Background/Aims: Prolonged fasting (PF) was shown to be of great potency to promote optimal health and reduce the risk of many chronic diseases. This study sought to determine the effect of PF on the endothelial progenitor cell (EPC)-mediated angiogenesis in the ischemic brain and cerebral ischemic injury in mice. Methods: Mice were subjected to PF or periodic PF after cerebral ischemia, and histological analysis and behavioral tests were performed. Mouse EPCs were isolated and examined, and the effects of EPC transplantation on cerebral ischemic injury were investigated in mice. Results: It was found that PF significantly increased the EPC functions and angiogenesis in the ischemic brain, and attenuated the cerebral ischemic injury in mice that was previously subjected to cerebral ischemia. Periodic PF might reduce cortical atrophy and improve long-term neurobehavioral outcomes after cerebral ischemia in mice. The eNOS and MnSOD expression and intracellular NO level were increased, and TSP-2 expression and intracellular O2- level were reduced in EPCs from PF-treated mice compared to control. In addition, transplanted EPCs might home into ischemic brain, and the EPCs from PF-treated mice had a stronger ability to promote angiogenesis in ischemic brain and reduce cerebral ischemic injury compared to the EPCs from control mice. The EPC-conditioned media from PF-treated mice exerted a stronger effect on cerebral ischemic injury reduction compared to that from control mice. Conclusion: Prolonged fasting promoted EPC-mediated ischemic angiogenesis and improved long-term stroke outcomes in mice. It is implied that prolonged fasting might potentially be an option to treat ischemic vascular diseases.

Stroke is one of the leading causes of mortality and permanent disability, with significant economic cost due to functional impairments [1]. Nowadays, urgent reperfusion of the ischemic brain by the thrombolytic drug tissue plasminogen activator (tPA) is the first target of stroke treatment, but only approximately 3% of the population suffering an ischemic stroke benefit from tPA, largely due to the drug's short therapeutic window [2]. Hence, it is imperative to develop potential new strategies targeting stroke treatment.

Endothelial progenitor cells (EPCs) are immature cells that can differentiate into mature endothelial cells, and could be recruited from bone marrow to the injury site to promote endothelial regeneration and neovascularization [3,4]. In addition, recent reports showed that EPCs may serve as a new marker for stroke outcomes, and EPCs have been used to successfully improve long-term stroke outcome in mice [3,4]. Hence it was implied that an improvement in EPC functions and EPC-mediated angiogenesis might potentially produce a protective action on ischemic stroke.

Dietary restriction could protect against age-related pathologies, like diabetes, neurodegeneration, cancer and cardiovascular diseases, in rodents and in human [5,6]. Especially, prolonged fasting (PF, lasting 48 to 120 hours) was implied to be more effective than calorie restriction or fasting lasting 24 hours or less, since PF could bring out more thorough switch in metabolism [7]. Cheng et al. showed that PF could promote hematopoietic stem cell-based regeneration and reverse immunosuppression from chemotoxicity in mice, in agreement with preliminary data on the protection of lymphocytes from chemotoxicity in fasting patients [8]. In addition, it was found that hematopoietic progenitor cells and endothelial cells could respond to granulocyte- macrophage colony-stimulating factor, thus improving vascularization in mice [9]. Thus, it can be logically speculated that PF might also promote the EPC functions and EPC-mediated angiogenesis.

On the basis of these findings, the present work sought to test the hypothesis that PF regimen may improve EPC-mediated angiogenesis and protect against cerebral ischemic injury in mice.

Animals

Male C57BL/6 mice (8 to 12 weeks old, 20-25g) were obtained from Sino-British SIPPR/BK Lab Animal Ltd (Shanghai, China). The animals were housed with controlled temperature (22-24°C) and lighting (8:00-20:00 light, 20:00-8:00 dark) and with free access to food and tap water. All animals received humane care, and the experimental procedures were in compliance with the institutional animal care guidelines. All the experiments were performed in a random and blind fashion.

Mouse stroke model and prolonged fasting

At 8 weeks of age, mice were subjected to permanent focal cerebral ischemia according to published protocols and our previous work [10,11]. Briefly, mice were anesthetized, and incised between the ear and the orbit on the left side. Then the stem of the left middle cerebral artery was exposed and occluded by electrocoagulation permanently. Body temperature was maintained at 37 ± 0.5°C by using a thermal blanket throughout the surgical procedure. The regional cerebral blood flow was monitored by Laser-Doppler flowmetry (moorVMS-LDF1, Moor Instruments Ltd, England) before and after occlusion. Mice with regional cerebral blood flow of more than 15% of the baseline were excluded.

Mice that endured cerebral ischemic injury were randomly divided to two groups: control (ad libitum diet for 4 days) and PF group. For PF group, mice were maintained on the ad libitum diet for 24 hours prior to its 48-hour PF treatment, followed by another ad libitum diet for 24 hours (Fig. 1A) [8]. No calories would be consumed during fasting: mice in any group had unlimited access to water; prior to the PF, animals were transferred into fresh cages to avoid feeding on residual chow and coprophagy.

