of VEGF and other angiogenic signals previously induced by hypoxia. An important stage of maturation is involvement of pericytes and smooth muscle cells of vascular walls. Pericytes adhere to endothelium and form the walls of capillaries and immature blood vessels. Accumulation of extracellular matrix proteins in basal membrane contribute to maturation of the vessel. Platelet-derived growth factor (PDGF) is very important in attracting pericytes. Increased expression of PDGF-β mRNA in tip cells during angiogenesis causes proliferation, directed migration and incorporation of pericytes into the vascular wall. PDGFR-β receptor is expressed on the surface of these cells [21]. Expression of PDGFR-β receptor was not observed in endothelial stalk cells. Therefore, PDGF-β does not influence these cells. Reduced number of vascular pericytes correlates with impaired integrity of AVM vessels and risk of intracranial hemorrhage [22]. Higher expression of PDGF-B was found in human cerebral AVMs compared with normal vessels [23].

Transforming growth factor β1 (TGF-β1) is essential in angiogenesis. This factor is activated by the contact between endothelial cells and pericytes. TGF-β1 activation under the influence of PDGF, ANG and TIE receptors inhibits endothelial proliferation and migration and induces differentiation of mesenchymal progenitor cells into pericytes. However, the role of TGF-β1 in angiogenesis is more complex, since there are 2 types of TGFβ receptors with multidirectional action. Endothelial-specific subtype of ALK1 receptor increases endothelial migration and proliferation while ALK5 receptor, on the contrary, inhibits their proliferation and migration. Endoglin gene (ENG) encodes proteins of TGFβ1 receptor complex. Normally, endoglin blocks ALK5 receptor (inhibition of endothelial proliferation) and enhances function of ALK1 receptor (activation of endothelial proliferation). Mutation of endoglin and/or ALK1 is accompanied by development of excessive AVM vasculature. Blocking of ALK5/endoglin enhances development of endothelium. This process is associated with impaired maturation of capillary plexus and development of arteriovenous fistulas. Thus, TGFβ may be crucial in growth and formation of AVMs de novo [7, 17].

Another signaling system is involved in regulation of complex interactions between endothelium and surrounding cells [24]. These are TIE2 tyrosine kinase receptor expressed by endothelial cells and its angiopoietin ligands (ANG 1 and 2). Both of these ligands bind to TIE2. However, the consequences of their interaction with the receptor are different. ANG1 stimulates TIE2 phosphorylation. ANG2 is a competitive inhibitor of ANG1. TIE2 activates transcription of PDGF and VEGF stimulating migration of mesenchymal cells to primitive endothelial vessel. TIE2/ANG1-dependent signaling pathway ensures association of pericytes and endothelium, reduces vascular permeability and stabilizes vascular system during its maturation [24]. Suppression of TIE2-associated signaling pathway by angiopoietin-2 inhibits migration of pericytes, disrupts vascular maturation and sensitizes vessels to stimulating effect of VEGF. Normally, ANG2 release is suppressed in a mature vasculature. However, overexpression is observed in perivascular area around the cerebral AVMs. This process ensures formation of abnormally dilated vessels without a mature wall structure. Concentration of ANG-1 in cerebral AVM is 30% lower than normal, while the concentration of ANG-2 mRNA is 40% higher compared to the control values [24, 25].

Matrix metalloproteinases (MMPs) is important aspect of VEGF and ANG-2 signaling pathways. These proteins promote angiogenic proliferation and rupture of AVMs. Proteolytic enzymes degrade pericellular substances and basal membrane that initiates the processes of angiogenesis. MMP-9 expression is higher in cerebral AVMs than in control tissue [26]. Starke et al. [27] found significantly increased serum MMP-9 prior to surgery in patients with ruptured AVMs compared to those without rupture. Moreover, these values were significantly increased immediately after surgery. SNP of MMP-9 and tissue inhibitor of MMP-4 (TIMP-4) is also associated with increased risk of rupture of cerebral AVM. These data were confirmed by genotyping analysis in patients with AVM [28].

