Extracellular Matrix in Tumor Angiogenesis

Abstract

Extracellular matrix (ECM) is a complex three-dimensional network that provides structure, strength, and contextual information for cellular growth, communication, differentiation, survival, adhesion, and migration. ECM basic proteins resist compressive forces and/or allow rapid diffusion, others strengthen the matrix, and give resilience or modulate cell-matrix interactions. ECM undergoes turnover and remodeling physiologically and during inflammation, wound repair and tumor invasion. Remodeling of the ECM is an integral component of the angiogenic process and depends on the composition of matrix molecules, soluble pro-angiogenic and anti-angiogenic factors, and their spatial regulation. This review will focus on the myriad roles of those molecules and will emphasize their involvement in critical points of angiogenesis.

1. Introduction

“Tumor progression is defined by irreversible change in the tumor characteristics reflecting the sequential appearance of a genetically altered subpopulation of cells with the new characteristics” [1]. The term, “tumor progression” is used to describe phenotypic changes in the preexisting neoplastic lesion. It is a coincidence of complex events characterized by morphological, molecular, and functional changes in tumor cells and their environment and encompasses a wide scale of mechanisms [2]. It is in part recognized as a product of evolving crosstalk between different cell types within the tumor and its surrounding supportive tissue or tumor stroma [3]. Invasive tumor cells interact with their microenvironment in a bidirectional manner and remodel it into a supportive context for tumor growth and progression. The composition of the tumor microenvironment varies between tumor types, but hallmark features include cellular components such as immune cells (T-cells, B-cells, NK-cells, macrophages, neutrophils, dendritic cells), stromal cells, blood vessels, cancer-associated fibroblasts, adipocytes, stellate cells, and noncellular components such as extracellular matrix (ECM) and exosomes [4].

2. Extracellular matrix: its composition and molecular profile

Extracellular matrix (ECM) is a noncellular, proteinaceous component of the stroma. It is a complex three-dimensional network of macromolecules. The ECM provides architectural structure, strength, and contextual information for cellular growth, adhesion, communication, differentiation, migration, and survival. Molecules that provide ECM structure are: glycosaminoglycans and proteoglycans (form a hydrated gel-like substance, resist compressive forces, and allow rapid diffusion) and fibrous proteins and collagens (strengthens the matrix and give resilience). They represent insoluble factors of the matrix [5]. Structural molecules are synthesized mainly by fibroblasts but also by other cells of connective tissue. ECM molecules named, “matricellular proteins” (e.g. thrombospondin-1 and -2, SPARC, tenascin-C, and osteopontin) do not function as structural elements but modulate cell-matrix interactions and cell functions [6]. ECM is in a dynamic state and undergoes turnover and remodeling in conjunction with signals and is enhanced during inflammation, wound repair, and tumor invasion. However, ECM can limit initiation of tumor at an early stage of its development, later, ECM stimulates tumor growth and progression and enhances its aggressiveness. Key enzymes which remodel ECM are matrix metalloproteinases (MMPs) and urokinase-type plasminogen activators (uPAs). They degrade components of the basement membrane as well as proteins and proteoglycans of connective tissue and liberate latent growth factors from their storage sites in the extracellular matrix. Factors that are activated in this fashion are, for example, fibroblast growth factors (FGFs), hepatocyte growth factor (HGF), and transforming growth factors (TGFs) [7]. Tumor growth-induced solid stress, matrix stiffness, increased interstitial fluid pressure, hypoxia and altered tumor pH have been established as a result of tumor growth and on the other hand, neoangiogenesis-supporting conditions. As structural and metabolic alterations of ECM can lead to the development or progression of disease, its molecules can serve as important targets for pharmacotherapy.

2.1 Collagens

Collagen represents 30% of dry weight in the human body and is the most abundant protein synthesized by fibroblasts and by several other cell types distinct by their molecular profile, morphology, distribution function, and involvement in pathologies [8]. Collagens play structural roles and contribute to mechanical properties, organization, and configuration of tissues. Some collagens have a restricted tissue distribution and hence specific biological functions [9]. Collagens are trimeric molecules composed of three polypeptide α chains, which contain the sequence repeat that allows the formation of a triple helix. Besides triple-helical domains, collagens contain non-triple-helical domains, used as building blocks by other extracellular matrix proteins. At present, 28 types of collagens are classified as fibrillar collagens, unconventional collagens including collagen VII, network-forming collagens (VI, VIII, and X), fibril-associated collagens with interrupted triple helix (IX, XII, XIV, XVI, and XIX), basement membrane collagens, transmembrane collagens, and multiplexins [10, 11]. Type I, II, III, V, XI, XXIV, and XXVII collagens belong to the classical fibrillar collagens [12]. Fibrillar collagens can assemble into supramolecular aggregates. Type I collagen is major collagen of tendons, ligaments, skin, cornea, and other connective tissues representing 90% of the total collagen in humans. It is mostly a part of the compound containing either type III collagen seen in skin and reticular fibers [13] or type V collagen found in bone [14]. The biomechanical properties of these compounds (e.g., torsional stability and stiffness or tensile strength) establish the stability and integrity of these tissues [15]. Bourgot and colleagues describe the evolution of fibrillar collagen organization during tumor progression where tumor-derived paracrine signals promote a desmoplasic reaction characterized by the activation of the resident fibroblasts into cancer-associated fibroblasts (CAFs) with enhanced secretory activity, reorganization of the collagen fibers (their cross-linking), augmenting the stiffness of the stroma. Tumor adjacent collagen fibers that promote invasive cancer cell migration can be organized parallel (Tumor Associated Collagen Signature—TACS-2) or perpendicular to the tumor border (TACS-3) [16]. Collagen fibers employ guidance signals for endothelial cell migration during regenerative angiogenesis. Inhibition of collagen cross-linking results in a 70% shorter regeneration area with 50% reduced vessel growth and disintegrated collagen fibers. The disrupted collagen scaffold impedes endothelial cell migration and induces the formation of abnormal angioma-like blood vessels [16]. Type I collagen, potently stimulates angiogenesis in vitro and in vivo [17]. Crucial to its angiogenic activity appears to be ligation and possibly clustering of endothelial cell surface α1β1/α2β1 integrin receptors by the GFPGER (502–507) sequence of the collagen fibril. Authors describe here genetically engineered “angiogenic superpolymers”, containing type I collagen, fibrillar collagens and collagen mimetics, possibilities of their modifications to display ideal angiogenic properties, and prove their usefulness for tissue engineering and human medicine [17].

