Hypoxia modulates paracrine signaling in mesenchymal stem cells

JIANG Lin，Sarah Beck，CAI Wen-feng，GAO Xiang，WANG Yi-gang

(Departments of Pathology and Laboratory Medicine，College of Medicine，University of Cincinnati Medical Center，231 Albert Sabin Way，Cincinnati，Ohio 45267-0529，USA.)

·高原医学国际论坛·

Hypoxiamodulatesparacrinesignalinginmesenchymalstemcells

JIANG Lin，Sarah Beck，CAI Wen-feng，GAO Xiang，WANG Yi-gang*

(Departments of Pathology and Laboratory Medicine，College of Medicine，University of Cincinnati Medical Center，231 Albert Sabin Way，Cincinnati，Ohio 45267-0529，USA.)

ObjectiveMesenchymal stem cells(MSCs)are adult multipotent cells of great interest in the areas of regenerative medicine and cell therapy.Recent advances in understanding of the paracrine mechanisms and cytokine modulation of MSCs suggest many potential clinical applications.For example，hypoxia affects several MSC activities，including the cell cycle，differentiation，paracrine signaling，and gene expression.Our understanding of MSCs has taken great strides forward，even to the creation of their niche by a complex of secreted cytokines.This review focuses on these advances，especially regarding the modulation of paracrine signaling by MSCs in hypoxic conditions.In addition，we have summarized recent studies on important MSC-related cytokines and their effects in vitro and in vivo.

Mesenchymal stem cell Hypoxia Cytokine paracrine

Introduction

Mesenchymal stem cells(MSCs)are a type of multipotent stem cell that is currently being studied with an eye to therapeutic applications such as immunomodulation and ischemic tissue repair[1].In addition to their multi-lineage differentiation potential，their strong paracrine capacity has been proposed as the primary mechanism of tissue repair after ischemic events like myocardial infarction and stroke[2，3].Although it has been reported that several soluble cytokines are responsible for MSC-induced chemotaxis and growth response，their role in the physiological signaling pathway remains unclear[4].An improved understanding of paracrine regulatory mechanisms in MSCs would be very valuable for translational medicine，because modulation of their paracrine activity affects not only their proliferation and differentiation efficiency，but also cell migration，homing，promotion of angiogenesis，and improvement of tissue function[5].The secretion of these cytokines is determined partly by the MSC’s conditionperse，and partly by the tissue microenvironment[6].

The hypoxic microenvironment has important physiological and pathological effects on tissue homeostasis.Hypoxia may has a great effect on several aspects of MSC biology，including stemness，metabolism，angiogenesis，and innate immunity[7].It is essential to cultivate them under hypoxic conditions because this simulates the ischemic environmentinvivo.The hypoxic environment prompts MSCs to release cytokines and stimulate the corresponding signaling pathways[8].Here we have reviewed reports of MSC cultureinvitrounder conditions of 1% to 10% oxygen.This review briefly discusses the effect of hypoxia on cytokine secretion and modulation of MSC signaling pathways，and considers new information about the relevant cytokines.

1 Effects of specific cytokines

1.1HIF-1(hypoxia-induciblefactor-1)

HIF-1 is a member of a subfamily of basic helix-loop-helix transcription factors that comprise α and β subunits.It plays an essential role in cellular and systemic responses to low oxygen availability[9].Reduction in oxygen tension leads to activation of HIF-1α in a variety of tissues，thus modulating the expression of hundreds of target genes，including vascular endothelial growth factor(VEGF)，stromal cell-derived factor-1(SDF-1)，hepatocyte growth factor(HGF)，basic fibroblast growth factor(bFGF)，tumor necrosis factor α(TNF-α)，and insulin-like growth factor 1(IGF-1)[10，11].This beneficial response may happen because when the MSC migrates into a hypoxic region，MSC-derived HIF-1 stimulates the production of therapeutic paracrine mediators，thus increasing cytokine concentration.Kenichi showed that hypoxic conditions(less than 20% O2)decrease osteogenic differentiation by activating the HIF signaling pathway，thus downregulating Cbfa1/Runx2，Osx，and increasing early progenitor MSC colony formation by maintaining the undifferentiated state and proliferation capacity[12].Specific inhibition of HIF-1α degradation，using hypoxia-mimicking agents，can modulate the human MSC’s survival，growth，migration，and role in revascularization[13].Moreover，accumulated evidence has revealed that expression of normoxic HIF is associated with increased expression of glycolytic HIF target genes，thus enhancing glycolysis accordingly.This suggests that HIF is the prominent regulator that responds to hypoxia，controlling the metabolic fate and multipotency of MSCs and enhancing cytokine secretion under hypoxic conditions.

