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Cardiovascular Research Advance Access first published online on January 10, 2008
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Cardiovascular Research, doi:10.1093/cvr/cvn002
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Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2008. For permissions please email: journals.permissions@oxfordjournals.org

The lysophospholipid mediator sphingosine-1-phosphate promotes angiogenesis in vivo in ischaemic hindlimbs of mice

Osamu Oyama1,2, Naotoshi Sugimoto1, Xun Qi1, Noriko Takuwa1,3, Kiyomi Mizugishi1,4, Junji Koizumi2 and Yoh Takuwa1,*

1 Department of Physiology, Kanazawa University Graduate School of Medicine, 13-1 Takara-machi, Kanazawa, Ishikawa 920-8640, Japan
2 General Medicine, Kanazawa University Graduate School of Medicine, Kanazawa, Ishikawa, Japan
3 Department of Health and Medical Sciences, Ishikawa Prefectural Nursing University, Kahoku, Japan
4 Japan Science and Technology Agency Innovation Plaza, Ishikawa, Japan

* Corresponding author. Tel: +81 76 265 2165; fax: +81 76 234 4223. E-mail address: ytakuwa{at}med.kanazawa-u.ac.jp

Received 16 July 2007; revised 25 December 2007; accepted 28 December 2007

Time for primary review: 36 days


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Funding
 References
 
Aims: The lysophospholipid mediator sphingosine-1-phosphate (S1P) acts on vascular endothelial cells to stimulate migration, proliferation, and capillary-like tube formation in vitro. It is unknown whether S1P stimulates in vivo angiogenesis induced under tissue ischaemia. We investigated the effects of both exogenously and endogenously overproduced S1P on post-ischaemic angiogenesis in murine hindlimbs.

Methods and results: The effects of locally injected S1P on blood flow recovery, angiogenesis, and vascular permeability in mouse ischaemic hindlimbs that underwent femoral arteriectomy were assessed by a laser Doppler blood flow (LDBF) analysis, anti-CD31 immunohistochemistry, and Miles assay, respectively, and compared with those induced by fibroblast growth factor (FGF)-2. Blood flow recovery and angiogenesis in sphingosine kinase 1-transgenic mice that overproduce S1P endogenously were also assessed and compared with wild-type mice. The LDBF analysis showed that daily intramuscular administration of S1P dose-dependently stimulated blood flow recovery, resulting in up to twice as much blood flow when compared with vehicle control, which was accompanied by 1.7-fold increase in the capillary density. The optimal S1P effects were comparable with those obtained with FGF-2. S1P injection did not increase vascular permeability. The post-ischaemic blood flow recovery and angiogenesis were accelerated in sphingosine kinase 1-transgenic mice, which showed 40-fold higher sphingosine kinase activity and 1.8-fold higher S1P content in skeletal muscle than in wild-type (WT) mice, without an increase in the vascular permeability when compared with WT mice.

Conclusion: These results indicate that either local exogenous S1P administration or endogenous S1P overproduction promotes post-ischaemic angiogenesis and blood flow recovery. These observations suggest potential therapeutic usefulness of S1P for tissue ischaemia.

KEYWORDS Angiogenesis; Ischaemia; Endothelial receptor; Lipid signalling; Transgenic animals


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Funding
 References
 
Exploring a novel and effective strategy for accelerating angiogenesis is a particularly important subject for developing an angiogenic therapy for vasoocclusive diseases in the light of high morbidity and mortality due to ischaemic diseases in the developed countries. To date, the therapeutic efficacy of angiogenic peptide growth factors, including vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2), hepatocyte growth factor, and their expression plasmids, have been tested with their topical and systemic administration.1–4 However, the therapeutic efficacy of these treatments was not satisfactory; some of these therapies were accompanied by several drawbacks including therapy-related issue oedema due to hyperpermeability of neo-vessels and proteinuria, which have hampered clinical application of these peptide growth factors.

