Concomitant overexpression of triple antioxidant enzymes selectively increases circulating endothelial progenitor cells in mice with limb ischaemia

Abstract Endothelial progenitor cells (EPCs) are a group of heterogeneous cells in bone marrow (BM) and blood. Ischaemia increases reactive oxygen species (ROS) production that regulates EPC number and function. The present study was conducted to determine if ischaemia‐induced ROS differentially regulated individual EPC subpopulations using a mouse model concomitantly overexpressing superoxide dismutase (SOD)1, SOD3 and glutathione peroxidase. Limb ischaemia was induced by femoral artery ligation in male transgenic mice with their wild‐type littermate as control. BM and blood cells were collected for EPCs analysis and mononuclear cell intracellular ROS production, apoptosis and proliferation at baseline, day 3 and day 21 after ischaemia. Cells positive for c‐Kit+/CD31+ or Sca‐1+/Flk‐1+ or CD34+/CD133+ or CD34+/Flk‐1+ were identified as EPCs. ischaemia significantly increased ROS production and cell apoptosis and decreased proliferation of circulating and BM mononuclear cells and increased BM and circulating EPCs levels. Overexpression of triple antioxidant enzymes effectively prevented ischaemia‐induced ROS production with significantly decreased cell apoptosis and preserved proliferation and significantly increased circulating EPCs level without significant changes in BM EPC populations, associated with enhanced recovery of blood flow and function of the ischemic limb. These data suggested that ischaemia‐induced ROS was differentially involved in the regulation of circulating EPC population.

tions using a mouse model concomitantly overexpressing superoxide dismutase (SOD)1, SOD3 and glutathione peroxidase. Limb ischaemia was induced by femoral artery ligation in male transgenic mice with their wild-type littermate as control. BM and blood cells were collected for EPCs analysis and mononuclear cell intracellular ROS production, apoptosis and proliferation at baseline, day 3 and day 21 after ischaemia. Cells positive for c-Kit + /CD31 + or Sca-1 + /Flk-1 + or CD34 + /CD133 + or CD34 + /Flk-1 + were identified as EPCs. ischaemia significantly increased ROS production and cell apoptosis and decreased proliferation of circulating and BM mononuclear cells and increased BM and circulating EPCs levels. Overexpression of triple antioxidant enzymes effectively prevented ischaemia-induced ROS production with significantly decreased cell apoptosis and preserved proliferation and significantly increased circulating EPCs level without significant changes in BM EPC populations, associated with enhanced recovery of blood flow and function of the ischemic limb.
These data suggested that ischaemia-induced ROS was differentially involved in the regulation of circulating EPC population.

| INTRODUC TI ON
Formation of new blood vessels (neovascularization) is an important mechanism in response to ischemic injuries/conditions such as ischemic heart disease and peripheral artery disease. 1,2 Bone marrow (BM)-derived endothelial progenitor cells (EPCs) play a critical role in vascular re-endothelialization, angiogenesis and prevention of neointima formation after vascular injury. 1,2 However, EPCs are a group of very heterogeneous cell population with a variety of different cell markers reported in the literature. 3 There are also multiple sources for EPCs with BM and blood as the two major EPC sources. 4 Reactive oxygen species (ROS) such as superoxide anion (O 2 -) and hydrogen peroxide (H 2 O 2 ) are critically involved in cell growth, migration, differentiation, apoptosis and senescence. 5,6 It has been reported that increased amounts of ROS is produced in response to tissue ischaemia. 8 Although high concentrations of ROS are involved in senescence and apoptosis of endothelial cells and stem/progenitor cells and associated with defective neovascularization, 9,10 low levels of ROS generated during tissue ischaemia serve as intracellular signals to trigger angiogenesis. 11,12 The effects of ROS following limb ischaemia on EPCs are reported to be mainly through Nox2containing NADPH oxidase. 10,13,14 However, the changes in specific subpopulations of EPCs in response to ROS formation during limb ischaemia have not been studied. It has been reported that ROS production in BM derived mononuclear cells is associated with the number of EPC. 14 Many antioxidant enzymes including copper-zine superoxide dismutase (SOD1) in the cytoplasm, 16 extracellular SOD (SOD3) 17,18 and glutathione peroxidase (Gpx-1) 19, 20 have been individually reported to have a beneficial effect on the mobilization of EPCs and neovascularization after limb ischaemia. However, it has been reported that SOD overexpression could be associated with increased levels of hydrogen peroxide (H 2 O 2 ) with increased oxidative stress in vitro and in vivo. 21  mice as the control as detailed previously. 27 Successful creation of the TG mice was confirmed with genotyping ( Figure S1A) and increased protein expression of the enzymes (using western blot analysis) ( Figure S1B) as well as increased enzyme activities as described. 22,23 Of note, the antibodies for SOD-1 (Invitrogen, Catalogue: MA1-105), SOD-3 (Santa Cruz, Catalogue: sc-376948) and Gpx-1 (Invitrogen, Catalogue: 702762) used for the present study were cross-reactive with the respective proteins from both human and mouse. Thus, the basal expression levels for these three enzymes were detectable in the wild-type littermate ( Figure S1B).