The vascular basement membrane represents an insoluble structural component of the wall of newly formed capillaries and undergoes several changes during tumor-induced angiogenesis. Initially, the membrane is degraded and disassembled by proteolytic activity of matrix metalloproteinases, mainly MMP2 and 9, but is finally after complex molecular crosstalk by regulation mainly via VEGF signaling, is reorganized to a native state around a newly formed capillary. Such vascular matrix changes during angiogenesis are associated with the expression of matrix proteins that can interact with vascular endothelium and provide endogenous angiogenic and anti-angiogenic signals. Basement membrane molecules play a role also in the process of the relapse of pathological angiogenesis [18]. Rapid relapse of tumor angiogenesis is hypothesized to be facilitated by the empty basement membrane sleeves (ebms) of previously regressed vessels, which are postulated to serve as scaffolding for endothelial cells during new angiogenic sprouting, following cessation of antiangiogenic treatment [19]. Type IV collagen is found in solid and soluble states in ECM, it is composed of three α(IV) chains [20]. The a1 and a2 isoforms are ubiquitously present in human basement membranes. Type IV collagen promotes cell adhesion, migration, differentiation, growth [21], and regulates endothelial cell proliferation and behavior during the critical steps of the angiogenic process. Studies have shown that the function of type IV collagen in the elongation and stabilization of microvessels was dose-dependent with low concentrations of type IV collagen promoting elongation, and high concentrations stabilizing them. Anti-angiogenic properties were associated with inhibitors of collagen metabolism and basement membrane collagen synthesis and deposition were crucial for blood vessel formation and survival [18]. There are six known bioactive peptides generated from collagen type IV [22]. These peptides are fragments of non-collagenous domains from the α1 (arresten), α2 (canstatin), α3 (tumstatin), α4 (tetrastatin), α5 (pentastatin), and α6 chains (hexastatin). Arresten, is an inhibitor of angiogenesis in squamous cell carcinoma, binding with α1β1 integrin in endothelial cells [22, 23, 24]. Carcinoma cells showing overexpression of arresten changed to an endothelial phenotype, suggesting inhibition of migrating carcinoma cells by inducing mesenchymal to endothelial (MET) transition [24]. Role of arresten is demonstrated in modulating the function of capillary endothelial cells and blood vessel formation using in vitro and in vivo models of angiogenesis and tumor growth [25]. Recently, the NC1 domain of the α2 chain of type IV collagen (canstatin) was also identified as an angiogenesis inhibitor. In the study by [25], Canstatin was first identified as vasculogenic mimicry (VM) inhibitor. Vasculogenic mimicry is a neovascularization phenomenon that was first reported in melanoma models. Distinct from classical tumor angiogenesis, VM provides a blood supply for tumor cells independent of endothelial cells and formed by deregulated tumor cells. VM is established in lung cancer [26], hepatocellular carcinoma [27], and glioma [28] and is associated with poor prognosis in cancer patients [29]. Vautrin-Glabik demonstrated that 13 amino acid sequence of tetrastatin decreases VEGF-induced-angiogenesis in vivo using the Matrigel plug model and decreases Human Umbilical Vein Endothelial Cells (HUVEC) migration and pseudotube formation in vitro [30]. Oskimaki et al. recently developed a bioinformatics-based approach to predict over 100 novel endogenous anti-angiogenic peptides [31]. An important peptides determined were tetrastatins, pentastatins, and hexastatins that were validated in vitro in cell proliferation and migration assays on HUVECs [32]. Using pentastatin-1 to an angioreactor-based directed in vivo angiogenesis assay (DIVAA), and in vivo NCI-H82 SCLC xenograft model strong potential for pentastatin-1 as a therapeutic agent for lung cancer was demonstrated [30].

2.2 Elastin

Elastin provides elasticity to the ECM. Elastin is roughly 1000 times more flexible than collagens. It is produced as tropoelastin, a 72 kDa precursor protein by fibriblasts, smooth muscle cells, chondrocytes, or endothelial cell and is secreted from the cell to the extracellular space, where it crosslinks with other elastin molecules. Elastin is the primary ECM protein present in arteries where it composes ~50% of their dry weight [33]. During aging, continuous mechanical stress and an increase in elastase activity contribute to the fragmentation of elastic fibers resulting in the release of elastin-derived peptides (EDPs) [34]. EDPs are matrikines—matrix fragments having the ability to regulate cell physiology and display a wide range of biological activities in a number of normal and transformed cells [35]. For example, they potentiate the migration and matrix invasion of tumor cells, stimulate the migration and proliferation of monocytes and skin fibroblasts and up-regulate MMP expression by fibroblasts inducing a remodeling program for melanoma invasion. Additionally, they are pro-angiogenic, chemotactic for inflammatory cells and promote elastase release [36]. Robinet and colleagues showed that elastin-derived peptides enhanced angiogenesis in the chick chorio-allantoic membrane in vivo, augmented pseudotube formation from human vascular and microvascular endothelial cells in the matrigel and promoted cell migration in wound healing assay [37].