Fig. 1

Prolonged fasting improved EPC functions in mice subjected to permanent focal cerebral ischemia. A. Illustration of experimental procedure. Mice were randomly divided to 2 groups. The Con group received ad libitum diet. For PF group, mice were maintained on the ad libitum diet for 24 hours prior to its 48-hour PF treatment, followed by another ad libitum diet for 24 h (Fig. 1A). EPCs were isolated at 4 days after cerebral ischemia, and cultured for 7 days. Then EPC functions were examined. B. EPC functions were improved in PF-treated mice compared with Con: the migration (a, b, g), tube formation (c, d, h), and adhesion (e, f, i) assays of EPCs **P<0.01 vs Con, n=9. Scale bar=100 µm. Con indicates control; PF indicates prolonged fasting.

Fig. 1

Prolonged fasting improved EPC functions in mice subjected to permanent focal cerebral ischemia. A. Illustration of experimental procedure. Mice were randomly divided to 2 groups. The Con group received ad libitum diet. For PF group, mice were maintained on the ad libitum diet for 24 hours prior to its 48-hour PF treatment, followed by another ad libitum diet for 24 h (Fig. 1A). EPCs were isolated at 4 days after cerebral ischemia, and cultured for 7 days. Then EPC functions were examined. B. EPC functions were improved in PF-treated mice compared with Con: the migration (a, b, g), tube formation (c, d, h), and adhesion (e, f, i) assays of EPCs **P<0.01 vs Con, n=9. Scale bar=100 µm. Con indicates control; PF indicates prolonged fasting.

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At 4 days after cerebral ischemia, the bone marrow-derived EPCs were isolated, cultured, and examined (Fig. 1A and 2A). In addition, the local angiogenesis in ischemic brain was assessed (Fig. 3A), Behavioral tests (including Body Asymmetry Test and Beam Test) were performed, and cerebral infarct volumes were determined (Fig. 4A).

Fig. 2

Prolonged fasting increased eNOS and MnSOD expression and intracellular NO level, and reduced TSP-2 expression and intracellular O2- level of EPCs in mice. A. Illustration of experimental procedure. Mice were randomly divided to 2 groups. The Con group received ad libitum diet. For PF group, mice were maintained on the ad libitum diet for 24 hours prior to its 48-hour PF treatment, followed by another ad libitum diet for 24 h. EPCs were isolated at 4 days after cerebral ischemia, and cultured for 7 days. Then EPCs and their culture supernatant were collected and examined. B. Western blot analysis showed that the expression levels of MnSOD (a) and eNOS (b) were increased in EPCs from PF-treated mice compared to control; **P<0.01 vs Con, n=13-14. The secreted TSP-2 (c) level was reduced in EPCs from PF-treated mice compared to control. **P<0.01 vs Con, n=8. C. a, Intracellular NO level in EPCs from PF-treated mice was enhanced compared with control. **P<0.01 vs Con, n=18. And intracellular superoxide level in EPCs from PF-treated mice was decreased compared with control. *P<0.05 vs Con, n=15. EPCs indicate bone marrow-derived EPCs; Con indicates control; PF indicates prolonged fasting.

Fig. 2

Prolonged fasting increased eNOS and MnSOD expression and intracellular NO level, and reduced TSP-2 expression and intracellular O2- level of EPCs in mice. A. Illustration of experimental procedure. Mice were randomly divided to 2 groups. The Con group received ad libitum diet. For PF group, mice were maintained on the ad libitum diet for 24 hours prior to its 48-hour PF treatment, followed by another ad libitum diet for 24 h. EPCs were isolated at 4 days after cerebral ischemia, and cultured for 7 days. Then EPCs and their culture supernatant were collected and examined. B. Western blot analysis showed that the expression levels of MnSOD (a) and eNOS (b) were increased in EPCs from PF-treated mice compared to control; **P<0.01 vs Con, n=13-14. The secreted TSP-2 (c) level was reduced in EPCs from PF-treated mice compared to control. **P<0.01 vs Con, n=8. C. a, Intracellular NO level in EPCs from PF-treated mice was enhanced compared with control. **P<0.01 vs Con, n=18. And intracellular superoxide level in EPCs from PF-treated mice was decreased compared with control. *P<0.05 vs Con, n=15. EPCs indicate bone marrow-derived EPCs; Con indicates control; PF indicates prolonged fasting.

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Fig. 3

Prolonged fasting enhanced angiogenesis in ischemic brain and attenuated cerebral ischemic injury in mice. A. Illustration of experimental procedure. Mice were first subjected to permanent focal cerebral ischemia, and then randomly divided to 2 groups. The control group received ad libitum diet. For PF group, mice were maintained on the ad libitum diet for 24 hours prior to its 48-hour PF treatment, followed by another ad libitum diet for 24 h. And the infarct volumes, neurobehavioral outcomes, and angiogenesis were assessed 4 days after ischemia. (Fig. 3A) B. The local angiogenesis in the ischemic brain in mice. a, CD31 immunostaining showed neo-microvessels in ischemic brain of mice treated with PF or control. b, The bar graph shows that the number of neo-microvessels in PF-treated mice was significantly increased compared with that in control mice. *P<0.05 vs Con, n=7. Scale bar= 50 µm. C. a, The representative images of 2,3,5-triphenyltetrazolium chloride (TTC)-stained brain sections. b, Cerebral infarct volumes. **P<0.01 vs con, n=9-11. c and d, Neurobehavioral outcomes (c, Body Asymmetry test; d, Beam Test). *P<0.05 vs Con, n=9-11. Con indicates control; PF indicates prolonged fasting.

