Follistatin-like 1 protects mesenchymal stem cells from hypoxic damage and enhances their therapeutic efficacy in a mouse myocardial infarction model
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Cell therapy remains the most promising approach against ischemic heart injury. However, poor survival of engrafted cells in ischemic sites diminishes its therapeutic efficacy. Follistatin-like 1 (Fstl1) is documented as a novel pro-survival cardiokine for cardiomyocytes, and it is protective during ischemic heart injury. In the present study, we characterize the potential of Fstl1 as an effective strategy to enhance hypoxia resistance of donor cells and optimize stem cell-based therapy.
Murine bone marrow-derived mesenchymal stem cells (MSCs) were expanded in monolayer culture and characterized by flow cytometry. MSCs were subjected to hypoxia to mimic cardiac ischemic environment. Expression of Fstl1 was monitored 0, 24, and 48 h following hypoxia. Constitutive expression of Fstl1 in MSCs was achieved by lentivirus-mediated Fstl1 overexpression. Genetically modified MSCs were further collected for cell death and proliferation assay following 48 h of hypoxic treatment. Acute myocardial infarction (MI) model was created by ligating the left anterior descending coronary artery, while control MSCs (MSCs-mCherry) or Fstl1-overexpressing MSCs (MSCs-Fstl1) were injected into the peri-infarct zone simultaneously. Subsequently, retention of the donor cells was evaluated on post-therapy 1, 3, & 7 days. Finally, myocardial function, infarct size, inflammation, and neovascularization of the infarcted hearts were calculated thereafter.
Expression of Fstl1 in hypoxic MSCs declines dramatically in a time-dependent manner. In vitro study further demonstrated that Fstl1 promotes survival and proliferation of hypoxic MSCs. Moreover, Fstl1 significantly prolongs MSC survival/retention after implantation. Finally, transplantation with Fstl1-overexpressing MSCs significantly improves post-MI cardiac function by limiting scar formation, reducing inflammatory response, and enhancing neovascularization.
Our results suggest Fstl1 is an intrinsic cardiokine promoting survival and proliferation of MSCs, thereby optimizing their engraftment and therapeutic efficacy during cell therapy.
KeywordsMesenchymal stem cells Myocardial infarction Transplantation Follistatin-like 1 Survival
Type I collagen
- LV vol;d
Left ventricular volume in diastole
- LV vol;s
Left ventricular volume in systole
Left ventricular internal diameter in diastole
Left ventricular internal diameter in systole
Multiplicity of infection
Mesenchymal stem cells
Polymerase chain reaction
Transforming growth factor-β
α-Smooth muscle actin
Myocardial infarction (MI) is the leading cause of morbidity and mortality worldwide. In terms of current therapeutic options for MI, stem cell-based therapies hold great promise for heart regeneration . Bone marrow-derived mesenchymal stem cells (MSCs) have unique properties that make them ideally suited for off-shelf clinical cell transplantation; they are easily extractable and are immune-privileged with multilineage potential . Nevertheless, their therapeutic efficacy has been hindered by poor survival, retention, and engraftment of transplanted cells due to insufficient blood supply, poor nourishment of cells, and generation of free radicals . Thus, it is reasonable to search for novel strategy promoting donor cell survival as well as optimizing their therapeutic effects.
Follistatin-like 1 (Fstl1), also known as TSC-36, is a secreted glycoprotein induced by transforming growth factor-β1 (TGF-β1) . Previous literature has demonstrated that Fstl1 exerts cardiovascular-protective activities in ischemia and pathological cardiac hypertrophy models [5, 6, 7]. Recently, we reported that Fstl1 protects cardiomyoblasts from cell death through Akt and Smad1/5/9 signaling . It is worthy of note that although cardiac Fstl1 level is markedly elevated in post-MI myocardium , retention and engraftment of donor cells in ischemic border zone are still very low . We therefore propose that supplementation of intrinsic Fstl1 may further improve survival and engraftment of donor MSCs.
In the present study, we demonstrated that Fstl1 is a critical molecule determining the fate of implanted MSCs. Overexpression of Fstl1 in MSCs enhances their resistance to hypoxia and hence their potential in vivo lifespan. It is worthy of note that Fstl1-overexpressing MSCs improve post-MI cardiac function more effectively, with reduced fibrosis, inflammatory cell infiltration, and enhanced neovascularization in peri-infarct zones. In summary, our results support the promise of Fstl1 as an effective strategy to optimize stem cell-based therapy in tissue injury.