| Hind limb ischaemia and blood flow measurement
FAL was performed to produce hind limb ischaemia in the mice as described. 28 Mice were anaesthetized with one dose of Ketamine 100 mg/kg (0.1 mg/g, ip) mixed with Xylazine 20 mg/ kg (0.02 mg/g, ip). 1.25% isoflurane/O 2 was inhaled to induce anaesthesia. The hind limbs were depilated. The animal's body temperature was maintained at 37 ± 0.5°C. The left femoral artery was exposed through a 2-mm incision without retraction and with minimal tissue disturbance. A 7-0 ligature was placed distal to the origin of the lateral caudal femoral and superficial epigastric arteries (the latter was also ligated) and proximal to the genu artery. The femoral artery was transected between the sutures and separated by 1-2 mm. The wound was irrigated with saline and closed. Laser Doppler perfusion imaging (LDPI, Moor Instruments, Devon, UK) was used to determine the total local blood perfusion in the limbs pre-operatively, immediately post-operatively and at day 3, 7, 14 and 21 post-operatively.
Excessive hair was removed from the limbs before imaging and the mice were placed on a heating pad at 37°C to minimize temperature variation. Right limb blood flow was also measured as control. The ratio of blood flow (left ischemic limb blood flow/ right normal limb blood flow) was used to monitor the blood flow recovery.

| Treadmill performance
Wild-type C57BL/6J and TG male mice were subjected to a treadmill running test following a protocol modified from that of Massett and Berk. 29 Mice were placed on a rodent treadmill equipped with an electric grid at the rear and were made to run continuously until exhaustion at day 7, 14 and 21 after limb ischaemia (indicated by falling on the electric grid twice) and the running time on the treadmill was recorded. Two separate trials with eight WT and eight TG mice per trial were conducted. Both TG and WT mice were able to run more than 400 minutes at baseline before limb ischaemia. scribed. 30,31 Capillaries were counted using the Image J software.

| Intracellular ROS detection
BM and blood cells were harvested from both WT and TG mice at day 3 and 21 after limb ischaemia. Healthy and age-matched mice without limb ischaemia were used as control. Red blood cells (RBC) were eliminated using RBC lysis as described. 33 The level of intracellular ROS formation in blood cells after limb ischaemia was determined using the ROS Detection Reagents-FITC (Invitrogen) as described. 34 The cells were incubated with the reagent for 10 minutes at 37°C. The labelled cells were washed twice with PBS and then suspended in warm PBS for analysis using flow cytometry. The fluorescence-positive cells were quantitatively evaluated using an LSRII (BD Bioscience, CA) at a wavelength of 525 nm as described. 35

| Analysis of mouse EPC populations and mononuclear cell apoptosis and proliferation
To determine the effect of limb ischaemia on the population of EPCs, BM and blood cells were harvested in the mice at day 3 and 21 after limb ischaemia. Healthy and age-matched mice without limb ischaemia served as control. After elimination of RBC with RBC lysis buffer, multicolour analysis for BM and circulating EPCs was performed using an LSRII system (BD Biosciences, CA). A variety of cell surface markers and their combinations for identification of BM and circulating EPCs were used as described, 36,37 including CD34 + /Flk-1 + , Sca-1 + /Flk-1 + , c-Kit + /CD31 + and CD34 + /CD133 + . The cell populations were carefully compensated (each cell population percentile was further confirmed with single antibody staining and fluorescence minus one with the isotype antibody as the control) and applied to all samples. The total cell population was gated and each EPC population with specific doublepositive markers was analysed using flow cytometry as described. 43,44 All antibodies were obtained from Biolegend (San Diego, CA) except

| Statistical analysis
All the data were presented as means ± standard deviation (SD), two way ANOVA (analysis of variance) (PRISM Version 4.0.; GraphPad Software, Inc, San Diego, CA) followed by Bonferroni post-tests was used for comparing the subgroups of data from TG and WT mice to minimize type I error as appropriate for all data. The differences were considered statistically significant when a two-tailed P < 0.05.

