Hypoxic Preconditioning of Human Umbilical Cord Mesenchymal Stem Cells Is an Effective Strategy for Treating Acute Lung Injury

Abstract

Acute respiratory distress syndrome (ARDS)/acute lung injury (ALI) is a severe clinical respiratory failure disorder associated with chronic pathology and disability and has a mortality rate of 40%–60%. However, the pathogenesis of ARDS/ALI remains unclear, and existing therapeutic options are insufficient for addressing the severity of the disease. Mesenchymal stem cells (MSCs) play an important role in the prevention and treatment of ALI, especially acute alveolar epithelial injury. However, the low survival rate of transplanted MSCs reduces their effectiveness. When human umbilical cord MSCs (hUC-MSCs) are transplanted directly, only a minority of cells migrate toward damaged tissues. Moreover, their maintenance time is short, leading to unsatisfactory therapeutic results. A moderate hypoxic environment can promote the proliferation of MSCs, inhibit apoptosis, and facilitate migration and chemotaxis. In summary, hypoxic culturing before transplantation improves the effectiveness of hUC-MSCs in treating ARDS/ALI and promises to provide novel diagnostic and therapeutic targets.

Introduction

Acute lung injury (ALI), and its most severe manifestation, acute respiratory distress syndrome (ARDS), are severe life-threatening medical conditions associated with refractory hypoxemia. ALI/ARDS is characterized by a widespread inflammatory response accompanied by the release of a huge pool of cytokines and chemokines, and damage to the endothelial and epithelial barriers of the lungs [1]. Risk factors for ARDS/ALI include serious infection, traumatic injury to the respiratory tract, shock, and toxicity [2]. Moreover, ALI/ARDS is associated with long-term illness and disability and has a high mortality rate of 40%-60% [3]. However, the pathogenesis of ARDS/ALI remains unclear. Given the unacceptably high mortality rate, additional treatment options are urgently needed [4]. A better understanding of ARDS/ALI may help to yield more effective treatment strategies.

Recent work has evaluated the effectiveness of cell therapy to reduce the ARDS/ALI pathogenesis. In particular, mesenchymal stem cells (MSCs) are thought to be a promising candidate for cell transplantation. MSCs are multipotent cells capable of self-renewal and can differentiate into multiple cell types, including chondrocytes, osteocytes, and adipocytes [5]. Bone marrow MSCs (BMSCs) and human umbilical cord MSCs (hUC-MSCs) are capable of differentiating into pulmonary epithelial cells, which exhibit characteristics specific to lung epithelial cells [6]. Compared to BMSCs, hUC-MSCs may represent a more practical source for MSCs due to their accessibility, pain-free procurement, and lack of ethical concerns [7].

Potential Mechanisms of Action of UC-MSCs in the Treatment of ALI

In ALI, alveolar epithelial cells (AECs) are often damaged and may even undergo apoptosis due to factors such as excessive inflammation, pathogenic bacteria, and the destruction of interepithelial tight junctions. Many studies have confirmed that hUC-MSCs can contribute to the prevention and treatment of ALI through both their multidirectional differentiation potential and paracrine functions. Through their potent immunomodulatory properties and cell proliferation ability, they can repair AECs and effectively control ALI.

Homing and differentiation of UC-MSCs

hUC-MSCs can migrate to the site of the injury. Indeed, in a mouse model of bleomycin or lipopolysaccharide (LPS)-induced lung injury, hUC-MSCs injected into the tail vein accumulated in the inflammatory and fibrotic lung tissues. Contrarily, hUC-MSCs did not aggregate in the lung tissue of normal mice [8]. Similarly, in a bleomycin-induced model of lung injury, 14 days after bleomycin injection (but not at 28 days), hUC-MSCs were detected only in fibrotic areas. This indicates that hUC-MSCs were attracted and retained within damaged lung tissues, a behavior which can be categorized as homing [9].