Fig. 3

Prolonged fasting enhanced angiogenesis in ischemic brain and attenuated cerebral ischemic injury in mice. A. Illustration of experimental procedure. Mice were first subjected to permanent focal cerebral ischemia, and then randomly divided to 2 groups. The control group received ad libitum diet. For PF group, mice were maintained on the ad libitum diet for 24 hours prior to its 48-hour PF treatment, followed by another ad libitum diet for 24 h. And the infarct volumes, neurobehavioral outcomes, and angiogenesis were assessed 4 days after ischemia. (Fig. 3A) B. The local angiogenesis in the ischemic brain in mice. a, CD31 immunostaining showed neo-microvessels in ischemic brain of mice treated with PF or control. b, The bar graph shows that the number of neo-microvessels in PF-treated mice was significantly increased compared with that in control mice. *P<0.05 vs Con, n=7. Scale bar= 50 µm. C. a, The representative images of 2,3,5-triphenyltetrazolium chloride (TTC)-stained brain sections. b, Cerebral infarct volumes. **P<0.01 vs con, n=9-11. c and d, Neurobehavioral outcomes (c, Body Asymmetry test; d, Beam Test). *P<0.05 vs Con, n=9-11. Con indicates control; PF indicates prolonged fasting.

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Fig. 4

Periodic prolonged fasting ameliorated long-term neurobehavioral outcomes and reduced cortical atrophy after cerebral ischemia in mice. A. Illustration of experimental procedure. Mice were first subjected to permanent focal cerebral ischemia, and then randomly divided to 2 groups. The Con group received ad libitum diet. Periodic PF treatment was conducted by maintaining mice on a 48-h fasting every 7 days for 4 cycles. And the body weight was measured at day 1, 4, 7, 14, 21, and 28 after ischemia. The neurobehavioral outcomes were tested at day 4, 7, 14, 21, and 28 after ischemia. Cortical atrophy was determined at 29 days after ischemia. B. Neurobehavioral outcomes (a, Body Asymmetry test; b, Beam Test). *P<0.05, **P<0.01 vs Con, n=11-13. C, Periodic PF did not significantly alter the body weight of mice. D, 2,3,5-triphenyltetrazolium chloride (TTC)-stained brain sections showed less atrophy in the brains from PF-treated group compared to control. E, The bar graph shows that quantitative analysis of the cortical atrophy. **P<0.01 vs con, n=11-13. Con indicates control; PF indicates prolonged fasting.

Fig. 4

Periodic prolonged fasting ameliorated long-term neurobehavioral outcomes and reduced cortical atrophy after cerebral ischemia in mice. A. Illustration of experimental procedure. Mice were first subjected to permanent focal cerebral ischemia, and then randomly divided to 2 groups. The Con group received ad libitum diet. Periodic PF treatment was conducted by maintaining mice on a 48-h fasting every 7 days for 4 cycles. And the body weight was measured at day 1, 4, 7, 14, 21, and 28 after ischemia. The neurobehavioral outcomes were tested at day 4, 7, 14, 21, and 28 after ischemia. Cortical atrophy was determined at 29 days after ischemia. B. Neurobehavioral outcomes (a, Body Asymmetry test; b, Beam Test). *P<0.05, **P<0.01 vs Con, n=11-13. C, Periodic PF did not significantly alter the body weight of mice. D, 2,3,5-triphenyltetrazolium chloride (TTC)-stained brain sections showed less atrophy in the brains from PF-treated group compared to control. E, The bar graph shows that quantitative analysis of the cortical atrophy. **P<0.01 vs con, n=11-13. Con indicates control; PF indicates prolonged fasting.

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Bone marrow-derived EPCs isolation and culture

Bone marrow-derived EPCs were isolated, and cultured in vitro as previously described [12,13]. In brief, bone-marrow mononuclear cells were isolated from mouse tibia and femur, suspended in EGM-2 (complete growth medium), and seeded in 6-well culture plates pre-coated with rat vitronectin (1 µg/mL, Sigma) at a density of 5×106 cells /well. After 4 days of culture, the adherent cells were further cultured for another 3 days with non-adherent cells removed.

In vitro cell function assays

For adhesion assays: Cell suspensions (1×104) EPCs were cultured in 96-well plates pre-coated with 1µg/ml rat vitronectin (Sigma-Aldrich) and incubated for 4 h at 37 °C in 5% CO2. Non-adherent cells were washed away and the adherent cells were fixed with 2% paraformaldehyde. Nuclei were stained with Hoechst 33258 (5×10-6mol/L, 10 min, Sigma, America). Adherent cells were counted at random under 5 high-power fields (magnifications ×100) per sample, and the mean number of the wells was determined for each sample [13].

For migration assay: Migratory capacity of EPCs was investigated using the modified Boyden chamber assay. Cell suspensions (5×104) EPCs were cultured in upper chamber, which were placed in 24-well culture dishes containing M199 and 50 ng/ml vascular endothelial growth factor (VEGF). The chamber was incubated for 24h at 37 °C in 5% CO2. Migrated cells were stained with Hoechst 33258 (5×10-6 mol/l, 10 min, Sigma, America). The number of migratory EPCs was counted at random under 5 high power fields (magnifications ×100) per sample, five different areas was determined for each sample [12,13,14,15].

For tube formation assay: EPCs of 5×104 cells/100µL were seeded into 96-well plates pre-coated with Matrigel (60µL, BD Bioscience). After 6 hours of incubation at 37°C with 5% CO2, tube formation was counted in 5 random high-power fields magnification per chamber under ×100 magnification [12,13].