2.3 Glycosaminoglycans

Glycosaminoglycans (GAGs) were primarily known as “space fillers” in the ECM, but later appeared as active signaling molecules in cell fate regulation via cytokine production, leukocyte recruitment, or inflammatory response [38]. GAGs are linear polysaccharides with two basic saccharide molecules that vary according to epimerization, sulfation, and deacetylation. Their specificity and functionality depend on the order of the carbohydrate chain and the other chemical modifications [38]. Hyaluronan is the simplest GAG since it is non-sulfated, does not undergo epimerization, and does not use typical covalent bonds for linking to proteins. Other GAGs—chondroitin sulfate, dermatan sulfate, keratan sulfate, and heparan sulfate usually use covalent bonds for attachment to proteins in proteoglycan molecules. Chondroitin and heparan sulfate are further remodeled by sulfation [33, 39]. Hyaluronan is synthesized at the plasma membrane by transmembrane enzymes of the HA synthase family (HAS1–3) [40, 41]. Chain length is dependent on polymerizing enzyme type, for example, HAS1 and HAS2 produce high molecular weight (~2000 kDa) HA and HAS3 produce lower molecular weight (100–1000 kDa) HA. After synthesis via HASes, extracellular HA can be rapidly altered due to its impressive turnover rate via a variety of hyaluronidases (mainly HYAL1 and 2) [40]. Despite the relative simplicity of its molecule, HA regulates a variety of cellular functions including wound repair, inflammation, cell migration, and angiogenesis [41, 42], and recently emerged as a key player in regulating the tumorigenic and inflammatory milieu [43]. Interestingly, its physiological sequel is largely related to the size of the molecule, for example, full-length HA mainly demonstrates anti-inflammatory property whereas its smaller fragments exert pro-inflammatory and pro-angiogenic features [40]. In cancer and other pathologic states, HA fragments are abundantly deposited in the extracellular environment that, in one hand, is a result of increased synthesis of HA via HASes and on the other hand—accelerated degradation via hyaluronidases, reactive oxygen species, and mechanical forces [44] creating a microenvironment supporting angiogenesis and inflammation [41, 45, 46]. Several evidence suggests that aberrant levels of HAS2 promote breast cancer growth, differentiation, lymph node involvement, and worse patient survival [47, 48]. HAS2 knockdown inhibited breast cancer growth and attenuated HA expression. Similarly, HAS2 has a regulatory effect on tumorigenicity and metastasis of prostate, colon, and ovarian tumors through excessive HA synthesis [49, 50]. Recently, Chen and colleagues [40] suggested a novel mechanism of angiogenesis regulation via autophagic degradation of HAS2 in endothelial cells. In [51], colleagues showed that the C-terminal module of perlecan, endorepellin, blocks VEGFR2 kinase activity, thereby evoking a strong pro-autophagic and anti-angiogenic response in vascular endothelial cells both ex vivo and in vivo. Bix and colleagues [52] have also shown that systemic delivery of recombinant endorepellin inhibits tumor growth and angiogenesis and increases tumor hypoxia in squamous and Lewis lung carcinoma xenograft models. Recently, HAS2 was degraded in vascular endothelial cells via autophagy evoked by nutrient deprivation, mTOR inhibition, or pro-autophagic proteoglycan fragments endorepellin and endostatin [40]. Autophagic degradation of HAS2 suppressed extracellular hyaluronan and inhibited ex vivo angiogenesis showed in aortic ring assay where they quantified the extent of active sprouting issued from the aortic rings and measured the radial distance of the newly-formed vessels where they found a significant reduction in angiogenesis [40]. The antiangiogenic activity of the role of endostatin and tumstatin was also emphasized, where tumor suppressor protein p53 prevented an incipient tumor from switching to the angiogenic phenotype mediated in part by endostatin and tumstatin [53].

The role of tumor-associated macrophages in angiogenesis is documented in [54]. TAMs induce tumor vascularization by releasing several factors, including VEGF which is the main angiogenic factor [55]. Monocytes (Mo) and monocyte-derived macrophages (MØ) can bind HA which induces intracellular signals [56, 57], however, the anti-tumor or pro-tumor role, is dependent on the size of HA in colorectal and breast carcinomas. As it is shown in [55] tumor necrosis factor (TNF)-stimulated gene 6 (TSG-6) was downregulated in Mo/MØ by high molecular weight hyaluronan, modulating their angiogenic behavior in breast carcinoma milieu, but not in colorectal carcinoma [55].

2.4 Proteoglycans

Next to collagens, proteoglycans (PGs) constitute a major class of extracellular matrix/cell surface components known to be involved in primary physiological and pathological phenomena; and due to the altered transcription/translation patterns that these PGs exhibit, they have been identified as potential diagnostic/prognostic and therapeutic targets in diverse disease states [58]. Based upon its direct involvement in cell-cell and cell-ECM interactions, this gene family has been strongly implicated in the regulation of cell movement. Assignment of diverse roles of PGs in promoting, or inhibiting, cell movement seems to be dictated by the biological system [58]. The proteoglycan superfamily now contains more than 30 molecules. They sustain the transparency of the cornea, the elasticity of blood vessels, the tensile strength of the skin, tendon, or cartilage, as well as compressive forces of the mineralized matrix of bones. PGs can alter the biology of growth factors and cytokines [59]. The basic proteoglycan unit consists of a “core protein” with one or more covalently attached glycosaminoglycan chain(s). Proteoglycans can be categorized depending upon the nature of their glycosaminoglycan chains and/or by size (kDa). Four major classes of PGs exist: (i) chondroitin sulfate/dermatan sulfate PGs (decorin, biglycan, versican); heparan sulfate/ chondroitin sulfate PGs (testican, perlecan); (ii) chondroitin sulfate (neurocan, aggrecan); (iii) keratan sulfate (fibromodulin, lumican). Among them, decorin, biglycan, testican, fibromodulin, lumican are small proteoglycans, and versican, perlecan, neurocan, and aggrecan are large proteoglycans. The small leucine-rich repeat proteoglycans (SLRPs) form a group of molecules on the basis of their relatively small protein core (36–42 kDa) [60, 61]. Some of these gene products are not classical proteoglycans. Despite being structural proteins, SLRPs constitute a network of signal regulation: being mostly extracellular, they are upstream of multiple intracellular signaling cascades. They affect intracellular phosphorylation and modulate pathways, including those driven by bone morphogenetic protein/transforming growth factor β superfamily members, receptor tyrosine kinases such as ErbB, and the insulin-like growth factor I receptor, and Toll-like receptors.