A distinctive feature of MSCs is their capacity to differentiate in vitro. One study found that MSCs can not only home to the lungs but can also differentiate into alveolar epithelial and pulmonary vascular endothelial cells [10]. Under different induction conditions, hUC-MSCs can undergo osteogenesis, chondrogenesis, or adipogenesis [11]. In addition, hUC-MSCs can express markers indicative of wound repair, typically expressed by wound repairing endothelial cells, fibroblasts, keratinocytes, peripheral vascular wall cells, and sweat gland cells [12].

Paracrine mechanisms of hUC-MSCs

MSCs can secrete various growth factors, including vascular endothelial growth factor (VEGF), insulin-like growth factor 1, prostaglandin E2, bone morphogenetic protein-2, fibroblast growth factor (FGF), hepatocyte growth factor (HGF), transforming growth factor-β1 (TGF-β1), and vascular secretion adhesion molecules. These factors play a role in repairing and protecting the AECs [13 –15].

The development and progression of a disease is associated with different cytokines. At the exudation stage, MSCs secrete anti-inflammatory proteins and antiapoptotic factors and thus elicit a strong anti-inflammatory and antiapoptotic response [16]. During the proliferation period in ALI, MSCs secrete paracrine antibacterial peptides and immune-regulatory factors, including VEGF, FGF, and platelet-derived cytokine growth factor, which can promote wound angiogenesis, proliferation, differentiation, and migration, and have antimicrobial activity. During the fibrotic phase of ALI, hUC-MSCs could effectively attenuate inflammation and fibrosis in bleomycin-induced pulmonary fibrotic mice through HGF secretion [17].

MUC5B is the main mucin in the airway and plays a role in protecting the respiratory tract. Indeed, the autoimmune function of MUC5B-deficient mice is impaired. However, an increase in MUC5B causes airway damage. In paraquat poisoning, chronic bronchitis caused by smoking, cystic fibrosis, and primary ciliary dyskinesia, MUC5B expression is significantly increased [18]. The expression of MUC5B can be modulated by various stimuli, including neuronal activity, and inflammatory mediators, and is related to interleukin (IL)-1β, IL-6, IL-9, IL-13, IL-17, and tumour necrosis factor-α (TNF-α).

Antifibrotic mechanism of hUC-MSCs

Pulmonary fibrosis is an indicator of poor prognosis of ALI. Importantly, hUC-MSCs could reduce inflammation and fibrosis in bleomycin-treated rats via a paracrine mechanism, and could also effectively restore normal lung structure [17]. Weiss et al. found that hUC-MSCs inhibited the expression of TGF-β, IL-1β, and TNF-α and reduced the concentration of collagen in the lung, thereby preventing fibrosis and supporting repair [19]. In addition, hUC-MSCs themselves have an antifibrotic effect. In rats with bleomycin-induced pulmonary fibrosis, hUC-MSCs were able to reduce the levels of MDA, GSSG, TNF-α, IFN-γ, TGF-β, IL-1, IL-2, IL-6, type I collagen mRNA, MMPs, TIMPs, and hydroxyproline, while increasing the expression of SOD, GSH, ACE2, and IL-10 [9].

Angiotensin-converting enzyme 2 (ACE2) is a homolog of human ACE and is mainly involved in the degradation of angiotensin II (AngII). ACE2 can prevent lung injury caused by inhaled acid, endotoxin shock, and sepsis. In contrast, AngII has been shown to exacerbate the pathology in models of bleomycin-induced pulmonary fibrosis. Loss of ACE2 caused severe lung damage in mice, while reinjecting it ameliorated lung damage. Thus, ACE2 can be used as a negative regulator to protect lung tissue in cases of ALI. Importantly, ACE2 inhibits the development of pulmonary fibrosis by reducing collagen deposition.

In a study of bleomycin-induced pulmonary fibrosis, ACE2 was transfected into hUC-MSCs using a lentiviral vector to construct recombinant ACE2-hUC-MSCs. Combining ACE2 with hUC-MSCs had a synergistic therapeutic effect on lung injury, eliciting a greater treatment response than the application of ACE2 or hUC-MSCs individually [20].