Intracellular nitric oxide (NO) and superoxide (O2-) measurement

Intracellular NO level was determined using membrane-permeable probes 4-amino-5- methylamine-2', 7'-difluorofluorescein (DAF-FM) diacetate (Molecular Probes) [12,13]. After 7 days of cultivation, bone marrow-derived EPCs were trypsinized and rinsed with EGM-2 twice, and then incubated with DAF-FM diacetate (10-6 mol/L) for 30 minutes at 37°C and an additional 30 minutes at room temperature in dark. After incubation, cells were washed with PBS twice and re-suspended in 300 µL 2% paraformaldehyde. The DAF-FM fluorescence intensity in cells was determined by flow cytometry.

Intracellular O2- level was determined using membrane-permeable dihydroethidium (DHE) (Molecular Probes) [12,13]. After 7 days of cultivation, bone marrow-derived EPCs were trypsinized and rinsed with EGM-2 twice, and then incubated with DHE (10-6 mol/L) for 30 minutes at 37°C in dark. After incubation, cells were washed with PBS twice and re-suspended in 300 µL 2% paraformaldehyde. The DHE fluorescence intensity in cells was determined by flow cytometry.

Western blot analysis

Cell homogenate was prepared as previously described [12,13]. The expression of eNOS and MnSOD was determined using western blotting. The following primary antibodies were applied: purified mouse anti-eNOS (BD Transduction Labs), mouse anti-MnSOD (BD Transduction Labs), at a dilution of 1:1000 overnight. Anti-mouse IgG secondary antibody (1:5000, CST) was used for both eNOS and MnSOD.

For TSP-2 secretion determination [13,16], the supernatant of EPCs was collected, condensed with an Amicon Ultra 4 centrifugal filter device with a 10,000 molecular weight cutoff (Millipore) according to the manufacturer's recommendations. The primary antibody used was mouse anti-TSP-2 (1:200, abcam Inc.) monoclonal antibody. Bands were visualized using Odyssey Imager with Odyssey 1.1 software (Li-Cor) and quantified using NIH image J software.

Infarct volume and neurobehavioral outcome assessment

To examine the neurobehavioral outcomes, body asymmetry test and beam test were performed at 4 days after cerebral ischemia with the examiner blind to the experimental groups, and then animals were euthanized and the brains were stained with 2,3,5-triphenyltetrazolium chloride (TTC) (Sigma, USA) to determine the infarct volume as previously reported (Fig. 3A) [10,11].

Body asymmetry test: the extent of head swing was recorded when being suspended by the tail of each mouse. The direction of the swing, either right or left, was recorded when the mice turned its head sideways by approximately at a 10° angle to the body's midline. After each swing, the mouse was allowed to move freely in a Plexiglas box for at least 30 seconds before taking the next test; the trials were repeated 20 times for each animal. The frequency of head swings toward the contralateral side was counted and normalized.

Beam test: One was beam walk test to assess motor coordination and balance after stroke injury. Mice were trained to traverse a narrow round beam (5 mm in diameter and 900 mm in length) to reach an enclosed escape platform. Mice were placed on one end of the beam and the latency to traverse the central 80% of the beam toward the enclosed escape platform at the other end was recorded. Data are expressed as mean latency to cross the beam (3 trials) [4]. The other was to measure motor asymmetry by elevated body swing test.

Histological and immunohistochemistry assessment

Mice were euthanized and the ischemic brains were fixed by transcardial perfusion with saline, followed by perfusion and immersion in 4% paraformaldehyde, before being embedded in paraffin [17]. A series of 6-µm-thick sections was cut from the block. Every 10th coronal section for a total three sections was used for immunohistochemical staining. Antibody against CD31 (BD Biosciences) immunostaining was performed to detect the angiogenesis in the ischemic brain (Fig. 3A) [18].

Mouse stroke model and periodic prolonged fasting

At 8 weeks of age, mice were subjected to permanent focal cerebral ischemia according to published protocols and our previous work [10,11], and the animals that endured cerebral ischemic injury were randomly divided to two groups: control (ad libitum diet) and periodic PF group. Periodic PF treatment was conducted by maintaining mice on a 48-h fasting every 7 days for 4 cycles (Fig. 4A) [8]. And the body weight was measured at day 1, 4, 7, 21, and 28 after cerebral ischemia. The neurobehavioral outcomes were tested at day 4, 7, 14, 21, and 28 after cerebral ischemia. Then, the cortical atrophy was determined at 29 days after cerebral ischemia (Fig. 4A).

Cerebral atrophy volume determination

Animals were euthanized after 29 days of permanent focal cerebral ischemia and the brains were stained with 2,3,5-triphenyltetrazolium chloride (TTC) (Sigma, USA) to determine cortical atrophy volume which were calculated by subtraction of the volume of ipsilateral hemisphere from the volume of contralateral hemisphere [4].

EPC transplantation and cerebral ischemic injury

EPCs were isolated from ad libitum diet and PF-treated mice, and were cultured for 7 days [12,13]. To track the injected EPCs in ischemic brain, the cells were labeled with 5-Bromo-2′-deoxyuridine (BrdU-labeling reagent, Invitrogen), as previously described [12]. Then the EPCs (1 × 106 cells/200µL PBS per mouse) were injected via the tail vein into mice just after cerebral ischemia, and control animals received equal volume of vehicle as previously described [4]. Briefly, mice were first subjected to permanent focal cerebral ischemia, and randomly divided to 3 groups (Fig. 5A and 6A). The vehicle group was intravenously injected with phosphate buffer saline (PBS), the control group was intravenously injected with EPCs from ad libitum diet-treated mice, and the PF group was injected with EPCs form PF-treated mice. The neurobehavioral outcomes were tested at 1 and 3 days after injection, and the infarct volume was measured at 3 days after injection (Fig. 6A) [4,10,11].

