mTOR signalling pathway in stem cell bioactivities and angiogenesis potential

Abstract The mammalian target of rapamycin (mTOR) is a protein kinase that responds to different stimuli such as stresses, starvation and hypoxic conditions. The modulation of this effector can lead to the alteration of cell dynamic growth, proliferation, basal metabolism and other bioactivities. Considering this fact, the mTOR pathway is believed to regulate the diverse functions in several cell lineages. Due to the pleiotropic effects of the mTOR, we here, hypothesize that this effector can also regulate the bioactivity of stem cells in response to external stimuli pathways under physiological and pathological conditions. As a correlation, we aimed to highlight the close relationship between the mTOR signalling axis and the regenerative potential of stem cells in a different milieu. The relevant publications were included in this study using electronic searches of the PubMed database from inception to February 2023. We noted that the mTOR signalling cascade can affect different stem cell bioactivities, especially angiogenesis under physiological and pathological conditions. Modulation of mTOR signalling pathways is thought of as an effective strategy to modulate the angiogenic properties of stem cells.


| INTRODUCTION
The existence of several signalling pathways consisting of multi-state effectors inside each cell can produce specific data in response to various intra-and extra-cellular clues.Following sequential enzymatic reactions and activation of varied scaffold proteins, cells can exhibit functional behaviour. 1,2The discovery and advent of stem cells with key biological properties have led to the advancement and progress of therapeutic approaches in human medicine. 3Using several mechanisms, stem cells and progenitors can contribute to therapeutic outcomes in injured sites. 4The regenerative outcomes are specified by engaging various effectors associated with different signalling cascades. 5The tight collaboration between effectors belonging to different signalling cascades activates a specific collection of factors that participate in certain stem cell behaviour. 6ong several molecular cascades, the critical role of the mammalian target of rapamycin (mTOR) pathway has been proved in the context of dynamic growths and the regulation of multiple biochemical reactions. 7Previous findings are consistent with the fact that the activation of the mTOR signalling axis is associated with the promotion of diverse biochemical reactions which per se are associated with the regulation of transcription, translation, protein synthesis and degradation via the modulation of p70S6K/S6 and 4EBPI/eIF4E. 8Metabolic status is a key factor that can affect the activity of the mTOR Hamid Lotfimehr and Narges Mardi are considered co-first authors of this study.signalling axis. 9The drop of intra-cellular glucose content to very low levels and the exposure of cells to starvation can provoke the mTOR pathway. 10The integration of the mTOR pathway with either intra-cellular or extra-cellular signals highlights the importance of this pathway in stem cell regenerative potential under physiological and pathological conditions. 11Of note, mTOR is a core-acting enzyme with a serine/threonine-protein kinase and belongs to the PI3K family (Figure 1). 8 has been shown that mTOR consists of two complexes, namely mTORC1 and two subunits.In terms of molecular structure, mTORC1 is a complex of mTOR, raptor, GβL and deptor.In contrast to mTORC1, mTOR, Rictor, GβL, PRR5, deptor and SIN1 are constituents of the latter complex mTORC2. 10From an evolutionary viewpoint, both mTORC1 and 2 are phylogenetically conserved with prominent participation in cell growth and proliferation. 12,13To be specific, mTORC1 partakes in the regulation of cell growth and autophagic response while mTORC2 is certainly an important factor in cytoskeletal re-modelling and actin re-arrangement (Figures 1 and 2). 14veral upstream effectors such as growth factors and insulin, low ATP ratio, hypoxia, Wnt, TNFα and amino acids (glutamine, leucine and arginine) can initiate the mTOR signalling pathway via different transduction pathways. 15Mechanistically, different stimulators can activate the mTORC1 signalling pathway.The presence of amino acids, insulin and growth factors can induce the recruitment of mTORC1 to the lysosome surface, and its association with small GTPases namely Rags.Four types of rags, including Rag-A, Rag-B, Rag-C and Rag-D, participate in the sense of nutrients by mTORC1. 16 be specific, Rag GTPases attach physically to the raptor and connects mTORC1 to the lysosome where Rheb, a mTORC1 activator, resides. 16Simultaneously, these factors are co-localized with tuberous sclerosis complex (TSC) as a suppressor of GTPase Rheb activity.The inhibition of the TSC complex and its dissociation coincides with the induction of GTPase activating protein on Rheb, resulting in the phosphorylation of mTORC1 on the lysosomal surface. 17The signalling axes associated with mTORC1 fulfil several bioactivities such as suppression of insulin-related pathways via GRB10, phosphorylation 4EBP1 and ribosomal S6 kinase (S6K) leading to protein synthesis and cell growth. 18 phosphorylation of S6K at Thr389, mTORC1 can induce phosphorylation by PDK1 in the kinase domain of S6K.Activated S6K promotes the phosphorylation of ribosomal protein S6 belonging to the ribosomal 40S subunit.It is suggested that mTORC1 can promote the transcription of rRNA via RNA pol I and RNA pol III and enhance ribosome biogenesis via the activation of S6K1. 