Aim: To determine the therapeutic effect of thy- mosin β4 (Tβ4) for treatment of ischemic limb disease in a mouse model. Methods: A mouse model of hindlimb ischemia was created by permanent ligation of femoral arteries and internal iliac artery. Tβ4 was dissolved in sterile saline and intramuscularly injected into the centre and periphery of ligation area in the treatment group (n = 10) starting from the surgery day until 4 weeks after surgery, while control animals received saline injection only (n = 9). All animals were sacrificed at 6 weeks after surgery and used for immunohistochemistry studies. Results: Tβ4 stimulated angiogenesis was evidenced by increased vascular density based on CD31 immunostaining, which was sig- nifycantly increased in Tβ4 group (562.5 ± 78.4/mm2) as compared with control group (371.1 ± 125.7/mm2; p < 0.05). The arteriole density based on CD31 and SMA dual immunostaining was similar between the Tβ4 (27.2 ± 16.9/mm2) and control (35.3 ± 6/mm2; p > 0.05) groups. Tβ4 increased Pax3/7+ skeletal muscle progenitor cell density. Pax3/7+ cell density of Tβ4 group (13.7% ± 2%) was significantly higher than that of the control group (4.3% ± 1.6%, p < 0.05). However, the numbers of central nuclei fiber and central nuclei per fiber were insignificantly increased in Tβ4 group as compared to control group. The numbers of central nuclei fiber were 8.9 ± 2.1 and 9.5 ± 1.6, and the central nuclei per fiber were 0.25 ± 0.07 and 0.48 ± 0.09 for control and Tβ4 groups, respectively. Conclusions: This preliminary study suggests that localized delivery of Tβ4 increased angiogenesis and skeletal muscle progenitor cell density in ischemic skeletal muscle, but failed to promote skeletal muscle regeneration.
Thymosin β4 (Tβ4), a ubiquitous 43 amino acid, is a 5 kDa polypeptide that plays an important role in mediating cell survival, migration, and proliferation [
In addition, Tβ4 has wound-healing and tissue-repair properties, which is associated with its ability to inhibit apoptosis and inflammation [5-8]. It can promote dermal healing in normal animals as well as in impaired models of dermal wound healing, including aged mice, diabetic mice, and steroid-treated rats [
Studies have shown that Tβ4 is up-regulated in regenerating muscle fibers after an injury [
All animals were handled in compliance with the Guide for the Care and Use of Laboratory Animals approved by the Institutional Animal Care and Use Committee at the National University of Singapore (NUS), Singapore. All animals were maintained by research animal resource (RAR) of NUS.
Eight-week old female Balb/C mice were purchased from the Centre for Animal Resources of NUS and were housed in vented cage system with a 12 hour light-dark cycle and received standard mouse chow. Balb/c mouse is a laboratory-bred strain of the house mouse and is one of the most widely used inbred strains used in animal experimentation. Mouse hind limb ischemia model was produced in 19 Balb/C mice (18 - 20 grams body weight) by permanent ligation of femoral artery [
Tβ4 was dissolved in sterile saline to a concentration of 2 µg/mL. Starting from the surgery day, 0.1 mL saline with Tβ4 was injected intramuscularly into the centre and periphery of femoral artery ligation area in the treatment group (n = 10), while control animals received 0.1 mL saline injection (n = 9). All animals received daily injection until 4 weeks after surgery. Then animals were sacrificed at 6 weeks after treatment and used for immunohistochemistry studies. No injection was made during the last two weeks. The aim of the additional two weeks was to exclude the immune reactions associated with injection, which may compromise the immunohistochemistry study of skeletal muscle.