Decorin was originally discovered as a collagen-binding protein necessary for fibrillogenesis [62, 63], hence related eponym of decorin [64]. Soluble decorin is a high-affinity antagonistic ligand for several key receptor tyrosine kinases resulting in protracted oncostasis and angiostasis [65]. Recently, decorin has emerged as a soluble pro-autophagic cue by initiating endothelial cell autophagy through activation of AMPK, an energy sensor kinase, and evoking tumor cell mitophagy as the mechanistic basis for the oncostatic effects [66]. Decorin, due to its role as a tumor repressor and anti-angiogenic factor was designated as “a guardian from the matrix” [67]. According to the review, decorin suppresses tumor growth and angiogenesis via EGFR and Met where decorin monomer binds a narrow region of an epitope that in part overlaps with the agonist binding site [68]. This binding further augments receptor dimerization, the consequence of which is rapid phosphorylation of the intracellular tails [69]. This event further recruits and activates downstream effectors, e.g. provides caveosome-mediated internalization of the decorin/receptor complex, and eventual degradation in lysosomes [70, 71]. The latter causes a protracted cessation of intracellular receptor signaling. As a major consequence of inhibiting Met, two potent oncogenes, β-catenin, and Myc, are targeted for sustained degradation via the 26S proteasome [72]. Decorin suppresses β-catenin signaling in a non-canonical fashion and the latter is targeted for degradation in a manner consistent with direct phosphorylation of β-catenin by an RTK, such as Met [73, 74, 75]. Wnt/β-catenin signaling activation and its member molecule mutations are well established in colorectal cancer and different epithelial tumor sprouting and nonsprouting angiogenesis. Wnt agonists (e.g., B cell Lymphoma 9 protein (BCL9) is the angiogenesis promoting, where antagonists such as the DKK-4 (also called the Dickkopf Wnt signaling pathway inhibitor 4), in particular, conditioned media from DKK-4 expressing cells promoted the migrative abilities of CRC and formation of capillary-like tubules of human primary microvascular endothelial cells [76].

Versican is a large chondroitin sulfate proteoglycan that forms aggregates with hyaluronan which connects it to the cell surface via hyaluronan receptors such as CD44 [77, 78]. Versican is implicated in many biological processes involving vasculature, such as atherosclerosis and vasculitis [79, 80]. There are five known versican splice isoforms; V0–V4 [81]. Each isoform except V3 has a glycosaminoglycan (GAG) domain with covalently attached chondroitin sulfate (CS) chains. Versican is highly expressed in the early stages of development but becomes downregulated after tissue maturation [82], interestingly, it is reexpressed during wound repair, arteriopathies, pulmonary fibrosis, or tumor formation [83]. Versican is anti-adhesive since it is a poor cell attachment and migration substrate and is excluded from focal adhesions [77, 84, 85]. Several clinical studies have suggested that high versican expression is a poor prognostic factor in gastric, pancreatic, head and neck squamous, or mammary cancers [77]. Increased versican immunostaining has been detected during tumor blood vessel formation [86]. Versican V2 isoform is the major type expressed in brain tissues, and brain tumors are greatly enriched in vascularization, therefore, authors hypothesized that the V2 isoform may play a role in angiogenesis in brain tumors. They injected U87 glioblastoma cells stably transfected with a versican V2 expression construct or a control vector into nude mice and showed that the tumors formed by the V2-transfected cells were visibly enriched in vascularisation, whereas the tumors formed by the vector-transfected cells did not exhibit this phenotype [86]. Furthermore, V2 expression facilitated endothelial-tumor cell interaction observed in tube-like structure formation in matrigel [82]. Koyama and colleagues demonstrated that basic fibroblast growth factor-induced neovascularization was elevated in the presence of either hyaluronan oligosaccharides or a hyaluronan aggregate containing versican, using the Matrigel plug assay. Administration of hyaluronan-versican aggregates, but not native hyaluronan alone, promoted stromal cell recruitment with the infiltration of endothelial cells, suggesting that hyaluronan overproduction accelerates tumor angiogenesis through stromal reaction, notably in the presence of versican [87]. Versican localized preferentially to the vicinity of tumor vasculature and macrophages in the tumor. However, the extracellular protease ADAMTS-generated versican fragment is uniquely localized to vascular endothelium. Members of the family of A disintegrin-like and metalloproteinase with thrombospondin type 1 motifs (ADAMTS) are involved in versican proteolysis and tumor progression [88, 89]. ADAMTS1 was first shown to display anti-angiogenic properties [90]. Later, it’s angiostatic (antiangiogenic) and tumor-suppressive properties have also been shown in model systems [91, 92], but controversial results about its relevance to metastasis and tumor growth have also gained attention [93]. ADAMTS family of secreted zinc-dependent metalloproteinases comprises at least 19 genetically distinct members in humans [94]. The expression of the majority of ADAMTS subtypes is associated with pre- and postnatal growth and onset and progression of cancer [95]. ADAMTS subtypes have been sub-classified as aggrecanases because of their ability to cleave large chondroitin sulfate. Despite their structural similarity to other matrix metalloproteinases, ADAMTS have a narrow substrate specificity. This feature could serve as an advantage for ADAMTS inhibitors in the treatment of cancer [95].

Asporin, also known as periodontal ligament-associated protein 1 (PLAP1) was identified in 2001 [96, 97]. Asporin mRNA was expressed primarily in the skeleton (perichondrium/periosteum of cartilage/bone) and other specialized connective tissues. Asporin blocks chondrogenesis and inhibits TGF-β1-induced expression of matrix genes and the resulting chondrocyte phenotypes [98]. Knockdown of asporin increases the expression of cartilage marker genes and TGF-β1; in turn, TGF-β1 stimulates asporin expression in articular cartilage cells, suggesting that asporin and TGF-β1 form a regulatory feedback loop. Asporin, like decorin, can bind collagen at the same site, but in contrast to decorin and biglycan, it drives collagen biomineralization [99]. Our laboratory has identified asporin as a novel cancer-related protein in invasive breast cancer [100]. Later, asporin was reported as an important player in tumor microenvironment [101] and experimentally proved that MDA-MB-231 and BT-549 cells invaded faster through collagen matrix which was prepared with the recombinant asporin. This finding was explained to be related to a less dense matrix due to the inhibition of collagen fibrillogenesis by asporin [102]. Recently, asporin was specifically reported in pancreas and prostate cancer by two additional groups [103, 104]. The direct role of asporin in angiogenesis/angiostasis is not been studied yet, however, a search of the Gene Expression Omnibus, revealed high levels of ASPN expression in white adipose-derived (WAT) CD34+ cells that are a very rich reservoir of CD45− CD34+ populations with endothelial differentiation potential/significantly increased levels of angiogenesis-related genes [101]. The multifaceted role of asporin was recently reviewed also in [105] where its emerging role in proliferation, migration, invasion, and angiogenesis through TGF-β, EGFR, and CD44 pathways was described [105].