1.2VEGF(vascularendothelialgrowthfactor)

VEGF is one of the most important angiogenic factors，promoting the proliferation and migration of smooth muscle and endothelial cells and mobilizing bone marrow-derived endothelial progenitor cells to home to ischemic tissue.Binding of VEGF to its specific receptor facilitates vasodilatation and angiogenesis by upregulating nitric oxide synthase(NOS)[14].VEGF is one of HIF-1’s target genes.Okuyama et al.exposed mouse MSCs to hypoxic conditions for 24 hours，which，combined with the transduction of HIF-1，significantly increased VEGF-1 receptor expression，angiogenesis and MSC migration in the HIF-1α-positive MSC group，as compared to HIF-1α-negative MSCs and control.MSC migration increased in proportion to the concentration of VEGF[17].Specifically，hypoxia stimulates HIF-1 to upregulate the expression of VEGF，which then gives rise to the signal transduction pathway of P13K，FAK and p38，promoting MSC migration.These results were also supported by aninvivostudy of a rat model of myocardial infarction(MI)[18，19].Moreover，in a rabbit model of MI，Reza et al.found that the transplanted MSCs were enriched in the peri-infarct border zone，accompanied by a significant augmentation of microvascular density and increased expression of VEGF and bFGF[20].This indicates that MSCs can change the milieu of the injured myocardium by secreting VEGF and other cytokines，which suggests new possibilities for clinical treatment of hypoxic-ischemic diseases.

1.3SDF-1(stromalcellderivedfactor-1)

SDF-1 is a member of the CXC chemokine family with high affinity for CXC chemokine receptor 4(CXCR4).The SDF-1/CXCR4 biological axis stimulates downstream signaling pathways such as PI3K/Akt，ERK，and MAPK[21].Up until now，experimental evidence has demonstrated that SDF1/CXCR4 participates in the repair process of damaged tissues and organs，including the heart，kidneys，liver and brain[22].Previous studies have shown that expression of SDF-1 correlates positively with the magnitude of hypoxia，and that the local concentration of SDF-1 may be dramatically increased beyond that of the surrounding areas，creating a niche suitable for MSCs and promoting the migration of CXCR4-positive MSCs to the injured tissue[23].Furthermore，SDF-1 is involved not only in MSC migration，but also in homing，recruitment，and engraftment.Hojjat’s recent study revealed that MSCs pretreated with SDF-1α significantly upregulate CXCR4 and lead to an increase in migration capacityinvitro.Such a response has also been observed in transplanted cells that release SDF-1αinvivo[24].Ma et al.reported that CXCR4-positive MSCs showed an enhanced migration capability toward SDF-1 and a protective effect in a model of acute ischemic liver failure，and even more soinvivo，where CXCR4-positive MSCs migrated in larger numbers than null-MSCs and colonized at a higher rate，making for a longer lifetime[25].Another study also found that SDF-1 promotes the degradation of the extracellular matrix by activating matrix metalloproteinase 1(MMP1)，thereby enhancing the migration ability of MSCs[26].However，because SDF-1 is sometimes expressed only transiently in ischemic-hypoxic tissues，current approaches to induce expression of the natural ligand for SDF-1 found on MSC surfaces and expression of CXCR4，via genetic modification，hypoxic preconditioning，and cytokine regulation，all of which require long-term MSC culture，have not been optimal for clinical translation.

1.4Othercytokines

Like VEGF，HGF is a widely-distributed cytokine that plays an important role in tissue generation，especially in cardiac and hepatic tissue.The binding of HGF with the receptor C-met phosphorylates tyrosine residues and activates the signaling pathway that plays a role in MSC biology[27].It has been demonstrated that HGF-positive MSCs express higher levels of EGF，bFGF，and VEGF than do null-MSCs.This can greatly improve mouse heart function post-MI，as shown by reduced cardiomyocyte(CM)apoptosis，enhanced angiogenesis，and increased proliferation of CMs bothinvitroandinvivo[28].bFGF participates in revascularization in hypoxic conditions primarily by promoting cell division in capillaries and endothelial venules，and then by stimulating capillary growth，leading to the establishment of collateral circulation in the ischemic area[29].IGF is a growth hormone-dependent peptide that is highly homologous to the precursor of insulin.It is involved in CM growth and differentiation，oxidative stress resistance，and anti-apoptotic or cardioprotective functions[30].It is reported that the rate of growth of fibroblasts was increased after exposure to MSC-derived hypoxic conditioned medium(HCM)，compared with the MSC-derived normoxic conditioned medium(NCM).Moreover，cell proliferation was significantly inhibited when IGF-1 was inhibited，which likewise decreased the HCM-enhanced mobility of fibroblasts.Grigory et al. have summarized seventeen cytokines whose receptors are expressed by hypoxia-cultured MSCs，thus mediating MSC migration. The most important among them are the EGF family，the FGF family，HGF，SDF-1α，VEGF-121，PDGF-AB，the IL family，and TNF-α.Presumably，the presence of these cytokines leads to a better homing of hypoxia-cultured MSCs，since their concentration is enriched at sites of injury[31].