17The over-expression of T cell differentiation protein 2 (MAL2), belonging to the MAL protein family, induces activation of the MAPK/mTOR signalling pathway, promotes ribosome biogenesis and facilitates proliferation in nonsmall cell lung cancer (NSCLC). 19The role of mTORC1 in the protein synthesis processes is associated with the phosphorylation of 4E-BPs.This factor acts as a translation repressor and inhibits translation via the regulation of eIF4E.The phosphorylation of 4E-BP1 by mTORC1 induces its release from eIF4E and promotes 5 0 cap-dependent translation of mRNAs. 17S6K phosphorylation by mTORC1 activates as a transcription factor namely sterol regulatory element-binding proteins (SREBP).It has been indicated that SREBP can induce the expression of key enzymes in the pentose phosphate pathway to accelerate nucleotide synthesis.It was suggested that phosphorylated S6K stimulates pyrimidine synthesis via the promotion of CAD activity. 20The activation of SREBP by mTORC1 induces lipogenic gene expression, via activating S6K or inhibits nuclear translocation of LIPIN1 via its phosphorylation. 20It has been indicated that mitochondrial elongation factor 2 promotes fatty acid synthesis and cholesterol biosynthesis via activation of ROS/Akt/mTOR signalling pathway and up-regulation of SREBP. 21e close relationship between the mTOR signalling pathway and autophagy has been indicated. 22Autophagy is defined as an early-stage self-degradation mechanism in response to several insulting conditions.This mechanism can sequestrate misfolded proteins into the intracellular vesicles namely autophagosomes.In the latter phases, the fusion of autophagosomes with lysosomes contributes to the formation of autophagolysosomes and the release of cargo to the cell outside. 23sed on the molecular analyses, multiple autophagy-related proteins (ATGs) are involved in the formation of autophagosomes and the activation of autophagy in a tightly controlled manner. 24The modulation of mTORC1 can lead to autophagic recycling of essential components under the situations of cells exposed to starvation and nutrition limitation. 22The activation of mTORC1 leads to anabolic reactions inside the cells.Along with these changes, the uptake of various nutrients is enhanced and the autophagic flux is prohibited. 25The inhibition of autophagy is orchestrated by phosphorylation of ULK1 at Ser758 and ATG13. 22The phosphorylation of ULK1/ATG13/FIP200 complex prohibits the formation of autophagosomes. 22Meanwhile, simultaneous phosphorylation of TFEB and MiTF-TFE family members intensifies these effects. 26It should not be forgotten that the cargo recycling by autophagy machinery can activate the mTORC1.These features indicate that autophagy is both upstream and downstream of mTORC1. 22 contrast, AMPK promotes autophagy via phosphorylation of ULK1 at different sites (Ser-555, Ser-777, Ser-317 and Ser-467) and thus triggers autophagic flux. 27In cells with energy stress and reduced ATP/AMP ratio, AMPK is stimulated due to allosteric changes which in turn increases the uptake of carbohydrates and lipid β-oxidation. 28e activated ULK1 promotes the phosphorylation of Beclin-1 in VPS34/Beclin-1/ATG14 complex.In an alternative way, mTORC1 inhibits autophagy indirectly via the suppression of lysosomal biogenesis, through phosphorylation and inhibition of nuclear translocation TFEB. 29like mTORC1, the activation of mTORC2-related signalling pathways via PKC can orchestrate several functions such as cell division, lineage trans-differentiation, autophagic and apoptotic responses and post-translational modification (Figure 1). 18 is postulated that in response to growth factors, the activation of mTORC2 contributes to the production of PIP3 via the phosphorylation and activation of AGC kinase family members (PKC, AKT and SGK).In the latter steps, mTORC2 binds to the ribosomes and co-translationally phosphorylates AKT at Thr450 residue.Alternatively, post-translational phosphorylation of AKT at Ser473 promotes the lipogenic pathway by activation of SREBP1c. 30Furthermore, mTORC2 promotes the production of insulin-like growth factor 2 (IGF2), by co-translation and phosphorylation of IGF2 mRNA-binding protein 1. 31 mTORC2 can phosphorylate and enhance the activity of YAP and the transcription of YAP-related genes, leading to the growth and invasion of glioblastoma cells. 32I G U R E 1 Mammalian target of rapamycin (mTOR) signalling pathway.The mTOR signalling pathway consists of two mTOR complexes, mTORC1 and mTORC2 with the ability to regulate different bioactivities inside the host cells.In upstream, several factors such as amino acids, Wnt, TNF-α, IGF and insulin can affect the activity of mTOR.The mTORC1 complex regulates microtubule re-organization, autophagy and lipolysis, lipid and protein synthesis while the mTORC2 complex is involved in cell survival and cytoskeletal re-modelling.
F I G U R E 2 Autophagy machinery following the mammalian target of rapamycin (mTOR) activity.mTOR inhibition can lead to the activation of ATG10, ATG13, ULK1 and FIP200 and formation of primary phagophores.This process is followed by elongation via the participation of the ATG7/ATG3/ATG4 complex and the conversion of LC3-I to LC3-II, leading to the formation of autophagosomes.After that, autophagosomes are fused with lysosomes to form autophagolysosomes.In the latter steps, the autophagic cargo is released to the outside of cells or recycled.
Considering the inevitable role of the mTOR signalling pathway in different cell lineages, one could hypothesize that this axis can also affect the dynamic activity of stem cells inside several niches under physiological and pathological conditions, leading to distinctive regenerative outcomes.Here, we aimed to highlight the possible role of the mTOR signalling axis on the dynamic growth of stem cells.

