MicroRNAs in Tumor Endothelial Cells: Regulation, Function and Therapeutic Applications

Tumor endothelial cells (TECs) are key stromal components of the tumor microenvironment, and are essential for tumor angiogenesis, growth and metastasis. Accumulating evidence has shown that small single-stranded non-coding microRNAs (miRNAs) act as powerful endogenous regulators of TEC function and blood vessel formation. This systematic review provides an up-to-date overview of these endothelial miRNAs. Their expression is mainly regulated by hypoxia, pro-angiogenic factors, gap junctions and extracellular vesicles, as well as long non-coding RNAs and circular RNAs. In preclinical studies, they have been shown to modulate diverse fundamental angiogenesis-related signaling pathways and proteins, including the vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR) pathway; the rat sarcoma virus (Ras)/rapidly accelerated fibrosarcoma (Raf)/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway; the phosphoinositide 3-kinase (PI3K)/AKT pathway; and the transforming growth factor (TGF)-β/TGF-β receptor (TGFBR) pathway, as well as krüppel-like factors (KLFs), suppressor of cytokine signaling (SOCS) and metalloproteinases (MMPs). Accordingly, endothelial miRNAs represent promising targets for future anti-angiogenic cancer therapy. To achieve this, it will be necessary to further unravel the regulatory and functional networks of endothelial miRNAs and to develop safe and efficient TEC-specific miRNA delivery technologies.