Materials and methods
C57BL/6J mice were obtained from the Experimental Animal Center of the Chinese Academy of Medicine Sciences of Soochow University. All animal protocols were approved based on the local ethics legislation with respect to animal experimentation.
Culture and characterization of bone marrow MSCs
Bone marrow-derived MSCs from C57BL/6J mice (Cyagen Biosciences) were expanded in monolayer culture with mesenchymal stem cell growth medium (Cyagen Biosciences) supplemented with 10% fetal bovine serum at 37 °C until the cells reached 80% confluence as described previously . The cells were then trypsinized and frozen in liquid nitrogen for later use. The enriched MSC population was characterized with antibodies against CD29, CD44, CD45, CD90, CD117, Sca-1, and their relative isotype controls on a flow cytometry (Millipore Guava easyCyte) as previously described .
Lentivirus transduction of MSCs
The recombinant lentivirus for Fstl1 was purchased from GeneChem (GV320, China) and designated as LV-Fstl1. Order of the vector elements is Ubi-MCS-3FLAG-SV40-mCherry. The mCherry empty vector was used as a control and designated as LV-mCherry. MSCs infected with LV-Fstl1 or LV-mCherry at a multiplicity of infection of 10 were designated as MSCs-Fstl1 and MSCs-mCherry, respectively. Transduction efficiency was determined based on observation under fluorescent microscope 72 h after infection. Overexpression of Fstl1 in MSCs-Fstl1 was further confirmed by qRT-RCR and western blot analysis.
Annexin V analysis and EdU incorporation assay
MSCs-mCherry and MSCs-Fstl1 were seeded in 6-well plates and incubated under hypoxic condition (94% N2, 5% CO2, and 1% O2) for 48 h. Cell death was measured by Annexin V-PE/7-AAD dead cell apoptosis kit (BD Pharmingen), and Annexin V-positive cells were quantified on a flow cytometry (Millipore Guava easyCyte) as described previously [11, 12]. Cells treated with 25 μM etoposide for 24 h were used as positive controls for apoptosis assay. Cell proliferation was assessed by click-it EdU flow cytometry kit (Life Technologies) according to the manufacturer’s instructions. Briefly, cells were incubated with 10 μM ethynyldeoxyuridine (EdU) for 2 h immediately after hypoxic treatment. Nuclear EdU was further marked by binding of azide group of click-it®Alexa Fluor 647 fluorophore to alkyne group of EdU. EdU incorporation was finally analyzed on a flow cytometry (Millipore Guava easyCyte).
Tube formation assay
For the in vitro tube formation assay, 100 μL thawed Matrigel (BD Biosciences) was coated on 96-well plates at 37 °C for 1 h to allow the matrix to polymerize. Next, 2 × 104 human umbilical vein endothelial cells (HUVECs) suspended in 50 μL endothelial growth medium-2 (EGM-2, Lonza) plus 50 μL MSC conditioned medium were seeded on the Matrigel and incubated at 37 °C for 12 h. Tube structures were inspected under an inverted light microscope. Tube length and branching per well were analyzed by ImageJ software as described previously [13, 14].
MI and implantation of MSCs
Permanent MI was established by ligation of the left anterior descending coronary artery (LAD) in male C57BL/6J mice as previously described . Briefly, animals were put under general anesthesia and ventilated by a rodent respirator. After a left thoracotomy between the third and fourth intercostal space, the left ventricle was exposed satisfactorily and LAD was ligated. Successful induction of MI was verified by a color change in the infarct region after ligation. Immediately after MI, 5 × 105 cells suspended in 20 μL PBS were intramyocardially injected surrounding the infarct zones at two different sites. Finally, a thoracic incision was carefully closed, and the mice were allowed to recover. Whereas in the sham-operated group, the needle was passed around the artery without ligation.
