Sphingosine 1-phosphate receptor 3 regulates recruitment of anti-inflammatory monocytes to microvessels during implant arteriogenesis

Abstract

Endothelial cells play significant roles in conditioning tissues after injury by the production and secretion of angiocrine factors. At least two distinct subsets of monocytes, CD45⁺CD11b⁺Gr1⁺Ly6C⁺ inflammatory and CD45⁺CD11b⁺Gr1⁻Ly6C⁻ anti-inflammatory monocytes, respond differentially to these angiocrine factors and promote pathogen/debris clearance and arteriogenesis/tissue regeneration, respectively. We demonstrate here that local sphingosine 1-phosphate receptor 3 (S1P₃) agonism recruits anti-inflammatory monocytes to remodeling vessels. Poly(lactic-co-glycolic acid) thin films were used to deliver FTY720, an S1P_1/3 agonist, to inflamed and ischemic tissues, which resulted in a reduction in proinflammatory cytokine secretion and an increase in regenerative cytokine secretion. The altered balance of cytokine secretion results in preferential recruitment of anti-inflammatory monocytes from circulation. The chemotaxis of these cells, which express more S1P₃ than inflammatory monocytes, toward SDF-1α was also enhanced with FTY720 treatment, but not in S1P₃ knockout cells. FTY720 delivery enhanced arteriolar diameter expansion and increased length density of the local vasculature. This work establishes a role for S1P receptor signaling in the local conditioning of tissues by angiocrine factors that preferentially recruit regenerative monocytes that can enhance healing outcomes, tissue regeneration, and biomaterial implant functionality.

Sphingosine 1-phosphate (S1P) is a naturally occurring bioactive lipid produced by red blood cells, activated platelets and endothelial cells (ECs). S1P signals pleiotropic cellular functions, including chemotaxis and recruitment of cells (1, 2), through the activation of five known G protein-coupled receptors (S1P₁–S1P₅) (3, 4). S1P also plays a significant role in the trafficking of stem and progenitor cells within extra medullary tissues (1) and regulates blood recirculation of osteoclastic precursors (5). Furthermore, S1P receptor signaling plays critical roles in the formation and stabilization of microvascular networks (6). Microvascular ECs signal to cells in their proximity through the production of growth factors and cytokines, such as MCP-1, SDF-1α, and VEGF (7, 8). Regulating the secretion of these “angiocrine” factors may be critical to improving the treatment of ischemic tissue diseases and enhancing regenerative capacity of biomedical implant materials. Local activation of S1P receptors 1 and 3, with the synthetic S1P analog FTY720, enhances microvascular remodeling through expansion of arterioles and capillary networks (9). Modulation of S1P receptors on ECs may be a unique way to control the localization of circulating cells that contribute to tissue regeneration and wound healing.

Monocytes are bone marrow (BM)-derived circulating immune effector cells that are recruited to infected or inflamed tissues (10) and exist in distinct populations. Geissmann et al. characterized two populations of monocytes with distinct migratory roles during inflammation in mice: a CD45⁺CD11b⁺Ly6C⁺Gr1⁺CX3CR1^lo inflammatory subtype of monocyte (IM) recruited to inflamed tissues and a CD45⁺CD11b⁺Ly6C⁻Gr1⁻CX3CR1^hi patrolling or anti-inflammatory subtype (AM) recruited to noninflamed tissues (11). IMs phagocytose debris and clear damaged cells, whereas AMs promote arteriogenesis/tissue regeneration. These monocytes are recruited by distinct cues into inflamed tissues, where they can differentiate into classically activated M1 or alternatively activated M2 macrophages, respectively (12, 13).

In this work, we explore how delivery of S1P receptor compounds can recruit regenerative AMs and lead to enhanced vascular remodeling. Our results demonstrate that S1P₃ activation on ECs is critical for SDF-1α and other proregenerative cytokine production and that S1P₃ receptor expression on both circulatory and tissue resident cells is necessary for FTY720-mediated vascular remodeling (Fig. S1). We show that AMs express higher levels of surface S1P₃ and show enhanced SDF-1α mediated chemotaxis after FTY720 treatment. Local AM recruitment to the perivascular niche in tissues after FTY720 treatment enhanced arteriogenesis in muscle and soft tissue.