2.5 Laminin

Laminins are major noncollagenous constituents of the basement membrane. The fragmentation or absence of BM structures seen in malignant tumors is due to active proteolytic degradation, decreased synthesis of BM components, and/or remodeling by the tumor cells [106]. There are 5α, 4β, and 6γ chains of laminin molecule [33]. It has three short and one long arm arranged in a cross-like structure. The α chains have a larger G domain at the C-termini, which is composed of 3 LG domains (LG1-LG3) connected by a binding region to other LG4 and 5 domains. Integrins bind to LG1–3. Heparan sulfate has been shown to bind to LG4 of the α1 chain. Certain laminin isoforms are predominant in vascular basement membranes and may be critical in maintaining the proper development as well as stability of the mature vessel [107]. LN-1 provoked angiogenesis in the chicken chorioallantoic membrane in the same manner as FGF-2, and vessel development in embryoid bodies was further enhanced in a synergistic mode by FGF-2 and LN-1. The latter significantly enhanced the differentiation of endothelial cells in a 3D collagen environment, either in the absence or presence of FGF-2 [108]. In tumors, as in normal tissues, the blood vessels express laminin α4, α5, β1, and γ1 chains, suggesting the presence of laminin-8 and -10, synthesized by VECs. Laminin-10 is more adhesive and migration promoting [109]. Microvessels are expected to express additional laminins α2, α3, and β2 [107]. The cellular origin of the laminin chains in the vessel should be carefully examined, since pericytes are also able to synthesize several laminins [110]. Lugassy and colleagues [111] in their work studied qualitative aspects of tumor cells and vasculature in melanoma and focused on the pericellular matrix. They demonstrated the angio-tumoral complex in which the tumor cell and endothelium are in direct contact via an amorphous matrix. This amorphous matrix lacks an organized lamina and contains predominantly laminin with noticeably less collagen type IV. Interestingly, this was absent in naevi. Authors regarded the laminin found in this amorphous matrix as “free” laminin, is distinct from laminin integrated into an organized lamina, and showed free laminin role in promoting the migration of melanoma cells in contact with vessels and suggested that this angio-tumoral complex represents a marker for metastasis [112]. During intravasation, tumor cells penetrate BM rich in laminin-8 and 10. When in circulation, large tumor cells and cell aggregates are often covered with platelets, that contain and, following stimulation, secrete laminin-8 and other laminin isoforms [113]. Tumor cell extravasation again requires penetration of the vascular BM to generate secondary tumors [107]. Interaction of tumor cells with endothelial cells and the basement membrane seems organ-specific, time and tumor type-dependent in the ultrastructural study on lung, liver, brain, kidney, and adrenal tissues. Study shows that endothelial cells of the lungs and liver can play a much more active role in the process of extravasation [114]. Laminin α3B chain normally expressed in vascular and epithelial basement membranes, was downregulated in skin cancers [115]. Notably, endothelial cell behavior during tumor progression is largely dependent on complex interactions between laminin molecules with integrins (please see also below).

2.6 Fibronectin

Fibronectin is a dimer with a molecular weight of ~270 kDa. There are two fibronectin forms, soluble plasma fibronectin (p-fibronectin), produced by hepatocytes and cellular fibronectin (c-fibronectin) produced in tissues where it is further deposited as a component of the fibrillar matrix. Many of the functions of fibronectin depend on the 3-dimensional structure of the protein and its assembly into a functional fibrillar matrix [116]. In ECM, fibronectin binds collagen, heparin, other fibronectin proteins, and cell surface integrins. Fibronectin binds integrins through the tripeptide motif of arginine, glycine, and aspartic acid (RGD)2,3, α5β1 integrin plays here a major role. Studies to elucidate the mechanisms of fibronectin fibrillogenesis in endothelial cells have revealed a determinant role for integrin beta subunit adaptor (ILK) in this process [117]. Example of how transient c-fibronectin expression participates in a “pro-angiogenic switch” comes from studies on vascular patterning in the developing retinal vasculature [118, 119]. During this process, blood vessels use the existing astrocyte network as a template, and fibronectin is the principal component of the astrocyte-derived extracellular scaffold. Bazigou et al. [120] showed that interaction between integrin α9 and fibronectin containing the EDA domain is required for fibronectin matrix assembly during lymphatic valve morphogenesis [120]. Targeted deletion of α4 in lymphatic vessels or pharmacological inhibition of α4β1 compromise growth factor- and tumor-induced lymphangiogenesis and suppressed metastatic spread in vivo. α4β1 and c-fibronectin were suggested as markers of proliferative lymphatic endothelium in malignant tumors [121]. Fibronectin is a Wnt target gene and lung vascularization and branching morphogenesis are dependent on Wnt and fibronectin signaling [122]. However, fibronectin level is weak in morphogenesis and quiescent vasculature and highly upregulated together with tenascin-C following vessel injury. Tenascin-C expression is also highly associated with angiogenesis in a wide range of disease states, including diabetes, aortic aneurysm, artherosclerosis, ulcerative colitis, inflammatory bowel disease, Crohn‘s disease, vasculitis, and cancer [122]. Both proteins were localized in the vessel wall, where fibronectin was more abundant on the luminal side and tenascin-C on the extraluminal side of the vascular BM. To note, tumor vessels were diversely positive for tenascin-C and oncofetal fibronectin, suggesting a temporally and spatially regulated expression of these ECM proteins in the tumor vasculature and may reflect different maturation states of the vessels. Re-expression of fibronectin occurs during pathological angiogenesis in various diseases such as cancer, late-stage atherosclerosis, and blinding ocular conditions [123, 124].