However, this genetic recombination method may be unsuitable for treating the coronavirus disease (COVID-19). The spike protein of the novel severe acute respiratory syndrome coronavirus-2 (nSARS-CoV-2) can specifically recognize the ACE2 receptor on the cell surface. Accordingly, the cells that express ACE2 may be attacked by nSARS-CoV-2. In a clinical trial on the treatment of COVID-19 from Beijing Youan Hospital, seven patients were injected with MSCs, and MSCs could significantly ameliorate patients' symptoms, while exhibiting no side effects. A gene expression analysis showed that the MSCs did not express ACE2, suggesting that MSCs may not be infected by nSARS-CoV-2 and should be safe and effective for patients [20].

Antiapoptotic mechanism of hUC-MSCs

LPS activates the Ras/MEK/ERK signaling pathway, promotes the phosphorylation of MEK and ERK, and results in a massive release of cytochrome C. After the formation of an apoptotic body, LPS activates caspase 3, a burst cascade reaction, and eventually leads to the apoptosis of type II AEC (AEC II) [21]. Studies have found that hUC-MSCs can reduce the LPS-induced apoptosis of AEC II by inhibiting the MEK/ERK pathway. MSCs can also secrete various growth factors, which play an important role in regulating prosurvival signaling pathways, such as the NF-κB and PI3K/AKT pathways, thereby reducing apoptosis [22]. hUC-MSCs can not only significantly reduce excessive inflammatory responses in the early stages of pathology but can also be effective in treating severe burn-induced lung damage and preventing apoptosis.

Other functions of hUC-MSCs

hUC-MSCs can directly participate in the structural repair of lung tissues by differentiating into replacement tissues. With immunomodulatory repair, hUC-MSCs regulate the permeability of epithelial cells through paracrine pathways, and thereby increase the clearance rate of the alveolar fluid. hUC-MSCs can treat and repair acute alveolar epithelial damage through potential differentiation, as well as through the secretion of cytokines and miRNA-containing exosomes and vesicles, which modulate the PI3K/AKT, Wnt, and NF-κB signaling pathways [23].

Stem cells have mechanisms to resist infection by pathogenic microorganisms. ALI mice experience severe infection due to sepsis. Thus, the use of MSCs can significantly reduce the mortality rate by increasing the clearance rate of bacteria [24]. These results prove that MSCs have the capacity to help kill pathogenic microorganisms. Moreover, treatment with hUC-MSCs can significantly reduce ALI resulting from H5N1 viral infection and thereby prolong survival [25]. Studies have shown that antagonizing the secretion of LL-37 by MSCs significantly reduces the antibacterial ability of MSCs. MSCs can also eliminate pathogens by increasing the phagocytosis of macrophages, and thus indirectly support the antibacterial activity [26].

MSCs can also inhibit the expression of the purinergic receptor P2X ligand-gated ion channel 7 via the exportation of miR-124-3p through exosomes. This serves to reduce the oxidative stress associated with injury and decrease the inflammatory response in cases of traumatic ALI [27]. MSCs can reduce the end-product of oxidative stress and therefore enhance the antioxidative stress activity [28,29].

MSCs exhibit a powerful two-way immune regulation mechanism, which can benefit the treatment of ALI. They can antagonize excessive immune responses and counteract cytokine storm syndrome by secreting anti-inflammatory factors. In addition, they can home damage tissues through chemotaxis, activate regulatory immune cells, and improve the targeting of the immune response.

Rather than the innate immune system, MSCs work by regulating the acquired immune system. They exhibit numerous regulatory functions, including restriction of excessive proliferation of T cells, inducing activation of CD4⁺CD25⁺FoxP³⁺ regulatory T cell, inhibiting undue proliferation, differentiation, and immunoglobulin production by B cells, inhibiting dendritic cell maturation, and promoting the polarization of macrophages to an anti-inflammatory phenotype [30]. Studies have shown that intravenous injection of hUC-MSCs can promote immune reconstruction in immunodeficiency nude mice and increase the concentration of T cells in the peripheral blood [31].

