Basic Sciences

Aldehyde Dehydrogenase-2 Is a Host Factor Required for Effective Bone Marrow Mesenchymal Stem Cell TherapySignificance

Hongming Zhu, Aijun Sun, Hong Zhu, Zheng Li, Zheyong Huang, Shuning Zhang, Xin Ma, Yunzeng Zou, Kai Hu, Junbo Ge
https://doi.org/10.1161/ATVBAHA.114.303241
Arteriosclerosis, Thrombosis, and Vascular Biology. 2014;34:894-901
Originally published March 19, 2014

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Abstract

Objective—Mesenchymal stem cell (MSC) therapy is a promising treatment for ischemic injury. However, the environmental regulatory mechanism is essentially unclear and thus greatly limits its application in clinical setting. Accumulating evidence suggests a vital role of aldehyde dehydrogenase-2 (ALDH2) in microenvironment homeostasis after ischemia. About 540 million people or 8% of population worldwide carry a loss-of-function allele of ALDH2. It is unknown whether ALDH2 functions as a host factor regulating the therapeutic potential of donor MSCs. Therefore, this study was designed to determine whether and how host ALDH2 regulates MSC retention and therapeutic efficacy after transplantation into ischemic tissues.

Approach and Results—Mice limb ischemia was performed by femoral artery ligation. A total of 106 MSCs were injected into the ischemic thigh muscles. One, 2, and 4 weeks after transplantation, MSC retention, blood perfusion recovery, limb necrosis, and fibrosis were analyzed. Compared with wild-type tissue, ALDH2 deficiency tissue significantly limited MSC retention and its perfusion recovery and limb salvage effects after ischemia. Importantly, local overexpression of ALDH2 optimized tissue microenvironment and significantly magnified all these MSC-induced improvement. Further study indicated that host ALDH2 regulated transplanted MSC survival and therapy as a microenvironment homeostasis mediator via local capillary density, energy supply, and oxidative stress regulating after ischemia.

Conclusions—Our study establishes ALDH2 as a key mediator of host stem cell niche for optimal MSC therapy and suggests that ALDH2 deficiency present in the general population is a limiting host factor to be considered for MSC therapy.

Introduction

Mesenchymal stem cell (MSC) therapy represents a promising approach for treating ischemic injury and promoting tissue regeneration.1 Despite significant advance in the field during the past decade, the hostile microenvironment resulted from the postischemic injury remains a major obstacle for a meaningful therapeutic effect.2–4 The effect of host factors, such as genetic variants, on stem cell therapy is largely unknown. Accumulating data from clinical trials using bone marrow stem cells have displayed some discrepancies in cardiac function improvement between the Asia patient5,6 and Europe patients.7–10 These data suggest that genetic variants in different ethnic patients may have different host environments affecting on stem cell retention and survival at the injury site, thereby different therapeutic outcomes.

Among common genetic mutants, a loss-of-function allele of aldehyde dehydrogenase-2 (ALDH2) is found in ≈8% world populations and 40% of East Asians resulting in the flushing responses to alcohol.11–14 ALDH2 gene encodes a key endogenous cytoprotection enzyme that protects ischemic injury partly through detoxification of toxic substances such as reactive aldehydes.15,16 However, it remains elusive whether ALDH2 plays a role in stem cell niche for tissue regeneration.

In this study, we investigated host ALDH2 roles in MSC therapy in a mouse hindlimb ischemia model using the loss- and gain-of-function approach. We showed that host ALDH2 deficiency attenuated ischemic tissue repair by MSCs, whereas local overexpression of ALDH2 improved tissue regeneration. These observations implicate that loss of function in ALDH2 through genetic mutation may be an unfavorable contributing factor for effective stem cell therapy.

Materials and Methods

Materials and Methods are available in the online-only Supplement.