Introduction
Cancer is a leading cause of global death [1]. Although the diagnosis and therapy of some tumor types have been considerably improved in recent years, novel efficient treatment options are still urgently needed. Such a promising option is the inhibition of angiogenesis, which may be performed as monotherapy or in combination with other therapeutic approaches [2,3].
Angiogenesis is defined as the growth of new blood vessels from pre-existing ones and is well known as one of the major cancer hallmarks, as defined by Hanahan and Weinberg [4]. It typically occurs when a tumor reaches 1-2 mm 3 in volume and can no longer be adequately supplied with oxygen and nutrients via diffusion [5]. During angiogenesis, endothelial cells (ECs) lining the lumen of blood vessels are activated by proangiogenic factors that are released from hypoxic tumor cells, as well as other components of the tumor microenvironment (TME) [6]. These pro-angiogenic factors include vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), angiopoietins, transforming growth factor (TGF)-β and placental-derived growth factor (PDGF) [7,8].
Upon their binding to cell surface receptors, ECs are stimulated to proliferate, migrate, form vascular sprouts and, ultimately, assemble into new microvascular networks within Compared to blood vessels within normal tissues, tumor vessels are irregularly organized, fragile and leaky [5]. Hence, it is reasonable that tumor ECs (TECs) differ from normal ECs in terms of morphology and function. TECs are highly heterogeneous and sensitive to certain growth factors, such as VEGF, but resistant to serum starvation and anti-cancer drugs, including 5-fluorouracil and paclitaxel [9]. Moreover, they are characterized by impaired endothelial barrier function, as well as increased angiogenic and metabolic activities [10]. All these features are probably due to their genetic abnormality. In fact, previous studies reported that TECs exhibit markedly different expression patterns of genes and non-coding RNAs when compared to normal ECs [11][12][13][14].
MicroRNAs (miRNAs), a specific type of non-coding RNA molecule of 18-24 nucleotides, have been shown to be implicated in the abnormality of TECs and the development of tumor vasculatures [15]. They are mainly transcribed by RNA polymerase II from miRNA genes, introns of protein-coding genes or polycistronic transcripts. A small subset of miRNAs can also be transcribed by RNA polymerase III [16,17]. The resulting long hairpin-like primary transcript (pri-miRNA) with thousands of nucleotides is further processed within the nucleus by RNase III Drosha into 70-90-nucleotide stem-loop precursor miRNA (pre-miRNA). The pre-miRNA is then exported by exportin 5 to the cytoplasm, where it is cleaved by RNase III Dicer into miRNA duplex. Finally, the miRNA duplex is loaded onto the RNA-induced silencing complex (RISC) and unwound into the singlestranded mature form and its complementary strand, which is normally degraded [18] ( Figure 2). The nomenclature of the mature miRNA is determined by the directionality of the miRNA strand. The 5p strand (miR-5p) arises from the 5′ side of the pre-miRNA, while Compared to blood vessels within normal tissues, tumor vessels are irregularly organized, fragile and leaky [5]. Hence, it is reasonable that tumor ECs (TECs) differ from normal ECs in terms of morphology and function. TECs are highly heterogeneous and sensitive to certain growth factors, such as VEGF, but resistant to serum starvation and anti-cancer drugs, including 5-fluorouracil and paclitaxel [9]. Moreover, they are characterized by impaired endothelial barrier function, as well as increased angiogenic and metabolic activities [10]. All these features are probably due to their genetic abnormality. In fact, previous studies reported that TECs exhibit markedly different expression patterns of genes and non-coding RNAs when compared to normal ECs [11][12][13][14].
MicroRNAs (miRNAs), a specific type of non-coding RNA molecule of 18-24 nucleotides, have been shown to be implicated in the abnormality of TECs and the development of tumor vasculatures [15]. They are mainly transcribed by RNA polymerase II from miRNA genes, introns of protein-coding genes or polycistronic transcripts. A small subset of miRNAs can also be transcribed by RNA polymerase III [16,17]. The resulting long hairpin-like primary transcript (pri-miRNA) with thousands of nucleotides is further processed within the nucleus by RNase III Drosha into 70-90-nucleotide stem-loop precursor miRNA (pre-miRNA). The pre-miRNA is then exported by exportin 5 to the cytoplasm, where it is cleaved by RNase III Dicer into miRNA duplex. Finally, the miRNA duplex is loaded onto the RNA-induced silencing complex (RISC) and unwound into the singlestranded mature form and its complementary strand, which is normally degraded [18] ( Figure 2). The nomenclature of the mature miRNA is determined by the directionality of the miRNA strand. The 5p strand (miR-5p) arises from the 5 side of the pre-miRNA, while the 3p strand (miR-3p) originates from the 3 side [19]. The mature miRNA in the RISC is able to guide the complex to its messenger RNA (mRNA) targets, usually by base pairing with their 3 untranslated regions (UTRs). This leads to the degradation or translation inhibition of target mRNAs depending on the degree of miRNA-mRNA complementarity [19] (Figure 2). Of note, non-canonical binding sites of miRNAs in mRNA regions have also been identified, including the 5 UTR, coding sequence and promoter regions [15,19]. Accordingly, each miRNA has the ability to target multiple genes and, thus, serves as a powerful regulator of diverse cellular processes, such as apoptosis, proliferation and migration [15]. Importantly, miRNAs play a pivotal role in maintaining physiological homeostasis, and their dysregulation has been strongly associated with a broad spectrum of human diseases, such as cancer [20]. the 3p strand (miR-3p) originates from the 3′ side [19]. The mature miRNA in the RISC able to guide the complex to its messenger RNA (mRNA) targets, usually by base pairi with their 3′untranslated regions (UTRs). This leads to the degradation or translation hibition of target mRNAs depending on the degree of miRNA-mRNA complementar [19] (Figure 2). Of note, non-canonical binding sites of miRNAs in mRNA regions ha also been identified, including the 5′UTR, coding sequence and promoter regions [15,1 Accordingly, each miRNA has the ability to target multiple genes and, thus, serves a powerful regulator of diverse cellular processes, such as apoptosis, proliferation and m gration [15]. Importantly, miRNAs play a pivotal role in maintaining physiological hom ostasis, and their dysregulation has been strongly associated with a broad spectrum human diseases, such as cancer [20]. MiRNA biogenesis and function. An miRNA gene is generally transcribed to pri-miRN by RNA polymerase II. Following the cleavage of pri-miRNA by Drosha, the resulting pre-miRN is transported by exportin-5 out of the nucleus into the cytoplasm. Further cleavage by Dicer resu in the generation of an miRNA duplex, which associates with the RISC. This association facilita the discarding or degradation of one strand of the duplex. The remaining mature miRNA then bin completely or partially to its target transcript, leading to mRNA degradation or translation repr sion. MiRNA biogenesis and function. An miRNA gene is generally transcribed to pri-miRNA by RNA polymerase II. Following the cleavage of pri-miRNA by Drosha, the resulting pre-miRNA is transported by exportin-5 out of the nucleus into the cytoplasm. Further cleavage by Dicer results in the generation of an miRNA duplex, which associates with the RISC. This association facilitates the discarding or degradation of one strand of the duplex. The remaining mature miRNA then binds completely or partially to its target transcript, leading to mRNA degradation or translation repression.
Although it is known that miRNAs in different cell types of the TME are capable of modulating tumor angiogenesis [15], we exclusively focus in this systematic review on miRNAs in ECs (also called endothelial miRNAs) that are involved in the regulation of TEC angiogenic activity. In detail, we elucidate the different mechanisms regulating their expression, describe their functions and targets in tumor angiogenesis modulation, illustrate their potential therapeutic applications along with associated challenges and provide insights into future directions of the field.