Sphingosine-1-phosphate (S1P) is an endogenous lipid mediator that exerts pleiotropic effects including cell migration, cell proliferation, and cell survival in diverse cell types through the specific G-protein-coupled receptors, S1P1–5.5–7 Vascular endothelial cells largely express S1P1 and S1P3, which mediate stimulation of endothelial cell proliferation, migration, and capillary-like tube formation in vitro.8–11 S1P stimulates angiogenesis in vivo via S1P1 and S1P3 in the Matrigel implant assay.8 S1P1-gene ablation in mice demonstrated that S1P1 in vascular endothelial cells is essential for the recruitment process of pericytes and smooth muscle cells to the nascent capillaries, i.e. vascular maturation.12,13 A recent investigation14 demonstrated that RNA interference-mediated S1P1 silencing inhibited tumour angiogenesis and tumour growth in vivo in an animal model of tumour implantation, indicating that S1P is involved in tumour angiogenesis via S1P1. S1P also acts on endothelial progenitor cells to activate CXCR4 chemokine receptor via S1P3 receptor, leading to potentiation of blood flow recovery in ischaemic limbs when S1P-stimulated progenitor cells are injected.15 In addition, S1P has a beneficial effect on ischaemia-induced myocardial damage through inhibiting leukocyte infiltration and apoptosis.16 S1P is released from activated platelets, red blood cells, and other cell types and present at a substantial concentration in the circulating plasma.17,18 Thus, S1P may be one of the key regulators that promote angiogenesis and vascular maturation under physiological and pathological conditions. However, it is totally unknown whether S1P has a beneficial, stimulatory effect on in vivo angiogenesis in ischaemic tissues.

The hindlimb ischaemic model19 is one of the well-established animal models for ischaemia-induced angiogenesis in vivo to evaluate the potential of angiogenic factors as a therapeutic agent. In this study, we examined how S1P affected post-ischaemic angiogenesis in the mouse hindlimb model, to evaluate whether S1P could be useful in stimulating angiogenesis. Our data show that either local administration of exogenous S1P or overproduction of endogenous S1P in the S1P-synthesizing enzyme sphingosine kinase1 (SPHK1)20-transgenic mice stimulated in vivo angiogenesis in ischaemic limbs, suggesting the potential usefulness of S1P as an angiogenic therapeutic agent.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Funding
 References
 
2.1 Animals
We studied C57BL/6J male mice of 8–12 weeks old (Nippon SLC, Shizuoka, Japan), male SPHK1 transgenic (SPHK1-Tg) mice, and their WT littermate male mice. We generated SPHK1-Tg mice by injecting the linearized SPHK1a expression DNA construct into the pronuclei of fertilized eggs (BDF2) from superovulated BDF1 females mated with BDF1 males. The SPHK1 expression construct took advantage of the CAG promoter that drives the expression of SPHK-1a gene in diverse tissues. SPHK1-Tg mice showed the SPHK1 transgene expression in a wide variety of tissues including brain, heart, lung, and skeletal muscle, as evaluated by northern and western analysis, and sphingosine kinase enzymatic activity (N. Takuwa et al., unpublished results). The SPHK1-Tg mice were back-crossed to C57BL/6J more than nine times. They are fertile and develop normally. The detailed phenotypes of the transgenic mice will be reported elsewhere. Mice were housed in a temperature-controlled conventional facility (24°C) under a 12:12 h light–dark cycle with free access to regular chow and water. Blood pressures and pulse rates were measured non-invasively in conscious mice using a computerized tail-cuff method (Softron BP98A, Softron Co, Ltd, Tokyo, Japan). All experiments using mice were approved by and performed according to the Guidelines for the Care and Use of Laboratory Animals in Kanazawa University, which strictly conforms to US National Institutes of Health guidelines.

2.2 Reagents
DL-erythrosphingosine-1-phosphate (S1P) (BIOMOL, Plymouth Meeting, PA, USA), sphingosine and human recombinant FGF-2 (Sigma, St Louis, MO, USA), pentobarbital (Kyoritsu, Tokyo, Japan), and fatty acid-free bovine serum albumin (BSA) (Sigma) were purchased.