Results

Host ALDH2 Influences the Retention of Transplanted MSCs

To examine the effect of ALDH2 on MSC therapy for tissue repairing, we used a mouse ischemic limb model. We first assessed the retention of transplanted cells in the ischemic limb. At 1, 2, and 4 weeks after cell injection, retention of the transplanted cells to the ischemic limb was tracked using in vivo imaging of green fluorescent protein (GFP; Figure 1A) and GFP intensity (Figure 1B). We found that ALDH2-knockout mice exhibited less GFP fluorescence signals in the ischemic limb compared with that from wild-type (WT) mice at all time points. To the contrary, ALDH2 overexpression (ALDH2 expression adenovirus [Ad.ALDH2] group) greatly enhanced MSC retention at these time points. Of note, GFP signal persisted in ischemic limbs from WT, control adenovirus (Ad.Null), and Ad.ALDH2 mice but not ALDH2-knockout mice 4 weeks after MSC injection. No GFP signal was detectable in nonischemic limbs of experimental mice.

Figure 1.
Figure 1.

Host aldehyde dehydrogenase-2 (ALDH2) regulates the retention of implanted mesenchymal stem cell (MSCs). A, Representative figures of in vivo green fluorescent protein (GFP) imaging of implanted MSCs. B, GFP signaling intensity in ischemic limb at different duration. C and D, Immunofluorescence of vascular-like structure by 4′,6-diamidino-2-phenylindole (DAPI; blue) and CD31 (red) and GFP (green) positive cells at 4 weeks after cell delivery. E, Quantitative polymerase chain reaction gene expression analyses. The mRNA levels of GFP sequence in ischemic muscle are presented. Mean±SD of ≥3 independent experiments per time point. *P<0.05 vs relative group of wild-type (WT) mice; #P<0.05 vs relative group of ALDH2 expression adenovirus (Ad.ALDH2) mice. Bar, 100 μm. Ad.Null indicates adenovirus control; and KO, knockout.

MSC retention was further confirmed using immunofluorescence for GFP protein (Figure 1C and 1D) and reverse transcription-polymerase chain reaction for GFP transcripts (Figure 1E). Our data revealed less cell retention in the injured limbs of ALDH2-knockout mice compared with that from the other 3 groups, whereas the best cell retention was noted in Ad.ALDH2 group. Four weeks after MSC transplantation, GFP-labeled MSCs were well detected in injured limbs of WT, Ad.Null, and Ad.ALDH2 but not ALDH2-knockout mice (Figure 1C and 1D). Consistently, the mRNA levels of GFP in ischemic limbs of ALDH2-knockout mice were 1.6-, 2.38-, and 4.28-folds lower than that in WT mice, and 7-, 7.67-, and 10-folds lower than that in Ad.ALDH2 mice, at 1, 2, and 4 weeks post injection, respectively (P<0.01; Figure 1E). Importantly, no GFP transcripts were detected in the control nonischemic limb. There was little difference between Ad.Null and WT groups, although both groups exhibited markedly low cell retention compared with the Ad.ALDH2 group. These observations demonstrate that the retention of MSCs in the ischemic limb is likely regulated by the presence of ALDH2 expression.

Host ALDH2 Regulates MSC Therapy for Ischemic Limb

The poor retention of MSCs in the ALDH2-knockout mice likely impedes the MSC-dependent tissue and function repair of the ischemic limb. Indeed, the laser Doppler perfusion analysis depicted a significantly decreased perfusion in the injured limb of ALDH2-knockout mice compared with the control WT mice at 1 and 2 weeks after MSC injection, whereas increased perfusion was noted from 1 to 4 weeks in all groups (Figure 2A and 2B). Quantitatively, at the 2-week point, the perfusion ratio in MSC-treated WT mice and Ad.ALDH2 group achieved 76.79% and 89.79%, respectively. In comparison, only 32.63% perfusion ratio was found in MSC-treated ALDH2-knockout mice, indicating a slow recovery of limb circulation. Consistently, the ATP content in Ad.ALDH2 group was significantly higher than the other groups. Reduced ATP content was found in the MSC-treated ALDH2-knockout mice (Figure 2C). There was no significant difference between Ad.Null group and WT groups.

Figure 2.
Figure 2.