Endothelial miRNAs Involved in Tumor Angiogenesis
In order to retrieve all published papers that focus on endothelial miRNAs regulating tumor angiogenesis, a systematic literature search was performed in the PubMed database until January 2023, as shown in Figure 3. The key words for this search included 'microRNA', 'miRNA' or 'miR' combined with 'endothelial cells' and 'angiogenesis', as well as 'tumor' or 'cancer'. Only original research articles written in English, focusing on miRNAs in ECs and investigating the effects of endothelial miRNAs on tumor angiogenesis were included.
Although it is known that miRNAs in different cell types of the TME are capable of modulating tumor angiogenesis [15], we exclusively focus in this systematic review on miRNAs in ECs (also called endothelial miRNAs) that are involved in the regulation of TEC angiogenic activity. In detail, we elucidate the different mechanisms regulating their expression, describe their functions and targets in tumor angiogenesis modulation, illustrate their potential therapeutic applications along with associated challenges and provide insights into future directions of the field.

Endothelial miRNAs Involved in Tumor Angiogenesis
In order to retrieve all published papers that focus on endothelial miRNAs regulating tumor angiogenesis, a systematic literature search was performed in the PubMed database until January 2023, as shown in Figure 3. The key words for this search included 'mi-croRNA', 'miRNA' or 'miR' combined with 'endothelial cells' and 'angiogenesis', as well as 'tumor' or 'cancer'. Only original research articles written in English, focusing on miR-NAs in ECs and investigating the effects of endothelial miRNAs on tumor angiogenesis were included.
We detected 81 original research articles, which fulfilled the above-mentioned inclusion criteria. These articles referred to 62 different endothelial miRNAs or miRNA clusters that are involved in tumor angiogenesis. The names of these miRNAs and the mechanisms regulating their expression, as well as their functional targets and effects, are listed in Table 1.  We detected 81 original research articles, which fulfilled the above-mentioned inclusion criteria. These articles referred to 62 different endothelial miRNAs or miRNA clusters that are involved in tumor angiogenesis. The names of these miRNAs and the mechanisms regulating their expression, as well as their functional targets and effects, are listed in Table 1.

Pro-Angiogenic Factors
Pro-angiogenic factors in the TME stimulate angiogenesis mainly by binding to their receptors on ECs and activating intracellular downstream signaling pathways. However, they can also exert their effects by regulating the expression of miRNAs in ECs.
VEGF is one of the most potent pro-angiogenic growth factors. Among the VEGF family members, which include VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E and placental growth factor (PlGF), VEGF-A (often abbreviated as VEGF) plays a dominant role in regulating angiogenesis and blood vessel permeability [107]. It has been reported that VEGF secreted by U87 glioblastoma cells downregulates the expression of miR-125b-5p (miR-125b) in human brain microvascular ECs (HBMECs), which consequently stimulates EC angiogenesis, because miR-125b acts as an angiogenesis inhibitor [27]. Moreover, the expression of anti-angiogenic miR-1-3p (miR-1) was downregulated in VEGF-stimulated ECs and ECs isolated from the lungs of VEGF transgenic mice [108], as well as ECs isolated from mouse NSCLC tumors [21]. EC-specific miR-1 overexpression mediated by lentivirus vectors or transgenic methods suppressed tumor growth and angiogenesis in several mouse models of NSCLC [21]. These findings suggest crucial clinical significance of miR-1 in the anti-angiogenic treatment of NSCLC. On the contrary, VEGF upregulated the expression of miR-296-5p (miR-296) in HBMECs in culture, which may explain the elevated level of this miRNA in TECs isolated from human gliomas. Furthermore, the inhibition of miR-296 with antagomirs reduced vascularization in tumor xenografts [74]. The upregulation of endothelial miR-296 by VEGF was later confirmed by Kim et al. in HUVECs [109].
Interleukin (IL)-1β, a well-known pro-inflammatory cytokine, serves as an important pro-angiogenic factor in the TME [110]. In ECs, it mediates the phosphorylation and degradation of the inhibitor of nuclear factor κB (IκB) kinase [111]. Subsequently, IκB-free nuclear factor κB (NF-κB) translocates into the nucleus, where it controls the transcription of mRNAs as well as miRNAs [112,113]. In a previous study, we found that IL-1β released

