Advances in the Pharmacological Intervention of Endothelial Progenitor Cells in the Treatment of Ischemic Stroke Free

Ischemic stroke is an irreversible injury of the central system based on atherosclerosis. It is caused by thrombosis that blocks the internal carotid, vertebral arteries, or brain blood vessels. This process reduces the blood supply, causes cell metabolic disorders and senescence, and further leads to vascular endothelial injury. Cerebral ischemic stroke (CIS) accounts for more than 80% of strokes [1]. After the occurrence of cerebral focal ischemia, the pathological progression of ischemic stroke can be roughly divided into the acute phase and chronic phase [2]. During the acute phase, danger signals released into the bloodstream by damaged brain cells may lead to systemic immune activation. After that, innate immune cells invade the brain and meninges, causing inflammation and irreversible damage to brain tissue. In the chronic phase, the process of antigen presentation marks the adaptive immune response of the brain, which may underlie the sequelae of neuropsychiatric diseases and is the main reason for the high incidence after stroke. Chronic cerebral ischemia is a reversible disease with a long intervention time window, and early intervention can prevent the occurrence of serious adverse events [3]. Therefore, for reducing pathological damage to ischemic tissue, restoring vascular damage caused by endothelial dysfunction is the main direction of the treatment of CIS. However, current neuroprotective drug therapy is minimally effective. Autologous vascular grafts are limited by the lack of available sources of autologous veins, arterial grafts, and the high morbidity of the donor area. Thrombolytic therapy has the disadvantages of a narrow treatment time window, the high risk of bleeding, and neurological deficits that remain in most patients after treatment [4]. Therefore, research and development of new drugs are imperative. Endothelial progenitor cells (EPCs) are precursors of the vascular endothelium [5]. EPCs migrate from the bone marrow (BM) to the peripheral circulation through physiological and pathological stimulation. Thereafter, they produce BM-derived cells such as CD133+, CD34+, vascular growth factor receptor 2 (VEGFR2)+/KDR+, von Willebrand factor, and so on, which are targeted and integrated into the damaged endothelium after ischemic injury [6]. EPCs are used in artificial blood vessels and implanted devices [7] for rapid endothelialization [8] and the promotion of patency, solving the problem of local tissue ischemia [9], making it a potential new treatment for CIS [10]. As a novel cell therapy, EPCs act a protector in ischemic stroke caused by atherosclerosis or arteriolar occlusion. Compared with large artery occlusion, stroke due to small artery occlusion is mainly caused by deep perforating artery lesions or diffuse cerebral arteriolar lesions [11]. Patients often have underlying diseases such as diabetes, long-term hypertension, and other injuries [12]. The risk factors of the two are very homologous, and the only differences (gender and ischemic heart disease) are with regard to the atherosclerotic factor [13]. Therefore, mobilization of EPCs is necessary to repair endothelial cell injury and avoid the further development of infarction. We herein review the progress related to new drug therapies to accelerate the capture, proliferation, and differentiation [14] of EPCs. Then, promote angiogenesis to treat CIS, aiming to provide theoretical support for the clinical application of new drug therapies for CIS.