| Concomitant overexpression of triple antioxidant enzymes significantly enhanced the recovery of blood flow and function of the ischemic limb
The recovery of blood flow and muscle function in the mice following acute hind limb ischaemia was evaluated. There was no measur-

| Concomitant overexpression of triple antioxidant enzymes effectively preserved the muscle fibres of the ischemic limb
As the induction of limb ischaemia leads to remarkable muscle degeneration, 48 the limb muscle morphology was examined. The muscle fibres reduced in size and became largely disconnected in the WT control mice with limb ischaemia. On the other hand, the muscle fibres appeared to remain long and large, as well as connected nicely in the limb muscles at day 21 after ischaemia in the TG mice with concomitant global overexpression of SOD1, SOD3 and Gpx-1 ( Figure 2). CD31 staining showed that the capillary density in the ischemic muscle gradually increased from day 3 to day 21 in WT mice ( Figure 2). Whereas in the TG mice, the muscle capillary density was significantly increased compared to the WT mice after limb ischaemia: 20.4 ± 1.1 (10 2 /mm 2 ) vs 16 ± 1.6 (10 2 /mm 2 ), 37 ± 2.9 (10 2 /mm 2 ) vs 20.4 ± 1.1 (10 2 /mm 2 ) and 48.2 ± 2.9 (10 2 /mm 2 ) vs 34.8 ± 2.4 (10 2 /mm 2 ) for day 3, 7 and 21 post ischaemia respectively (n = 8, P < 0.01). The capillary density appeared to be largely intact in the TG mice at day 21 after ischaemia (similar to the non-ischemic limb) ( Figure 2).

| Concomitant overexpression of triple antioxidant enzymes effectively prevented intracellular ROS formation after limb ischaemia
It is known that limb ischaemia significantly increases ROS production 49

| Concomitant overexpression of triple antioxidant enzymes selectively maintained circulating EPCs at elevated level without significant change in bone marrow EPC population after limb ischaemia.
To evaluate the role of ROS in the regulation of individual EPC populations, both BM and blood cells were collected to analyse the EPC populations. Flow cytometry analysis showed that BM EPC levels including CD34 + /Flk-1 + and Sca-1 + /Flk-1 + were significantly increased up to 7-and 4-fold at day 3 after ischaemia respectively, and stayed at the same levels up to day 21 in the WT mice (Figures 4 and 5). Although there was no change in the population of BM c-Kit + /CD31 + cells at day 3 after limb ischaemia, this cell population was significantly increased at day 21 ( Figure 4C). The population of BM CD34 + /CD133 + cells slightly increased at day 3 after ischaemia (0.02 ± 0.01%) and then dropped at day 21 (0.02 ± 0.01%) ( Figure 4D). Very interestingly, the populations of circulating Sca-1 + /Flk-1 + cells and CD34 + /CD133 + cells initially increased at day 3, but significantly decreased at day 21 ( Figure 4B,D). On the other hand, the CD34 + /Flk-1 + cell population stayed at an extremely low level shortly after limb ischaemia and only slightly increased at day 21 ( Figure 4A).
All circulating EPC populations in TG mice were significantly increased up to 3 folds at day 21 over the WT mice following hind limb ischaemia ( Figure 5). And the ischaemia-induced ROS production was also inhibited by TG mice (Figure 3). The blood c-Kit + /CD31 + cell population was elevated to 6.5 ± 1.13% in the TG mice (4.3 ± 1.1% in WT mice) at day 3 after ischaemia ( Figure 5C). However, there were no significant differences in the populations of BM EPCs including CD34 + /Flk-1 + , Sca-1 + /Flk-1 + and c-Kit + /CD31 + in TG mice compared to the WT mice ( Figure 4). However, CD34 + /CD133 + was significantly increased at day 3 after ischaemia and then decreased to the same level as WT mice at day 21 ( Figure 4D).