| mTOR PATHWAY ROLE IN DIFFERENTIATION AND STEMNESS MAINTENANCE
As a common belief, stem cells exhibit eminent self-renewability and differentiation capacity towards different cell lineages. 33Because of their quiescent status under physiological conditions, it is thought that stem cells possess low-rate translation properties despite high cytosolic ribosome levels. 34A low-rate translation makes these cells eligible to maintain themselves in an undifferentiated state. 35It has been indicated that high-rate mRNA expression and translation do necessitate several proteins at the post-translational level. 35Molecular investigations have revealed that either orientation towards certain cell types or maintenance of stemness can be orchestrated via the regulation of ribosome activity and protein synthesis capacity (Table 1). 34To be specific, each cell type, especially stem cells, harbour heterogeneous ribosomes with the potential to translate specific kinds of mRNAs in response to protein changes. 36Such heterogeneity can lead to distinct interactions between the mRNA-ribosomal RNA and mRNA-ribosomal protein which pre-determines different patterns for translation. 36Due to the close association between the mTOR signalling pathway and metabolic activity, mTOR signalling can be tightly involved in the procedure of protein synthesis in stem cells to regulate growth, differentiation and viability. 35It was indicated that mTORC1 positively regulates the transcription of ribosomal RNA and proteins and factors associated with ribosome assembly. 37The activation of mTOR stimulates global translation rate by engaging effectors 4E-BP and S6K.Along with these changes, the expression of eIF, eEF and ribosomal proteins are triggered, leading to activation and differentiation of stem cells. 38Besides, other possible mechanisms have been proposed for the involvement of the mTOR signalling pathway in the dynamic growth of stem cells.For instance, recent studies have shown that the interaction of Akt with mTOR signalling triggers tendon differentiation of MSCs towards tenocyte-like cells via the synthesis of type I collagen and ECM deposition. 39Akt can activate mTORC1 via the phosphorylation and inhibition of TSC2 and PRAS40. 40The inhibition of Akt and mTORC1 impaired the collagen synthesis and tenogenesis of MSCs. 39In another experiment conducted by Yao and co-workers, they showed that the over-expression of miR-29a-3 in human umbilical cord MSCs after activation of PTEN/ mTOR/TGF-β1 signalling led to the secretion of exosomes (Exos) and the expression of tendon-specific markers. 41These features indicated that mTOR signalling can also regulate the paracrine activity of MSCs.
It confirmed that miR-29a-3 regulates the tenogenesis of MSCs by targeting PTEN and direct interaction of Akt with mTOR. 41 was indicated that MSCs can limit the differentiation of CD4 + T cells towards T helper 17 cells (Th17) via the regulation of the mTOR pathway.The modulation of mTOR signalling is associated with regulatory T cell (Treg) and Th17 differentiation through the activation of STAT3 or STAT5 and HIF-1α. 42PI3K/AKT/mTOR/p70S6K axis plays a role in the differentiation of Th2 and Treg cells, and inhibition of the mTOR pathway, involving in the maintenance of Th2/Treg balance. 43Importantly, the suppression of mTORC1 kinase activity reduces the differentiation of intestine Caco-2 cells into functional enterocytes.Selective activation of upstream effector AMPK can inhibit mTORC1 via phosphorylation of Ser1387 residue. 44Therefore, one could hypothesize that mTORC1, in contrast to mTORC2, is not essential to the promotion of cell differentiation towards specific lineages.Whether and how underlying mechanisms are engaged after the promotion of the mTOR signalling pathway is under investigation.
Ionic stimulation of the mTOR pathway via magnesium supplementation has been shown to trigger myogenic differentiation of aged muscle progenitor cells. 45It is thought that there is a close relationship between the tyrosine kinase activity of the IGF-1 receptor and the AKT/PKB-mTOR pathway, leading to the promotion of stem cell differentiation towards myocytes. 46It is important to mention that the mTOR pathway is touted as a pleiotropic cascade in the regulation of stem cell differentiation towards several lineages.For example, in a study increased intra-membrane distribution of GPR30 provoked PI3K/AKT/mTOR and enhanced osteogenesis in periodontal ligament stem cells. 47Shikonin, one of the derivatives of naphthoquinone, induces odontoblastic differentiation of dental pulp stem cells through the AKT-mTOR signalling pathway and expression of CD44. 48Using anti-CD44 monoclonal antibody, the levels of phosphorylated-mTORC1, phosphorylated-mTORC2 and phosphorylated-Akt were reduced, leading to enhanced differentiation of HL60, THP-1 and KG1a leukaemic cells and tumour growth suppression. 49These data reinforce the idea that the regulation of CD44 and inhibition of PI3K/ Akt/mTOR pathway are strategic plans to control the propagation of leukaemic cells.