3. Cell-extracellular matrix interactions and angiogenesis

3.1 Integrins

Integrins are the main receptors involved in cell-matrix contacts. They contain transmembrane subunits α and β, large extracellular domain, and intracellular domain that interacts with cytoskeleton proteins. Subunits form 24 integrins. Integrins provide transmission of chemical and mechanical signals, which results in rearrangement of the cell cytoskeleton and activation of pathways that control cell survival and motility, angiogenesis, differentiation, and apoptosis. The ability of the cell to survive without contact with a substrate is a feature of tumor cells. Integrin expression changes significantly during carcinogenesis and different tumors express different integrins. Integrin α6β4 in cooperation with epidermal growth factor receptor (EGFR) is expressed mostly in breast carcinoma [125], while integrin αVβ3 in cooperation with platelet-derived growth factor (PDGF) and EGFR are expressed in glioblastomas and melanomas [126]. The role of integrins in tumor angiogenesis has been partially discussed above in relation to laminins and will also be discussed below.

Matrix metalloproteinases (MMPs), also known as matrixins, are members of the metzincin protease superfamily of zinc-endopeptidases. There are 187 members of MMPs which are encoded in the human genome and 28 members are secreted MMPs. They can degrade every protein in ECM and basement membranes. Several MMPs are membrane-type which contribute to the precise localization of protease activity, as this is required at the edge of migrating cells. Several MMPs—collagenase, gelatinase, matrilysin degrades collagen, gelatin, and fibronectin, respectively. Stromelysin degrades structural proteins and proteoglycans. MMP activity is regulated by tissue inhibitors of MMPs (TIMPs 1–4) which are produced by more cells than MMPs themselves [127]. MMPs are directly implicated in embryonic growth and tissue morphogenesis that require disruption of ECM barriers for microenvironment remodeling and cell migration and contribute to the formation of a complex microenvironment for tumor development and progression through activation of growth factors, suppression of tumor cell apoptosis, destruction of chemokine gradients developed by host immune response, or release ECM-sequestered angiogenic factors [128]. For example, MMP-11 (human stromelysin-3, hST-3) favored the release of insulin-like growth factor 1 that is bound to specific binding proteins (IGFBPs) [129]. MMP-9 can proteolytically activate TGF-β and promote tumor invasion and angiogenesis [130]. Several other pro-angiogenic factors such as VEGF and basic fibroblast growth factor (bFGF) are induced/activated by MMPs. MMP-14 overexpression by cancer cells increases VEGF synthesis and promotes angiogenesis in glioblastomas [131] and breast carcinomas [132, 133]. VEGF expression was also inspired by MMP-2 in A549 lung adenocarcinoma cells through the binding to αvβ3 and activated integrin signaling [134]. Cancer cell-derived MMP-13 (collagenase-3) also induced VEGF synthesis by endothelial cells and fibroblasts and initiated tumor angiogenesis in vivo [135]. MMP-1, -8, and -13 are collagenases associated with angiogenesis and their loss leads to irreversible rupture of the matrix [136]. The fragmentation of basement membrane type IV collagen is carried out by MMP-2 and MMP-9. Type IV collagenase activity is important in the early steps of endothelial cell morphogenesis/capillary formation. Interstitial collagenase (MMP-1) is a membrane-type 1 matrix metalloproteinase (MT1-MMP) that can also break down collagen types I–III, gelatin, laminin, and other ECM components. MT1-MMP is expressed by endothelial cells and it may regulate angiogenesis by activating pro-MMP2 and by cleaving collagens on the cell surface at a highly localized site [136]. Tissue inhibitors of metalloproteinases regulate them, playing a key role in angiogenesis regulation by inhibiting neovascularization.

3.2 Matrix topology, stiffness, and solid stress

Physical and chemical features of the tumor environment determine matrix topology (architecture) and stiffness that depends on the size of biopolymer fibers and the density of the fiber network [137]. Connective tissue is characterized by different fiber arrangements. Different combinations and densities of the cells, fibers, and other ECM components as well as different fiber arrangements ranging from loose or random to highly aligned structures, produce graded variations of connective tissue. ECM topology can represent an important regulator of cell motility through physical signals that geometrically impel adhesion foci to conduct directional migration [138]. Cancer cells perform contact guidance mediated by mechanosensory integrins through which they, using contractile force, actively remodel the ECM fibers surrounding a tumor (align them perpendicularly to the tumor) [137, 138, 139]. Dense fibrillar collagen that is characteristic of breast cancer stroma forms radial patterns extending away from tumors. On the other hand, the reticular arrangement of the collagen matrix surrounding mammary glands may anchor and/or hinder cells. Thus, ECM topography, in particular, its non-linear pattern reduces invasion while linear structure promotes it. Matrix concentration and post-translational modifications such as glycosylation and cross-linking affect the mechanical properties, including viscoelasticity or stiffness. Tumors exhibit a higher degree of stiffness than their normal adjacent counterpart. For example, the healthy mammary gland is highly compliant (elastic modulus E = ~200 Pa), while the average tumor is stiffer (E = ~4000 Pa). Both the tumor-surrounding stroma and vasculature exhibit increased stiffness (E = ~800–1000 Pa and ~450 Pa [140].