Host aldehyde dehydrogenase-2 (ALDH2) regulates mesenchymal stem cell (MSC)–induced perfusion and ATP recovery. A, Representative laser Doppler perfusion images of experimental groups. Hindlimb ischemia was performed by femoral artery ligation and excision in male wild-type (WT) C57 BL/6 or ALDH2 genetic modification mice (7–8 weeks; n=12 for each group). B and C, Perfusion ratio and ATP content in ischemic muscles from MSC-treated mice. Untreated controls are displayed in Figure IV in the online-only Data Supplement. Mean±SEM. *P<0.05 vs relative group of WT mice; #P<0.05 vs relative group of ALDH2 expression adenovirus (Ad.ALDH2) mice. Ad.Null indicates adenovirus control; and KO, knockout.

Using morphological assessment of limb necrosis, we found comparable tissue necrosis, eventually leading to partial limb loss, despite the increased perfusion recovery over time in all groups (Figure 3A and 3B). However, MSC treatment alleviated foot loss in 5 of 8 (62.5%) WT mice, whereas only 1 of 4 (25%) MSC-treated ALDH2-knockout mice show similar improvement. In contrast, significant therapeutic improvement occurred in 8 of 9 Ad.ALDH2 mice (88.9%). In addition, calf muscle weight was higher in MSC-treated Ad.ALDH2 mice compared with that in MSC-treated ALDH2-knockout mice (P<0.05; Figure 3C). Analysis fibrosis of ischemic limbs also revealed that local ALDH2 overexpression suppressed fibrosis (Figure 3D and 3E). Taken together, these data demonstrate that transplanted MSCs directly incorporate into host ischemic muscles and improved function recovery of ischemic limbs. Such beneficial effect was significantly enhanced by ALDH2 overexpression although it was attenuated by ALDH2 deficiency, suggesting important roles of host ALDH2 in MSC therapy.

Figure 3.
Figure 3.

Host aldehyde dehydrogenase-2 (ALDH2) regulates mesenchymal stem cell (MSC) repair of ischemic muscle. A, Respective morphological images of ischemic foot. B, Graded assessment of necrosis, the following 3 grades were used to measure the degree of limb necrosis: toe necrosis, necrosis limited to the toes; foot necrosis, necrosis extending to the dorsum pedis; and limb necrosis, necrosis extending to the crus. C, Gastrocnemius muscle weight in different groups. D, Representative Masson staining images of ischemic muscle. Dark gray represents muscle fiber and light gray represents collagen formation (fibrosis). Scale bar, 0.2 mm. E, The ratio of fibrosis to muscle fiber was quantified. Four weeks after cell transplant, MSC significantly decreased muscle fibrosis in wild-type (WT) mouse compared with in knockout (KO) mouse (*P<0.05). *P<0.05 vs relative group of WT mice; #P<0.05 vs relative group of ALDH2 expression adenovirus (Ad.ALDH2) mice. Ad.Null indicates adenovirus control; and HI, hindlimb ischemia.

Host ALDH2 Regulates Vascular Niche Formation After Ischemia

Reduced MSC retention and therapy in ALDH2-knockout mice indicate a disruptive or attenuated stem cell microenvironment in mutant mice. We thus performed immunostaining using CD31 antibodies to label vascular capillary cells in particular vasculature of injured limbs from control and ALDH2 mice. At 4 weeks, we noted a significantly decreased capillary density and number of CD31+ cells in ischemic regions from limbs of ALDH2-knockout mice compared with ischemic limbs from WT mice (P<0.01). The capillary density in ischemic limbs of Ad.ALDH2 mice exhibited the best capillary recovery among all groups (Figure 4A and 4B).

Figure 4.
Figure 4.

Host aldehyde dehydrogenase-2 (ALDH2) regulates microvascular niche in postischemia. A, Respective immunofluorescence (IF) images of capillary staining using CD31 positive (gray). B, Capillary density based on the number of CD31 positive cells. C, Angiogenic protein level in ischemic tissues. Data exhibiting levels of some angiogenic protein regulated by ALDH2. *P<0.05 vs relative group of wild-type (WT) mice; #P<0.05 vs relative group of ALDH2 expression adenovirus (Ad.ALDH2) mice. Ad.Null indicates adenovirus control; CYR61, cysteine-rich angiogenic inducer 61; EGF, eidermal growth factor; FGF, fibroblast growth factor; IGFBP, insulin-like growth factor binding protein; KO, knockout; MMP, matrix metallopeptidase; and VEGF, vascular endothelial growth factor.

