FoxC1-Induced Vascular Niche Improves Survival and Myocardial Repair of Mesenchymal Stem Cells in Infarcted Hearts

Aims Forkhead box C1 (FoxC1) is essential for maintaining the hair follicle stem cell niche. The role of FoxC1 in maintaining mesenchymal stem cell (MSC) niches after myocardial infarction (MI) has not been directly determined to date. In this study, we determined to explore the possible roles and mechanisms of FoxC1 on MSC survival and function in the ischemic niche. Methods and Results MI model was established in this study, and expression level of FoxC1 was overexpressed or knocked down through efficient delivery of FoxC1 transfection or siFoxC1. Fifteen days later, the animals were allocated randomly to receive phosphate-buffered saline (PBS) injection or MSC transplantation. We identified FoxC1 as a key regulator of maintaining the vascular niche in the infarcted hearts (IHs) by driving proangiogenic and anti-inflammatory cytokines while repressing inflammatory and fibrotic factor expression. This vascular niche improved MSC survival and capacity in the IHs. Importantly, FoxC1 interacted with MSCs and was required for vessel specification and differentiation of engrafted MSCs in the ischemic niches, promoting myocardial repair. Inhibiting FoxC1 abolished these effects. Conclusion These results definitively implicate FoxC1 signaling in maintaining ischemic vascular niche, which may be helpful in myocardial repair induced by MSC therapy.


Analysis of the FoxC1-mediated niches
The base myocardial tissues were used for qRT-PCR, western blot, and immunohistochemistry evaluation of the expression of FoxC1 and proangiogenic cytokines (Ang-1, bFGF, VEGF). To quantify blood vessel density, additional slides were prepared from the animals in each subgroup. The sections were stained with anti-factor VIII antibody and counterstained with an immunoperoxidase kit (Vector Labs). Ten fields per slide were randomly chosen from infarct portions that were bordered by non-infarct portions along the LAD. Blood vessel density was expressed as the number of factor VIII + ECs per square millimeter 3 . The vessels were defined as round or elliptical structures with a central lumen lined by cells staining positively for factor VIII. A pathologist blinded to the group identities evaluated the capillary density by counting vessels in the selected areas.
Enzyme-linked immunosorbent assays (ELISA) 3 The levels of FoxC1, Ang-1, bFGF, and VEGF in supernatant of heart tissues were measured by ELISA using a duoset methodology (R&D Systems; Minneapolis, MN).
Briefly, after standard procedures, the tissue-free samples were pipetted into the wells of the microtitre plates, specific horseradish peroxidase-linked polyclonal antibodies were added and immunoreactive levels of FoxC1, Ang-1, bFGF, and VEGF were determined. Values below the detection limit were assumed as zero.

Real-time RT-PCR
Total RNA was isolated from homogenized heart tissue or cultured cells using TRIzol (Gibco BRL). The RNA was reverse-transcribed with a TaqMan cDNA Synthesis Kit (Applied Biosystems) and amplified on a TaqMan 7500 (Applied Biosystems).
Supplementary Tab. S1 lists the primer and probe sequences. Expression was calculated via the comparative threshold cycle (Ct) method and normalized to that of GDPDH, which was included as an internal control; thresholds for the individual reactions were determined using ABI Prism SDS 2.0 data processing software (Applied Biosystems).

Western blotting
To confirm the protein expression of the genes of interest, western blotting was performed as described previously 4

EGFP labelling
Before transplantation, MSCs were transfected with a lentiviral vector containing enhanced GFP (EGFP) cDNA, as described previously 7 . More than 70% of MSCs were EGFP-positive, as determined by flow cytometry. 5

Cell transplantation and animal grouping
15d after establishing the FoxC1-induced vascular niche models, the animals were randomized to receive PBS injection or MSCs therapy. The total volume of PBS or cell suspension injected was ~100 µL, and each animal in the cell treatment groups

Follow-up
All rats underwent echocardiography to evaluate cardiac functions before transplantation and at 30 days post-cell therapy, and were closely observed up to death to evaluate their survival curves.