Changes in ECM topology and stiffness can shape mechanosensing events and activate intracellular signaling processes involved in cell migration. Among signaling pathways/genes involved in directionally persistent migration are, for example, vinculin, talin, FAK, p130CAS, and filamin A. Integrin receptors and the physical arrangement of adhesions assure orientation of the cytoskeleton while leading-edge protrusions can be stabilized by matrix orientation [137, 138]. When cancer cells experience an increase in ECM stiffness, they respond to the change by generating increased traction forces on their surroundings by regulating growth factor signaling and focal adhesion formation. For this purpose, the cell has several alternatives: for example, it can either force the network fibers apart and remodel the shape, form trails of variable caliber until it can pass through the pore, or the tumor cell degrades the fiber matrix via multistep pericellular proteolysis that was observed in individual and collective cancer cell migration [140]. Increased tumor tissue stiffness has been linked to tumor progression, direct stem cell differentiation, cell-cell and cell–matrix adhesion, hyaluronan synthesis, and expression of genes that play important roles in invasion and metastasis [128, 141, 142, 143]. A computational model was used to investigate the effect of ECM topography on vascular morphogenesis and explanation of mechanisms that control cell shape and orientation, sprout extension speeds, and sprout morphology. Sprout extension speed and morphology depend on matrix density, fiber network connectedness, and fiber orientation and varying matrix fiber density affect the likelihood of capillary sprout branching. The authors calculated optimal density for capillary network formation and suggested matrix heterogeneity as a mechanism for sprout branching. The density of the matrix fibers has a strong effect on the extension speed and the morphology of a new blood vessel pointing to new targets for pro- and anti-angiogenesis therapies [144].

Another important tumor characteristic is tumor growth-induced solid stress. As tumor cells proliferate they sequentially create new solid material (i.e. cells and matrix components) which pushes against the surrounding tumor microenvironment. Uncontrolled proliferation of cancer cells leads to ignorance of contact inhibition, their expansion imposes elastic tension on the surrounding tumor microenvironment, storing stress through the deformation of adaptable structures, and collapsing delicate structures, such as blood and lymphatic vessels. Interestingly, solid stress is accumulated within the tumor and is still sustained after the tumor excision [145]. Collagen and hyaluronan molecules are the main contributors of the ECM to solid stress. Collagen, as it becomes stiffer when stretched, is responsible for tensile stress. This observation is valid for both capsular and interstitial collagen. When hyaluronan resists compression, its negatively charged chains are pushed away, owing to electrostatic repulsion and trap water, therefore matrix becomes poorly compressible [145]. The compression of vessels by solid stress may create potential obstacles to drug delivery: the collapse of blood vessels hampers access to systemically administered drugs. This collapse might explain the fact that neoplasias with more ECM might be more resistant to treatment. For instance, chondrosarcomas, chordomas, or pancreatic ductal adenocarcinoma (the latter has the highest solid stress magnitude 7 kPa = 52.5 mmHg) are tumors rich in ECM and refractory to chemotherapy [146, 147, 148]. Further, the lack of lymphatic vessel function induces drainage compromise, leading to uniformly elevated interstitial fluid pressure. As a result, the transport of therapeutics, like antibodies and nanoparticles, is reduced because the dominant mode of transport becomes diffusion which is an inadequately slow process for large particles [149]. In this sense, decreasing solid stress by the angiotensin inhibitor, losartan, decompress tumor blood vessels, enhances drug delivery, and potentiates chemotherapy effects [150].

As stated above, endothelial activation is believed to be predominantly related to biochemical signals. However, mechanical forces have more recently also been demonstrated to regulate endothelial cell phenotype and function. Recent work has shown that mechanical forces control endothelial cell proliferation, survival, and migration [151, 152] and fluid shear stress from blood flow plays a critical role in regulating vessel morphogenesis, sprouting, and barrier function [153, 154]. To convert mechanical forces and biophysical signals into intracellular biochemical reaction cascades, endothelial cells employ a complex system of mechanosensors (actin cytoskeleton, integrins, cell-cell adhesion receptors, receptor tyrosine kinases, ion channels, and G-protein-coupled receptors) to sense and respond to mechanical forces [155]. Matrix stiffening enhanced integrin-mediated Rho/Rho-associated protein kinase (ROCK) activity and contraction in tumor epithelial and endothelial cells [156, 157, 158]. Tumor endothelial cells have abnormal mechanosensitivity to uniaxial cyclic strain transmitted through the ECM, which is mediated by vigorous regulation of Rho activity and cytoskeletal tension. Normal and tumor endothelial cells express similar levels of active β1 and β3 integrins [159]. Tumor endothelial cells demonstrate constitutively high baseline activity of Rho and ROCK, thicker stress fibers, higher adhesion strength, and augmented cytoskeletal tension. Logically, described features are mainly due to higher intrinsic Rho and ROCK-related cytoskeletal tension in the background of unchanged levels of integrins. These dynamics between normal and tumor endothelial cells in response to mechanical impulses suggest that the aberrant mechanical forces from the tumor microenvironment may cause tumor endothelial cells to gradually obtain an altered phenotype. Such alteration may further enable tumor endothelial cells to spread over a wider range of matrix stiffness [155, 158]. Specific integrins have been demonstrated to contribute to non-tumor and tumor angiogenesis. The expression of α1β1 and α2β1 integrins is upregulated by VEGF in endothelial cells [160], and the combined antagonism of α1β1 and α2β1 reduced human squamous cell carcinoma growth and angiogenesis [161]. The α5β1 integrin is selectively expressed in angiogenic vasculature. Upregulated αvβ3 and αvβ5 integrins in endothelial cells are necessary for the growth and survival facilitation of neovessels [162]. As already mentioned, αv integrins are also involved in cytokine-dependent pathways of angiogenesis. Integrin αvβ3 is incumbent in pathways activated by FGF or TNFα while integrin αvβ5 is necessary for angiogenic pathways activated by VEGF or TGFα [163]. Specifically, the αvβ5 integrin pathway downstream of VEGF causes activation of FAK and Src kinase [164]. The αvβ3 integrin has also been associated with VEGFR2 and the binding of αvβ3 to its corresponding ECM ligands has been shown to increase VEGF signaling [165]. Integrin αvβ3 is overexpressed in newly developed vasculature of mammary carcinoma [166], the expression level of αvβ3 and αvβ5 integrins in tumor neovessels were found to be associated with the neuroblastoma grade [167]. The experimental inhibition of αvβ3 integrin suppressed angiogenesis and related breast tumor growth in immunodeficient (SCID) mouse/human chimera [166] and resulted in tumor reduction in human clinical trials [168]. Combined inhibition of αvβ3 and αvβ5 integrins also significantly reduced growth of human melanoma xenografts in SCID mice [169]. Integrin α6β4 signaling has similarly been involved in incipient invasive phase of pathological angiogenesis. The β4 substrate domain promotes bFGF-mediated angiogenesis in matrigel plug assay and hypoxia-inducible factor VEGF-mediated angiogenesis in the retinal neovascularization model regulates sprouting angiogenesis by forced nuclear translocation of activated ERK and NF-κB in migrating endotheliocytes [170]. Furthermore, targeted deletion of the signaling domain of the integrin β4 significantly reduced the size and microvascular density in various tumors including melanoma, lung cancer, lymphoma, or fibrosarcoma [170]. These data demonstrate the role of cytoskeletal- and integrin-mediated mechanosensory pathways in facilitating tumor angiogenesis.