Next, we performed an angiogenic protein array to identify the candidate proteins underscoring angiogenesis. At 4 weeks after surgery, our result showed downregulated levels of a collection of angiogenic factors, including cysteine-rich angiogenic inducer 61, endoglin, eidermal growth factor, fibroblast growth factor-acidic, angiogenin, angiopoietin-1, tissue factor, matrix metallopeptidase-3/-9, and insulin-like growth factor binding protein-1/-2 in limbs from ALDH2-knockout mice (Figure 4C; P<0.05). Vascular endothelial growth factor, matrix metallopeptidase-9, and fibroblast growth factor-acidic, were upregulated in Ad.ALDH2 mice. In vitro angiogenesis showed that artery ring or microvascular endothelial cells from each group underwent angiogenesis equally well under normoxia. However, ALDH expression significantly altered artery ring sprouting and tubular network formation under hypoxia (Figure 5). These changes in revascularization may affect stem cell functional niche by regulating supply nutrition and cytokines.

Figure 5.
Figure 5.

Aldehyde dehydrogenase-2 (ALDH2) regulates microvascular genesis in vitro. A, Representative images of aortic ring sprouting under normoxia or hypoxia. B, Surface area analysis of sprouting. C, Representative images of tube-like construction formation under normoxia or hypoxia. D, Tube formation count, the closed endothelial networks of vessel-like tubes were counted at a magnification ×50. Mean±SEM. *P<0.05 vs relative group of wild-type (WT) mice; #P<0.05 vs relative group of ALDH2 expression adenovirus (Ad.ALDH2) mice. Ad.Null indicates adenovirus control; and KO, knockout.

Host ALDH2 Regulates Antioxidant Activities After Ischemia

To further determine the host ALDH2 function in maintaining the microenvironment homeostasis, biochemical analysis of antioxidative capacity was performed (details in the Table). We found that limb tissue superoxide dismutase activities were decreased in all groups after ischemic injury. However, the hindlimb ischemia group of ALDH2-knockout mice showed relatively low superoxide dismutase activity at 4-week post surgery compared with the hindlimb ischemia group in WT mice (6.37±2.00 versus 10.05±2.22; P<0.05). Also at 4 weeks, tissue malondialdehyde contents of the ischemic limbs of all 4 groups’ mice were higher than those of the nonischemic limbs. However, the malondialdehyde content of the ischemic limb of ALDH2-knockout mice was significantly higher than that of WT mice (11.03±1.68 versus 7.48±1.71; P<0.05), whereas the malondialdehyde level in the ischemic limbs of Ad.ALDH2 mice was significantly decreased. Moreover, the tissue glutathione/glutathione disulfide ratio was decreased in the ischemic limbs of the WT and ALDH2-knockout mice; the ratio was partially restored by ALDH2 overexpression (P>0.05). In addition, ALDH2-knockout increased ischemia-induced 4-hydroxy-2-nonenal (4-HNE) accumulation (Figure 6A). In vitro cell death assay showed that 4-HNE accumulation resulted in death of 83.21%, 29.42% or 5.12% MSCs at 5, 10, and 30 nmol/L (P<0.01; Figure 6B and 6C). Pathway study indicated that 4-HNE induced MSC death through activating c-jun n-terminal kinase and p53 pathways (Figure 6D–6F). ALDH2 overexpression markedly inhibited postischemic 4-HNE accumulation, thus enhancing MSC survival and therapy. Together, these results demonstrated that host ALDH2 expression influences tissue antioxidant capacity, which is essential for local tissue homeostasis for MSC therapy.

View this table:
Table.

Antioxidant Activities of Muscle Tissue

Figure 6.
Figure 6.