| Concomitant overexpression of triple antioxidant enzymes inhibited apoptosis and promoted proliferation of bone marrow and circulating mononuclear cells
To further explore the mechanisms on how AON in the TG mice maintained the circulating EPCs at a higher level compared to the WT mice, BM and blood cells were collected at day 3, 7 and 21 following limb ischaemia to analyse the mononuclear cell apoptotic and proliferation rate. As shown in Figure 6, in the BM, both early F I G U R E 4 Concomitant overexpression of triple antioxidant enzymes did not significantly change the bone marrow EPCs population after limb ischaemia. Flow cytometry analysis showed that bone marrow EPCs (BM EPCs) level including CD34 + /Flk-1 + and Sca-1 + /Flk-1 + were significantly increased up to 7-and 4-fold at day 3 after ischaemia respectively, and maintained at the same level at day 21 in WT mice (A and B). Although there was no change in the population of BM c-Kit + /CD31 + cells at day 3 after limb ischaemia, this cell population was significantly increased at day 21 (C). The population of BM CD34 + /CD133 + cells slightly increased at day 3 after ischaemia (0.02 ± 0.01%) and then dropped at day 21 (0.02 ± 0.01%) (D). However, no significant changes were observed in the populations of BM EPCs including the cells positive for CD34 + /Flk-1 + , Sca-1 + /Flk-1 + and c-Kit + /CD31 + in TG mice compared to the WT mice (A, B and C), except for CD34 + /CD133 + cells that were significantly increased at day 3 after ischaemia and then decreased to the same level as WT mice at day 21 (D). BL: blood. BM: bone marrow. *Day 3 or 21 vs Ctrl, P < 0.05, n = 8; $ Day 21 vs 3, P < 0.05, n = 8; #WT vs TG, P < 0.05, n = 8. *Day 3 or 21 vs Ctrl, P < 0.05, n = 8; $ Day 21 vs 3, P < 0.05, n = 8; #WT vs TG, P < 0.05, n = 8.

| D ISSCUSS I ON
In the present study, we demonstrated that limb ischaemia increased ROS production in both circulating and BM mononuclear cells and significantly increased EPCs levels in BM and blood as expected. Flow cytometry analysis showed that the CD34 + /Flk-1 + cell population stayed at an extremely low level shortly after limb ischaemia and only slightly increased at day 21 (A). On the other hand, circulating Sca-1 + /Flk-1 + cells and CD34 + /CD133 + cells were initially increased at day 3, but significantly decreased at day 21 (B and D). When ischaemia-induced ROS production was inhibited in TG mice, all the circulating EPCs populations in TG mice were significantly increased up to 3-fold at day 21 after ischaemia over the WT mice. The blood c-Kit + /CD31 + cell population was elevated to 6.5 ± 1.13% in the TG mice (4.3 ± 1.1% in WT mice) at day 3 after ischaemia (C). TG mice maintained circulating EPCs at elevated level compared to the WT mice. BL: blood. BM: bone marrow. *Day 3 or 21 vs Ctrl, P < 0.05, n = 8; $ Day 21 vs 3, P < 0.05, n = 8; #WT vs TG, P < 0.05, n = 8 in the circulating CD34 + /Flk-1 + cell population in mice. 23 On the other hand, fine particulate matter (PM 2.5 ) exposure could lead to a significant increase in ROS production and significant decrease in the population of circulating CD34 + /CD133 + cells that was effectively prevented when ROS formation was inhibited in mice. 56 In the present study, we observed a significantly increased ROS production after limb ischaemia that persisted for up to 21 days. The BM CD34 + /CD133 + , blood CD34 + /CD133 + and Sca-1 + /Flk-1 + cell populations were initially increased at day 3 after limb ischaemia, then Further studies are needed to address this complex situation.
Three antioxidant enzymes SOD1, SOD3 and Gpx-1 were reported to attenuate oxidative stress and might be essential for SCs and EPCs function and reparative neovascularization after ischaemia. 10,16,17 However, SOD1, SOD3 and Gpx-1 function as a team to reduce ROS production and minimize oxidative stress. Briefly, O 2 is readily dismutated by SOD into H 2 O 2 , which can then be converted into hydroxyl radicals by a Fenton-type reaction or scavenged by either catalase or Gpx-1. 22 However, there is a major concern that SOD overexpression itself could increase the level of hydrogen peroxide (H 2 O 2 ) with increased oxidative stress in vitro and in vivo, 21  one study showed that infusion of the antioxidant ebselen into WT mice significantly blocked the increase in blood flow and capillary density after ischaemia. 13 Clearly, more studies are needed to address this inconsistent observation in the future.
It is known that certain level of ROS could be proangiogenic.
However, excessive levels of ROS lead to vascular disease through direct and irreversible oxidative damage to macromolecules and disruption of redox-dependent vascular wall signalling processes. 60 It is also known that angiogenesis is a complex process and involves a vari- In conclusion, our data demonstrated that concomitant overexpression of SOD1, SOD3 and Gpx-1 significantly attenuated ischaemia-induced ROS production and selectively maintained an elevated level of the circulating EPCs, not the bone marrow EPCs, associated with enhanced recovery of blood flow and function of the ischemic limb. Further studies are needed to investigate the mechanisms for the selective beneficial effects of ROS reduction on circulating EPCs.

ACK N OWLED G EM ENTS
This work was supported by US NIH grants to ZL (NIH HL124122 and ES026200).

CO N FLI C T S O F I NTE R E S T
None.