Fig. 5

Prolonged fasting increased the effect of EPCs on angiogenesis promotion in ischemic brain of mice. A. Illustration of experimental procedure. EPCs were isolated from ad libitum diet and PF-treated mice, 5-Bromo-2-deoxyUridine (BrdU) were added into culture medium at day 5 of culture. Mice were first subjected to permanent focal cerebral ischemia, and randomly divided to 3 groups. The vehicle group was intravenously injected with phosphate buffer saline (PBS), the Con-EPC group was intravenously injected with EPCs from ad libitum diet-treated mice, and the PF-EPC group was intravenously injected with EPCs form prolonged fasting-treated mice. Immunohistochemical staining was performed at 3 days after EPC transplantation. B, Photomicrographs show BrdU-labeled EPCs migrated into ischemic area after 3 days of injection. Inserted boxes in each picture show the large magnification of cells. Scale bar = 25µm. C, a, CD31 immunostaining shows neo-microvessels in ischemic brain of mice 3 days after injection. b, The bar graph shows that the number of neo-microvessels in ischemic brain of mice injected EPCs from PF-treated mice was significantly increased compared to the mice injected EPCs from ad libitum diet-treated mice or vehicle; **P<0.01, n=5. Scale bar = 50µm. Vehicle indicates phosphate buffer saline; Con-EPC indicates EPCs from ad libitum diet-treated mice; PF-EPC indicates EPCs from prolonged fasting-treated mice.

Fig. 5

Prolonged fasting increased the effect of EPCs on angiogenesis promotion in ischemic brain of mice. A. Illustration of experimental procedure. EPCs were isolated from ad libitum diet and PF-treated mice, 5-Bromo-2-deoxyUridine (BrdU) were added into culture medium at day 5 of culture. Mice were first subjected to permanent focal cerebral ischemia, and randomly divided to 3 groups. The vehicle group was intravenously injected with phosphate buffer saline (PBS), the Con-EPC group was intravenously injected with EPCs from ad libitum diet-treated mice, and the PF-EPC group was intravenously injected with EPCs form prolonged fasting-treated mice. Immunohistochemical staining was performed at 3 days after EPC transplantation. B, Photomicrographs show BrdU-labeled EPCs migrated into ischemic area after 3 days of injection. Inserted boxes in each picture show the large magnification of cells. Scale bar = 25µm. C, a, CD31 immunostaining shows neo-microvessels in ischemic brain of mice 3 days after injection. b, The bar graph shows that the number of neo-microvessels in ischemic brain of mice injected EPCs from PF-treated mice was significantly increased compared to the mice injected EPCs from ad libitum diet-treated mice or vehicle; **P<0.01, n=5. Scale bar = 50µm. Vehicle indicates phosphate buffer saline; Con-EPC indicates EPCs from ad libitum diet-treated mice; PF-EPC indicates EPCs from prolonged fasting-treated mice.

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Fig. 6

Prolonged fasting promoted the therapeutic effect of EPCs on cerebral ischemic injury in mice. A. Illustration of experimental schedule. EPCs were isolated from control and PF-treated mice, and cultured for 7 days. Mice were first subjected to permanent focal cerebral ischemia, and randomly divided to 3 groups. The vehicle group was intravenously injected with phosphate buffer saline (PBS), the Con-EPC group was intravenously injected with EPCs from ad libitum diet-treated mice, and the PF-EPC group was intravenously injected with EPCs form prolonged fasting-treated mice. The neurobehavioral outcomes were tested at 1 and 3 days after injection, and the infarct volumes were measured at 3 days after EPC injection. B, a, The representative images of 2,3,5-triphenyltetrazolium chloride (TTC)-stained brain sections 3 days after injection; b, Cerebral infarct volumes. *P<0.05, **P<0.01, n=12-13. c and d, Neurobehavioral outcomes (c, Body Asymmetry test; d, Beam Test). *P<0.05, **P<0.01, n=12-13. Vehicle indicates phosphate buffer saline; Con-EPC indicates EPCs from ad libitum diet-treated mice; PF-EPC indicates EPCs from prolonged fasting-treated mice.

Fig. 6

Prolonged fasting promoted the therapeutic effect of EPCs on cerebral ischemic injury in mice. A. Illustration of experimental schedule. EPCs were isolated from control and PF-treated mice, and cultured for 7 days. Mice were first subjected to permanent focal cerebral ischemia, and randomly divided to 3 groups. The vehicle group was intravenously injected with phosphate buffer saline (PBS), the Con-EPC group was intravenously injected with EPCs from ad libitum diet-treated mice, and the PF-EPC group was intravenously injected with EPCs form prolonged fasting-treated mice. The neurobehavioral outcomes were tested at 1 and 3 days after injection, and the infarct volumes were measured at 3 days after EPC injection. B, a, The representative images of 2,3,5-triphenyltetrazolium chloride (TTC)-stained brain sections 3 days after injection; b, Cerebral infarct volumes. *P<0.05, **P<0.01, n=12-13. c and d, Neurobehavioral outcomes (c, Body Asymmetry test; d, Beam Test). *P<0.05, **P<0.01, n=12-13. Vehicle indicates phosphate buffer saline; Con-EPC indicates EPCs from ad libitum diet-treated mice; PF-EPC indicates EPCs from prolonged fasting-treated mice.