Hypoxia
Hypoxia is a key microenvironmental feature of the majority of solid tumors [102]. It drives tumor angiogenesis, metastasis, immunosuppression and treatment resistance by regulating various cell types of the TME, including tumor cells, fibroblasts and ECs [102,103]. The hypoxic TME stimulates EC angiogenesis mainly via the activation of hypoxia-inducible factors (HIFs), which are highly conserved transcriptional factors regulating a multitude of genes and non-coding RNAs [104,105]. Recently, we could demonstrate in vitro that HIF1α activation in human dermal microvascular ECs (HDMECs) exposed to hypoxia inhibits the transcription of miR-186-5p (previous name: miR -186), which may explain the downregulation of this miRNA in TECs of human non-small-cell lung cancer (NSCLC) samples [49]. This finding is consistent with a recent study reporting that the expression level of miR-186 in human umbilical vein ECs (HUVECs) is decreased under hypoxic conditions [106]. The downregulation of miR-186 due to hypoxia, in turn, promoted the angiogenic activity of ECs by upregulating protein kinase C, a bona fide target of miR-186 [49].

Pro-Angiogenic Factors
Pro-angiogenic factors in the TME stimulate angiogenesis mainly by binding to their receptors on ECs and activating intracellular downstream signaling pathways. However, they can also exert their effects by regulating the expression of miRNAs in ECs.
VEGF is one of the most potent pro-angiogenic growth factors. Among the VEGF family members, which include VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E and placental growth factor (PlGF), VEGF-A (often abbreviated as VEGF) plays a dominant role in regulating angiogenesis and blood vessel permeability [107]. It has been reported that VEGF secreted by U87 glioblastoma cells downregulates the expression of miR-125b-5p (miR-125b) in human brain microvascular ECs (HBMECs), which consequently stimulates EC angiogenesis, because miR-125b acts as an angiogenesis inhibitor [27]. Moreover, the expression of anti-angiogenic miR-1-3p (miR-1) was downregulated in VEGF-stimulated ECs and ECs isolated from the lungs of VEGF transgenic mice [108], as well as ECs isolated from mouse NSCLC tumors [21]. EC-specific miR-1 overexpression mediated by lentivirus vectors or transgenic methods suppressed tumor growth and angiogenesis in several mouse models of NSCLC [21]. These findings suggest crucial clinical significance of miR-1 in the anti-angiogenic treatment of NSCLC. On the contrary, VEGF upregulated the expression of miR-296-5p (miR-296) in HBMECs in culture, which may explain the elevated level of this miRNA in TECs isolated from human gliomas. Furthermore, the inhibition of miR-296 with antagomirs reduced vascularization in tumor xenografts [74]. The upregulation of endothelial miR-296 by VEGF was later confirmed by Kim et al. in HUVECs [109].
Interleukin (IL)-1β, a well-known pro-inflammatory cytokine, serves as an important pro-angiogenic factor in the TME [110]. In ECs, it mediates the phosphorylation and degradation of the inhibitor of nuclear factor κB (IκB) kinase [111]. Subsequently, IκB-free nuclear factor κB (NF-κB) translocates into the nucleus, where it controls the transcription of mRNAs as well as miRNAs [112,113]. In a previous study, we found that IL-1β released by NSCLC cells activates NF-κB and, thus, suppresses the expression of miR-22-3p (miR- 22) in HDMECs co-cultured with NSCLC cells. This mechanism possibly contributes to the observed downregulation of miR-22 in TECs of human NSCLC samples [59]. These findings are in line with previous studies showing that IL-1 downregulates the expression of miR-22 in primary cultured chondrocytes [114]. Moreover, NF-κB directly binds to the miR-22 promoter and inhibits the transcription of this miRNA in 182R-6 breast cancer cells [115]. In addition, the overexpression of miR-22 in ECs resulted in the inhibition of NSCLC angiogenesis and growth, suggesting that this miRNA holds promise as a therapeutic target for anti-angiogenic cancer treatment [59].
TGF-β is a prominent member of a large family consisting of 33 multifunctional cytokines, including TGF-β isoforms, activins and bone morphogenetic proteins [116]. It regulates a plethora of cellular processes, such as proliferation, motility and differentiation during organ development and homeostasis, while its dysregulation has been linked to multiple diseases, including fibrosis, vascular pathologies and cancer [116]. It is noteworthy that TGF-β has also been proposed to modulate angiogenesis. Its angiogenic and angiostatic effects on ECs are dose-and context-dependent in vitro [117]. However, it serves as a potent angiogenesis inducer in vivo [117]. McCann et al. [76] recently reported that TGF-β is capable of reducing the transcription of miR-30c-5p (miR-30c) in ECs. The vascular tropic nanoparticle-mediated delivery of miR-30c antagomirs promoted E0771 mammary tumor angiogenesis and growth, whereas miR-30c mimics showed the opposite effects in vivo. It is worth noting that the downregulation of miR-30c by TGF-β has also been observed in other cell types, including primary hepatic stellate cells [118], renal tubular epithelial cells [119], cardiac fibroblasts [120] and ovarian cancer cells [121].