Results

Inflammation-Associated Microvascular Network Growth.

Dorsal skinfold window chambers (“backpacks”) were surgically placed on mice after removal of the superficial layer of dermis to expose the underlying vasculature (14). Poly(lactic-co-glycolic acid) (PLAGA) thin films were placed in the window chamber directly after surgery (acute treatment), and were either unloaded (PLAGA) or FTY720-loaded (Fig. 1A). We evaluated the concentration of local chemokines MCP-1 and SDF-1α over a 7-d period. Inflammatory chemokine MCP-1 expression peaked at day 1 with a 10-fold elevation and decreased on days 3 and 7 (Fig. 1B). In contrast, SDF-1α remained low after surgery but was significantly elevated by day 3 and peaked at day 7 (Fig. 1B).

Fig. 1.

FTY720 enhances inflammation-associated microvascular growth. (A) Schematic of acute and delayed PLAGA film implantation. (B) MCP-1 and SDF-1α in backpack tissue over 7 d. Arteriolar diameter expansion (C) and length density (D) significantly enhanced after acute (Left) and delayed (Right) FTY720 implantation. (E) Microvascular networks surrounding implants at 0 and 7 d after delayed implantation. Delayed FTY720 promotes growth of new vessels (arrowheads), vessel tortuosity (arrows), and arterial diameter enlargement (red arrowheads). (F) Cytokine quantification (fold change from sham) in tissue surrounding FTY720-loaded implants 3 d after implantation. (G) FTY720 reduces ratio of MCP-1:SDF-1α in tissue around implant 1 d after implantation. (H and I) AM (H) and IM (I) in blood before and after backpack surgery. FTY720 significantly increases the proportion of AMs in blood 3 d after surgery (H), and surgery results in an increase in IMs after 1 and 3 d (I). (J) AMs and IMs in backpack tissue 3 d after surgery. (K) FTY720 significantly enhances the fold change of AMs in tissue 3 d after surgery. (L) CD206⁺ and MHCII⁺ cells in backpack tissue. *P < 0.05 compared with sham, PLAGA, or day 0. (Scale bar: 100 μm.)

Vascular parameters were assessed by intravital imaging on days 0, 3, and 7 after acute implantation of the films. Sham and acute-PLAGA mice had reduced luminal arteriole diameter over the first 3 d whereas acute-FTY720 film placement resulted in diameter expansion relative to day 0 (Fig. 1 C, Left). Similarly, sham and acute-PLAGA groups had reduced vascular length density, a measure of network expansion/regression. The acute-FTY720 group had smaller reductions (Fig. 1 D, Left). By 7 d after implantation, luminal diameter expansion was observed in sham and PLAGA-treated mice; however, FTY720 significantly increased arteriolar diameter expansion (Fig. 1 C, Left). Length density was reduced at 7 d in sham and acute-PLAGA groups, whereas there was an increase in length density with acute FTY720 (Fig. 1 D, Left).

To determine whether FTY720 also enhances remodeling after the peak of the inflammatory response, we investigated vascular parameters after delayed placement of the polymer films 7 d after surgery (delayed PLAGA or delayed FTY720) (Fig. 1A). Images were then acquired 7, 10, and 14 d after surgery (0, 3, and 7 d after placement of the film). Indeed, delayed-FTY720 treatment also significantly increased both arteriolar diameter expansion and length density relative to delayed-PLAGA mice 3 and 7 d after film implantation (Fig. 1 C, Right; D, Right; and E). Enhanced vessel tortuosity (arrows), new vessel growth (arrowheads), and diameter enlargement (red arrowheads) are classic signs of arteriogenesis (15, 16) that were observed with delayed-FTY720 treatment (Fig. 1E). The arteriolar remodeling effect in response to either acute or delayed biomaterial release of FTY720 has been termed “implant arteriogenesis.”