Preclinical and preliminary clinical data on hUC-MSCs for the treatment of COVID-19 indicate that hUC-MSCs enhance tissue repair through anti-inflammatory functions and immune modulation, as well as through direct antiviral activity [32]. Therefore, we strongly believe that hUC-MSCs have not exerted a single mechanism, but through the constellation of factors in the development and progression of ALI. The possible mechanisms of hUC-MSCs in the treatment of ALI are shown in Fig. 1.

FIG. 1.

Possible mechanisms of hUC-MSCs in the treatment of ALI. (A) hUC-MSCs can home to damaged lung tissues; (B) hUC-MSCs can differentiate into alveolar epithelial cells and pulmonary vascular endothelial cells; (C) hUC-MSCs can secrete various growth factors such as VEGF, IGF-1, and PGE2; (D) hUC-MSCs can inhibit the expression of TGF-β, IL-1β, and TNF-α, thereby having antifibrosis effect; (E) hUC-MSCs can improve apoptosis partly by inhibiting the MEK/ERK pathway; (F) hUC-MSCs not only can directly participate in the structural repair of lung tissues but also regulate the permeability of epithelial cells through paracrine in immunomodulatory repair; (G) hUC-MSCs have the ability to kill pathogenic microorganisms directly, which can also eliminate pathogens by increasing the phagocytosis of macrophages, thus indirectly show their antibacterial ability; (H) hUC-MSCs can regulate acquired immune system in ALI, including inhibiting excessive proliferation of T cells; (I) hUC-MSCs can be used as antioxidant in traumatic ALI. ALI, acute lung injury; hUC-MSCs, human umbilical cord mesenchymal stem cells; IGF-1, insulin-like growth factor 1; IL-1β, interleukin-1β; PGE2, prostaglandin E2; TGF-β, transforming growth factor-β; TNF-α, tumour necrosis factor-α; VEGF, vascular endothelial growth factor. Color images are available online.

Drawback and Solutions for Using Umbilical Cord Stem Cells for the Treatment of ALI

The beneficial effects of hUC-MSC transplantation open new avenues for the use of cell therapy in treating ALI and other inflammatory lung diseases. At the same time, many unanswered questions remain. The use of MSCs is restricted by their lower incorporation rate within the vivo environment. Most of the graft cells are lost within 1 month due to progressive pathological changes in the microenvironment or local inflammation of the graft site. The use of MSCs in ALI is limited by the substantial apoptosis of transplanted cells. Moreover, oxidative stress at the graft site also promotes MSC death. With the widespread use of MSCs in the clinical setting, a number of studies have reported that injecting MSCs directly affects their homing and retention in damaged tissue, making the treatment less effective and resulting in poor engrafting, which has become a bottleneck in the continuous development of MSCs.

Besides, hUC-MSCs do not express MHC II antigen. The expression rate of MHC I antigen was lower than BM-MSCs. hUC-MSCs do not express the costimulatory surface antigens CD40, CD80, and CD86, thereby developing immune tolerance [33]. In addition, hUC-MSCs express IL-6 and VEGF, which play a role in MSC immune regulation, in other words, immunosuppressive properties. These data, taken together with observations xenotransplantation of hUC-MSCs, suggest that they would be tolerated in allogenic transplantation [19].

Currently, it is paramount to improve the survival rate of transplanted cells. Current strategies include genetic engineering, tissue engineering, using exosomes derived from hUC-MSCs, and pretreating the hUC-MSCs with biological factors (eg, growth factors, drugs, and hypoxia). In a bleomycin-induced lung fibrosis injury model, hUC-MSCs were modified to express ACE2; it was found that ACE2 synergistically improved the therapeutic efficacy of hUC-MSCs [34]. Moreover, hUC-MSCs overexpressing SOD2 were more effective than normal hUC-MSCs in reducing paraquat-induced lung injury in rats [35]. Pretreating hUC-MSCs by a low level of TGF-β1 increased the expression of various extracellular matrix components, such as fibronectin, which thereby improved survival rate and the possible therapeutic effects of hUC-MSCs [36]. Hypoxic preconditioning is considered to be an intense cell stimulation and an effective strategy to enhance the viability of stem cells after transplantation into the injured area.

