Extracellular vesicles are subcellular structures that are heterogeneous in nature and surrounded by a lipid bilayer membrane that exerts multiple functions in intercellular communication
[10]. Based on how they are delivered from the original cell to the extracellular medium, EVs can be released by inward budding of the endosomal membrane or outward budding of the cellular membrane
[11]. The recipient cell can then internalize EVs through endocytosis or membrane fusion to unload their contents into the cell cytoplasm (
Figure 1)
[12]. Since their discovery in the early 1980s, many biomolecules have been identified as cargo in EVs, including proteins, amino acids, signaling lipids, and different genetic molecules like DNA, RNA, and non-coding RNAs, promoting both physiological and pathological processes
[13]. Based on their biogenesis, EVs are generally classified into microvesicles and exosomes
[10], although some authors suggest a further division into apoptotic bodies and proteasomes
[14]. Microvesicles are generated by outward budding of the plasma membrane and range from 50 nm to 1000 nm. In contrast, exosomes are membrane vesicles smaller in size (30–100 nm) and are formed by inward budding of the endosomal membrane to be later secreted by fusion with the cell membrane
[14,15][14][15]. The role of EVs as cell-to-cell mediators in respiratory disease
[16], neurodegenerative disease
[17], kidney disease
[18], cardiovascular disease
[19], and cancer progression and metastasis
[20,21,22][20][21][22] has been documented to ameliorate the understanding of the behavior or these subcellular structures.
2. Mechanism of Angiogenesis
The angiogenesis process embodies forming new blood vessels from existing vessels in response to physiological and pathological mechanisms
[41]. During embryogenesis, the vascular network develops through a combination of vasculogenesis, referred to as the de novo formation of the heart and new blood vessels from stem endothelial cells, namely, angioblasts, and angiogenesis, which expands the initial primitive vascular plexus
[42]. Although most blood vessels remain quiescent under physiological conditions, tissue repair and regeneration through wound healing, ovulation, and endometrial thickening throughout the menstrual cycle are based on angiogenesis for proper functioning
[43,44,45][43][44][45]. While vascular growth varies depending on where angiogenesis is initiated and the tissue to which they will provide a new blood supply, several mechanisms are common in forming these vessels
[46]. In a hypoxic state, the recruitment of cells that promote inflammation; angiogenic growth factor production; degradation of the basement membrane; and endothelial cells (ECs) sprouting, migrating, proliferating, differentiating, and modulating vascular support cells are some of the shared characteristics in angiogenesis
[47].
The angiogenic process (
Figure 3) comprises several stages involving the sprouting, migration, and proliferation of ECs guided by the vascular endothelial growth factor (VEGF)
[48]. Following VEGF stimulation, pericytes from the vessel wall detach, and the basal membrane is weakened by proteolytic degradation. At the same time, ECs adopt an invasive and motile phenotype called tip cells that send out filamentous pseudopodia to guide vascular budding
[49]. The cells behind the tip cells are denominated stalk cells, which proliferate to maintain the integrity of the structure and function of the nascent vessels, mainly expanding the vascular lumen
[50]. ECs modify their shape by negatively charging glycoproteins on the apical surface to repel each other and open the lumen while redistributing cell-to-cell adhesion to the periphery
[51]. For maturation to occur, pericytes must be recruited by the platelet-derived growth factor subunit B (PDGF-B) and angiopoietin 1 (Ang1) signaling along with the strengthening and consolidation of the adhesion between ECs with junctional molecules such as VE-cadherin, while a basement membrane is deposited by tissue inhibitors of metalloproteinases (TIMPs)
[52,53][52][53].
Figure 3. Stages of the angiogenic process. (
A) Angiogenic signals, such as VEGF, promote pericyte detachment from the basement membrane and weaken the extracellular matrix. (
B) Endothelial cells display characteristic phenotypes after VEGF stimulation: migratory tip cells or proliferating stalk cells. (
C) Attractive and repulsive forces control endothelial cells, forming a vessel lumen to initiate blood flow. (
D) PDGF-B and Ang1 signaling lead to pericyte recruitment, while junctional molecules consolidate EC–EC adhesion. Modified from Viallard et al.
[47].