2.3 Unilateral hindlimb ischaemia model of the mouse
Mice were subjected to surgical procedures to achieve unilateral hindlimb ischaemia after intraperitoneal injection of pentobarbital (60 mg/kg), according to the method described previously.19 In brief, following a skin incision at the left paracenter of the lower abdomen, the femoral artery, which originated from the external iliac artery and terminated to bifurcate into the saphenous and the popliteal arteries, was exposed. The femoral artery was ligated with 8-0 silk, and the whole length of the femoral artery was excised and the skin incision sutured.

2.4 Drug administration
S1P was dissolved in dimethylsulphoxide (DMSO) at the concentration of 2 x 10–3 M, aliquoted, and stored at –20°C. S1P solutions at indicated concentrations were prepared by diluting in a small amount of DMSO and then dissolving into Ca2+- and Mg2+-free Dulbecco's phosphate-buffered saline (PBS) containing 0.1% fatty acid-free BSA. The final concentration of DMSO did not exceed 0.1%. For vehicle control, the same concentration of DMSO without S1P was adopted. FGF-2 was dissolved in PBS at a concentration of 10 µg/mL, aliquoted, and stored at –20°C. It was diluted to the final concentration of 3.3 ng/mL in PBS containing 0.1% fatty acid-free BSA. The concentration of FGF-2 has been shown to be effective in promoting ischaemia-induced angiogenesis in animal models in previous reports.19,21 Ten microlitres each of the solutions of either S1P or FGF-2 or the vehicle solution was daily injected intramuscularly into four sites in the medial portion of the thigh muscle and two sites in the calf muscle of the ischaemic limb for 14 or 28 days. The total daily doses of S1P were 0.06, 0.6, or 6 pmol per mouse with each solution of 10–9, 10–8, and 10–7 M in Figure 1 and 0.6 pmol per mouse in Figure 2, and that of FGF-2 was 198 pg per mouse with 3.3 ng/mL solution in Figure 2.


Figure 1
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  		Figure 1 Daily local S1P injection enhances the blood flow recovery and increases the capillary density in mouse ischaemic hindlimbs. The LDBF pseudocolour images (A) and the ischaemic/non-ischaemic limb LDBF ratio value (B), as determined with an LDBF analyser, in mice that received either vehicle or various concentrations of S1P solutions. Sixty microlitres of S1P solutions or vehicle solution was daily injected into hindlimb muscle for 14 days. In (B), closed circle, vehicle; open box, S1P 10–9 M; open circle, S1P 10–8 M; and closed box, S1P 10–7 M. In each group, seven mice were analysed. *P < 0.05 and **P < 0.01 when compared with the vehicle control. (C) Immunohistochemical staining of the capillaries with anti-CD31 antibody (red) at day 14. **P < 0.01 when compared with vehicle control. In each group, four or five mice were analysed. (D) Vascular permeability in the muscle of the ischaemic limbs of mice that received daily injections of S1P (10–7 M) or vehicle solutions, as evaluated with the Miles assay. Evans Blue dye was injected into the tail vein, and the amount of extravasated Evans Blue in the calf muscle was determined. The ratio of the amounts of the dye in ischaemic to non-ischaemic control muscle in each mouse was determined. n.s. denotes statistically not significant. In each group, four mice were analysed.

 


Figure 2
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  		Figure 2 The angiogenic effects of S1P are comparable with e induced by FGF-2. The LDBF pseudocolou images (A) and the ischaemic/non-ischaemic limb LDBF ratio value (B) on days 14 and 28 after femoral arteriectomy, as determined with an LDBF analyser in the mice that received daily injections of 60 µL of vehicle, S1P or FGF-2 solutions for 14 or 28 days. In (B), open box, vehicle; filled box, S1P (10–8 M); and hatched box, FGF-2 (3.3 ng/mL). In each group, six or seven mice were analysed. *P < 0.05 and **P < 0.01 when compared with the vehicle control. (C) Immunohistochemical staining of capillaries with anti-CD31 antibody at day 14. In each group, six or seven mice were analysed. **P < 0.01 when compared with the vehicle control.