Under starvation conditions, accumulated pyruvate with an increased AMP/ATP ratio expedites the mesodermal differentiation of human ESCs via the stimulation of AMPK. 50Data indicated that elevated pyruvate can blunt glycolytic capacity and glycolysis.In line with these changes, the components of tricarboxylic acid were increased, indicating stimulated mitochondrial oxidative phosphorylation. 50on myogenic differentiation, syndecan-3 reduces the activity of insulin receptor after interaction with AKT/mTOR pathway. 51It was suggested that modulation of miR-143-3p and miR-100-5p via the mTOR pathway is involved in driving human MSC fate for tissue regeneration. 52Both miRNAs act as mechanosensitive factors and their expression can be changed in response to matrix stiffness and RhoA activity. 52It was shown that co-transfection of encapsulated MSCs with co-transfection of miR-100-5p and miR-143-3p inside 3D gelatin-polyethylene glycol hydrogel diminished protein levels of mTOR, Rictor and Larp1 and exerted rapamycin-like effects. 52The inhibition of mTOR and its effectors increases the osteogenic potential of MSCs with the deposition of hydroxyapatite and alkaline phosphatase activity. 52In an experiment, it was indicated that an Indian Hedgehog (IHH) shortage leads to skewed differentiation in bone marrow-derived mesenchymal stem cells (BMSCs), which can be modified by inhibition of mTOR and ROS. 53The incubation of mouse MSCs with IHH siRNA led to premature ageing (β-galactosidase") and skewed differentiation. 53The activation of mTOR and accumulation of ROS is associated with senile changes and skewed differentiation in MSCs. 53Concerning several bioactivities, recent investigations have confirmed the crucial role of the mTOR signalling pathway in the orientation and phenotype acquisition of several stem cell types. 7Cs are a small fraction of cells within tumour parenchyma with prominent tumourigenesis and metastatic activity. 54These cells exhibit unique self-renewal and stemness features via the expression of certain factors such as CD44 + /CD24 À and CD133 + , resulting in resistance to chemotherapeutic agents. 54Using tissue microarray, it was suggested that mTOR is highly expressed in 74% of patients with NSCLC. 55Likewise, inevitable roles of PI3K/Akt/mTOR and IL-8 were indicated in CSCs isolated from hepatic tissue. 56Based on the data, rapamycin can diminish the positivity of liver CSCs in terms of CD133 and EpCAM and spheroid formation. 56Treatment of pancreatic tissue CD133 + CSCs with rapamycin reduces survival rate and stemness features with simultaneous loss of CD133. 57The down-regulation of PI3K/AKT/mTOR with the loss of clonogenic capacity was achieved in breast CSCs after exposure to flavonoid quercetin. 58These studies T A B L E 1 mTOR signalling pathway and stem cell differentiation and survival.In an experiment conducted by Katsuno and colleagues, deprivation of HMLE cells from TGF-β blunted EMT rate with the reduction of CD44 + /CD24 À cells, and downregulation of certain genes such as NANOG, POU5F1 and SOX2. 59Data have pointed to the fact that the inhibition of mTOR in CSCs is an effective way to control tumour propagation via the reduction of CSCs and stemness removal.It should not be neglected that mTOR signalling pathway has synergism with other molecular pathways to control dynamic activity of CSCs. 60iedman and co-workers indicated that treatment of glioblastoma U87 and LN18 cells with rapamycin and all-trans retinoic acid increases differentiation of CSCs and loss of nestin.These features coincided with the reduction of neurosphere diameter and migration properties. 60In line with this statement, the inhibition of mTOR using rapamycin improved the suppressor activity of Fbxw7 gene. 61The activity of Fbxw7 is associated with the ubiquitination of different oncoproteins in which homozygous or heterozygous ablation of this gene can contribute to several malignancies. 61Liu et al. claimed that temporal inhibition of mTOR delayed tumour development and blunted the loss of Fbxw7 factor in Fbxw7 +/À mice. 61Simultaneous inhibition of mTOR and treatment of human breast cancer specimens with tamoxifen led to stemness reduction, promotion of mesenchymal to epithelial transition (MET), and sensitivity to chemotherapeutic agents. 62Some data revealed that inhibition of multiple mTOR signalling pathway effectors is another strategy for achieving regenerative outcomes.For instance, dual inhibition of mTOR and PI3K contribute to the reduction of colorectal SW620 CSC population and MET rate. 63mTORC1/S6K1 pathway is also involved in the regulation of CSC stemness. 64,65The activation of mTORC1/S6K1 axis promotes the phosphorylation of Gli1 belonging to the Hedgehog pathway and increases the release of Gil1 from an endogenous inhibitor SUFU.The mTORC1/S6K1 axis can also reduce the degradation of Gil1 via GSK3β.Nuclear translocation of GSK3β is tightly controlled after the activation of mTORC1/S6K1 pathway, leading to the stabilization of stemness factor c-Myc. 64,65 Like mTORC1, the regulatory role of mTOC2 has been documented in terms of CSC stemness.It was suggested that mTORC2 can inhibit FoxO upon phosphorylation of Akt.Following Akt phosphorylation, nuclear translocation of FoxO is decreased, resulting in the release of c-Myc.5][66] Hypoxic conditions within the tumour niche preserve the stemness features of CSCs.The elevation of HIF-2α in hypoxic tumour cells increases the number of CD44 + /CD24 À MDA-MB-231 cells via the modulation of the PI3K/ AKT/mTOR axis. 67Taken together, these data confirm the key role of mTOR and relevant effectors in the maintenance of stemness and CSC population within the tumour niche.Of course, due to the complexity and existence of various effectors, it is postulated that the mTOR pathway can directly or in collaboration with other molecular pathways affect the dynamic growth and differentiation capacity of CSCs.