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In addition, the histological and immunohistochemical staining was analyzed at 3 days after EPC transplantation (Fig. 5A). At 3 days after EPC injection, mice were euthanized and the ischemic brains were fixed by transcardial perfusion with saline, followed by perfusion and immersion in 4% paraformaldehyde, before being embedded in paraffin. A series of 6-µm-thick sections was cut from the block. Antibody against CD31 (BD Biosciences) immunostaining was performed to detect the angiogenesis in the ischemic brain [17,18]. To detect the in vivo EPC integration, slides were stained with anti-CD31 antibody (BD Biosciences), followed by BrdU antibody (Santa Cruz Bio-technology Inc.) incubation. The secondary antibodies were Alexa Fluor 488 (Abcam) or Cy3 (Abcam). The nucleus was counterstained with DAPI (Cell Signaling Technology) [12].

EPC-conditioned media (CM) therapy and cerebral ischemic injury

EPCs were isolated from ad libitum diet and PF-treated mice, and were cultured for 7 days [12,13]. At day 6 of culture, growing EPCs were washed twice with endothelial basal medium-2 without growth factors and serum (EBM-2, Lonza). Then, the fresh EBM-2 (1.5 mL/well) was added to obtain conditioned media (CM) that was collected 24 hours later. 4 mL CM was concentrated for 40 min using a 10 kDa filter unit (Millipore, Ireland), for a final volume of approximately 200 µl [19]. Vehicle (EBM-2) and control/PF-CM (200 µL of CM collected from control or PF-treated mice EPCs) were injected through the tail vein into the mice just after cerebral ischemia. The infarct volume was assessed, and the behavioral tests were conducted to evaluate the neurobehavioral outcomes at 1 day after CM injection (Fig. 7A) [4,10].

Fig. 7

Prolonged fasting promoted the therapeutic effect of EPC-conditioned media (CM) on cerebral ischemic injury in mice. A. Illustration of experimental schedule. EPCs were isolated from control and PF-treated mice, and the conditioned media (CM) were then used for in vivo experiments as indicated. Mice were first subjected to permanent focal cerebral ischemia, and randomly divided to 3 groups. The vehicle group was intravenously injected with EBM-2, the Con-CM group was intravenously injected with EPC-CM from ad libitum diet-treated mice, and the PF-CM group injected with EPC-CM form prolonged fasting-treated mice. The neurobehavioral outcomes and infarct volumes were assessed at 1 day after CM injection. B. a, The representative images of 2,3,5-triphenyltetrazolium chloride (TTC)-stained brain sections 1 day after CM injection. b, Cerebral infarct volumes. **P<0.01, n=12. c and d, Neurobehavioral outcomes (c, Body Asymmetry test; d, Beam Test). *P<0.05, **P<0.01, n=12. Vehicle indicates EBM-2; Con-CM indicates EPC-CM from ad libitum diet-treated mice; PF-EPC indicates EPC-CM from prolonged fasting-treated mice.

Fig. 7

Prolonged fasting promoted the therapeutic effect of EPC-conditioned media (CM) on cerebral ischemic injury in mice. A. Illustration of experimental schedule. EPCs were isolated from control and PF-treated mice, and the conditioned media (CM) were then used for in vivo experiments as indicated. Mice were first subjected to permanent focal cerebral ischemia, and randomly divided to 3 groups. The vehicle group was intravenously injected with EBM-2, the Con-CM group was intravenously injected with EPC-CM from ad libitum diet-treated mice, and the PF-CM group injected with EPC-CM form prolonged fasting-treated mice. The neurobehavioral outcomes and infarct volumes were assessed at 1 day after CM injection. B. a, The representative images of 2,3,5-triphenyltetrazolium chloride (TTC)-stained brain sections 1 day after CM injection. b, Cerebral infarct volumes. **P<0.01, n=12. c and d, Neurobehavioral outcomes (c, Body Asymmetry test; d, Beam Test). *P<0.05, **P<0.01, n=12. Vehicle indicates EBM-2; Con-CM indicates EPC-CM from ad libitum diet-treated mice; PF-EPC indicates EPC-CM from prolonged fasting-treated mice.

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Statistical Analysis

Data are expressed as mean ± SEM. Statistical significance of difference between 2 groups was performed using the Student's unpaired t test. When more than 2 groups were compared, one-way ANOVA followed by Tukey post hoc analysis was used. A value of P<0.05 was considered statistically significant.

PF regimen significantly enhanced EPC functions in mice

To testify whether PF could enhance EPC function, mice were fasted or fed an ad libitum diet (AL) and then bone marrow-derived EPCs were isolated for in vitro cell function assays (Fig. 1A). It was found that EPC function (including adhesion, migration, and tube formation functions) were significantly enhanced (+71.2%, +60.6%, +77.5%, P<0.01) in PF-treated mice compared with control (Fig. 1B).