 
2.5 Laser Doppler blood flow analysis
The blood flow of the ischaemic (left) and contra-lateral non-ischaemic (right) limbs was measured with a laser Doppler blood flow (LDBF) analyzer (Moor Instruments, Devon, UK) before and after operation. Before each measurement, mice were anaesthetized with pentobarbital (60 mg/kg intraperitoneally) and placed upon a plate warmed at 37°C for 15 min. After scanning, the stored data were analysed to quantify the mean LDBF per unit two-dimensional area on the en-face image of each entire hindlimb in mice at the supine position, which was determined by the software provided by the manufacturer (Moor Instruments). For each animal, the values were expressed as the ratio of LDBF values in ischaemic (left)/non-ischaemic control (right) limb at a given time point.

2.6 Immunohistochemical detection of a capillary
The calf muscle was excised, embedded in OCT compound (Sakura Fine Chemical, Tokyo, Japan), and frozen in the dry ice/ethanol bath. Six micrometre sections were subjected to immunohistochemical detection of endothelial cells by sequential incubation with a rat monoclonal anti-mouse CD31/PECAM-1 antibody (PharMingen, San Diego, CA, USA; 1:100 dilution), a biotinylated rabbit anti-rat IgG antibody, and streptoavidin-conjugated peroxidase (Vectastain ABC kit, Vector Laboratories, Burlingame, CA, USA), and visualized with 3,3'-diaminobenzidine tetrahydrochloride. Capillary densities were counted in randomly chosen 10 high power fields per mouse and expressed as the number of capillaries per mm2.

2.7 Miles assay
Five days after surgery, 0.1 mL of 1% Evans Blue was injected via the tail vein. Thirty minutes later, mice were euthanized by pentobarbital anaesthesia and perfused with physiological saline through a cannula inserted into the descending aorta with an incision in the right atrium to allow outflow. Calf muscle was removed, weighed, and Evans Blue was extracted in 0.5 mL formamide/100 mg wet weight tissue by incubating at 60°C for 24 h. Absorbance of 620 nm (Evans Blue) and 740 nm (haemoglobin) was measured, and the absorption of Evans Blue dye was corrected for that of haemoglobin by the following equation: A620 (corrected) = A620–(1.426 x A740 + 0.030).22

2.8 Determinations of sphingosine kinase activity and S1P content of skeletal muscle
SPHK activity was measured essentially, as described previously.23 Muscle homogenates were incubated with 50 µM sphingosine prepared in mixed micelles with Triton X-100, 10 µM of [{gamma}-32P]ATP (1 mM), and 10 mM MgCl2. Labelled lipids were extracted and resolved by thin layer chromatography, as described earlier.23 Labelled S1P was quantified with a FujiBAS2000 Bioimage analyser.

For determination of S1P content, lipids from muscle homogenates were extracted by adding 1 mL of 25 mM HCl–1 M NaCl, 1 mL of methanol, 1 mL of chloroform, and 100 µL of 3 NaOH, and the phases were separated.24 The aqueous phase containing S1P, devoid of sphingosine and the majority of phospholipids, was transferred to a siliconized glass tube. The organic phases were re-extracted with 1 mL of methanol–1 M NaCl (1:1, vol/vol) and the aqueous fractions combined. Mass levels of S1P in the pooled aqueous phases were determined exactly as described previously.24

2.9 Statistics
All data are shown as mean ± SEM. Analysis of variance was followed by Bonferroni's test to determine the statistical significance of difference between mean values for Figures 1B and C, 2B and C, and 3B and C. Unpaired t-test was performed for the comparison between two groups for Figures 1D and 3A, D, and E. For all statistical comparisons, P < 0.05 was considered significant.