Echocardiography
Under general anesthesia as described above, rats underwent echocardiography in a LVID is LV internal dimension, s is systole, and d is diastole. All measurements were averaged for three consecutive cardiac cycles.

MPO and ROS detection
At the end of each study, the heart was removed and divided into three transverse slices from the base, mid-region, and apex. The apical slices were homogenized in saline (per 100 mg tissue in 0.9 ml saline). After 15-min centrifugation at 3000 ×g, the supernatant was collected and stored at -70°C until used. Myocardium myeloperoxidase (MPO) and reaction oxygen species (ROS) were detected according to the manufacturer's instructions (A044, A018, NJBI). The peroxide-sensitive fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate (Molecular Probes) was used to assess the generation of intracellular ROS as described previously 8 . This compound is converted by intracellular esterases to 2′,7′-dichlorodihydrofluorescein (DCF), which is then oxidized by hydrogen peroxide to highly fluorescent DCF.
Differential interference contrast images were obtained simultaneously using an Olympus inverted microscope with ×40 Aplanfluo objective and an Olympus 7 fluoview confocal laser-scanning attachment. The DCF fluorescence was measured with an excitation wave length of 488 nm of light, and its emission was detected using a 510-550-nm bandpass filter. The change in MPO absorbance was measured at 460 nm using a spectrophotometer. One unit of MPO was defined as the quantity of enzyme required to hydrolyze peroxide at a rate of 1 mmol/min at 25°C.

Assessment of inflammation, myocardial infarct size, and collagen content
The mid-region areas in each group were used to evaluate the myocardial infarct size and collagen density by triphenyltetrazolium chloride (TTC) staining (Sigma) and Masson's trichrome stain as previously described 9 . The sections were stained at 37°C for 15 min with 1.5% TTC in PBS and fixed with 4% paraformaldehyde solution overnight. The respective TTC-stained (red, normal) and non-stained (white, infarct) areas were measured digitally using Image-Pro Plus 6.0 software (Media Cybernetics).
The percentage infarct area was used to estimate the extent of each myocardial infarct.
In Masson's trichrome staining, the collagen-rich myocardial scar in the infarcted wall stains blue, whereas viable myocardium stains red. The collagen density was expressed as the proportion of collagen deposition or scar tissue to normal LV myocardium. H&E staining was performed on each LV cross section to examine myocardial injury and inflammatory responses. Inflammation cells and viable myocardium were quantified from 4 separate fields/animal using Image-Pro Plus analysis software (version 6.2, MediaCybernetics, Bethesda, MD) and expressed as the percent area stained.
Immunohistochemistry, immunohistofluorescence, and immunocytofluorescence 8 The peri-infarct region was paraffin-embedded and cut into serial 10-µm slices. The sections were stained with antibodies against the following (Supplementary Tab. S2): FoxC1, Ang-1, bFGF, VEGF, factor VIII, IL-6, and MMP2, and counterstained with an immunoperoxidase kit (Vector Labs) or 4,6-diamidino-2-phenylindole (DAPI; Roche Diagnostics, Basel, Switzerland). A pathologist blinded to treatment group identity counted the vessels in 10 randomly selected fields per section at 10 sections per heart. Vascularity was expressed as the number of factor VIII + vessels per square millimeter 6 .
Light microscopy images were obtained for each coverslip. For immunocytofluorescence staining prepare a cell culture dish plate with glass cover slips and coated with laminin solution (final concentration 50 μg/ml laminin in PBS).
For immunofluorescence staining, a cell culture dish plate was prepared with glass cover slips and coated with laminin solution (final concentration 50 μg/ml laminin in PBS). The isolated myocytes or EGFP + cells were plated by using a microscope. The cells were cultured adhere to cover slip for 12h at 37 °C in 2 % CO 2 . Then, the solution was removed, and immunocytofluorescence staining was immediately started with a standard staining procedure protocol as previously described 10

FACS of engraftment, proliferation, differentiation of transplanted MSCs
Cells were collected from the left ventricles of five randomly selected hearts per experimental group, as previously described 4 . Briefly, the heart was excised and