3.1. Role of Exosomes in Endothelial Metabolism
Endothelial cells that line the innermost part of all blood vessels and lymphatic vessels perform numerous functions, such as the maintenance of vascular integrity, transcellular transport of nutrients and oxygen, leukocyte adhesion, platelet aggregation, interstitial fluid formation and the secretion of vasoactive substances. Bone marrow endothelial cells are involved in the development of hematopoietic stem cells and hematopoiesis [
126]. Multiple metabolic pathways, such as glycolysis, fatty acid oxidation and amino acid metabolism, are used by endothelial cells to carry out their functions efficiently [
127]. Glycolysis, in which glucose is converted to lactate, rather than oxidative phosphorylation is responsible for the production of 85% of ATP in endothelial cells. 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase-3 (PFKFB3) and hexokinase 2 (HK2) are the rate-limiting enzymes driving glycolysis in endothelial cells. Fatty acid oxidation, which is mainly controlled by carnitine palmitoyltransferase 1 (CPT1), is necessary for biomass (proteins, lipids, nucleotides) synthesis and redox homeostasis. Metabolism of amino acid glutamine is important for endothelial cells, which are bathed in a high-oxygen environment, in order to withstand oxidative stress [
128]. Cysteine metabolism supports endothelial functions, such as cell adhesion, mechano-transduction and flow-induced vasodilatation, by S-sulfhydrating target proteins (e.g., integrins) [
129]. Thus, it is not surprising that endothelial cell metabolism is significant for normal physiological processes. Endothelial dysfunction is seen during disease states, such as diabetes mellitus, atherosclerosis, pulmonary arterial hypertension and cancer, as metabolism is perturbed or becomes excessive in these disorders [
130].
Exosomes have an impact on endothelial metabolism and bioenergetics (
Figure 4). Exosomes isolated from human umbilical vein endothelial cells (HUVECs) exposed to high glucose levels influence the protein expression and functionality of endothelial cells. They increase the expression of endothelial nitric oxide synthase (eNOS) and ICAM-1 in HUVECs, as well as endothelial wound healing [
131]. Metabolism in endothelial cells is influenced by signals from other cell types in their proximity. In this context, rat cardiomyocytes, under glucose deprivation, secrete more exosomes that are loaded with functional glucose transporters (GLUT1, GLUT4) and glycolytic enzymes (lactate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase). Upon transfer to cardiac microvascular endothelial cells, the internalized exosomes augment glucose uptake, glycolytic activity and pyruvate synthesis in endothelial cells [
132]. In addition, cardiomyocyte-derived exosomes activate eNOS, which is responsible for nitric oxide production in endothelial cells, conferring protection against ischemia/reperfusion injuries [
133]. This metabolic cross-talk also occurs between endothelial cells and immune cells. Mixed exosomes produced by monocytes and endothelial cells under high glucose conditions favor endothelial inflammation by increasing the endothelial expression of ICAM-1 [
134]. Likewise, exosomes derived from mature dendritic cells are involved in the progression of endothelial inflammation via vesicular tumor necrosis factor-α (TNF-α)-mediated NFκB pathways [
135]. Oxidized low-density lipoprotein-stimulated macrophages suppress tube formation in endothelial cells via exosomes [
136]. Together, these studies provide strong evidence that exosomes can modulate endothelial metabolism and functions.