Figure 3
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  		Figure 3 Stimulated blood flow recovery and an increase in the capillary density in ischaemic hindlimbs of SPHK1-Tg mice without vascular hyperperpeability. (A) Sphingosine kinase activity in the muscle of SPHK1-Tg and WT mice. Sphingosine kinase activity in calf muscle of hindlimbs from four each of SPHK1-Tg and WT mice was determined, as described previously. (B) The limb blood flow after femoral arteriectomy as determined with an LDBF analyser. The mice did not receive administration of S1P or any other angiogenic factor. Closed circle, control mice (n = 4) and open circle, SPHK1-Tg mice (n = 8). *P < 0.05 when compared with the WT mice. (C) S1P content in muscle of ischaemic and control non-ischaemic limbs in WT and SPHK1-Tg mice. S1P content in muscle of hindlimbs from four each of SPHK1-Tg and WT mice at day 9 after femoral arteriectomy was determined as described previously. (D) Immunohistochemical staining of capillaries with anti-CD31 antibody (red) at day 9. **P < 0.01 when compared with the WT mice. (E) Vascular permeability in the muscle in the ischaemic limbs of mice. Vascular permeability was determined as in Figure 1D with the Miles assay. n.s. denotes statistically not significant.

 

   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Funding
 References
 
3.1 Local injections of S1P stimulate blood flow recovery and angiogenesis in ischaemic hindlimbs
We determine the in vivo effect of S1P on the blood flow recovery after surgical arteriectomy of a mouse femoral artery. Just after the operation and every day thereafter, the mice received intramuscular injections of various concentrations of S1P or vehicle solutions into the thigh and the calf muscles of the ischaemic hindlimb. The blood flow in the ischaemic (left) and non-ischaemic (right) hindlimbs was analysed by an LDBF image analyser and expressed as pseudocolour images (Figure 1A) and also as the ischaemic and non-ischaemic LDBF ratio (Figure 1B). After arteriectomy, the blood flow fell down to a nadir that was less than 10% of the value in the contra-lateral non-ischaemic limb. In control mice that received the vehicle solution, the blood flow recovered slowly, with the LDBF ratios being approximately 0.2 and 0.3 on 7 and 14 post-operative days, respectively. Daily injections of S1P (10–7 M) significantly enhanced the blood flow recovery in the ischaemic limb. The LDBF ratios in S1P-treated mice were approximately two-fold higher than those in control mice at these time points. S1P at lower concentrations also tended to stimulate blood flow recovery, although statistically not significant. We examined whether S1P had any stimulatory effect on angiogenesis by determining the capillary density in the calf muscle. S1P injections resulted in dose-dependent increases (maximally 1.7-fold increase over the vehicle control at 10–7 M S1P) in the capillary density (Figure 1C). Daily S1P (10–7 M) injections did not affect body weight, blood pressure, heart rate, and blood leukocyte and erythrocyte counts (Table 1).


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  		Table 1 Effects of daily S1P injections on body weight, cardiovascular parameters, and blood cell counts in mice

 
3.2 Local injections of S1P do not increase vascular permeability in ischaemic hindlimbs
It was previously shown that local administration of an angiogenic peptide was accompanied by regional tissue oedema as a result of vascular hyperpermeability in newly formed vessels.1–4 We evaluated the effect of S1P on vascular permeability by using the Miles assay, which determined the amount of the Evans Blue dye that had extravasated after intravenous injection. Figure 1D shows comparison of the amounts of Evans Blue dye extracted from calf muscles, which are expressed as the ratio of the value for ischaemic muscle to that for non-ischaemic control muscle in each animal (permeability ratio). In S1P-treated group and vehicle control group, the mean values of the permeability ratio were approximately 2.4 and 2.3, respectively, with no statistically significant difference.

3.3 S1P and FGF-2 exert comparable stimulatory effects on blood flow recovery and angiogenesis in ischaemic hindlimbs
We compared the effects of S1P and FGF-2, the latter being a well-established angiogenic growth factor that was tested in clinical trials,1–3,21,25 on both blood flow recovery and angiogenesis. The stimulatory effects of S1P on the blood flow were nearly equal in magnitudes to those of FGF-2 on both 14 and 28 days after the operation (Figure 2A and B). The capillary density in the ischaemic limb was similar between S1P and FGF-2 treatment groups, which was ~60% greater than vehicle control (Figure 2C).