In 1997, Asahara et al. [15] isolated EPCs from human peripheral blood for the first time. Currently, EPCs can be obtained from circulating monocyte populations, umbilical cord blood, and BM. In experimental mouse models [6], it was found that the cerebral infarction area of ischemic stroke after perfusion of EPCs was significantly reduced, together with an improvement in behavioral function. This was because when blood vessels were stimulated by ischemia and hypoxia, BM-derived EPCs were targeted from the BM to the periphery and chemotactically migrated to the ischemic injury site. There, they differentiated into mature vascular endothelial cells, forming neovascularization during angiogenesis, or indirectly playing the role in angiogenesis in a paracrine manner by secreting cytokines that promote angiogenesis, including vascular endothelial growth factor (VEGF), basic fibroblast growth factor, and angiotensin [16]. Current studies have shown that extracellular vesicles secreted by EPCs can act independently as a bioactive substance to transmit information in cells and participate in the process of angiogenesis [6]. The functions of EPCs correlate positively with the degree of vascular damage, including proliferation, migration, adhesion, differentiation, and tubulogenesis. Therefore, the EPCs can represent a new approach for the treatment of CIS. Dysregulation of cerebral blood flow (CBF) due to dysfunctions of neurons, glial cells, and vascular endothelial cells often accompanies the pathologic development of ischemic stroke. Biochemical cycle product is the main impact factor in the function of cerebrovascular and small arteries, such as nitric oxide (NO), reactive oxygen species (ROS), and peroxynitrite. Based on these bioactive substances, myogenic tension and neurovascular coupling injury are the most common pathological reactions. As a novel cell therapy, EPCs act as the protector in this process. For example, Wang et al. [17] reported that EPCs can reduce ROS production to protect cerebral endothelial cells. Conversely, many studies have reported that these substances also accelerate the aging of EPCs. Thus, it is necessary to target EPCs in peripheral blood. In addition, myogenic tension is a purinergic-dependent mechanism where purinergic signals play an important role in regulating myogenic responses. Recently, the endothelial cell panx1 channel may be a new purine release channel expressed in the whole vascular system, which can regulate the vascular function of veins and arteries. After the cerebral ischemia-reperfusion injury, ecpanx1 reduces the number of leukocytes infiltrating into ischemic brain tissue from the venous system in a non-subtype-specific manner and reduces the myogenic tension of the cerebral artery, so as to reduce vasoconstriction, increase CBF in cerebral vessels, and improve the prognosis of stroke [18]. Therefore, it is a good strategy for improving myogenic tension to reconstruct endothelial cell pannexin 1 by EPCs, without changing the passive properties of cerebrovascular cells. The blood-brain barrier is composed of endothelial cells on the basal plate and interacts with astrocytes and pericytes. EPCs and pericytes are two important cells in blood-brain barrier repair under an ischemic stroke. Endothelial dysfunction leads to the extravasation of plasma components and the formation of harmful edema, which is considered to be the reason for the increase in blood-brain barrier permeability during cerebral ischemia [19]. However, the EPCs could improve endothelial dysfunction, prevent extravasation of plasma components, reduce the formation of harmful edema, and reduce the permeability of the blood-brain barrier. For example, the EPCs transplantation leads to white matter recovery, enhances neurogenesis, and reduces the permeability of the blood-brain barrier [20]. Therefore, EPCs can repair the blood-brain barrier of ischemic stroke. In the environment of chronic inflammation and oxidative stress, adiponectin (APN) EPCs of aging rats act on the damaged area of vascular endothelium through a variety of paracrine cytokines, participating in vascular repair and regeneration [21]. With the recovery of microvessel density and the increase of blood-brain barrier-related proteins, the integrity of the blood-brain barrier was restored [22].

The αvβ3 integrin ligand LXW7 is an effective and specific EPC/EC-targeting ligand [8] and is noncombinable with inflammatory cells [23]. However, it has a strong and specific EPC/EC capture capacity, which can improve EPC/EC function to achieve rapid endothelialization. In the rat carotid artery bypass graft model, lXW7-modified small-caliber vascular grafts significantly promoted EPC/EC recruitment and endothelization. Six weeks after implantation, the patency rate of LXW7-modified grafts was 83% and that of untreated grafts was 17% [24]. In vitro studies showed that the downstream signaling pathway of EC binding was formed by activating extracellular regulated kinase (ERK)1/2 and phosphorylating VEGFR2 to promote expression of αvβ3 [8]. With its strong binding affinity to EPCs, vascular grafts modified by LXW7 can quickly produce a structurally stable endothelial interface between the graft surface and the circulation, which significantly improves the adhesion, proliferation, and differentiation of endothelial cells, and inhibits platelets production. In addition, LXW7 contains unnatural amino acids, which makes it more resistant to protein hydrolysis and more stable in vivo, allowing LXW7 to maintain its function over the long term during application [23]. In a rat carotid artery bypass model, LXW7-modified small-diameter vascular grafts significantly promoted EPC/EC recruitment, resulting in rapid in situ endothelialization and long-term high patency.