To investigate the role of S1P receptors in the early phase of the inflammatory response, the following studies are all acute film implantation. Analysis of cytokine levels in backpack tissue 3 d after acute film implantation suggests that FTY720 significantly decreased the expression of many factors associated with implant rejection and poor wound healing, including TNF-α, MIP-1β, regulated on activation normal T cell expressed and secreted (RANTES), and MIP-1α relative to unloaded PLAGA films (Fig. 1F and Fig. S2). Additionally, FTY720 elevated proregenerative cytokines [i.e., IL-5, -10, and granulocyte colony-stimulating factor (GCSF)] (Fig. S2). Interestingly, FTY720 film implantation reduced the MCP-1:SDF-1α ratio relative to sham and PLAGA-treated tissues after only 1 d (Fig. 1G), suggesting a shifting balance from inflammatory to regenerative factors.

FTY720 Recruits Anti-Inflammatory Monocytes to Tissue.

Exploratory studies showed that S1P-loaded films caused a significant reduction in CD11b⁺ inflammatory cell adhesion to local endothelium compared with PLAGA films (Fig. S3A). To determine how local S1P receptor agonism affects monocyte localization during an inflammatory stimulus, flow cytometry analysis of two monocyte subsets, IM and AM was performed on digested peri-implant tissue (Fig. S3B) and on the blood over the course of surgery and healing (Fig. 1 H and I). AMs were detectable in the blood before surgery and 1 and 3 d after surgery (Fig. 1H). Local FTY720 release in tissue resulted in a small but significant increase in circulating AMs 72 h after surgery. IMs were also detectable in circulation before and after surgery, increased 24 h after surgery, and partially receded after 72 h independent of treatment group (Fig. 1I). FTY720 animals had an increasing trend in the percent of AMs in tissue 3 d after surgery (Fig. 1J). FTY720 produces a significant 1.7-fold increase in the AM proportion of monocytes compared with PLAGA when normalized to blood (Fig. 1K). To assess macrophage profile of the tissue surrounding PLAGA implants, dorsal skin sections were stained for CD206 (M2a/c) and MHCII (M1). Representative images suggest that FTY720 may increase CD206⁺ and decrease MHCII⁺ cells relative to PLAGA 7 d after surgery (Fig. 1L).

To further support these findings, backpacks were implanted on an established mouse model for tracking AMs and IMs, the CX3CR1–eGFP^+/− mouse (Fig. 2A). The two subsets of monocytes were distinguished with flow cytometry (11) (Fig. S3C). Although FTY720 did not change the proportion of myeloid cells that were IMs relative to unloaded PLAGA (Fig. 2B), FTY720 did significantly increase the proportion of AMs relative to unloaded PLAGA in peri-implant tissue (Fig. 2C and Fig. S3D). Intravital microscopy 1 h after surgery found fewer CX3CR1–eGFP monocytes flowing, rolling, or adherent around FTY720-loaded polymer films (Fig. 2D) compared with sham and PLAGA-treated. This trend persisted at 24 h after surgery (Fig. 2E). Secondary single-cell analysis of the flowing and adherent cells surrounding the implants at 24 h demonstrates that the individual cell CX3CR1 fluorescence intensity of adherent cells is higher in the FTY720 group (Fig. 2 F and G). Monocyte subset proportions were not altered in the BM or spleen by FTY720 treatment, but did enhance the number of circulating AMs in the blood (Fig. S3E and Fig. 1H). Together, these results suggest that local FTY720 delivery enhances the recruitment of AM cells that are able to firmly adhere to the endothelium, a key step before extravasation into the tissue.

Fig. 2.

FTY720 regulates AM rolling and adhesion in tissue surrounding implants. (A) Schematic for CX3CR1–eGFP mice surgeries, imaging, and tissue harvest. (B and C) FTY720 does not change the proportion of IMs (B) but increases AM infiltration 3 d after surgery (C). (D and E) FTY720 decreases the number of flowing, rolling, and adherent monocytes around implants 1 h (D) and 1 d (E) after surgery. (F and G) Single-cell CX3CR1 expression of flowing (F) and adherent (G) monocytes 24 h after surgery. Adherent cells around FTY720-loaded implants express significantly higher amounts of CX3CR1 1 d after surgery (G). S, sham; P, PLAGA; F, FTY720. *P < 0.05 compared with sham or PLAGA.