2 Mechanism of cytokines in hypoxia

Hypoxia has a major impact on molecular metabolism and signaling pathways in MSCs.When MSCs are exposed to low oxygen tension，MMP-1 and MMP-3 are initially regulated by HIF-1[32].Another enzyme detected is secreted lysyloxidase(LOX)，which takes part in cell migration via focal adhesion and matrix adhesion kinase activity.Activated LOX stimulates transcription of TWIST，a factor that mediates the epithelial-mesenchymal transition[33].Secondly，signaling pathways can also be controlled by hypoxia.NF-κB is a common transcription factor that regulates the expression and secretion of VEGF and bFGF in the eukaryotic cell，and also involved in cell proliferation，differentiation，and migration[34].Crisostomo et al. found that when human MSCs were added to a NF-κB inhibitor and cultured in a 1% O2environment for twenty-four hours，expression of VEGF，bFGF，HGF，and IGF was significantly decreased in the hypoxic NF-κB inhibitor group compared to control[35].In addition，the TGF-β/SMAD signaling pathway participates in the fibrinolytic system of MSC recruitment，where urokinase plasminogen activator receptor(uPAR)and plasminogen activator inhibitor-1(PAI-1)are expressed[36，37].Thirdly，chromatin modifiers are involved in epigenetic variation by targeting numerous downstream enzyme activities and gene expressions under hypoxia.For example，the ablation of histone lysine-specific demethylase 4B(KDM4B)brings about adipogenic differentiation and reduction of osteogenic differentiation in hypoxic MSCs[38].Finally，the cooperation of cytokines has a role in the secretion of MSCs.Kong et al. found that expression of VEGF and bFGF were significantly increased in SDF-1 pretreated hypoxic MSCs compared to non-pretreated hypoxic MSCs[39].Likewise，it is also revealed that pretreating MSCs with bFGF and G-CSF leads to increased expression of HGF while inhibiting expression of TGF-β[40].These cooperating factors can induce cascade amplicification that has a still greater impact on MSC secretion.

3 Conclusion

MSCs display various paracrine responses to oxygen depletion.Hypoxia not only impacts features of MSCs themselves，including proliferative capacity，inhibition of apoptosis，migration，and homing，but also acts upon the surrounding injured tissue via paracrine signaling，further improving organ function.The debate about the role of oxygen concentration and the specific mechanism of hypoxia’s action on MSCs indicates the need for further study，which will lead to clinical applications.The question whether MSC responses to hypoxiainvitroare consistent with thoseinvivoalso needs further discussion.

ConflictofInterests

The author declares that there is no conflict of interests regarding the publication of this paper.

Acknowledgments

This work was supported by NIH Grants，R56HL130042 and HL136025 WANG Yi-gang.

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低氧对间充质干细胞旁分泌信号的调节

姜 霖，萨拉·贝克，蔡文锋，高 翔，王义刚*

(美国辛辛那提大学病理与实验医学系，美国，俄亥俄州，45267-0529)

间充质干细胞(mesenchymal stem cells，MSCs)是一类多功能干细胞，在细胞治疗和再生医学领域受到研究者的极大关注。最近在MSCs的旁分泌机制和细胞因子对MSCs的调控方面取得的重大研究进展提示，MSCs在临床应用方面具有多种潜能。例如，低氧从多方面影响MSCs功能(细胞周期，细胞分化，旁分泌信号通路和基因表达)。MSCs构建细胞龛调节细胞局部微环境是通过复杂的分泌细胞因子方式实现的，这使得我们对MSCs的认识取得了巨大飞跃。这篇综述集中在上述研究进展上，尤其是低氧条件下MSCs对旁分泌信号通路的调节。除此之外，本文还回顾了近期关于间充质干细胞相关的重要细胞因子和它们在体内、体外作用的相关研究。

间充质干细胞 低氧 细胞因子 旁分泌

R329

A

主持嘉宾简介：王义刚教授任职于美国辛辛那提大学病理与实验医学系，是再生医学研究部主任暨终身教授，为美国心脏协会资深会员(FAHA)、美国中风协会(ASA)和国际心脏研究协会(ISHR)会员，担任多种国际性学术刊物编委工作。同时任美国国立卫生研究院(NIH)和美国心脏协会(AHA)等多个重大基金机构的项目审核人。他拥有三十多年的临床及基础研究经验，长期担任多项NIH课题研究首席科学家，在众多国际著名学术出版物发表重要论著。王教授所带领的团队主要从事心脏再生的相关机制研究并延伸至干细胞的多潜能性、增值与分化，以及重编程效应等诸多方面研究。王教授在心血管病生方面的研究建树独到，主要研究内容包括：1)干细胞治疗心肌梗塞研究；2)缺血性心肌损伤和修复的分子机制及信号通路研究；3)心脏停跳液成分对心脏功能保护的影响研究。

10.13452/j.cnki.jqmc.2017.03.001

#：Corresponding author：MD.，Ph.D& Professor，Tel：001-513-558-5798，E-mail：yi-gang.wang@uc.edu