Under physiological conditions, angiogenesis is strongly regulated by factors ranging from metabolites to hormones
[54]. Various molecular pathways have been extensively studied that comprise the angiogenesis process, including the VEGF-VEGFR, Angiopoietin-Tie, Delta-Notch, and Ephrin-Eph
[55]. Angiogenesis, vasculogenesis, and lymphangiogenesis are mostly regulated by six VEGF members encoded in the human genome, namely, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, and the placenta growth factor (P1GF)
[48,56][48][56]. According to their molecular configuration and affinity, the aforementioned factors may bind to different tyrosine kinase receptors VEGFR-1, VEGFR-2, and/or VEGFR-3; VEGF-A binds to VEGFR-2 to contribute to angiogenesis, whereas VEGF-C and VEGF-D enhance lymphatic vessel sprouting by binding to VEGFR-3
[57]. Another group of angiogenesis inducers are the platelet-derived growth factors (PDGF), which induce ECs proliferation and migration by binding to two tyrosine kinase receptors, PDGFR-α and PDGFR-β
[58]. Another angiogenesis factor is the fibroblast growth factor (FGF) 2, responsible for inducing metalloproteinase (MMP) secretion to degrade the basement membrane and promote vessel sprouting along with VEGF
[59]. Angiopoietin 1, interleukin 8 (IL-8), epidermal growth factor (EGF), and tumor necrosis factor α (TNF-α) also exert a pro-angiogenic effect in ECs through several signaling pathways
[60,61,62,63][60][61][62][63]. Conversely, angiopoietin 2, angiostatin, endostatin, vasostatin, and TIMPs inhibit angiogenesis and play an important role in achieving vascular homeostasis
[64,65][64][65].
Extracellular vesicles produced by many cellular lineages under specific circumstances can be taken up by ECs to promote and regulate angiogenesis
[66]. For instance, a study in endometrial stromal cells (HESCs) found active secretion of EVs during decidualization in a controlled manner by the hypoxia-inducible factor 2 alpha (HIF2α)–Ras-related protein Rab-27B (RAB27B) cascade, revealing a cargo with a variety of growth regulators, signaling molecules, metabolic modulators, and factors that control the expansion and remodeling of ECs
[67]. In a myocardial infarction animal model, stem cell-derived small extracellular vesicles (sEVs) loaded with miR-486-5p promoted cardiac angiogenesis via fibroblastic MMP19-VEGFA cleavage signaling
[68]. Moreover, Gregorius et al.
[69] evaluated the effects of mesenchymal stromal cell (MSC)-derived sEVs on the proliferation, migration, and tube formation of cerebral microvascular ECs. Interestingly, hypoxic preconditioning enhanced angiogenesis and increased post-ischemic endothelial survival by regulating several miRNAs through the uptake of protein-enriched sEVs cargo. Another study demonstrated that HS-1 protein X-1 (HAX1), a major regulator of myeloid homeostasis, was present in EVs secreted by nasopharyngeal carcinoma (NPC) tumors, promoting an angiogenesis phenotype by activating the focal adhesion kinase (FAK) pathway in ECs by increasing the expression level of integrin subunit beta 6 (ITGB6)
[70]. Conversely, EVs produced by bone marrow MSCs were found to be enriched in the cluster of differentiation 39 (CD39), TIMP-1, and CD73, inhibiting tumor angiogenesis by targeting the extracellular matrix remodeling and the endothelial cell migration
[71].
3. Exosomal Long Non-Coding and Circular RNAs in Cancer Angiogenesis
LncRNAs in EV cargo have been demonstrated lately, mainly in cancer
[72,73,74][72][73][74]. A recent study launched an online repository of EV long RNAs (exLRs) in diverse human body fluids, comprising 19,643 mRNAs, 15,645 lncRNAs, and 79,084 circRNAs obtained from human blood, cerebrospinal fluid, bile, and urine samples. The database provides novel exLR signatures to help discover new biomarkers that could aid in diagnosing and treating many diseases
[75]. Based on available recent research, Casado-Díaz et al.
[76] concluded that lncRNAs and other RNAs included in MSC-derived EVs can be applied in chronic skin ulcers to improve accelerated healing and decrease scar formation due to immunosuppressive and immunomodulatory properties. Conversely, in a diabetic wound-healing animal model, upregulated lncRNAs packed in EVs from fibroblasts enhanced keratinocyte MMP-9 expression to induce collagen degradation, delaying wound healing
[77]. Recently, the long non-coding repressor of NFAT (NRON) was detected in BMSC-derived EVs, inhibiting osteoclast differentiation and osteoporotic bone loss in vitro and in vivo
[78].
In tumors, the high rate of cell proliferation forces the formation of new blood vessels
[79]. However, in most cases, these blood vessels are dilated, tortuous, and immature, leading to excessive permeability and increased hypoxia
[80]. In addition, vascular disorganization causes heterogeneity in the tumor blood vessel network, creating highly vascularized tumor areas and other hypoxic areas with low vascular density
[47]. Thus, hypoxia becomes a major driver of tumor angiogenesis, along with other mechanisms promoted by activated oncogenes or loss of tumor suppressor genes, in which lncRNAs play an important role, mainly through acting as competing endogenous RNAs for miRNAs
[81]. Similarly, circRNAs have been extensively studied in cancer, elucidating important roles in tumor development, growth, and angiogenesis
[82]. For instance, VEGFR-related pathways have been linked to circRNAs by affecting tumor angiogenesis by sponging miRNAs
[83,84][83][84]. The landscape of exosomal lnc- and circRNAs in angiogenesis in cancer is summarized in
Table 1 and
Figure 4.
Figure 4.
Molecular landscape of exosomal lnc- and circRNAs in angiogenesis in cancer.
Table 1.
The landscape of exosomal lnc- and circRNAs in angiogenesis in cancer.