AMs Are Recruited from Circulation by FTY720.

To determine whether monocytes in circulation are recruited to the dorsal skin tissue, labeled IMs or AMs from donor mice were delivered into circulation by adoptive transfer before backpack placement (Fig. 3A). Both adoptively transferred IMs and AMs were detectable in the blood before and after backpack surgery (Fig. 3 B and D). IMs were detectable in the dorsal tissue of PLAGA- and FTY720-treated mice 3 d after surgery. Arnold et al. reported that IMs are recruited to inflamed tissues and differentiate to AMs in situ (17). Consistent with this observation, mice with a labeled IM graft had labeled AM in the tissue, suggesting a potential local differentiation (Fig. 3C). There was no difference between PLAGA and FTY720 with respect to phenotype switching, suggesting that FTY720 does not increase the tissue AM content by differentiation of IM cells.

Fig. 3.

FTY720 recruits AMs from blood to tissue surrounding implants and alters cytokine secretion. (A) Schematic for AM/IM adoptive transfer, surgery, and tissue harvest. (B) DiI⁺ IMs in blood. (C) DiI⁺ IM-derived AMs/IMs in dorsal tissue 3 d after surgery. (D) DiI⁺ AMs gradually removed from blood. (E and F) DiI⁺ AMs in dorsal tissue. FTY720 significantly enhanced AMs from circulation (E) and DiI⁺ macrophages (F) in tissue. (G) CD206⁺ and MHCII⁺ macrophages derived from DiI⁺ cells. (H) Representative montages of DiI⁺ cells from IM (Upper) or AM (Lower) adoptive transfers 3 d after surgery. (I and J) DiI⁺ cells from IM (I) and AM (J) adoptive transfer were significantly closer to vessels with FTY720. (K and L) Inflammatory cytokine secretion is decreased from AMs (K) and IMs (L) treated for 1 h with FTY720 or SEW2871. (M) HUVECs treated with FTY720 for 1 h increased regenerative cytokine secretion relative to SEW2871 and vehicle. (N) SDF-1α production from HUVECs cultured with VPC0101 was ablated relative to FTY720. *P < 0.05 compared with PLAGA/SEW2871/VPC01091.

Adoptively transferred AM decreased in blood from day 0 to 3 (Fig. 3D). AMs were detected in the dorsal tissue of PLAGA- and FTY720-treated mice. FTY720 significantly increased the proportion of labeled AMs in tissue 3 d after surgery (Fig. 3E). Monocytes that enter the tissue can differentiate into macrophages (13). To determine whether adoptively transferred AMs differentiated into tissue macrophages, dorsal tissue was digested and stained for F4/80. FTY720 significantly enhanced the proportion of adoptively transferred F4/80 macrophages relative to PLAGA (Fig. 3F). FTY720 also produced a trend of enhanced CD206⁺ macrophages and reduced MHCII⁺ macrophages in dorsal tissue (Fig. 3G).

Single cell analysis of the localization of labeled cells in the tissue derived from either the AM or IM adoptive transfer demonstrates that circulation-derived cells are significantly closer to vessels in the FTY720 group (Fig. 3 J–L and Fig. S4). These data suggest that AMs are recruited from circulation and migrate toward FTY720-conditioned environment.

Secretion of Inflammatory and Regenerative Cytokines.