Gap Junctions
Gap junctions, which are composed of transmembrane connexin hexamers, represent membrane channels that mediate the direct transfer of small molecules, such as ions, amino acids, secondary messengers and metabolites, between adjacent cells in solid tissues [122]. They play pivotal roles in a wide range of both physiological and pathological processes [123]. In particular, it has been shown that gap junctions mediate the interaction between tumor cells and ECs and are therefore directly involved in the induction of tumor angiogenesis [124,125]. This view is further supported by a recent study showing that miR-5096 is transported from glioblastoma cells to ECs via gap junctions, leading to EC tube formation [86]. Of interest, gap junctions also play a role in the transfer of miR-5096 from glioblastoma cells to astrocytes. As a consequence, miR-5096 promotes glioma invasiveness, while the underlying mechanism needs further elucidation [126].

Extracellular Vesicles (EVs)
EVs are lipid bilayer-encapsulated particles that are released by almost all types of cells [127]. They serve as vesicles for the exchange of proteins, lipids and nucleic acids between cells, which is considered as an important mechanism of intercellular communication [127]. Based on their size and origin, EVs are generally categorized into exosomes and microvesicles [128]. Exosomes originate from endosomes, and their size ranges from 50 to 150 nm. In contrast, microvesicles with a diameter of up to 1000 nm emerge from the plasma membrane [128]. Out of the 62 miRNAs or miRNA clusters listed in this review, 37 have been demonstrated to be transferred from epithelial cells, tumor cells or cancer stem cells into ECs through EVs, which subsequently modulate tumor angiogenesis. Examples of such miRNAs include miR-1229-3p (miR-1229) [24], miR-1246 [26], miR-21-5p (miR-21) [52], miR-221-3p (miR-221) [62], miR-25-3p (miR-25) [69], miR-9-5p (miR-9) [93] and miR-92a-3p (miR-92a) [95], which can be delivered from colorectal cancer cells to ECs via EVs and promote angiogenesis. It is important to note that the origin of miRNA-containing EVs is often not limited to a single type of cancer cell. For instance, microvesicles derived from NSCLC cells, melanoma cells, pancreatic cancer cells and glioblastoma cells have also been shown to upregulate endothelial miR-9 [93]. Moreover, the intratumoral injection of miR-9 antagomirs inhibited the vascularization and growth of the HM7 colorectal tumor and LLC lung carcinoma [93]. These findings suggest that EVs derived from the TME play a significant and major role in regulating endothelial miRNAs and tumor angiogenesis.