A rabbit atherosclerosis model experiment showed that PJ34 could prevent the oxidative damage to rabbit EPCs in vitro [25]. Related research used H₂O₂ to establish a stress-induced premature aging model. PJ34 treatment of EPCs was found to have an important role in improving stress-induced premature aging of EPCs and preventing endothelial dysfunction by inhibiting poly(ADP-ribose) polymerase 1 (PARP1) activation of PAR and thereby maintaining intracellular NAD+ levels and increasing sirtuin 1 (SIRT1) activity [26]. However, in the case of pathological DNA damage, overuse of PJ34 will cause excessive consumption of NAD+ and damage EPCs; therefore, the most suitable concentration of PJ34 remains to be confirmed. Zha et al. [26] used H₂O₂ to establish a stress-induced premature aging model to determine whether PJ34 can inhibit EPC aging. Cells treated with different concentrations of PJ34 were used to detect the effect of PJ34 on young EPCs, and H₂O₂ was used to induce premature aging of cells. Cells treated with H₂O₂ + PJ34 were compared with cells treated with H₂O₂ to verify the effect of PJ34 on senescent EPCs. Untreated cells were used as negative controls. Finally, SIRT1 short hairpin RNA (AD-SH-SIRT1) was used to silence SIRT1 expression in EPCs, and the effect of PJ34 on SIRT1 expression was detected again, and the activity of PARP1 was measured and evaluated by analyzing the production of PAR. The results showed that PJ34 might inhibit PARP1, maintain intracellular NAD+ level, enhance SIRT1 activity without increasing SIRT1 expression, and thus improve the function of senescence EPCs [26].

20-Hydroxyeicosapentaenoic acid (20-HETE) is an important component of arachidonic acid ω-carbon hydroxylation products. It widely exists in the brain. It is the main eicosenoic acid in the kidney and cerebral microcirculation which is usually used as a vasoconstrictor and plays an important role in regulating myogenic tension. 20-HETE acts upstream of the hypoxia-inducible factor 1 alpha/VEGF pathway induced by hypoxia to promote ischemia-induced angiogenesis. In a mouse hindlimb ischemic angiogenesis model, after the injection of the 20-HETE synthesis inhibitor dibromo-dodecenyl-methylsulfimide or a 20-HETE antagonist, the blood flow recovery in the ischemic response of the mice was reduced significantly [27]. Besides, the 20-HETE acts as a “double blade” in stroke. Inhibition of 20-HETE can reduce arterial myogenic tension and increase CBF, which has a good effect on ischemic stroke. Ischemia-induced compensatory neovascularization can be regulated by the combined effect of 20-HETE promoting EPC and local preexisting EC response [28].

Salidroside (SAL) is a polyphenol compound isolated from Rhodiola rosea. With its established chemical structure and metabolism, SAL is used to treat atherosclerotic coronary heart disease through anti-inflammatory and antioxidant mechanisms, thus affecting CIS [29]. Exposing cultured human coronary artery ECs to oxidized low-density lipoprotein resulted in decreased BCL2-like 1 (Bcl-xL) levels and an increased rate of apoptosis. A series of experimental methods, including 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, confirmed that SAL reduces the transcription level of BCL2L1 (encoding Bcl-xL) and inhibits oxidized low-density lipoprotein production by downregulating microRNAs (miRNA)-133a, which protected the endothelium from apoptosis. However, the mechanism by which SAL regulates miR-133a deserves further study [30]. SAL can protect BM-EPCs and EPCs from angiogenesis by activating the protein kinase B (Akt)/mechanistic target of rapamycin kinase (mTOR)/ribosomal protein S6 kinase B1 (P70S6K) and mitogen-activated protein kinase (MAPK) signaling pathways. SAL stimulates the phosphorylation of Akt and ERK1/2, the mammalian targets of mTOR, and P70S6K and promotes cell migration and angiogenesis. In addition, SAL reversed H₂O₂-induced JUN N-terminal kinase and p38 MAPK phosphorylation, and H₂O₂ stimulated a low expression ratio of BCL2-associated X, apoptosis regulator (Bax)/Bcl-xL to restore the function of EPCs [31]. Tang et al. [31] evaluated VEGF secretion and NO production in BM-EPCs supernatant treated with or without SAL treatment. After 24 and 48-h incubation, 40 and 80 μM SAL promoted the secretion of VEGF by 11.3% (24 h, 40 μM), 17.5% (24 h, 80 μM), 18.9% (48 h, 40 μM), and 21.0% (48 h, 80 μM), respectively. In addition, NO production of EPCs increased significantly after SAL stimulation of cells 2, 5, 8, and 10 days. These results suggest that the enhanced ability of SAL to induce EPCs tube formation is mediated by increased VEGF and NO production [31].