Thus, we analyzed the possible roles of angiogenesis factors in pathogenesis of cerebral AVMs. VEGF induces endothelial mitogenesis. Mutations of genes associated with AVMs (ALK1, ENG, MPG) activate an altered Notch signaling pathway. This cascade subsequently reduces PDGF signaling while angiogenic response leads to formation of the vessels with abnormal wall structure. Experimental trials show that either correction of Notch signals [14] or increased expression of PDGF [21] restore abnormal vascular structure of AVMs. Therefore, new methods of AVM management may be developed through modulation of these two pathways.

Endothelial expression of VEGF and its receptors is observed in almost 80% of AVMs resected after incomplete embolization. At the same time, only one quarter of AVMs expresses these factors if preoperative embolization is absent [20]. These data can explain growth of AVMs after partial embolization. The following mechanisms for stimulating angiogenesis after partial embolization of AVM are discussed:

1. Hypoxia-induced angiogenesis. Incomplete embolization of AVM is followed by restructuring of blood flow and local hypoxia. These processes increase expression of pro-angiogenic factors, in particular, hypoxia-inducible transcription factor (HIF-1). HIF-1 promotes secretion of SDF-1 (stromal-derived factor-1) in endothelial cells and increases VEGF expression by about 30 times within a few minutes after hypoxic exposure [29]. In hypoxic environment, HIF-1 permeates to the nucleus and accelerates transcription of VEGF, PDGF, TGF, Notch. At the same time, VEGF stability is increased that results 4-fold prolongation of its half-life [30]. This is necessary because VEGF ensures angiogenesis and vascular proliferation until tissue metabolic needs will be satisfied.

2. Inflammation-induced angiogenesis. Histological examination revealed aseptic inflammation and angionecrosis in AVM one day after onyx embolization [31]. Inflammatory reaction in AVM vascular wall increases endothelial release of interleukin-6 (IL-6) and activates expression of MMP-9. The last one damages AVM endothelium and can lead to its rupture. The effect of inflammation on angiogenesis is enhanced by polymorphism in genes of proinflammatory cytokines in patients with AVM (allele IL6-174G). Local intranidal increase of IL-6 mRNA level correlates with augmentation of mRNA level of matrix metalloproteinases (MMP-3, MMP-9). This correlation confirms that IL-6 is essential in angiogenic cascade [31].

3. Angiogenesis associated with hemodynamic changes in AVM. Hemodynamic changes following AVM embolization include enlargement of high-flow afferent arteries and shift of intraluminal tension of blood flow in unembolized segment of AVM. These changes stimulate expression of VEGF, TGF, MMP-9, ENG in AVM endothelium [29, 32].

Modern surgical, endovascular and radiosurgical treatment of high-flow large AVMs is accompanied by relatively high disability and mortality rates [33]. Therefore, development of new cellular and molecular biological treatment methods is advisable [34]. The current paradigm for AVM treatment does not consider any special genetic or molecular factors of a particular patient. It is time to think that better understanding of AVM growth mechanisms may be valuable to change their natural course and optimize treatment strategy [35].

It is believed that ABM rupture mechanism is associated with local vascular remodeling. Therefore, drugs impeding this process may be theoretically attractive as therapeutic agents. For example, doxycycline in very low doses inhibits expression of MMP-9 mRNA and reduces activity of cerebral MMP-9. Moreover, this drug is accompanied by acceptable rates of complications [36]. Some researchers have hoped for intra-arterial administration of bevacizumab (monoclonal antibody to VEGF) [37]. The advantage of this administration over intravenous injection is higher local concentration in the area of interest and significantly reduced total dose. As a result, reduced systemic toxicity and cost of treatment are observed. Some authors consider that direct anti-VEGF therapy may be ineffective or even harmful due to damage of normal vascular cells although VEGF is still a potential target for antiangiogenic therapy [38]. Thrombospondin-1 (TSP-1) as natural antagonist of VEGF is characterized by extremely low concentration in AVM and may be of interest as alternative to anti-VEGF therapy. It has been recently proven that TSP-1 augmentation can block VEGF in AVMs while microRNA-18a involved in post-transcriptional regulation of gene expression increases concentration of TSP-1 in AVMs. This results significant decrease of the level of VEGF in AVM endothelium, reduced endothelial proliferation and improved structure of the vascular wall. At the same time, there is no effect on the endothelium of normal vessels [38]. Moreover, intravenous infusion of miRNA-18a is followed by its successfully penetration into target cells without additional reagents. Therefore, it is perspective therapeutic approach.