3.2. Role of Exosomes in Angiogenesis and Tumor Microenvironment
Angiogenesis is a complex biological process by which new blood vessels are formed. This process requires coordinated activities, such as the proliferation and migration of endothelial cells. Endothelial cells are quiescent in normal states, but proliferate in a rapid manner under ischemia and hypoxic conditions or in response to injury [
128]. During the growth of new vessels, endothelial cells dynamically adapt their metabolism to the increased demands of energy substrates and biomass synthesis. Glycolysis is likely to be the main metabolic pathway used by migrating tip cell phenotypes to sustain energy during vascular sprouting. Fatty acid oxidation provides proliferating stalk cells with the substrates necessary for biomass synthesis. After the formation of new vessel sprouts, endothelial cells differentiate into quiescent phalanx cells in which glycolytic rates and mitochondrial respiration are lower compared to tip cells and stalk cells [
137]. Vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) and other angiogenic signals are key factors in regulating the angiogenic process [
128]. In recent years, exosomes have been shown to play a role in the angiogenic process. For instance, exosomes from adipose-derived stem cells (ASCs) promote angiogenesis in vitro, as well as in a mouse model of hindlimb ischemia. They also induce the polarization of M1 macrophages to M2 macrophages, which secrete angiogenic cytokines and growth factors. This study also highlighted the possible synergistic impact of ASC exosomes and M2 macrophages on angiogenesis [
138]. Another study also showed that ASC exosomes promote vascularization. They explained the underlying mechanism in which those exosomes that overexpress miR-21 upregulate the expression of hypoxia-inducible factor-1α (HIF-1α), VEGF, stromal cell-derived factor-1 (SDF-1), Akt and the extracellular signal-regulated kinases ERK1/2 in HUVECs [
139].
Tumor endothelial cells and cancer cells are important components of the tumor microenvironment. Their metabolic cross-talk, dynamic interaction and competition for nutrients influence the progression of cancer [
140]. Cancer cells use massive amounts of glucose for their proliferation and survival. At the same time, cancer cells can suppress glucose utilization in neighboring cells by transferring exosomes loaded with miR-122, which mediates the inhibition of glycolytic enzyme pyruvate kinase [
141]. Furthermore, cancer cells can absorb metabolic cargos that are carried by CAF-derived exosomes [
142]. In hypoxic environments, cancer cells gain the ability to promote cancer progression by increasing the release of exosomes to neighboring cells. Hypoxic breast cancer cells promote tumor spreading by releasing exosomes, which induce mitochondrial reprogramming, ILK-Akt activation and malignant morphogenesis in normal mammary epithelial cells [
123]. Meanwhile, hypoxic cancer cells increase the rate of glycolysis, leading to the release of lactate, which can be taken up by nearby cells, including tumor endothelial cells. Unlike normal endothelial cells, tumor endothelial cells exhibit a hyper-proliferative feature, with an increase in glycolysis, fatty acid synthesis, glutamine metabolism and mitochondrial respiration [
130]. Therefore, the metabolite lactate secreted by cancer cells is likely to be important for endothelial cells in glucose-deprived tumor environments, leading to abnormal proliferation. Lactate has been shown to play a significant role in promoting angiogenesis by increasing VEGF, HIF-1α and IL-8 signaling in endothelial cells [
143]. Previous studies have suggested that the metabolism of hyper-glycolytic tumor endothelial cells can be inhibited, and this blockage has shown advantageous effects in pre-clinical tumor models. For instance, inhibiting PFKFB3 in hyper-glycolytic tumor endothelial cells has beneficial effects, such as tumor vessel normalization, reduced metastasis and improved chemotherapy [
144]. Thus, targeting endothelial metabolism can be an alternative option or an additive treatment to suppress tumor angiogenesis, as there are limitations in the efficacy and usefulness of anti-angiogenic drugs (e.g., VEGF antagonists) in the treatment of cancer [
130].