Animal study design
Mice were randomized into four groups: (1) sham, (2) MI/PBS, (3) MI/MSCs-mCherry, and (4) MI/MSCs-Fstl1. MSC engraftment was monitored by mCherry signal on post-therapy 1 day and by CM-DiI labeling on post-therapy 3 and 7 days (n = 3–10). Moreover, cardiac function was assessed by echocardiography on post-therapy 7 and 14 days (n = 6–10). Masson’s trichrome and immunofluorescent staining for vimentin, CD68, BS1 lectin, CD31, and α-SMA was performed on post-therapy 7 days to evaluate fibrosis, inflammatory cell infiltration, and neovascularization. Finally, total RNA from host myocardium proximal to transplanted cells was used to analyze the expression of fibrotic and inflammatory genes after cell transplantation (n = 3–4).
Detection of MSCs recruitment
For detection of engrafted cells, MSCs were pre-labeled with 1 μg/mL chloromethylbenzamido (CM-DiI, Invitrogen) before cell therapy as described previously . Alternate sets of serial vertical sections around the injection site were prepared and further monitored by fluorescent microscopy. Engrafted MSCs were also identified by co-localization of mCherry fluorescence (red) and immunofluorescent signal from anti-mCherry (green). Briefly, serial slides were sequentially incubated with rabbit anti-mCherry primary antibody (Abcam), FITC-conjugated anti-rabbit IgG (Senta Cruz), and DAPI-containing anti-fade medium and imaged under a fluorescent microscope.
Cardiac function of mice was evaluated by transthoracic echocardiography on Vevo 2100 system (VisualSonics, Canada) equipped with an 80-MHz probe as described previously . All parameters were measured from M-mode recoding. Left ventricle ejection fraction (EF) and fractional shortening (FS) were automatically calculated by the echocardiography software using the following formulas: EF (%) = (LVID;d3 − LVID;s3)/LVID;d3 × 100% and FS (%) = (LVID;d − LVID;s)/LVID;d × 100%, respectively.
Assessment of infarct size
Scar formation was analyzed using a Masson’s Trichrome Stain Kit (Sigma) as described previously . In brief, hearts were collected and cut into frozen sections of 7 μm. Stained slides were photographed and quantified with ImageJ software. The percentage of infarct size was calculated as (fibrosis area/total LV area) × 100%.
Immunofluorescent staining was performed according to the standard protocol as previously reported . Briefly, the heart sections were incubated with anti-vimentin, anti-CD68, anti-CD31 (Abcam), and anti-α-SMA (Santa Cruz) to detect fibroblasts, macrophages, and blood vessels. Then, FITC-conjugated anti-mouse IgG or anti-rabbit IgG was added and incubated for 1 h before observation. For detection of neovascularization, 1 mg/mL Griffonia (Bandeiraea) simplicifolia lectin 1 (BS1 lectin; Vector) was injected into the left ventricle via direct cardiac puncture 15 min before the sacrifice of mice. Slides were sequentially stained with goat anti-BS1 lectin antibody (Vector) and Alexa Fluor 488-conjugated anti-goat IgG (Jackson ImmunoResearch). Finally, the heart sections were mounted with DAPI-containing anti-fade medium and imaged.
Reverse transcription PCR and quantitative RT-PCR