Marrow-derived AM/IM (Fig. S3B) and human umbilical vein ECs (HUVECs) were treated with either FTY720 or SEW2871 (a selective S1P₁ agonist) for 1–24 h (Fig. 3 K–M). Changes in cytokine release were compared. Changes by FTY720, but not SEW2871, support a role for S1P₃-mediated regulation, whereas similar activity of FTY720 and SEW2871 suggests a dominant role for S1P₁. Agonism of both S1P₁ and S1P₃ reduced the secretion of many inflammatory cytokines 1 h after treatment of both AMs and IMs (Fig. S5 D and E). Of the seven inflammatory cytokines that were significantly decreased in tissue treated with FTY720 (Fig. 1F), S1P₃ activation on AMs resulted in significant decreases in five (Fig. 3K) and did not change two. S1P₃ activation on IMs also resulted in significant decreases in five cytokines (Fig. 3L), whereas S1P₁ activation significantly reduced the secretion of only MIP-1β and RANTES from IMs (Fig. 3L). FTY720 treatment of ECs enhanced proregenerative cytokines (refs. 18–20; Fig. 3M and Fig. S5) relative to SEW2871. S1P₃ antagonism with an established S1P₁ agonist/S1P₃ antagonist, VPC01091 (21), abrogated the secretion of SDF-1α from HUVEC, suggesting a role for S1P₃ in the secretion of regenerative cytokines (Fig. 3N).

SDF-1α–Mediated Chemotaxis of AMs and IMs.

CXCR4⁺ cells potently induce angiogenesis (22, 23). Although both subsets of monocytes express CXCR4, the SDF-1α receptor, Ly6C⁺ IMs gradually lose CXCR4 expression as they egress from the BM (32). To assess whether S1P receptor signaling regulates monocyte chemotaxis toward SDF-1α through activation of S1P₁ or S1P₃, AMs and IMs were sorted. AMs increased migration toward SDF-1α over basal media by 2.03-fold (Fig. 4A), whereas IM did not (Fig. 4B). Pretreatment for 1 h with FTY720 enhanced AM chemotaxis toward SDF-1α by 3.72-fold (Fig. 4A) but did not affect the chemotaxis of IM (Fig. 4B). Pretreatment with SEW2871 did not enhance SDF-1α–mediated chemotaxis of AMs, supporting our hypothesis that these responses are mediated through S1P₃ (Fig. 4 A and B). Furthermore, FTY720 did not enhance SDF-1α–mediated chemotaxis of AMs harvested from S1P₃^−/− mice (Fig. 4C). FTY720 preferentially promotes the chemotaxis of AMs toward SDF-1α in an S1P₃-dependent manner.

Fig. 4.

S1P₃ activation promotes SDF-1α chemotaxis of AM and S1P₃ is elevated on AM. (A–C) FTY720 enhances chemotaxis of AM (A), but not IM (B) toward SDF relative to vehicle but not of S1P₃^−/− AM or IM. (D) PCR of mRNA from AM and IM shows a trend toward increased expression of S1P₃ in AM. (E) Murine IM and AM were treated for one hour with FTY720. Membrane S1P₃ protein was higher in AM and enhanced with FTY720. (F) RAW264.7 macrophages were polarized to M1 and M2 phenotypes and treated with FTY720 for 1 hour. Membrane S1P₃ expression was higher in RAW264.7 M2 macrophages and elevated with FTY720. (G) Polarized THP-1 were stained with antibodies against S1P₃ (red), CD14 (green), and DAPI (blue). M2 macrophages express higher levels of membrane S1P₃. *P < 0.05 compared to vehicle-vehicle; ^#P = 0.09 compared with IM (Scale bar: 10 μm.).

S1P₃ Expression in AM and IM.

PCR revealed that AMs had increased S1P₃ mRNA over IMs (Fig. 4D). In agreement with this finding, S1P₃ protein expression was increased 2.63-fold in AMs relative to IMs. One-hour FTY720 treatment resulted in an increase in S1P₃ in AMs and IMs relative to IM control (Fig. 4E). In murine RAW264.7 macrophages polarized to M1 and M2 phenotypes, M2 cells expressed more S1P₃ protein (1.6-fold over M0) compared with M1-polarized cells (Fig. 4F). FTY720 also enhanced the expression of S1P₃ in M0 and M2 macrophages but not M1 macrophages. S1P₁ was higher in both M1 and M2 phenotypes relative to M0 but was not different or elevated with FTY720 treatment (Fig. 4F). To assess whether human macrophages shared these phenotypic profiles, human THP-1 macrophages were polarized to M1 and M2 phenotypes, and cells were fixed and stained with antibodies against S1P₃. M2 polarized macrophages express less CD14, an M1 marker, and significantly more S1P₃ on their surface (Fig. 4G). S1P₁ expression did not change between M0, M1, or M2 cell types. Together, these results reveal S1P₃ as a unique marker of AMs.