Aldehyde dehydrogenase-2 (ALDH2) regulates oxidative stress–induced mesenchymal stem cell (MSC) death. A, 4-Hydroxy-2-nonenal (4-HNE) content in ischemic tissue. B and C, Dose-dependent response and representative images for 4-HNE–induced MSC death. After treatment with different concentrations of 4-HNE for 24 hours, the intensively fluorescent calcein acetomethoxy derivate of calcein (gray cell) indicated the live cell and the nucleic acids–EthD-1 complex (gray dot) indicated the dead cell. Cell images are representations from >3 independent experiments, Bar, 200 μm. D to F, Mechanism of 4-HNE–indcued MSC death. SP600125 was used as c-jun n-terminal kinase (JNK) inhibitor. D and E, 4-HNE–induced MSC death by JNK signal activation. Cell viability was test by cell counting kit-8 analysis. JNK protein phosphorylation was evaluated using Western blot. F, p53 mediated JNK-dependent MSC death. *P<0.01. Ad.ALDH2 indicates ALDH2 expression adenovirus; Ad.Null, adenovirus control; KO, knockout; and WT, wild type.

Discussion

The salient findings from our study include (1) host ALDH2 regulates the retention of engrafted MSCs in ischemic limbs and mediates MSC therapy and (2) host ALDH2 level influences tissue microenvironment, including angiogenic and antioxidant functions necessary for energy supply and oxidative stress relief. Based on these findings, it is plausible to conclude that ALDH2 may serve as a key host factor maintaining stem cell niche necessary for MSC therapy (Figure 7).

Figure 7.
Figure 7.

Scheme summarizing the proposed mechanism of action behind aldehyde dehydrogenase-2 (ALDH2)-exerted response in mesenchymal stem cell (MSC) cell therapy. Arrow, Upregulation or promotion; line with short rod in front, inhibition. 4-HNE indicates 4-hydroxy-2-nonenal.

Poor retention of implanted cells is a major problem in stem cell treatment of ischemic diseases.2,4,17 Various strategies, including tissue engineering scaffolds,18 genetic modifications,19 and hypoxia-based preconditioning,20 are used to prevent loss of the implanted cells in the ischemic region. However, these strategies only moderately improve the outcome.21 Endogenous factors that regulate or maintain function of host stem cell niche have been studied extensively in recent years. Among these factors, microcirculatory damage,22 insufficient nutrient supply, and oxidative stress23 are currently considered to be the major host factors critical for successful stem cell therapy.

Our earlier study suggested that host vascular niche and cytokine production govern the therapeutic efficacy of engrafted stem cells.24 Here, we found that most of the implanted cells were located around blood vessels rather than distributed evenly throughout injured muscles. In the ALDH2-knockout mice, however, impaired ischemia-induced revascularization directly resulted in the poor nutrient supply to the ischemic tissue, thus limiting its ATP production to impede the retention of the implanted MSCs in injured tissues. Angiogenic factors facilitate intracellular survival mechanisms25 as well as vessel regeneration that contributes to cell survival via oxygen recovery and nutrient supply. Indeed, our protein array analysis revealed that ALDH2-knockout tissues expressed low level of angiogenic factors, including cysteine-rich angiogenic inducer 61, endoglin, eidermal growth factor, fibroblast growth factor-1, angiogenin, angiopoietin-1, tissue factor, matrix metallopeptidase-3/-9, insulin-like growth factor binding protein-1/-2.26–30 Of note, Cyr61 is a key polypeptide required for the angiogenic-induced function of the MSC secretome31 and eidermal growth factor increases proliferation, spreading, and survival of MSCs.25 Further, endoglin participates in stem cell differentiation.32 Fibroblast growth factor signaling acts as a major player to promote self-renewing proliferation and inhibit cellular senescence of MSCs in both tissue homeostasis and repair.33 Upregulation of angiogenin enhances the tolerance of engrafted MSCs to hypoxia injury during vasculogenesis in vitro and in vivo.34 In addition, matrix metalloproteinases remodel extracellular matrix for better stem cell migration and recruitment.35 Therefore, downregulation of these proangiogenic factors in injured ALDH2-knockout tissues may contribute to poor revascularization, leading to reduced MSC retention and survival.