The Importance of Senescence and Physiological (Hypoxic) Culture Conditions in MSCs

Stem cell senescence has been suggested to be a key driver of the aging process, and, in particular, MSC senescence a contributor to aging-related disease. In the process of in vitro culture, MSCs inevitably undergo replicative senescence or premature senescence due to the oxidative stress of cells under the influence of various external factors, which results in the dysfunction of MSCs themselves and seriously restricts their application in tissue engineering medicine and clinical practice. Senescent cells show significant structural changes, including, but not limited to, larger size, irregular shape, becoming multinucleate, and more prominent stress fibers associated with metabolic changes, DNA damage, and autophagic failure [37]. At the same time, the expression of CD44, CD90, CD105, and Stro-1, the surface-specific markers of MSCs, decreased [38]. At present, more and more scholars have tried to retard the aging process of MSCs by adding bioactive substances or drugs and other methods, but the validity has yet to be verified.

Traditional cell culture is carried out at an oxygen concentration of 21%; however, in vivo, stem cells are usually not exposed to such high oxygen levels. Due to limitations associated with blood supply and metabolic activity in local tissues, oxygen concentration in tissues ranges from 3% to 5% [39]. The culture in hypoxic conditions (1%–3% O₂) may also be beneficial for the MSC, as this oxygen tension is more similar to the physiologic niche for MSC in the bone marrow (2%–7% O₂) [40]. This suggests that culturing cells with relatively lower oxygen concentrations in vitro will more closely replicate their natural physiological environment.

Hypoxic treatment before MSC transplantation has shown positive results in many studies. In vitro, an appropriate low oxygen concentration can reduce oxygen free radicals in cells and improve their metabolic efficiency. It is therefore believed that proper hypoxia treatment before MSC transplantation can enhance their viability and tissue regeneration potential [41]. A moderate hypoxic environment can promote the proliferation of MSCs, reduce their apoptosis [41], and facilitate migration and chemotaxis. MSCs cultured under either normoxia or hypoxemia undergo accelerated apoptosis over the first 12 h. On the contrary, hypoxic preconditioning increases cell survival. Specifically, hypoxia can downregulate proapoptotic genes and upregulate antiapoptotic genes, thus improving the antiapoptotic ability of hUC-MSCs.

Studies have shown that hypoxia not only increases the survival rate of transplanted cells but also enhances their ability to secrete cytokines and engage in paracrine/autocrine signaling. In vivo, Hu et al. found that transplanting hypoxic preconditioned BMSCs into a rat model of myocardial infarction enhanced the paracrine impact of MSCs, reduced the apoptosis of transplanted cells, and strengthened tissue repair [42].

In addition, under physiological conditions of oxygen concentration, hUC-MSCs upregulate miR-145, which modulates their conversion to AEC II [43]. Interestingly, more exosomes are extracted from MSCs preconditioned with hypoxia. In addition to the quantitative impact on exosome secretion, the content of exosomes released by cells under hypoxic condition also undergoes significant changes [44]. Hypoxia-preconditioned MSCs have superior effect in ameliorating renal function on acute renal failure animal model [45]. One study revealed that the behavior of hUC-MSCs is donor-dependent, meaning that sensitivity to the effects of hypoxia differs between cells originating from different donors, which ultimately affects their capacity to contribute to angiogenesis [46]. Specific information on the culture conditions of MSCs for cellular therapy is shown in Table 1.

Table 1.