Refined Qingkailing (RQKL) is made by selecting baicalin, geniposide, and cholic acid of bezoar, hyodeoxycholic acid (bezoar) from the Chinese patent medicine Qingkailing in a ratio of 4.4:0.4:3:2.6 and is used to prevent CIS [32]. Experiments have shown that baicalin isolated from Huang-Lian-Jie-Du-Tang activates signaling pathways, including MAPK, phosphoinositide 3-kinase (PI3K), HIF-1, nuclear factor kappa B, and forkhead box pathways, to resist CIS [33]. In a transient MCAO rat model, after merging the protein-protein interaction networks of CIS and RQKL-related targets [33], it was found that enhancing the activity of Akt could promote the anti-apoptosis of EPCs. Thus, RQKL activates the PI3K/Akt signaling pathway and inhibits the apoptosis process of EPCs [32]. Ma et al. [32] conducted a neurological deficit score to determine whether RQKL treatment improved neurological function after ischemic stroke. The results showed that there were obvious neurological deficits in the ischemic group. In addition, 15 mg/kg RQKL significantly decreased the neurological deficit score, while 30 and 60 mg/kg RQKL significantly decreased the neurological deficit score, and the difference was not statistically significant (p > 0.05). The infarct volume ratio was assessed by TTC staining, with normal tissue stained red and infarcts unstained (white). Twenty-four hours after transient MCAO, the infarct rate of 30 and 60 mg/kg groups was significantly lower than that of the ischemic group. No neuroprotective effect was observed in the lowest dose group [32].

Resveratrol is a nonflavonoid organic compound that has a protective effect on EPCs [34]. Studies have shown that resveratrol-mediated upregulation of endothelial NO synthase can increase the production of NO in EPCs, inhibit the synthesis of endothelin-1, and reduce the oxidative stress of EPCs and the activation of nuclear factor kappa B to prevent the upregulation of vascular cell adhesion molecule 1 and intercellular adhesion molecule 1 induced by tumor necrosis factor alpha [35]. Targeting of VEGF2 by miR-542-3p can induce EPC production [36]. Through the peroxisome proliferator-activated receptor gamma/heme oxygenase 1 pathway, resveratrol can prevent EPCs from aging and reduces oxidation [37]. Resveratrol also targets focal adhesion kinase, improves EPC function through miR-138, and promotes thrombolysis in vivo [38]. Resveratrol-induced upregulation of Kruppel-like factor 2 can protect advanced EPCs from tumor necrosis factor alpha-induced inflammatory injury [39]. In order to study whether the PI3K/Akt/mTOR pathway is involved in the neuroprotective effect of resveratrol, Hou et al. [40] used Western blots to carefully detect the protein expression of the PI3K/Akt/mTOR pathway. The results showed that after 24 h brain I/R, the protein expressions of P-Akt (Ser473) and P-MTOR (Ser2448) in resveratrol group were 1.66 times higher than those in the VEH group (*p < 0.05; Res vs. Veh), and the protein expressions of P-Akt (Ser473) and P-MTOR (Ser2448) in the resveratrol group were 1.66 times higher than those in the Veh group (*p < 0.05). In addition, PI3K inhibitor LY294002 significantly inhibited resveratrol-induced protein expression of P-Akt (Ser473) and P-MTOR (Ser2448) by 0.52% and 0.63%, respectively (*p < 0.05; Res LY294002 vs. Res). The total protein expression of Akt and mTOR was not significantly changed. These observations support the hypothesis that resveratrol may play a neuroprotective role by activating the PI3K/Akt/mTOR pathway [40].