To determine the potential mechanisms underlying the EPC protection in PF-treated mice, we assessed the eNOS, MnSOD and TSP-2 (a potent angiogenesis inhibitor) expression levels and intracellular NO and O2- levels of EPCs in mice (Fig. 2A). It was found that, compared with control, eNOS and MnSOD expression levels were significantly increased (+42.0%, +26.1%, P<0.01), while TSP-2 expression level was reduced (-49.4%, P<0.01) in EPCs from PF-treated mice (Fig. 2B). And intracellular NO levels was significantly increased (+29.1%, P<0.01) while O2- level decreased (-11.6%, P<0.05) in EPCs from PF-treated mice (Fig. 2C).

These results suggest that PF regimen could enhance EPC functions, increase eNOS and MnSOD expression and intracellular NO level, and decrease TSP-2 expression and intracellular O2- level of EPCs in mice.

PF enhanced angiogenesis in ischemic brain and attenuated cerebral ischemic injury in mice

To access whether PF can enhance local angiogenesis in ischemic brain and attenuate cerebral ischemic injury, mice were first subjected to permanent focal cerebral ischemia, and randomly allocated to 2 groups, then, the control group received ad libitum (AL) diet and the PF group were fed AL for 24 hours followed by fasting for 48 hours and the infarct volumes, neurobehavioral outcomes, and angiogenesis in ischemic brain were determined 4 days after ischemia (Fig. 3A). It was found that capillary density was significantly higher (+31.9%, P<0.05) in the PF-treated group compared with control (Fig. 3B). In addition, we found that the infarct volumes were significantly decreased (-30.7%, P<0.01) and the corresponding neurobehavioral outcomes were markedly ameliorated in the PF-treated group compared with control (Fig. 3C). These results suggested that PF for 48 hours was able to increase angiogenesis in ischemic brain and reduce cerebral ischemic injury in mice.

Periodic PF ameliorated long-term neurobehavioral outcomes and reduced cortical atrophy after cerebral ischemia in mice

To investigate the potential long-term neuroprotection of PF against ischemic brain injury, mice were first subjected to permanent focal cerebral ischemia, and randomly allocated to 2 groups, then, the control group received ad libitum diet and the PF group were fed 24 hours AL followed by multicycle fasting for 48 hours and the body weight and neurobehavioral functions of mice were tested at different days after ischemia, and the ischemic brains were collected 29 days after ischemia to access the cortical atrophy (Fig. 4A). We observed that there was no significant difference in body weights between PF-treated and control mice (Fig. 4C). Beam test showed that ischemia mice with multicycle PF-treated had significant improvements compared with control, which is confirmed by the body asymmetry test (Fig. 4B). The overall atrophy volume of the brain hemisphere in the multicycle PF-treated mice was significantly reduced (-31.7%, P<0.01) compared with the control group at 29 days after ischemia (Fig. 4C). These results indicated that periodic PF might improve long-term neurobehavioral outcomes and reduce cortical atrophy after cerebral ischemia in mice.

PF promoted the therapeutic effect of EPCs on cerebral ischemic injury in mice

To assess whether the enhancement of EPC function can promote local angiogenesis and reduce cerebral ischemic injury, EPCs from ad libitum diet-treated or PF-treated mice were labeled with 5-Bromo-2′-deoxyuridine (BrdU) and then intravenously injected into ischemic mice just after cerebral ischemia. The neurobehavioral outcomes were tested 1 and 3 days after EPC transplantation, and the local angiogenesis and cerebral infarct volumes were analyzed 3 days after EPC transplantation (Fig. 5A and 6A). Photomicrographs showed that some BrdU-labeled EPCs were localized with CD31 microvessels in the ischemic brain of mice that received EPC transplantation. It suggests that EPCs might home into the ischemic brain after 3 days of injection (Fig. 5B). In addition, it was found that capillary density in the ischemic brain was higher in PF-EPC group (injected with EPCs from PF-treated mice) compared to Con-EPC group (injected with EPCs from ad libitum diet-treated mice; +17.5%, P<0.01) or Vehicle group (injected with PBS; +37.1%, P<0.01; Fig. 5C).

Furthermore, we found that infarct volumes in PF-EPC group were significantly reduced compared to Con-EPC group (-22.7%, P<0.01) or vehicle group (-34.2%, P<0.01), and the corresponding neurobehavioral outcomes were markedly ameliorated in PF-EPC group compared with Con-EPC or Vehicle group at 1 and 3 days after cerebral ischemia (Fig. 6B).

These results suggested that transplanted EPCs might home into ischemic brain, and the EPCs from PF-treated mice had a stronger ability to promote angiogenesis in ischemic brain and reduce cerebral ischemic injury compared to the EPCs from ad libitum diet-treated mice.

PF promoted the therapeutic effect of EPC-conditioned media on cerebral ischemic injury in mice

To study the potential paracrine effect of EPCs on cerebral ischemic injury, the conditioned media (CM) of EPCs were collected and injected into mice via the tail vein just after cerebral ischemia (Fig. 7A). It was found that the infarct volumes were significantly decreased in the PF-CM group (injected with CM of EPCs from PF-treated mice) compared to the Con-CM group (injected with CM of EPCs from ad libitum diet-treated mice; -21.8%, P<0.01) or Vehicle group (injected with EBM-2; -38.5%, P<0.01), and the corresponding neurobehavioral outcomes were markedly ameliorated (Fig. 7B). These results suggested that the CM of EPCs from PF-treated mice had a stronger ability to reduce cerebral ischemic injury compared to the CM of EPCs from ad libitum diet-treated mice.