3.4 Stimulated angiogenesis in transgenic mice that overproduce S1P
To evaluate the effect of increased endogenous production of S1P on post-ischaemic angiogenesis, we studied and compared post-ischaemic blood flow recovery and angiogenesis in both mice transgenic for the S1P-synthesizing enzyme SPHK1 (SPHK1-Tg) and their WT littermates. SPHK enzymatic activity in the calf muscle was approximately 40 times greater in SPHK1-Tg mice when compared with that in the WT littermates (Figure 3A). After arteriectomy, SPHK1-Tg mice showed accelerated blood flow recovery when compared with WT littermates, with a significant 1.4-fold increase in the LDBF ratio value on day 9 after the operation (Figure 3B). The LDBF ratio values in SPHK1-Tg mice on 7 and 14 post-operative days were also greater than WT littermates with the P values of 0.061 and 0.101, respectively. The S1P level in non-ischaemic, control calf muscle was approximately 1.8-fold higher in SPHK1-Tg mice than in WT mice (Figure 3C). The S1P level in the ischaemic muscle tended to be higher in SPHK1-Tg mice than in WT mice (P = 0.08). In addition, there was a slight tendency of higher S1P content in ischaemic muscle than in non-ischaemic muscle in either WT or SPHK1-Tg mice. We also observed an increased capillary density in the ischaemic limb in SPHK1-Tg mice when compared with WT littermates on day 14 (Figure 3C). There was no difference in the capillary density of the non-ischaemic control limbs in WT and SPHK1-Tg mice. The ischaemic/non-ischaemic permeability ratio was comparable between SPHK1-Tg and WT littermates (Figure 3D), indicating that enhancement of post-ischaemic angiogenesis in SPHK1-Tg mice was not accompanied by higher vascular permeability when compared with WT mice.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Funding
 References
 
Previous investigations8–11 showed that S1P acts on vascular endothelial cell cultures via S1P1 and S1P3 receptors to stimulate migration and capillary-like tube formation in vitro, to the similar extents as the potent angiogenic peptide growth factors including VEGF and FGF-2. Exogenous S1P induces angiogenesis in the implanted matrices such as the Matrigel and agarose in the subcutaneous tissue of animals, indicating the stimulatory activity of S1P on angiogenesis in vivo.8,14,26 Very recently, blockade of the angiogenic S1P receptor stimulation by either siRNA-induced receptor knockdown16 or FTY720-induced receptor downregulation was shown to suppress tumour angiogenesis and tumour growth.26 Apart from these in vivo studies in the matrix implantation assays and tumour angiogenesis, there were very few studies that showed an in vivo angiogenic activity of S1P.

The present study is the first report to show the stimulatory effects of S1P of both exogenous and endogenous origins on in vivo angiogenesis and resultant blood flow recovery in an experimental ischaemic hindlimb model. Our data show that local administration of S1P stimulated blood flow recovery in ischaemic limbs after femoral arteriectomy in a dose-dependent manner (Figure 1A and B). The stimulation of the blood flow recovery was accompanied by an increase in the capillary density in the ischaemic hindlimb muscle (Figure 1C), suggesting that S1P-induced stimulation of blood flow recovery was due to neovessel formation. In addition, SPHK1-Tg mice that overproduced S1P endogenously showed increased blood flow recovery and capillary density after ischaemia when compared with WT littermates (Figure 3), suggesting that endogenously overproduced S1P serves to stimulate post-ischaemic angiogenesis. The stimulatory effects on the blood flow and angiogenesis by the optimal dose of S1P was similar in the magnitudes as those induced by the potent angiogenic peptide growth factor FGF-2 (Figure 2), suggesting that the angiogenic activity of S1P may be comparable with the known angiogenic factors in strength. In addition, daily local administration of S1P exhibited no adverse effect in mice, in terms of general conditions, the blood cell count, or the cardiovascular parameters (Table 1). These data collectively suggest potential usefulness of S1P for therapeutic angiogenesis in ischaemic conditions.