miRNAs play an important role in the regulation of cell migration, proliferation, apoptosis, and angiogenesis [41]. These small noncoding RNAs promote or inhibit angiogenesis by targeting positive or negative regulators of angiogenesis, which has an important impact on vascular diseases. Targeting miRNAs or their targets represent a new treatment strategy for the prediction, diagnosis, and prognosis of CIS [42].

APN is an endogenous protein synthesized and secreted by adipose tissue, which can act on the blood-brain barrier and reduce the release of inflammatory factors in EPCs, thereby reducing blood vessel damage and enhancing anti-atherosclerosis [43]. EPCs transfected with the APN gene were transplanted into a mouse MACO model, and the infarct size, microvessel density, and vascular repair were improved to protect the mice from CIS [44, 45]. APN can promote the migratory activity of EPCs by activating the PI3K/cell division cycle 42 (Cdc42)/Rac family small GTPase 1 (Rac1 axis). Besides, APN increases X-linked inhibitor of apoptosis protein (XIAP), which increases the resistance of EPCs to apoptosis or inhibits the p38 MAP kinase/cyclin-dependent kinase inhibitor 2A (P16INK4A) signaling pathway. Both of these APN-related effects prevent EPCs from aging and undergoing apoptosis [46]. Western blotting data indicated that APN binding to the receptor (APN 1 and APN 2) activates the protein kinase AMP-activated catalytic subunit alpha 1 (AMPK)/Akt/endothelial NO synthase signaling pathway through phosphorylation, which can induce NO synthesis in ECs, thus enhancing the ability of EPCs to reproduce, migrate, and form blood vessels, and exerting anti-atherosclerotic effects [47]. Therefore, APN-transduced EPC transplantation is a potential therapeutic method for vascular diseases.

Silencing of NRF2 (encoding nuclear factor, erythroid 2 like 2) can enhance the degree of apoptosis of EPCs and induce the production of ROS, which impairs the function of EPCs. However, sestrin 2 increased the upregulation of the Nrf2 protein level by enhancing p62-dependent autophagy and weakening the apoptosis-promoting effect of angiotensin II on EPCs [48].

Apelin-13 promotes the differentiation of advanced EPCs by regulating Kruppel-like factor 4 (KLF4). In the control culture experiment of EPCs in the KLF4 upregulated group and apelin-13 upregulated group, it was found that the VEGFR2 promoted the migration ability of EPCs [49]. That study provided valuable information to explore the mechanism of EPC differentiation and could help to develop more effective treatments in the future. Apelin is involved in the proliferation of EPCs. Studies have shown that the binding of VEGFR2 to its ligand VEGF-A and hypoxic conditions can activate apelin signaling via hypoxia-inducible factor 1 alpha in EPCs. This is followed by activation of PI3K/Akt signaling to upregulate the production of EPCs and promote their proliferation [50]. In contrast, apelin signaling is downregulated by miRNAs and small interfering RNAs to inhibit the proliferation of EPCs. As the downstream effector of apelin, MAPK signaling can regulate the proliferation of EPCs. Inhibiting the ERK-MAPK signaling pathway downregulated the proliferation of EPCs transfected with an apelin-expressing plasmid. Pretreatment of EPCs with two inhibitors of MAPK, sb-239063 or PD98059, eliminated the proliferation of EPCs induced by upregulation of apelin signaling [51]. In addition, calcitonin gene-related peptides can promote the proliferation and inhibit the apoptosis of EPCs by inhibiting MAPK-related signals [52]. Therefore, the effects of apelin signaling and downstream MAPK signaling on the proliferation of EPCs suggest that apelin and MAPK can be used as therapeutic targets to prevent hypoxic/ischemic brain injury.