S1P₃ on Circulating and Local Cells.

To investigate the role of S1P₃ in vivo on FTY720-implant arteriogenesis, we created S1P₃ BM chimeras by lethally irradiating wild-type mice and injecting S1P₃^−/− (global knockout) marrow-derived cells into the tail vein (S1P₃^−/− BM) 6 wk before backpack surgery (Fig. 5A Upper). Three days after polymer implantation, S1P₃^−/− BM mice treated with FTY720 showed a significant reduction in arteriolar diameter expansion and length density relative to wild-type chimeras (Fig. 5 B and C). S1P₃^−/− mice reconstituted with wild-type marrow (Fig. 5A Lower) did not recover this reduction in growth, and mice treated locally with PLAGA eluting VPC01091 showed similar reductions in diameter expansion and length density (Fig. 5 B and C). Surprisingly, delivery of a specific S1P₁ agonist, Compound 26 (C), in the backpack model resulted in a significant increase in arteriolar diameter expansion (Fig. 5B) but not length density (Fig. 5C), which suggested an S1P₁-dependent mechanism for arteriolar remodeling. S1P₁ activation enhances the recruitment of pericytes, which enlarge small arterioles and capillaries (6). To assess the role of S1P₁ activation in the proliferation of pericytes, primary human microvascular pericytes were treated with 1 μM FTY720 or SEW2871 and assessed longitudinally for proliferation. Although S1P₁ activation may promote pericyte proliferation (Fig. S6), without S1P₃ activation, microvascular networks do not undergo significant network-wide growth.

Fig. 5.

FTY720-induced microvascular growth is dependent on S1P₃ activation on local and circulatory cells. (A) Schematic of S1P₃^−/− BM and WT BM chimera generation. (B and C) S1P₃ activation is critical for maximum arteriolar diameter expansion (B) and length density (C) expansion on both marrow-derived cells and local vascular cells. *P < 0.05 relative to WT-PLAGA. P, PLAGA; F, FTY720; V, VPC01091; C, Compound 26.

AM Recruitment and Arteriogenesis in Muscle Ischemia.

To further examine the role of monocytes in arteriolar remodeling, muscle ischemia was produced in transgenic mice expressing eGFP on monocytes and DsRed on pericytes (Ds-RedNG2/CX3CR1-eGFP). Primary feeder arterioles were ligated, promoting consequent arteriolar remodeling (Fig. S7A). These mice express DsRed on pericytes and eGFP on monocytes. FTY720-loaded scaffolds enhanced the growth of lectin-positive capillaries from remodeling arterioles relative to unloaded PLAGA scaffolds (Fig. 6A). The percent increase in tortuosity of vessels surrounding FTY720-loaded PLAGA thin films was significantly greater than that surrounding unloaded PLAGA films (Fig. S7B). Small increases in tortuosity can significantly enhance the surface area for oxygen and nutrient transport, which is critical for regeneration in ischemic tissue (15, 16, 24). Significantly more CX3CR1–eGFP^hi cells were observed within one cell length away from vessels around FTY720-releasing implants relative to unloaded PLAGA implants. Closer examination revealed significantly more AMs directly associated with remodeling vessels downstream of arterial ligation, per field of view, in FTY720-treated tissues relative to unloaded PLAGA implants (Fig. 6 A and B). Interestingly, the tortuosity of vessels in the remodeling watersheds was positively correlated with CX3CR1–eGFP^hi cell association, and both were elevated with FTY720 treatment (Fig. S7B). To further characterize the phenotype of the CX3CR1⁺ cells recruited to remodeling vessels, we stained for CD68, a pan-macrophage marker, and CD206, an M2a and M2c marker. Paralleling FTY720-dependent recruitment of CD206⁺ macrophages in inflamed tissue (Figs. 1L and 3G), muscles treated with FTY720 resulted in a significant recruitment of CD206⁺ cells to the perivascular space around remodeling vessels (Fig. 6C).