In addition to metabolic cross-talk between cancer cells and endothelial cells, they communicate with each other via exosomes. Therefore, it is tempting to focus on how cancer cell-derived exosomes influence endothelial metabolism and angiogenesis, since they can be considered as crucial mediators in tumor microenvironments. Exosomes derived from acute myeloid leukemia (AML) cells influence the activities of HUVECs, such as proliferation, migration and tube formation by enhancing glycolysis, as well as by inducing VEGF receptor expression in endothelial cells [
145]. Exosomes from rat pancreatic adenocarcinoma cells can promote the proliferation and migration of rat aortic endothelial cells by regulating the endothelial expression of chemokine CXCL5, chemokine receptor CCR1, VEGF receptor 1, von Willebrand factor and tissue factor. It is worth noting that endothelial–exosome interactions and exosome-mediated angiogenic effects depend on the exosomal expression of the Tspan8-integrin α4β1 complex [
110]. The impact of exosome-associated integrins on the angiogenic potential of endothelial cells is also demonstrated in prostate cancer progression. As described above, PrCa exosomes promote angiogenesis by transferring exosomal integrin αVβ6 to endothelial cells, which do not normally express epithelial-specific integrin αVβ6. The uptake of exosomal αVβ6 is related to the upregulation of angiogenesis-promoting survivin levels and the downregulation of angiogenesis-inhibiting pSTAT1 in endothelial cells [
102]. Moreover, tumor exosomes can carry and transport VEGF to endothelial cells to promote angiogenesis. Angiogenic effects are mediated via VEGF receptor 2 signaling, and VEGF-bound tumor exosomes confer resistance to treatment with VEGF antibody bevacizumab [
146]. It is well known that tumor exosomes favor metastatic spread by preparing pre-metastatic niches. The study by Hoshino et al. demonstrated that pre-metastatic niche formation by different cancer cells follows distinct expression patterns of exosomal integrins [
101]. Intriguingly, colorectal cancer-derived exosomes have been implicated in pre-metastatic niche formation by inducing vascular permeability and angiogenesis. These effects are mediated by cancer-promoting miR-25-3p, which is loaded in exosomes [
147]. In the same way, exosomes from metastatic breast cancer and hypoxic lung cancer destroy the vascular barrier and promote metastasis by downregulating tight junction proteins, such as zonula occludens-1 (ZO-1) [
148,
149]. Endothelial-to-mesenchymal transition (EndoMT), in which endothelial cells are differentiated into CAFs, is an important mechanism underlying tumor growth and metastasis. Exosomes from different types of cancer cells can efficiently induce EndoMT, in which endothelial cells undergo significant changes in phenotype, genotype and behavior. CAFs, in turn, gain the ability to remodel the ECM, to disrupt the vascular barrier and to stimulate the migration and invasion of cancer cells [
150]. Conversely, mesenchymal stem cell-derived exosomes have the ability to reverse the EndoMT process via the recovery of CAFs back to endothelial cells [
151]. Tumor-associated macrophages (TAMs), which are major immune cells in tumor microenvironments, are involved in cancer progression (e.g., HCC) by releasing their exosomes into tumor cells [
120]. The specific participation of TAM-exosomes in tumor angiogenesis was shown in one study in which M2-polarized TAMs induced angiogenesis in pancreatic ducal adenocarcinoma by transporting miR-155-5p- and miR-221-5p-loaded exosomes to endothelial cells [
152]. Collectively, these data strongly suggest that tumor exosomes have the ability to reprogram endothelial cells within the tumor microenvironment, thereby promoting tumor growth and metastasis.
Another alternative strategy of tumor exosomes to promote tumor progression is mediated through their exosomal expression of programmed death ligands (PDL-1 or PDL-2). Programmed cell death protein 1 (PD-1), which is predominantly expressed in T-cells and tumor-infiltrating lymphocytes, interacts with PDL-1 or PDL-2 to suppress T-cell activation and to induce tumor immune escape [
153]. The genetic blockage of exosomal PDL-1 is suggested to confer anti-tumor immunity and to extend the lifespan in a syngeneic mouse model of prostate cancer [
154]. In melanoma, tumor cells escape from immunosurveillance by delivering PDL-1 into their exosomes and thereby inhibiting the functions, such as proliferation, cytokine secretion and cytotoxicity of CD8
+ T-cells. In the clinical setting, the plasma level of exosomal PDL-1 in patients of metastatic melanoma is found to be significantly higher than that of control subjects. Additionally, the level of PDL-1 in circulating exosomes is suggested to be helpful in stratifying clinical responders from non-responders to anti-PDL-1 therapy [
155]. Thus, it is prominent that exosomal PDL-1 secreted by tumor cells plays a major role in promoting tumor growth through an immune-dependent mechanism.