Total RNA was reverse transcribed to cDNA using the PrimeScript RT reagent Kit (Takara, Japan). qRT-PCR was performed using the SYBR Premix Ex Taq reaction mix (Takara, Japan) on a StepOne Plus real-time PCR system (Applied Biosystems) as previously reported . The reaction conditions included 95 °C for 10 min and then 40 cycles of 95 °C for 15 s, 65 °C for 30 s, and 72 °C for 10 s. Expression of target genes was determined by comparative ΔΔCt method and GAPDH or 18S was used as an internal control gene. The sequences of specific primer pairs are described below: Fstl1, 5′-TTATGATGGGCACTGCAA-3′ and 5′-ACTGCCTTTAGAGAACCAG-3′; Fsp1, 5′-AGGAGCTACTGACCAGGGAGCT-3′ and 5′-TCATTGTCCCTGTTGCTGTCC-3′; α-SMA, 5′-GCTGGTGATGATGCTCCCA-3′ and 5′-GCCCATTCCAACCATTACTCC-3′; CTGF, 5′-GGCCTCTTCTGCGATTTCG-3′ and 5′-GCAGCTTGACCCTTCTCGG-3′; Col1a1, 5′-CCAAGAAGACATCCCTGAAGTCA-3′ and 5′-TGCACGTCATCGCACACA-3′; Fn1, 5′-GTGTAGCACAACTTCCAATTACGAA-3′ and 5′-GGAATTTCCGCCTCGAGTCT-3′; TNF-α, 5′-AAACCACCAAGTGGAGGAGC-3′ and 5′-ACAAGGTACAACCCATCGGC-3′; IL-6, 5′-CGTGGACCTTCCAGGATGAG-3′ and 5′-CATCTCGGAGCCTGTAGTGC-3′; IL-1β, 5′-TGTAATGAAAGACGGCACAC-3′ and 5′-CTCCACTTTGCTCTTGACTTC-3′; VEGF, 5′-GCACATAGAGAGAATGAGCTT-3′ and 5′-CCCTCCGCTCTGAACAAGGCT-3′; PDGF-BB, 5′-TCCGGCTGCTGCAATAACC-3′ and 5′-GGCTTCTTTCGCACAATCTCAAT-3′; IGF-1, 5′-TCTGAGGAGGCTGGAGATGT-3′ and 5′-GTTCCGATGTTTTGCAGGTT-3′; Ang-1, 5′-ATCTTGATAACCGCAGCCAC-3′ and 5′-TGTCGGCACATACCTCTTGT-3′; bFGF, 5′-CCAGTTGGTATGTGGCACTG-3′ and 5′-CAGGGAAGGGTTTGACAAGA-3′; GAPDH, 5′-TGCCCAGAACATCATCCCT-3′ and 5′-GGTCCTCAGTGTAGCCCAAG-3′; and 18S, 5′-GTAACCCGTTGAACCCCATT-3′ and 5′-CCATCCAATCGGTAGTAGCG-3′.
Western blot analysis
Protein lysates were processed for western blot analysis following the standard protocol [21, 22]. The following primary antibodies were used to recognize the proteins: p-Akt (Ser473), Akt, p-GSK-3β (Ser9), GSK-3β (Cell Signaling Technology), Fstl1 (R&D), Vimentin (Abcam), α-SMA, and GAPDH (Santa Cruz). Immunoreactivity was detected by routine enzymatic chemiluminescence.
Data were expressed as mean ± SEM. Statistical analysis was performed using ANOVA for multiple comparisons and two-tailed Student’s t test for comparisons between the two groups. P < 0.05 was considered statistically significant.
Fstl1 expression declines dramatically in hypoxic MSCs
To unveil the potential role of Fstl1 in hypoxic MSCs, a time-course study was conducted on 0, 24, and 48 h to quantify its expression pattern. Notably, a rather dramatic decline of Fstl1 expression was observed in a time-dependent manner. As illustrated in Fig. 1h, Fstl1 expression in hypoxic MSCs is decreased to 23.71% at 24 h (P < 0.001 vs 0 h) and to 8.04% at 48 h (P < 0.001 vs 0 h).
Efficient transduction of functionally active Fstl1 in MSCs
Fstl1 enhances survival and proliferation of MSCs against hypoxic challenge
Fstl1 promotes retention of engrafted MSCs in ischemic myocardium
MSCs-Fstl1 implantation preserves post-MI heart function more effectively
MSCs-Fstl1 ameliorates fibroblasts accumulation and ECM production in ischemic myocardium
MSCs-Fstl1 treatment reduces myocardial infiltration of inflammatory cells in peri-infarct zones
Macrophages play both beneficial and detrimental roles in the wound healing process after MI [29, 30]. It is well documented that polarized macrophages can be classified mainly into two different phenotypes: proinflammatory (M1) and anti-inflammatory (M2). To further validate whether altered macrophage polarization is involved in MSCs-Fstl1-treated ischemic myocardium, we measured mRNA levels of both M1 (iNOS, CD80) and M2 (Argnase-1, CD206) markers on post-therapy 7 days. As illustrated in Additional file 6: Figure S6, expression of iNOS, CD80, Argnase-1, and CD206 all remain unchanged between MI/MSCs-mCherry and MI/MSCs-Fstl1, suggesting that MSCs-Fstl1 regulates post-MI cardiac remodeling via a mechanism that may be distinct from macrophage polarization.