Long Non-Coding RNAs (lncRNAs) and Circular RNAs (circRNAs)
Endothelial miRNAs can also be regulated by lncRNAs and circRNAs. lncRNAs are a class of single-stranded RNA that lack protein-coding capacity, with a length longer than 200 nucleotides [129], while circRNAs are single-stranded non-coding RNAs with a covalently closed-loop structure [130]. Both lncRNAs and circRNAs possess binding sites for miRNAs and serve as miRNA sponges. Sponged miRNAs are incapable of interacting with their target mRNAs. As a consequence, the target genes of miRNAs are positively regulated by lncRNAs and circRNAs [131,132]. It has been reported that endothelial miR-29a-3p (miR-29a) is sponged by lncRNA H19, which is upregulated in glioma microvessels and ECs cultured in glioma cell-conditioned medium. Accordingly, the knockdown of lncRNA H19 resulted in miR-29a upregulation and the downregulation of its target, vasohibin 2 (VASH2), in ECs, ultimately leading to the inhibition of gliomainduced EC angiogenesis in vitro [73]. The sequestration of miR-29a by lncRNA H19 has also been observed in many cancer cell types, such as breast cancer cells [133], clear cell renal cell carcinoma cells [134] and osteosarcoma cells [135]. In addition, endothelial miR-138-5p (miR-138) was targeted by circ_002136 and downregulated in ECs cultured in U87 glioblastoma cell-conditioned medium (GECs). It suppressed GEC angiogenesis by targeting SOX13 and subsequentially increasing SPON2 transcription [33].

Function of Endothelial miRNAs in Tumor Angiogenesis
Endothelial miRNAs are considered potent regulators of tumor angiogenesis due to their capacity to target multiple genes associated with angiogenesis. Indeed, they exert proor anti-angiogenic effects by regulating diverse angiogenesis-related signaling pathways and proteins, as outlined in the following subsections by means of selected, exemplary miRNAs, and summarized in Figure 5.

Krüppel-Like Factors (KLFs)
KLFs are a family of zinc finger-containing transcription factors that regulate basic cellular processes, including apoptosis, proliferation, migration, differentiation, inflammation and metabolism [157]. They are involved in the pathophysiology of diverse diseases, such as obesity and cancer [157]. So far, researchers have identified 18 different KLFs, among which KLF2, KLF4, KLF5 and KLF10 play important roles in regulating angiogenesis [158][159][160][161]. Previous studies have shown that exosomal miR-182-5p (miR-182) secreted by hypoxic glioblastoma cells and exosomal miR-25 derived from colorectal cancer cells induce angiogenesis and increase vascular permeability by targeting KLF2 and KLF4 in ECs [47,69]. The direct binding of miR-25 to KLF4 was further confirmed by Lu et al. [162] using a dual-luciferase reporter assay. Additionally, KLF2 is also a bona fide target of pro-angiogenic miR-3157-3p and miR-92a [78,97]. Ling and colleagues first proved that miR-92a targets KLF2 [163]. In addition, a recent study has reported that miR-141 secreted by small-cell lung cancer cells is able to be delivered to ECs via exosomes and promote EC angiogenic activity by targeting KLF12 [35]. However, the specific role of KLF12 in tumor angiogenesis needs further elucidation.

Suppressor of Cytokine Signaling (SOCS)
SOCS proteins are negative regulators of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway [173]. The evolutionarily conserved JAK/STAT pathway regulates a variety of developmental and homeostatic processes, such as the development of the immune system, hematopoiesis and stem cell maintenance [174]. Growing evidence suggests that this pathway significantly contributes to tumor angiogenesis by promoting EC survival, proliferation and migration [175,176]. The JAK/STAT pathway is initiated upon the binding of cytokines or growth factors to their specific receptor subunits. This leads to the multimerization of the receptor subunits and the transphosphorylation of receptor-associated JAKs. Activated JAKs, in turn, phosphorylate the cytoplasmic tyrosine residues of receptors to provide docking sites for STATs. Phosphorylated STATs dimerize and translocate to the nucleus, where they regulate the transcription of diverse genes [174]. SOCS is capable of downregulating the JAK/STAT pathway via different mechanisms. These include blocking the binding of STAT to receptor, directly inhibiting the kinase activity of JAK and promoting the degradation of JAK or STAT [173]. Accordingly, SOCS acts as an angiogenesis inhibitor. The targeting of SOCS3 and SOCS5 by endothelial miR-221, miR-141-3p (miR-141) and miR-9, respectively, activates the JAK/STAT pathway and consequently stimulates the formation of new blood vessels [36,62,93]. Additional studies further confirm the binding relationship between miR-221 and the 3 UTR of SOCS3 [177,178], as well as between miR-9 and the 3 UTR of SOCS5 [179,180].