Specific Information on the Culture Conditions of Mesenchymal Stem Cells for Cellular Therapy

Model	Materials	Treatment	Hypoxia preconditioning	Results	Ref.
Hind limb ischemia injury model	NOD/SCID/β-2-microglobulin-deficient mice or NOD/SCID mice	BMSC (5 × 10⁵ cells per mouse), injected into the left ventricle	1%–3% oxygen	Mice that had received hypoxic preconditioned MSC recovered faster than that had received normoxic MSC	[39]
Myocardial infarction	Wild-type C57B6 mice	BMSC (1 × 10⁶ cells, 100 μL), intraventricular injection	Cells were exposed to anaerobic medium (0% oxygen)	Preconditioning of BMSCs enhanced survival and ability to attenuate LV remodeling	[40]
Myocardial ischemia	Male Wistar rats	BMSC (1 × 10⁶ cells, 150 μL), directly injected into the ischemic peri-infarct region	0.5% oxygen for 24 h	Improves infarcted heart function	[41]
Gentamicin-induced ARF	Male Wistar rats	hUC-MSCs (1 × 10⁶ cell), intraperitoneally	5% oxygen for 24 h	HP-MSCs have a superior beneficial effect in improving renal function	[44]
Limb ischemia	Male BALB/c nude mice	hUCB-MSCs (1 × 10⁶ cells), injected into the quadriceps muscle	1% oxygen for 24 h or 2 weeks	Sensitivity to hypoxic conditions is different between cells originating from different donors	[45]

ARF, acute renal failure; BMSC, bone marrow mesenchymal stem cell; HP, hypoxia preconditioning; hUC-MSCs, human umbilical cord mesenchymal stem cells; hUCB-MSCs, human umbilical cord blood mesenchymal stem cells; LV, left ventricular.

The Role of Hypoxia-Inducing Factor-1α and Related Pathway Proteins in Hypoxic Preconditioning of UC-MSCs in the Treatment of ALI

To understand the beneficial proliferative and differentiation inducing effects of hypoxia, many studies have focused on the activity of the hypoxia sensor hypoxia-inducing factor-1α (HIF-1α). In particular, HIF-1α, the more significant effector for cells, can promote cell proliferation and enhance viability. Thus, many studies have focused on the activation of HIF-1α and its related signaling pathways as a key mediator of hypoxia-based biological characteristics of MSCs.

Previous research has demonstrated that the antiapoptotic mechanisms associated with HIF-1α are mediated by the combination of downstream hypoxia response elements, which increase the BNIP3 transcriptional activity, downregulate the expression of Bax, and increase the ratio of Bcl-2/Bax, thereby promoting cell survival. In recent years, some studies have found that HIF-1α may be involved in the regulation of the PI3K-Akt pathway, however, the specific mechanism remains unclear. Some studies have shown that Akt, as a kinase, can act on HIF-1α to change the activity of its downstream molecules. However, other studies have shown that high expression of HIF-1α in turn promotes the activation of Akt [47]. Despite the above inconsistencies, Akt and HIF-1α are undoubtedly the key molecules that regulate apoptosis. It remains of great significance to clarify the specific roles of hypoxic preconditioning for facilitating the survival of hUC-MSCs.

Conclusion

The pathogenesis of ALI remains unclear. Nevertheless, this study focused on the role of hUC-MSCs in treating ALI. hUC-MSCs represent a promising therapeutic option to test in the clinic for the treatment of ALI. We aim to conduct a series of experiments to interrogate the mechanisms by which hUC-MSCs ameliorate ALI at the molecular, cellular, tissue, and animal levels. We expect to further explore the possible targets of hUC-MSCs on the alveolar epithelium. Understanding the molecular mechanism of hUC-MSCs in the treatment of ALI will provide a theoretical basis for understanding pathogenesis and inform new directions for designing future targeted treatment.

Footnotes

Acknowledgment

Thanks also for the editorial work of Editage Ltd.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

This study was funded by Department of Science and Technology of Jilin Province (Grant No. 20190201279JC); and The Provincial Special Fund for Industrial Innovation from Jilin Province (Grant No. 2017C058-3); and the Department of Finance of Jilin Province (Grant No. 2020SC2T005).

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