MSCs-Fstl1 transplantation accelerates neovascularization in peri-infarct region
Stem cell therapy for the repair of damaged myocardium has evolved into a promising treatment for ischemic heart diseases. MSC-based therapy, originating from BM-, adipose tissue-, or umbilical cord-cells, continues to gain consent and appeal, because of the large body of preclinical evidence supporting higher paracrine cardio-reparative potential . MSC-mediated tissue repair has been reported to dampen inflammation as well as promote neovascularization in ischemic myocardium through paracrine mechanisms [34, 35]. However, poor survival of donor cells and failure of their subsequent engraftment occur within the first days after delivery, posing a significant challenge in the field . Finding a new method to improve survival and engraftment of MSCs in the injured myocardium is therefore imperative to optimize their therapeutic application.
There are several major findings in this study. First, intrinsic Fstl1 expression in MSCs declines dramatically following hypoxia. Second, MSCs-Fstl1 is more tolerant than MSCs-mCherry to the hypoxic challenge. Third, compared with MSCs-mCherry, MSCs-Fstl1 exhibits better retention and pro-angiogenic capacity following MI. Finally, MSCs-Fstl1-mediated cardiac repair is associated with reduced post-MI fibroblasts accumulation, ECM deposition, and inflammatory cell infiltration.
As a pro-survival cellular factor, we and others have demonstrated that Fstl1 inhibits cell death of cardiomyocytes , H9c2 , and endothelial cells  under various conditions. We validated here that restoration of intrinsic Fstl1 in MSCs improves their behavior and subsequent in vivo engraftment. In accordance with our observations, Holmfeldt et al. identified Fstl1 as one of the 17 novel regulators of hematopoietic stem cell repopulation . Additionally, periodic secretion of Fstl1 by feeder cells also facilitates telomere maintenance and long-term self-renewal of mESCs by enhancing sporadic Zscan4 expression . Importantly, we also validated activation of the pro-survival Akt/GSK-3β signaling in hypoxic MSCs-Fstl1, consistent with the previous report that retroviral-mediated overexpression of Akt1 also enhances survival of MSCs in an ischemic setting .
Various, seemingly contradictory effects of Fstl1 on cell proliferation and growth have been reported. On one hand, the results of our study demonstrated that Fstl1 promotes MSCs proliferation under hypoxic conditions (12.30% versus 3.00% in MSCs-mCherry). Similarly, restoration of the epicardial Fstl1 also enhances proliferation of immature cardiomyocytes, and consequently, activates regeneration of the adult mammalian heart, and reverses post-MI remodeling . On the other hand, Fstl1 seems to inhibit pathological cell proliferation and therefore benefit proliferative diseases. For example, Fstl1 inhibits proliferation and migration of vascular smooth muscle cells  and, consequently, attenuates neointimal formation in response to arterial injury through an AMPK-dependent mechanism . Moreover, Fstl1 has also been identified as a tumor suppressor in ovarian and endometrial tumors partially through inhibition of cell proliferation, migration, and invasion .
In conclusion, our current data demonstrated a favorable role of Fstl1 in stem cell-based therapy for experimental myocardial infarction. Fstl1 enhances survival and engraftment of transplanted cells, thereby promoting neovascularization as well as alleviating myocardial ECM deposition and inflammatory cell infiltration in ischemic hearts. Our data support the promise of Fstl1-overexpressing MSCs as a novel strategy to improve MSCs-based therapy for ischemic diseases.
This work was supported by Jiangsu Province’s Key Discipline / Laboratory of Medicine (XK201118), National Key R&D Program of China (2017YFA0103700), National Natural Science Foundation of China (NSFC-81770258), Science and Technology Project of Suzhou (SYS201705), National Clinical Key Specialty of Cardiovascular Surgery, Jiangsu Clinical Research Center for Cardiovascular Surgery (BL201451), National Natural Science Foundation of China (No. 31500944), Natural Science Foundation of Jiangsu Province (BK20150687 and BK20160321), and Taishan Scholar Project of Shandong Province of China (tsqn20161066 to Wencheng Zhang).
Availability of data and materials
All data generated or analyzed during this study are included in this published article.
WC and ZS conceived the project. HS, GC, YL, YS, and ZZ were responsible for the experimental design and application. JL performed the myocardial infarction surgery. WC wrote and revised the manuscript. WY standardized the figures. GX and XZ provided technical support. YZ, WZ, and ZH provided valuable suggestions for the manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate
All animal experiments were approved by the Ethics Committee of Soochow University (Ref: SZUM2008031233) and were conducted according to institutional animal ethics guidelines for the Care and Use of Research Animals established by Soochow University, Suzhou, China.
Consent for publication
The authors declare that they have no competing interests.
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