| ROLE OF mTOR SIGNALLING ON ANGIOGENESIS CAPACITY
The term angiogenesis or neovascularization refers to the formation of nascent blood vessels from the pre-existing network. 68It is thought that angiogenesis is a fundamental procedure with the participation of multiple cells.In response to the secretion of several pro-angiogenesis factors, endothelial cells (ECs) proliferate and generate nascent blood vessels under physiological and pathological conditions. 69In a complementary mechanism known as vasculogenesis, the differentiation of bone marrow endothelial progenitor cells, namely EPCs, into mature ECs and the formation of primitive vascular plexus can support blood into certain sites. 70Irrespective of vessel formation via neoangiogenesis or vasculogenesis, the activation of certain signalling pathways and balances between pro-and anti-angiogenic factors can pre-determine the fate of vascular generation inside the body.
Previous findings are consistent with the fact that persistent activation of mTOR promotes angiogenesis.In support of this notion, the inhibition of mTOR1 and 2 via the specific blocker can prohibit angiogenesis in mouse models of retinoblastoma xenograft. 71In vitro data Ser2448.Along with these changes, phosphorylation of S6K1 and 4EPB1, and AKT was blunted in both cell types. 71e reciprocal interaction between EPCs, ECs and haematopoietic progenitor cells (HPCs) is critical in EC activity.Recent data indicated that ageing-related changes can alter crosstalk between endothelial niche and haematopoietic cells via the inhibition of the mTOR signalling pathway. 72Ramalingam and co-workers indicated that conditional deletion of mTOR in young mice ECs promotes the ageing of HPCs.Along with these changes, the levels of VEGF were reduced and the RTK signalling pathway was inhibited.Interestingly, the transplantation of wild-type HPCs to mTOR À / À mice restored bone marrow activity. 723 These data coincided with the reduction in the phosphorylated form of Akt, Erk and S6 factors.
It was suggested that cytoskeletal re-modelling and morphological adaptation are critical issues involved in the angiogenesis capacity and the formation of aligned EC layers. 74In this regard, the collaboration and cross-talk between VEGF, Notch and mTOR signalling pathways are important. 74Of note, co-inhibition of PI3K-Akt and mTORC1 in the presence of angiogenic factors such as VEGF can promote endothelial lineage adaptation with significant elongation. 74To be specific, the inhibition of these pathways in ECs can induce elongated forms of ECs even in the presence of low levels of VEGF.These data support the notion that PI3K-Akt and mTORC1 have negative effects on EC elongation and induce specific morphology.Of course, it should not be forgotten that the inhibition of mTOR can affect other adaptive molecular signalling pathways leading to hypermetabolic activity in certain stem cell lineage like haematopoietic stem cells (HSCs). 75In mTOR knockout mice, the stimulation of ERK-MNK-eIF4E-RNA polymerase II-c-Myc can compensate for mTOR-related activity in HSC proliferation capacity by influencing global gene expression. 75Fan and co-workers indicated that mTOR deficiency can inhibit the colony-forming properties of HSCs.In line with these changes, the number of HSC subsets at G0 was significantly reduced while active cells at phases of G1 and S/G2/M increased, indicating the uncontrolled expansion of HSCs. 75RNA sequencing showed that the expression of genes associated with proliferation such as c-Myc, Pim1, Fos, Dusp1 and Jun was stimulated in mTOR-deficient HSCs.
Recent findings extended our understanding of mTOR's impact on angiogenesis potential via the regulation of cell metabolism and biochemical reactions. 76The inhibition of AKT and mTOR expression can negatively control the activity of enzymes related to the glycolytic pathway and angiogenesis in tumour cells. 76For example, in bladder cancer cells the inhibition of Hepatitis B X-interacting protein (HBXIP), involved in centrosome duplication, suppressed the angiogenesis capacity via the inhibition of VEGF and erythropoietin.Liu and colleagues indicated that phosphorylation and expression of mTOR and AKT were reduced in HBXIP-inhibited tumour cells.
Besides, the activity of glycolytic enzyme proteins was diminished, leading to a reduction of glucose consumption and lactate content. 76 tumour ECs, both aerobic oxidation and most probably glycolytic pathways are activated.Therefore, it is logical to hypothesize that the inhibition of glycolysis, the main source of energy production mechanism in cancer cells, can lead to the suppression of angiogenesis and thus EC atresia. 77It is well-recognized that the activity of certain glycolysis enzymes such as enolase-1 (α-enolase) can affect angiogenesis.