Matrix Metalloproteinases (MMPs)
MMPs, a family of zinc-dependent endopeptidases, facilitate tumor angiogenesis and metastasis by degrading components of the extracellular matrix (ECM), resulting in the release of ECM-sequestered pro-angiogenic factors and exposure of the integrin-binding sites of ECM proteins [181,182]. On the contrary, tissue inhibitors of MMPs (TIMPs) are known to inhibit the activity of MMPs and act as negative regulators of angiogenesis [183]. Liu et al. recently reported that miR-526b-3p targets MMP2 and VEGF in ECs cultured in glioma cell-conditioned medium (GECs), causing a significant decrease in GEC viability, migration and tube formation [87]. In addition, the downregulation of TIMP2 by miR-3157-3p in ECs contributed to its pro-angiogenic effects in NSCLC [78].

Therapeutic Applications of Endothelial miRNAs
So far, several anti-angiogenic agents have been approved by the United States Food and Drug Administration (FDA) for the treatment of metastatic cancers, such as colorectal cancer, renal cell carcinoma, hepatocellular carcinoma and thyroid cancer. These agents include humanized monoclonal antibodies against VEGF/VEGFR (e.g., bevacizumab and ramucirumab), the soluble VEGF decoy receptor aflibercept as well as tyrosine kinase inhibitors (e.g., sunitinib and sorafenib) [184]. Unfortunately, their clinical efficiency is quite low due to the onset of innate or acquired resistance [184,185]. This resistance is mediated by different mechanisms, including the elevation of intratumoral hypoxia, the upregulation of alternative angiogenic pathways and increased tumor metastasis [184]. Therefore, it is necessary to search for more effective strategies for anti-angiogenic cancer therapy.
Given their potent regulatory function in TEC activity, endothelial miRNAs represent promising novel targets for the development of second-generation anti-angiogenic therapeutics. In this context, two major approaches have been suggested in endothelial miRNA-based therapy [186,187]. There is the possibility of introducing anti-angiogenic miRNAs into TECs. On the other hand, TECs can be treated with miRNA antagonists (also called antagomirs or anti-miRNAs) that inhibit pro-angiogenic miRNAs. However, the cellular uptake of miRNAs or antagonists is hampered by their charge repulsion and high vulnerability to serum RNase degradation. To overcome this problem, chemical modifications and sophisticated delivery systems have been established in recent years [188].
Chemical modifications of miRNAs or anti-miRNAs include phosphorothioate backbone modification, 2 -O-methyl conjugation or locked nucleic acid (LNA) modification [189]. Unfortunately, these chemical structure optimizations only slightly improve the stability and cellular penetration of RNA oligonucleotides. In contrast, non-viral (i.e., lipids, polymers, inorganic compounds and extracellular vesicles) and viral delivery systems (i.e., lentivirus and adeno-associated virus (AAV)) successfully protect oligonucleotides from nuclease degradation and transport them to different organs, such as the liver and the kidneys [189,190]. Based on these delivery systems, considerable progress has been made in the selective transport of miRNAs or anti-miRNAs to ECs, which are particularly difficult to transfect or transduce.
EC-targeting peptides conjugated to lipid-and polymer-based nanoparticles are most widely used. For instance, the systemic administration of anti-miR-132-3p (miR-132), anti-miR-296 and miR-7 loaded in nanoparticles modified with cyclic RGD has been shown to increase the endothelial uptake of oligonucleotides and inhibit the angiogenic activity of ECs in vitro and in vivo [191][192][193]. Of note, RGD is a peptide that binds to integrin αvβ3 and αvβ5 on the membrane of ECs. More recently, RGD-modified exosomes overexpressing miR-92b-3p (miR-92b) were found to inhibit ovarian cancer angiogenesis and growth [98]. Similarly, the Ala-Pro-Arg-Pro-Gly (APRPG) peptide, which has an affinity to VEGFR1 on ECs, was utilized to generate APRPG-polyethylene glycol (PEG)-modified lipoplexes for the in vivo delivery of miR-499-5p (miR-499) to tumors via intravenous injection. These miRNA-carrying lipoplexes accumulated in tumor blood vessels and inhibited the growth of colon carcinoma [194]. Moreover, the integrin α4β1 ligand Arg-Glu-Asp-Val (REDV) was linked to trimethyl chitosan via a PEG linker. This modified polyplex selectively delivered miR-126 to ECs and consequently enhanced their proliferation [195].
The screening of random peptide libraries on the surface of AAV capsids has been performed to identify vectors that enable high transduction efficiency in ECs. One successful example of such a vector is the modified AAV9 capsid plasmid displaying peptide SLRSPPS [196]. By using this modified vector, the overexpression of miR-92a significantly inhibited endothelium-dependent relaxation in mouse aortas [197].
Nonetheless, despite the above-mentioned achievements, the efficient, specific and safe delivery of miRNAs or anti-miRNAs to ECs, especially TECs, still remains a big challenge to date.