The effects of small-molecule compounds and biological macromolecules on EPCs have brought new hope to the diagnosis and treatment of CIS (see in Fig. 1). At present, the study by Steinle et al. [53] found that EPCs transfected with mRNA significantly increased the production of VEGFA, SDF1A, and ANG1, among which ANG-1 (112 ± 6.1 ng/mL), VEGF-A (142 ± 6.7 ng/mL), and stromal cell-derived factor 1 (SDF-1)α (17 ± 1.4 ng/mL), and all EPCs transfected with a single mRNA or mRNA mixture showed comparable migratory activity. Combination therapy comprising recombinant human granulocyte colony-stimulating factor and SDF-1 may also be the best therapeutic strategy to prevent stroke-induced damage. Furthermore, EPC-derived extracellular vesicles play important roles in tissue repair and regeneration [54]. On the other hand, Zeng et al. [55] performed in vitro experiments in which BMECs were isolated from cortical tissues and treated with microvesicles from rat spleen EPCs, showing increased BMEC proliferation, migration, and tube formation, suggesting that EPC-derived microvesicles are attractive potential application in the treatment of brain injury. In addition, EPCs expressing synthetic recombinant genes, such as VEGFA, SDF1A, and ANG1, can significantly improve their chemotactic activity. Combination therapy comprising recombinant human granulocyte colony-stimulating factor and SDF-1 might also be the best treatment strategy to prevent stroke-induced damage. In addition, EPC-derived extracellular vesicles play an important role in tissue repair and regeneration. These observations provide new ideas and applications for EPCs. Therefore, the therapeutic effects of various EPCs and the molecular mechanism by which they promote angiogenesis can be further clarified for clinical application. These observations provide new ideas and applications for EPCs. Therefore, the therapeutic effects of various EPCs and the molecular mechanism by which they promote angiogenesis can be further clarified for clinical application. As basic drugs to treat CIS, small-molecule compounds often involve multiple pathological mechanisms, including inflammation and oxidative stress. They can only act on a single target, and some of them have limitations regarding liver and kidney toxicity; therefore, intensive research is needed to promote the reduction of their toxicity and side effects for their better use. The specific molecular mechanisms of the proprietary Chinese medicines that have been studied in vitro are not clear. In addition, their low solubility, unstable chemical properties, and short biological half-life limit their clinical application; however, they can be used in combination with other drugs. Although EPCs provide a novel potential therapy for the treatment of vascular diseases including ischemic stroke, so far, their clinical treatment still lacks effective treatments and drugs, and the existing conventional treatment methods cannot quickly and effectively restore local blood supply, and nerve damage cannot be avoided. At the same time, the regeneration ability of EPCs is also affected by a series of exogenous factors and drugs, as well as vascular risk factors. Therefore, it is of great clinical significance to explore the potential application of new drug therapies for EPC mobilization, proliferation, and differentiation. We expect that the future exploration of more effective cytokines, signal pathways, and other drugs, and the determination of their specific mechanisms will clarify the biological functions EPCs and achieve their efficient amplification, which would improve their therapeutic effect on CIS.

The authors have no conflicts of interest to declare.

This study was financially supported by the Zhejiang provincial basic public welfare research program (LGF21H200007) and the Major horizontal projects of Zhejiang Shuren University (202111842040).

Lu Lu: formal analysis, methodology, and writing – original draft; Jia-Jie Zhu: data curation, methodology, and writing – original draft; Hai-Yan Zhang: data curation and methodology; Xiao-Ping Li: conceptualization and writing – review and editing; Ke-Da Chen: conceptualization and writing – review and editing.

Lu Lu, Jiajie Zhu, and Haiyan Zhang contributed equally to this work.