Shu et al. claimed that the inhibition of α-enolase is associated with
the loss of stemness properties, migration and dynamic growth in pulmonary CSCs. 77This enzyme can stimulate the stemness features in CSCs via the phosphorylation of 4EBP1, S6K and mTOR.Commensurate with these data, α-enolase is proposed as a valuable target molecule in cancer therapy.Whether and how mTOR modulation can preserve stemness features or induce mature phenotype in certain lineages needs further investigation. 78Besides the fundamental role of glycolysis in angiogenesis, the reduction of mitochondrial respiration after the accumulation of harmful free radicals and insidious oxidants occurs.Under such circumstances, the activity of Akt/mTOR is prominently diminished via the phosphorylation of AMPK. 79In a similar work, the incubation of amniotic-derived epithelial cells Exos with fibroblasts or ECs induced angiogenesis potential via PI3K/AKT/ mTOR axis under diabetic conditions, indicating the integration of PI3K pathway with mTOR signalling cascade. 80Based on recent data, it seems that an array of signalling biomolecules can affect the activity of the PI3K/AKT/mTOR axis.For instance, the expression of type I and II polycystin proteins following in polycystic renal tissue correlates with hyper-phosphorylation of Akt, activation of the p110γ subunit of PI3K and VEGF. 81Like mature ECs, the importance of the PI3K/AKT/ mTOR axis has been recently clarified in the vasculogenic capacity of EPCs after exposure to Celastrol belongs to quinone methides. 82The axis. 83Peng and co-workers indicated the elevation of pS6, a mTORC1 activator, along with increased CD31 + ECs in the cutaneous tissue of rosacea-like mouse models and human counterparts. 84e inhibition of mTOR using rapamycin resulted in the reduction of excessive CD31 + vascular beds.Interestingly, the number of CD31 + ECs and VEGF levels were high in the skin of TSC2 +/À mice when compared to the TSC2 +/+ control group (Figure 3).These features indicated that the loss of inhibition on mTOR can lead to aberrant angiogenesis under pathological conditions.In an experiment conducted by Ding and co-workers, they explored the role of Deptor, a mTORC1 suppressor, which was investigated on angiogenesis potential. 85It has been indicated that the levels of VEGF, HIF-1α and CD31 were increased in cardiac, hepatic and renal tissues in Deptor À / À mice. 85Despite the existence of scientific documents regarding the stimulatory effects of mTORC1 on angiogenesis, in some circumstances like cancer niche, the sole inhibition of mTORC1 cannot lead to sufficient blockade of blood supplementation. 86In this regard, the application of KU0063794 with dual inhibition of mTORC1/mTORC2 can be an efficient strategy to inhibit the angiogenesis potential of ECs. 86In ECs exposed to KU0063794, EC elongation and tubulogenesis capacity were significantly diminished.
These features coincided with abnormal accumulation of F-actin filaments.Likewise, KU0063794 has the potential to abort the angiogenic potential of murine ESCs via simultaneous inhibition of mTORC1/mTORC2. 87Similarly, the inhibition of mTORC1/mTORC2 using ZJQ-24, an indole hydrazide compound, led to the inhibition of eIF4F, dephosphorylation of AKT and vascularization in tumour parenchyma. 88Previous data have indicated the collaboration of the mTOR signalling pathway with other molecular cascades is important in terms of angiogenesis.For example, the attachment of ligand CXCL12 to cognate receptor CXCR4 promotes CXCR4/PI3K/ mTORC2/Akt and stimulates the angiogenesis and migration of ECs via the mTORC1 axis. 89der inflammatory conditions, several cytokines such as prostaglandin E2 exert pro-angiogenic effects via the activation of mTORC2.
This prostaglandin stimulates the pro-angiogenic factors such as VEGF, FGF and so forth, and certain adhesion molecules like PECAM-1 in tumour ECs. 90There are controversial data related to the exact role of mTORC1 and/or mTORC2 on angiogenesis.For example, it was suggested that the suppression of mTORC2, but not mTORC1, can blunt the angiogenesis properties in ECs. 91A large number of pro-angiogenic factors and receptors such as VEGF, IGF-1, FGF, VEGFR-2 and insulin can orchestrate angiogenesis capacity in ECs via engaging mTORC2 independent of mTORC1 activation.From molecular aspects, the attachment of growth factors to relevant RTKs promotes the phosphorylation of CD146 and leads to the interaction of CD146 KKGK motif with the Rictor of mTORC2.This physical interaction stabilizes mTORC2 and stimulates angiogenesis in PI3K/ mTORC1 independent manner. 92e molecular identities of stem cells in different conditions are associated with the crosstalk of the mTOR signalling pathway with other molecular cascades. 93For instance, the neural differentiation The role of mammalian target of rapamycin (mTOR) in cutaneous tissue angiogenesis (A-H).In vivo angiogenesis was induced using an active form of Cathelicidin namely LL37.Immunofluorescence staining of CD31 and pS6 was performed in rosacea patients and compared to the normal samples (HS) (A, B).Immunostaining of CD31 and pS6 in mice cutaneous samples after treatment with LL37 and rapamycin (C, D; White arrows: CD31 + cells).Semi-quantitative measuring of CD31 vessels in LL37-treated rosacea-like mice after treatment with rapamycin (E, F).Semi-quantitative measuring of CD31 + vessels in control and TSC2 +/À knockdown mice after treatment with LL37 (G, H).PTEN effectors in tendon regeneration via paracrine activity. 