3.3 Hypoxia and interstitial fluid pressure

Hypoxia is another feature of the abnormal tumor microenvironment that is intrinsically linked to the formation of neovasculature and clinically manifests with metastatic progression and worse patient survival [171, 172]. Diffusion-limited hypoxia is a sequel of tumor cells located distantly from the blood-supplied areas. Such cells “suffer” from prolonged hypoxia and tumor cells are kept viable for hours to a few days in such environment [173]. Within the cell, hypoxia induces oncogenes, enhances DNA mutation chance, and selects for cells with increased apoptotic rate [171, 174]. Extracellularly, hypoxia supports tumor progression by increased matrix deposition, turnover, cross-linking, and remodeling [175]. HIF-1α increases vascularization in hypoxic areas and allows for the survival and proliferation of cancer cells, its inhibition prevents the expansion of neoplasia [176]. Along with known angiogenic factors, novel ones and their receptors include VEGF, VEGFR-1, -2, bFGF, platelet-derived growth factor B (PDGF), insulin-like growth factor II (IGF2), adrenomedullin, and epidermal growth factor (EGF) are targets of the HIF transcription factors. Several of these angiogenesis-related gene products, including iNOS, endothelin, adrenomedullin, and heme oxygenase 1, are also implicated in the modulation of local blood flow by regulating the vascular tone [177]. The well-known EMT activators such as Snail, Slug, and Twist are also induced by hypoxia [178]. Hypoxia also affects stem cells [179] that become pluripotent and aggressive with high metastatic potential. Resistance to anti-angiogenic therapy thus may be mediated by HIF-1α activated genes. Therapeutical targeting of hypoxia includes bioreductive prodrugs, HIF-1 targeting, and genetic engineering of anaerobic bacteria [180].

Abnormal metabolism in the tumor is further characterized by a decrease in extracellular pH. The known sources of H ⁺ ions in tumors are by- or end-products of anaerobic glycolysis, such as lactic acid and carbonic acid [181, 182]. The dysbalance between production and removal of H ⁺ ions lowers the extracellular pH in tumors. The level of pH also decreases in tumors with increasing distance from nearest blood vessels. Low extracellular pH causes stress-induced alteration of VEGF and IL-8 gene upregulation and relevant protein expression in three different tumor cells in vitro [183]. When the possible relationship between pH, pO2, and their effect on VEGF expression in vivo was examined using GFP imaging of tissues, pO2 and pH appear to regulate VEGF transcription in tumors independently. For example, in the hypoxic state or neutral pH, VEGF-promoter activity increased, with a decrease in pO2 and independent of pH. In decreased pH or oxygenated conditions, VEGF-promoter activity increased, with a decrease in pH and independent of pO2 [184]. To conclude, these key microenvironmental factors regulate angiogenic profiles in a complementary mode.

Another pathophysiologic feature of the tumor microenvironment is elevated interstitial fluid pressure (IFP) in the range of 10–100 mmHg [185, 186]. IFP of normal tissue is around zero [187]. The driving force in increasing tumor IFP is the tumor vasculature [188, 189]. In contrast to normal vessels which are characterized by dichotomous branching, tumor vasculature is chaotic, with trifurcations and branches with unsteady calibers, larger inter-endothelial junctions, multiple fenestrations, vesicles, vesico-vacuolar channels and a disruption of normal basement membrane [190]. Due to described ultrastructural alterations, vascular permeability in solid tumors is generally higher compared to normal counterparts. Tumors, also either lack lymphatics or the intratumoral vessels are non-functional [191], as a result, excess fluid accumulates in the interstitium resulted in elevated IFP. In IFP regulation model, fibroblasts actively regulate the tension applied to the ECM through integrins which enable fibroblasts to modify collagen fiber tension and modulate the elasticity of the ECM in response to hyaluronan and proteoglycan expansion. According to [192], interestingly, a significantly dense and stiffer collagen framework and related higher IFP is also a result of the synthesis of another important proteoglycan fibromodulin by stromal fibroblasts, which is mainly promoted by emerged inflammatory processes in malignant tumors. Interstitial fluid pressure may serve as another target for cancer therapy. Roh and colleagues [193] reported an inverse relationship between tumor IFP and degree of tissue oxygenation and suggested IFP’s role in predicting radiotherapy effect. Increased tumor IFP can also act as an obstacle to drug delivery, which makes questionable their efficacy. Several studies have also demonstrated advanced amelioration of chemotherapeutics following a reduction in tumor IFP [150].

4. Conclusions

The extracellular matrix in non-tumor states regulates tissue development and homeostasis, and its deregulation imparts to neoplasia and its progression. It serves not only as the mechanical milieu upon which cells/tissues inhabit but creates and exerts critical biochemical and biomechanical messages that drive cell growth, survival, differentiation, migration, and manage neoangiogenesis and immune scaffold. The cellular mechanisms inducing both angiogenesis and immunosuppressive responses are often reached by the same cell types and soluble factors. Studies point out that combinatorial strategies toward many potential targets with emphasis on angiogenesis should be adapted as a useful therapeutic approach to hinder/reverse tumor progression.