Our in vitro characterization of microvascular endothelial cells supported these in vivo observations, consistent with previous findings.36,37 We found that ALDH2 deficiency attenuated the sprouting and formation of endothelial networks under hypoxic but not normoxic condition in vitro. Hypoxia leads to the stabilization of hypoxia-inducible factor-1, the main regulator of the cellular response to hypoxia,38 to further regulate levels of hypoxia-responsive genes. In this study, we found that expression of angiopoietin-1, a known target of hypoxia-inducible factor-1 in the vascular cells, was downregulated in the injured ALDH2-knockout limbs. This finding suggests a deregulation of hypoxia-inducible factor-1 in the injured limbs resulted from the ALDH2 deficiency. Future studies are warranted to attest this hypothesis. A pioneering study39 reported that ALDH2 knockout influences hematopoietic stem and progenitor cell pool in bone marrows. ALDH2 deficiency exacerbates genotoxicity accumulation in hematopoietic stem cell via negating the acetaldehyde detoxification capability. This finding provided the rationale support for our current study. Impaired angiogenesis results in oxidative stress that hinders stem cell adhesion, causes the death and anoikis of the grafted cells, thereby reducing the efficacy of stem cell repairing.40 In the present study, we found that ALDH2 deficiency increased the toxic substance 4-HNE accumulation in the ischemic tissue. 4-HNE damaged implanted MSC by activated c-jun n-terminal kinase/p53 pathway.

Murohara and colleagues5 examined the safety and efficiency of therapeutic angiogenesis with autologous bone marrow stem cell (TACT) in patients with limb ischemia. A cohort of patients displayed no therapeutic effects.41 We observed the similar result in our clinical trial of bone marrow stem cell delivery in myocardium infraction patients.42 Both clinical trials in Asia have shown a limited efficacy of the implantation of bone marrow stem cells for angiogenesis of ischemic injury. Our current study establishes that MSC therapeutic effect is dependent on the host ALDH2, which regulates the microenvironment of engrafted cell retention and function. Given the wide distribution of ALDH2 loss-of-function allele mutation in Asian population, the unfavorable effect of ALDH2 deficiency in stem cell niche needs to be evaluated for the overall outcome of stem cell therapy in future clinical practice. In this study, we also addressed the question whether overexpression of ALDH2 would augment MSC transplantation. Our data indicate that ALDH2 overexpression enhances MSC retention and therapy for ischemia. This finding suggests a patient-specific gene modification strategy to improve the stem cell niche for cell-based therapy in the patients with ALDH2 mutation. Considering the key role of ALDH2 in alcohol metabolism, our data may imply a potential relationship between alcohol intoxication and stem cell therapeutic efficacy.

In summary, host ALDH2 regulates stem cell niche that may contribute to the retention and therapeutic effect of implanted MSCs. Further study of this model may help to identify other wide distribution or population-specific mutants and develop patient/allele-specific strategies in stem cell therapy for ischemia diseases.

Sources of Funding

This work was supported, in part, by the National Natural Science Foundation of China (30971250), National Basic Research Program of China (2011CB503905), and Program for New Century Excellent Talents in University (NCET).

Disclosures

None.

Footnotes

  • Received September 23, 2013.
  • Accepted February 10, 2014.

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Significance

Our work described a novel finding that aldehyde dehydrogenase-2 regulates the retention and therapeutic potential of transplanted mesenchymal stem cells as a host niche regulator in limb ischemia. This study is of particular clinical importance. First, this study may advance our understanding in that genetic variant serves as a host regulatory factor in stem cell therapy. Furthermore, it provides experimental evidence, which explains possible suboptimal outcomes in certain clinical trials using bone marrow cell. Finally, given the large portion of aldehyde dehydrogenase-2 mutant carrier and the high prevalence of stem cell therapy, this study should shed some lights in individualized stem cell therapy.

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  • Aldehyde Dehydrogenase-2 Is a Host Factor Required for Effective Bone Marrow Mesenchymal Stem Cell TherapySignificance
    Hongming Zhu, Aijun Sun, Hong Zhu, Zheng Li, Zheyong Huang, Shuning Zhang, Xin Ma, Yunzeng Zou, Kai Hu and Junbo Ge
    Arteriosclerosis, Thrombosis, and Vascular Biology. 2014;34:894-901, originally published March 19, 2014
    https://doi.org/10.1161/ATVBAHA.114.303241
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