41In glioma CSCs, the close collaboration between BTK and mTOR/VEGF axis can reduce stemness potential and angiogenesis capacity via the regulation of CD31 + cells, indicating the critical role of BTK/mTOR/ VEGF in the progression of tumour mass via vasculogenesis (Figure 4). 94e modulation of autophagy is another mechanism by which mTOR can affect the angiogenesis potential in stem cells. 70More in-depth investigations regarding the role of autophagy on the angiogenesis capacity of stem cells have led to controversies in the field. 95cubation of ECs with MSC-derived Exos promotes angiogenesis capacity via the promotion of tubulogenesis and EC migration. 96In a study conducted by Xia and colleagues, the injection of MSC Exos promoted ischemic stroke injury via the inhibition of autophagy response following the activation of STAT3. 96It was shown that the inhibition of Apelin, an endogenous ligand with a G protein-coupled receptor; accelerates MSC senescence indicated by the over-activity of beta-galactosidase activity. 97It was suggested that the stimulation of Apelin rejuvenated senile MSCs and promoted angiogenic capacity via phosphorylation of AMPK, inhibition of mTOR, and induction of autophagy. 97Along with these changes, an intra-cellular increase of ATG7 and 12 coincided with an increased LC3 II/I ratio occurs to exclude exhaust materials from the host cell. 95It is believed that the stimulation of autophagy in MSCs can increase their resistance against short-and long-term hypoxic conditions after transplantation into the target sites.In line with these statements, pre-treatment of MSCs with autophagy stimulators such as rapamycin can increase cell retention number, activate paracrine mechanisms and promote angiogenesis capacity via the secretion of VEGF, HGF, IGF, SCF and SDF-1α (Figure 5). 98Likewise, Jeong and co-workers indicated that the promotion of autophagy in ECs can increase blood supply and vessel growth into the ischemic area in the mouse hind limb. 99ke autophagy, the integrity of the mTOR complex and lipolysis has been previously indicated. 100To be specific, the activity of the mTOR complex, especially mTORC1, restrains the lipolysis rate in adipocytes and prohibits systemic hypercholesteremia and hyperlipidemia. 100Cs transplantation can diminish post-hepatectomy liver failure via the regulation of fatty acid β-oxidation and intra-hepatocyte lipid content.These effects were tailored after the activation of the mTOR sig- the formation of de novo blood vessels in the target sites. 102Emerging data have confirmed the close association between mTOR signalling and the release of factors via nano-sized vesicles namely Exos. 103os with endosomal origin range between 30 and 100 nm and are produced via the invagination of the vesicular membrane into the lumen of MVBs.Molecular investigations revealed that Exos contains certain cargoes with several types of growth factors that are involved in inter-cellular communication. 104Due to the complexity of the mTOR signalling cascade and Exos biogenesis, data have confirmed the interplay between these two pathways via shared signalling effectors. 105,106To be specific, continuous activation of mTOR1 can lead to intra-cellular accumulation of CD63 + vesicles and inhibition of Exos release from origin cells in in vitro and in vivo conditions.By contrast, the suppression of mTOR1 activity via rapamycin or other growth factors can yield the opposite outcome. 106In an experiment, the inhibition of mTOR1 with a specific inhibitor, Torin-1, increased MVBs interaction with lysosomal marker LAMP1 and Exo release.In line with these changes, the co-localization of mTOR1 with VPS16 and LAMP1 is inhibited, suggesting a critical role of ceramide-mTOR signalling in Exo secretion. 103In addition to the modulatory effect of mTOR on the Exo abscission rate, emerging data have proved that Exos can also regulate the function of the mTOR axis after entry into the target cells. 107It has been indicated that Exos isolated from miR-26a-expressing MSCs promoted axonal regeneration in a rat model of spinal cord injury via the modulation of the PTEN/AKT/mTOR axis. 107is strategy can be used by cancer cells to stimulate angiogenesis into the tumour parenchyma.In an experiment, it was suggested that hepatocellular carcinoma development and expansion are mediated by angiogenesis via the activation of the PI3K/Akt/mTOR pathway in HUVECs.To this end, cancer cells can release Exos enriched in SNHG16 which can increase the expression of resistant factor GALNT1. 108 Of note, the close interaction of the mTOR signalling pathway with angiogenesis effectors can facilitate the modulatory effects of Exos.For instance, miR-100-loaded Exos can efficiently regulate the mTOR/HIF-1α/VEGF pathway and blunt angiogenesis in breast cancer cells. 109In an experiment conducted by Qu and colleagues, they examined the angiogenic effect of human cord blood MSC Exos on a rat model of premature ovarian failure (Figure 6). 110os isolated from miR-126-3p over-expressing MSCs promoted