Concluding Remarks and Perspectives
Over the last two decades, there has been significant interest in the role of miRNAs in tumor angiogenesis, leading to intensive research in this field. Our comprehensive search of the literature on PubMed revealed that approximately 80% of publications focus on the indirect effects of miRNAs in tumor cells on EC angiogenesis. However, ECs are the primary cell type responsible for angiogenesis. Therefore, our systematic review specifically focused on endothelial miRNAs that play crucial roles in regulating the aberrant angiogenic activity of TECs. The definitions of TECs in publications can be categorized into different groups: (i) ECs cultured with tumor cell-conditioned medium; (ii) ECs co-cultured with tumor cells without direct contact on a Transwell plate; (iii) ECs co-cultured with tumor cells with direct contact and subsequently isolated from tumor cells; (iv) ECs isolated from fresh human or mouse tumor tissues; and (v) ECs isolated from formalin-fixed paraffinembedded (FFPE) human tumor samples using laser capture microdissection. Although all the TEC types mentioned above have been considered in this review, it is important to note that the in vitro experimental settings used to study TECs only monitor a fraction of the TME. The TME, characterized by hypoxia, acidity and nutrient deficiency, contains not only tumor cells but also immune cells, fibroblasts, macrophages and the extracellular matrix [6]. In our view, ECs dissected from FFPE human tumor tissues best capture the features of TECs in the TME. Even freshly isolated TECs from tumor tissues, while still valuable for analysis, are no longer considered true TECs as they have been removed from the TME.
Previous studies have shown that the intracellular levels of endothelial miRNAs involved in tumor angiogenesis are mainly determined by the TME via hypoxia, proangiogenic factors, cell-cell transfer and sponging by lncRNAs and circRNAs. Moreover, these miRNAs target key angiogenesis-related signaling pathways or proteins, including the VEGF/VEGFR, Ras/Raf/MEK/ERK, PI3K/AKT and TGF-β/TGFBR pathway, as well as KLFs, SOCS and MMPs/TIMPs. While we present these mechanisms of endothelial miRNA regulation and function separately in this review for organizational purposes, it should be noted that they interconnect with each other and form a complex network. For instance, studies have shown that hypoxia can stimulate the expression of pro-angiogenic factors such as VEGF, IL-1β and TGF-β in a variety of cell types [198][199][200][201][202][203]. Therefore, it is not surprising that miR-1, miR-22 and miR-30c, which have been reported to be downregulated by VEGF, IL-1β and TGF-β in ECs, respectively [21,59,76], could also be inhibited by hypoxia in certain scenarios [204][205][206][207][208]. Moreover, KLF2 has been shown to suppress the expression of VEGFR2 by inhibiting its promoter activity [158]. Accordingly, KLF2-targeting miR-182 and miR-25 upregulate VEGFR2 in ECs and consequently promote angiogenesis [47,69].
This systematic review highlights the critical roles of endothelial miRNAs in regulating tumor angiogenesis. Furthermore, the multiple-gene-targeting capacity of miRNAs may help to prevent acquired therapy resistance. Therefore, targeting endothelial miRNAs holds promise as a novel approach for developing second-generation anti-angiogenic cancer treatments. Combining endothelial miRNA-targeting therapy with other anti-cancer treatments, such as chemotherapy, radiotherapy and immunotherapy, may enhance clinical outcomes. However, due to the nascent stage of therapeutic applications for endothelial miRNAs, several scientific and technical challenges must be addressed. To facilitate clinical translation, a better understanding of the regulatory and functional networks of endothelial miRNAs is a critical prerequisite. Moreover, the development of efficient and safe miRNA delivery systems specific to TECs is required. In addition, it is essential to assess the effects, dosage, pharmacokinetics, side effects and acquired resistance of endothelial miRNA-targeting treatments in appropriate animal models. Rapid and significant progress in RNA sequencing technologies enabling the discovery of new miRNAs, high-throughput approaches for miRNA target identification, chemical modifications of miRNAs, nanotechnology and viral vector development, as well as tailored animal models for drug discovery and development, may help researchers achieve these goals in the near future.