Fig. 6.

FTY720 recruits AMs to ischemic vessels in the spinotrapezius ligation model and enhances arteriogenesis. (A) Diagram of smooth muscle actin-stained whole-mounted spinotrapezius muscle with ligation (red cross) and film implantation (blue circle). (B) Immunohistochemistry images of the spinotrapezius vasculature surrounding PLAGA film (Left) or FTY720-loaded film (Right) 7 d after ligation and implantation in a DsRed–NG2 CX3CR1–eGFP mouse. CX3CR1–eGFP+ cells can be seen in the interstitial space proximal to the “remodeling” artery (white arrow, Left) induced by ligation. Delivery of FTY720 results in CX3CR1–eGFP+ cell recruitment (green arrows), together with increased sprouting and remodeling of microvascular networks (blue, lectin). (C) FTY720 significantly increased overall CX3CR1⁺ cell content as well as CX3CR1⁺ cells directly associated with remodeling vessels. (D) Further analysis of remodeling arterioles revealed significant CD206⁺ cell recruitment with FTY720. P < 0.05. (Scale bars: 50 μm.)

Discussion

Distinct subsets of monocytes contribute to wound healing: IMs through pathogen and debris clearance and AMs through repair processes (25, 26). For example, IL-4 propagates T helper type 2 (TH2) (anti-inflammatory) immune responses by stimulating the proliferation of AMs (20), and S1P receptor signaling skews immune responses toward a TH2-cell response (27). Together, the recruitment and polarization of cells by angiocrine factors can spatially concentrate regenerative cells and enhance tissue repair, vascular remodeling, and implant integration. The results presented here reveal that local S1P₃ agonism can enhance tissue regeneration through the recruitment of AMs during tissue injury and ischemia (Fig. S1).

FTY720-loaded PLAGA films significantly reduced many proinflammatory cytokines in inflamed tissues (Fig. 1F and Fig. S2A). S1P₃ activation on HUVEC increased the secretion of regenerative cytokines, and S1P₃ antagonism abrogated SDF-1α secretion (Fig. 3 M and N and Fig. S5). S1P₁ agonism alone did not recapitulate this proregenerative change. Similarly, FTY720 enhanced the tissue ratio of SDF-1α relative to the inflammatory chemokine MCP-1 (Fig. 1G). FTY720 treatment enhances migration of BM progenitor cells to SDF-1α (28). In our study, SDF-1α is a more potent chemoattractant for AMs than IMs, and FTY720 further enhanced chemotaxis of AMs, but SEW2871 did not (Fig. 4 A and B). The shift in chemotactic signals resulting in enhanced AM recruitment is consistent with our observation that there are more AMs in FTY720-treated inflamed tissues (Figs. 1 J–L, 2C, and 3 F and G). In single-cell fluorescence analysis, the adherent cells around FTY720-loaded implants 1 d after implantation expressed significantly higher levels of CX3CR1–eGFP, which mediates firm adhesion (29).

To support the idea that the AMs measured in dorsal tissue are derived from circulation, we have shown that adoptive transfer of labeled AMs into donor mice results in enhanced recruitment of labeled cells in the injured tissue in the FTY720-treated group. A portion of labeled cells within the inflamed tissue expressed mature F4/80⁺ macrophage markers and a trend of increased CD206⁺ cells in the FTY720 group (Fig. 3 E–G), suggesting that the monocytes may differentiate into macrophages in the tissue. FTY720 enhances the secretion of SDF-1α from ECs and the chemotaxis of AMs toward SDF-1α, resulting in a higher proportion of AMs in injured tissue (Figs. 1K, 2C, and 3 E and F).