Mouse medial thigh muscle was explanted. Cryo-sections of 6 µm thickness were cut and used for fluorescence immunostaining. To quantify the muscle fibers with central nuclei, tissue sections were fluorescence immunostained with rabbit anti-dystrophin (Santa Cruz Biotech, USA) and 4,6-diamidino-2-phenylindole (DAPI, Sigma Aldrich, USA). To quantify the undifferentiated skeletal muscle progenitor cells, mouse tissue sections were dual immunostained with goat anti-Pax3/7 (Santa Cruz Biotech, USA) and rabbit anti-dystrophin. Eight non-over-laping microscopic fields/muscle (n= 5 animal each group) were calculated in control and Tβ4 group muscle sections.
Dual fluorescence immunostaining for CD31 (Santa Cruz Biotech, USA) and smooth muscle actin (SMA) (Sigma Aldrich, USA) was carried out to quantify vascular density at 6 weeks after treatment. For each tissue section, the number of blood vessels was counted from 4 - 8 randomly selected microscopic fields at 200× magnification. A total of 25 tissue sections from five animals per group were used to measure the final average vascular density.
All data are expressed as mean ±SED. All statistical analyses were performed with SPSS (version 17). The difference between groups was analyzed by the method of student t-test. All tests were performed with a significance level of 5%.
There was no significant change of body weight between groups: they were 18.7 ± 0.4 g and 19 ± 0.4 g of control and Tβ4 group animals before treatment, respectively and increased to 19.6 ± 0.5 g and 19.2 ± 0.3 g after treatment. All mice survived the experiments. However, two mice in Tβ4 group each had one nail fall off from the ischemic limb.
The vascular density based on CD31 immunostaining was significantly increased in Tβ4 group (562.5± 78.4/mm2) as compared with control group (371.1 ± 125.7/mm2; p < 0.05) at 6 weeks after treatment (Figures 1(A), (D) and (G)). The arteriole density based on CD31 and SMA dual immunostaining was similar between the Tβ4 group (Tβ4 group=27.2 ± 16.9/mm2) and control group (35.3 ± 6/mm2; p>0.05) (Figures 1(B), (C), (E), (F) and (H)). This suggests that Tβ4 increases angiogenesis only.
Skeletal muscle histology demonstrated that Tβ4 insignificantly increased number of central nucleated fiber and central nuclei per fiber compared to control mouse (
Immunostaining for Pax3/7 expression showed that Tβ4 significantly increased Pax3/7+ nuclei (13.7 ± 2%) of total nuclei as compared to control group (4.3 ± 1.6%) per high magnification field (p < 0.05) (
The current preliminary study demonstrates that Tβ4 increased angiogenesis and skeletal muscle progenitor cell density, but failed to promote skeletal muscle regeneration in ischemic skeletal muscle.
Tβ4 is the most abundant member of the β-thymosin family in mammalian tissue [
Our previous study demonstrated that Tβ4 increased human skeletal myoblast (hSkM) migration, proliferation, and decreased cell injury under hypoxia in vitro [
Significantly increased Pax3/7 expressing nuclei were found in Tβ4 treated skeletal muscle. This indicates that Tβ4 increased Pax3/7 expressing progenitor cell density in ischemic limb muscle. Recent studies have identified Pax3+/Pax7+ somatic cells as the source of muscle progenitors [17,18]. Pax3+/Pax7+ cells are the common progenitors responsible for all embryonic, fetal, and adult myogenesis in axial and limb muscles. Pax3 is sufficient to induce MyoD and Myf5 in vitro [
Though Pax3/7 progenitor cell density was increased, there is no significantly increased skeletal muscle regeneration between Tβ4 and control groups. The central nucleated fiber and the central nuclei per fiber of Tβ4 were insignificantly increased as compared with control group. This finding is inconsistent with Spurney’s study [
In summary, the current study demonstrates that Tβ4 stimulates angiogenesis and increased skeletal muscle progenitor cell density, but failed to increase regenerating muscle fiber in ischemic limb muscle. Further study should be performed to investigate the underlying mechanism that the role of Tβ4 on homing and migrating of Pax3/7+ muscle progenitor cells has.
The project was funded by Singapore National Medical Research Council (NMRC) grant EDG09may058.