This study showed, for the first time, that PF regimen significantly promoted EPC function and EPC-mediated angiogenesis in mice.

Prolonged fasting inhibited progrowth signaling and activated pathways that enhance cellular resistance to toxins in mice and human [5,20,21]. The enhanced pathways included increased circulation and cardiovascular disease protection, and modulation of reactive oxygen species and inflammatory cytokines [5,6]. Our data indicated that PF intervention could significantly improve EPC functions in mice. The discovery of EPCs has dramatically altered the vision of postnatal revascularization, indicating that adult vessels could be repaired, not exclusively by recruiting, proliferation, and migration of neighboring mature and terminally differentiated ECs [22], but also by incorporation of bone marrow-derived EPCs in sprouting new blood vessel [23]. Several studies demonstrated that circulating EPCs are mobilized in response to ischemic stimuli and localized at the site of vascular damage where they proliferate, differentiate, and adhere to the vessel wall, promoting reendothelialization of damaged vessels and inducing angiogenesis in the ischemic areas [24,25,26,27]. Consistent with the enhanced EPC functions by PF, it was found that the intervention could also significantly increase local angiogenesis in the ischemic brain of mice.

Our data also indicate that (periodic) PF regimen could significantly ameliorate cerebral ischemic injury and improve long-term neurobehavioral outcomes after cerebral ischemia in mice. The infarct volumes were significantly decreased, and the corresponding neurobehavioral outcomes were markedly improved in PF-treated mice compared with control. Evidence is accumulating that angiogenesis was a major rejuvenation mechanism post-stroke [28,29,30]. The observed EPC function enhancement and subsequent increment of the local angiogenesis might partly contribute to the mitigated cerebral ischemic injury and the improved long-term neurobehavioral outcomes brought by PF regimen.

Previous reports showed that transplantation of exogenous EPCs were able to home to local injured tissue, promote vascular revascularization, and improve tissue repair and regeneration [31]. Innate EPCs might not provide sufficient supply for severe injuries, although they could be recruited into peripheral blood in response to ischemic injury. In addition, EPCs in the peripheral blood was also decreased in patients with ischemic stroke. Therefore, the rationale of EPC therapy in brain ischemia was to provide sufficient EPCs to participate in brain repairing by transplant exogenous EPCs [32]. We observed that intravenously transplanted EPCs homed into the ischemic site, and significantly reduced cerebral infarct volumes in mice. Notably, the EPCs from PF-treated mice had enhanced ability to migrate into ischemic zone, to promote angiogenesis, and to reduce cerebral ischemic injury, compared to the EPCs from control mice. The protection against ischemia was also proved by Xu et al. [33], who showed that recruitment of EPCs could stimulate the angiogenesis in transplanted autologous free fat tissue, thus preventing its ischemic necrosis. Besides, other type of stem cell also exhibited excellent proliferative capacity [34], implying the potency of progenitor cells for clinical application.

Other authors have already postulated that a novel cell-free therapeutic approach for angiogenesis with EPC-conditioned medium could be a potent alternative to progenitor cell therapies [35]. These authors demonstrated that an injection of EPC-CM was as effective as cell transplantation for promoting tissue revascularization in a rat model of chronic hindlimb ischemia [35]. Similar results have been reported for the treatment of diabetic dermal wounds. The injection of EPC-CM into wounded diabetic mice promoted wound healing and increased neovascularization to a similar extent to EPC transplantation [36]. In the present study, we demonstrated that cerebral ischemic injury was mitigated by the administration of EPC-CM in a mouse stroke model. In addition, we found that EPC-CM harvested from PF-treated mice was more effective in reducing cerebral ischemic injury compared to EPC-CM from control mice. Taken together, our data suggest that PF intervention could enhance the protective effect against cerebral ischemia of EPC-CM in an angiogenesis-independent route.

We further investigated the mechanisms underlying EPC function enhancement produced by PF intervention. A significant decrease of secreted TSP-2, a key inhibitor of endothelial cell and EPC function [37,38], was observed in BM-EPCs harvested from PF-treatment mice. It has been demonstrated in our previously study that decreased expression of eNOS and MnSOD, together with increased intracellular O2- and reduced intracellular NO levels, might be associated with EPC dysfunction [13]. In addition, Cui et al. demonstrated that apoptosis of EPCs could be associated with increased reactive oxygen species production [39]. We observed, in the present study, that PF treatment induced a significant increase in MnSOD and eNOS expression, and intracellular NO level and a reduction in intracellular O2- level. Thus, the decreased TSP-2 secretion, and the increased expression in MnSOD and eNOS, added by enhance NO bioavailability, might partly contribute to the enhanced EPC function by PF treatment.

In summary, PF intervention significantly improved EPC-mediated angiogenesis, and improved neurobehavioral outcomes in the long run after cerebral ischemia in mice. These results might not only extend the findings in fasting regimen, but also indicate a novel strategy to improve EPC functions and EPC-mediated angiogenesis, thereby providing a potential therapy for patients with ischemic vascular disease.

This work was supported by the National Key Basic Research Program of China (973 Program; 2014CB542403 to A.F. Chen and H.H. Xie), the National Natural Science Foundation of China (81370253 and 81170115 to H.H. Xie; 81603097 to X.H. Dong), the National Science Foundation of China Key Research Project (81130004 to A.F. Chen).

None.

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B. Xin and C.-L. Liu contributed equally to this work.

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