S1P₃ activation consistently reduced the secretion of inflammatory cytokines from AMs and IMs but did not affect the phagocytic activity of macrophages (Fig. 3 K and L and Fig. S8). The expression of membrane S1P₃ in murine and human monocytes was significantly elevated in AMs and M2 macrophages (Fig. 4). Keul et al. showed that the recruitment of monocytes that contributed to atherosclerosis was dependent on S1P₃ (30). Because S1P₃ acts as a modulator of SDF-1–mediated chemotaxis, differential expression is likely responsible for the differences in SDF-1α–mediated chemotaxis between the two subtypes. IM can differentiate into AM (31) but FTY720 did not alter this differentiation (Fig. 3C).

The analysis of the vascular remodeling in the chimeric S1P₃ mice support a role for S1P receptor signaling in both marrow-derived and peripheral cells. Within the tissue, monocytes were found in proximity to remodeling vasculature. Single cell analysis of labeled AMs in tissue revealed that AMs recruited from blood were significantly closer to vessels surrounding FTY720-releasing films (Fig. 3 H–J). Similarly, in the spinotrapezius model in vessels surrounding FTY720-loaded PLAGA scaffolds in ischemic muscle, there was a significant increase in cells expressing CX3CR1, possibly owing to increased SDF-1α in response to ischemia (25). This finding was accompanied by an increase in capillary expansion (Fig. 6 A and B). Upon closer observation, these cells adopted a peri-vascular location and were in direct contact with vessels, especially at the arteriolar bases of new collaterals (Fig. 6A). Additional staining revealed that CD68⁺/CD206⁺ cells (M2 macrophages) were significantly recruited to remodeling vessels with FTY720 treatment and not unloaded PLAGA (Fig. 6C). Tortuosity, a classic sign of arteriogenesis, was also significantly increased in vessels surrounding FTY720-loaded implants (Fig. S7B).

In addition to ECs and monocytes, pericytes participate in many events during inflammation (32) and vascular remodeling (33). S1P₁ activation enhances the recruitment of pericytes to remodeling vessels (6, 34). Recent evidence by Stark et al. identifies interactions between arterial pericytes and CX3CR1⁺ monocytes during inflammation (32), which may suggest that pericytes play multiple roles in regulation of vascular and inflammatory modulation. We cannot rule out a role of vascular mural cells in angiocrine-type signaling. To further examine the roles of S1P₁ and S1P₃ in pericytes, we examined the proliferation of human microvascular pericyte cultures. Agonism of either S1P_1/3 (FTY720) or S1P₁ alone (SEW2871) induced statistically significant increases in cell number over 7 d in vitro (Fig. S6). Although our data imply that S1P₁ activation alone is sufficient in promoting arteriolar diameter expansion (Fig. 5B), when S1P₃ is antagonized in the presence of S1P₁ activation, as with S1P₃^−/− BM chimeras treated with FTY720, significant arteriolar diameter expansion is not observed (Fig. 5B).

These findings provide unique and exciting insight into the mechanism of monocyte-supported implant arteriogenesis during ischemia and wound healing. S1P₃ plays a critical, nonredundant role in conditioning local tissues with angiocrine factors like SDF-1α and preferentially recruiting AMs (Fig. S1). These cells localize to inflamed tissue and contribute to arteriogenesis, which has the potential to promote tissue regeneration and enhance biomaterial implant integration and functionality. These data also provide support for the use of FTY720, and other S1P₃-activating compounds for local therapeutic induction of arteriogenesis.

Methods

Wild-type C57BL/6 and NG2–DsRed mice were obtained from The Jackson Laboratories; heterozygous CX3CR1–eGFP mice were a generous gift from Klaus Ley (La Jolla Institute for Allergy, La Jolla, CA); and S1P₃^−/− mice were a kind gift of Richard Proia (National Institutes of Health, Bethesda). NG2–DsRed mice were crossed with the CX3CR1–eGFP mice to generate DsRed–NG2 CX3CR1–eGFP mice. Mice in all studies were male, 8–12 wk old, and weighed 18–25 g. The murine dorsal skinfold window chamber model and spinotrapezius ligation model with polymer implantation were performed as described (15) and are explained in detail in SI Methods. All surgical procedures and animal care protocols were approved by the University of Virginia Animal Care and Use Committee. Further methods are in SI Methods.