One of the major adverse effects in therapeutic angiogenesis is local oedema because of vascular leakage at the site of neo-angiogenesis.1–4 Previous studies2,21,25 showed that angiogenic therapy using administration of VEGF and FGF and their gene transduction-mediated overexpression was accompanied by local tissue oedema. In the present study, the enhanced angiogenesis induced by both exogenous and endogenous S1P was not associated with increased vascular leakage when compared with control animals (Figures 1D and D). S1P was previously shown to organize endothelial tight junctions8,27 and promote vascular mural cell recruitment.12,28,29 These S1P effects likely lead to the protection of the neo-vessels from hyperpermeability. The angiogenic activity of S1P without evidence of concomitant hyperpermeability suggests that the combination of S1P and other angiogenic peptides including VEGF may obviate the problem of local tissue oedema due to vascular leakage in the angiogenic therapy.

Recent studies1,3,30 showed the contribution of bone-marrow-derived circulating endothelial precursor cells to the new blood vessel formation in ischaemic tissues. CD34+ vascular endothelial progenitors express S1P3 receptor.15 Stimulation of progenitor S1P3 receptor with S1P or synthetic analogue FTY720 activates the CXCR4 chemokine receptor, which is essential for the effectiveness of progenitor cell therapy for angiogenesis. Therefore, S1P by itself may stimulate angiogenesis through not only direct stimulatory effects on pre-existing endothelial cells but also the recruitment of circulating endothelial precursor cells. It is possible that S1P could interact with other angiogenic peptides to stimulate angiogenesis, because S1P may stimulate the expression of VEGF and other angiogenic peptides in vascular and non-vascular cells31,32 and trans-activate VEGF receptors in endothelial cells.33 Alternatively, S1P and angiogenic peptides may in concert stimulate angiogenesis by synergistically activating a downstream effector critical for angiogenesis, for example, the endothelial type of nitric oxide synthase (eNOS) by stimulating Ca2+ mobilization and Akt-dependent eNOS phosphorylation.34 In addition, the production and/or release of S1P in ischaemic tissues may be stimulated through mechanisms involving growth factor stimulation, hypoxia-induced upregulation of sphingosine kinase, and platelet activation.17,35 In fact, we observed the tendency that S1P content was higher in ischaemic muscle than in non-ischaemic muscle both in WT and SPHK1-Tg mice (Figure 3C).

We recently showed that S1P2 negatively regulates angiogenesis in marked contrast to S1P1 and S1P3.36 The S1P2-mediated inhibitory signal likely counteracts the stimulatory actions of S1P1 and S1P3 on angiogenesis, although the regulation of the expression of these S1P receptor subtypes in the vascular endothelium in ischaemic tissues is not well understood. Hence, the selective stimulation of S1P1 and S1P3 or blockade of S1P2 or the combination of these may confer a better strategy for the angiogenic therapy to target S1P receptors.

In conclusion, we provide evidence that S1P is an effective angiogenesis stimulator in post-ischaemic condition in vivo, which accelerates blood flow recovery and neo-vessel formation without local oedema or any other adverse effect and with a relatively low cost.


   Funding
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Funding
References
 
This work has been supported by Practical Application Research program of JST (Japan Science and Technology Agency) Innovation Plaza Ishikawa, grants from the Ministry of Education, Science, Sports, and Culture of Japan, the Japan Society for the Promotion of Science, and funds from Chugai Pharmaceuticals.


   Acknowledgements
 
We thank Drs J. Imagawa, T. Koga, M. Fukazawa, and O. Kuromaru in Chugai Pharmaceuticals for useful discussion about the experiments, and Ms S. Murakawa, M. Ushiro, and C. Hirose for technical and secretarial assistance.

Conflict of interest: all authors have no potential conflict of interest.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Funding
 References
 

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  9. Kanda T, Kobayashi I, Ohta H, Ui M, Okajima F. Sphingosine 1-phosphate stimulates proliferation and migration of human endothelial cells possibly through the lipid receptors, Edg-1 and Edg-3. Biochem J (2000) 348:71–76.[CrossRef][Web of Science][Medline]
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