have indicated that TAK-228, an FDA-approved mTOR compound, can reduce the tubulogenesis activity of human retinal ECs in a dosedependent manner.Further analyses have revealed that TAK-228 exerted a dual inhibition of retinoblastoma cell growth and angiogenesis properties.Upon the incubation of retinoblastoma RB355 cells and retinal ECs with TAK-228, phosphorylation of mTOR was reduced at of phosphorylated PI3K/AKT/mTOR can blunt vascular mimicry in Celastrol-treated EPCs coinciding with inhibition of the VEGFR-2/Angiopoietin-2/VEGF axis, indicating the critical role of this molecular axis in the function of progenitor and adult endothelial lineage. 82Stem cell-derived Exos harbour several signalling molecules which are important in the regeneration of damaged tissues.Of these factors, miRNAs are short-length genetic materials that can be transferred via Exos and alter the activity of certain effectors inside the acceptor cells.It has been documented that injection of BMSC Exos containing miR-29c into a mouse model of experimental infarction led to an improved regeneration of the ischemic area via the stimulation of autophagy and activation of the PTEN/Akt/mTOR Real-time PCR analysis of VEGF in normal and TSC2 +/À knockdown mice treated with LL37.Scale bar: 50 μm.Student's t-test (B).One-way ANOVA with Bonferroni's post-hoc test (D, F, H and I).*p < 0.05; **p < 0.01 84 (Copyright 2021, Frontiers in Cell and Developmental Biology).capacity of small peptide ghrelin was indicated on adipose-derived MSCs orchestrated via AKT/mTOR and β-catenin pathway.The activation of the AKT/mTOR axis can stimulate nuclear β-catenin and phosphorylated GSK-3β. 93Of note, the suppression of the AKT/mTOR axis or β-catenin can deteriorate the neurogenesis capacity of ghrelin on MSCs.These data show that the collaboration of the F I G U R E 4 Pro-angiogenesis properties of rat bone marrow mesenchymal stem cells (MSCs) in cardiac ischemia/reperfusion model.GFPexpressing MSCs were pre-treated with rapamycin and transplanted into the affected sites in cardiac tissue.Immunofluorescence imaging revealed that GFP + MSCs can express CD31 (white arrows) and align in the vascular lumen, indicating the stimulatory role of mammalian target of rapamycin inhibition on endothelial lineage differentiation of MSCs.Scale bar = 100 μm.*p < 0.01 related to control; # p < 0.01 related to MSC group (n = 6) 94 (Copyright 2021, Pharmaceuticals).mTORsignalling pathway is vital to orient the stem cells towards certain lineages.In another study, the application of EC Exos loaded with miR-29a-3p-specific agonist promoted the healing procedure in Achilles tendon injury in a rat model.Molecular investigation revealed the participation of TGF-β1 and mTOR signalling pathways as well as nalling pathway.Besides, local production of interleukin-10 via injected MSCs is a compensatory mechanism to regulate immune cell function and promote angiogenesis in the injured areas.101 4 | mTOR AND PARACRINE ANGIOGENESIS ACTIVITY OF STEM CELLSIn addition to the differentiation capacity of stem cells towards ECs, the production and release of several angiogenic factors can promote F I G U R E 5 Pro-angiogenesis properties of rat bone marrow mesenchymal stem cells (MSCs) in cardiac ischemia/reperfusion model.GFPexpressing MSCs were pre-treated with rapamycin and transplanted into the affected sites in cardiac tissue.Immunofluorescence imaging revealed that GFP + MSCs can express CD31 (white arrows) and align in the vascular lumen, indicating the stimulatory role of mTOR inhibition on endothelial lineage differentiation of MSCs.Scale bar = 100 μm.*p < 0.01 related to control; # p < 0.01 related to MSC group (n = 6) 98 (Copyright 2020, Stem Cell Reviews and Reports).

F I G U R E 6
Evaluation of angiogenesis and apoptosis rate in ovarian tissue of rats with premature ovarian failure (POF) (A-G).Immunofluorescence imaging revealed that the number of TUNEL + cells was reduced in the POF rats that received miR-126-3p-bearing Exo (A).Immunohistochemistry staining indicated increased PCNA cell number in ovarian follicles, indicating the number of proliferating cells (B).In line with these changes, the number of CD31 + cells was increased in the POF rats that received 126-3p-bearing Exo (C).Western blotting indicated the suppression of apoptosis-related Bax, Caspase 3, and induction of anti-apoptotic factor Bcl-2 (D, E). 126-3p-bearing Exo increased protein levels of FGF, VEGF and IGF-1 in the ovarian tissue (n = 10).One-way ANOVA analysis.hucMSC, human umbilical cord MSCs derived exosomes; NC, negative control; PCNA, Proliferating cell nuclear antigen.*p < 0.05110 (Copyright 2022, Stem Cell Research & Therapy).
angiogenesis via the acceleration of the PI3K/Akt/mTOR signalling axis.These Exos induced the phosphorylation and synthesis of mTOR, Akt and PI3K in granulosa cells.Along with these changes, the administration of miR-126-3p-loaded Exos can increase the levels of CD31 vessels and PCNA + cells in the ovarian tissue matrix coinciding with the reduction of TUNEL-positive cells.110These data show the close relationship between the mTOR signalling pathway and Exo abscission.Due to the existence of numerous signalling biomolecules and factors inside Exos, it is mighty that Exos can easily modulate the effectors related to the mTOR pathway.5 | CONCLUSIONIt is thought that the mTOR signalling pathway can affect the bioactivity of stem cells, such as dynamic growth, differentiation capacity and angiogenesis potential under physiological and pathological conditions.Due to the complex entity of the mTOR signalling pathway and reciprocal cross-talk with other signalling cascades, it is difficult to interpret the exact role of this pathway in stem cells in in vitro and in vivo conditions.It would be better for further studies to focus on the determination of angiogenesis capacity and address the exact role of mTORC1 and mTORC2 complexes in the angiogenic potential of progenitors and stem cells.Whether and how these subunits and downstream effectors collaborate and/or blunt their effects remain unknown.Besides, the exact role of the mTOR signalling pathway in normal stem cells and CSCs should be addressed in terms of angiogenesis.