Methods for the treatment of tissue lesions with CCR2 agonists

Abstract

The present invention relates to methods and pharmaceutical compositions for the treatment of tissue lesions. The inventors showed that CCR2 is expressed on FMCs, especially on a subpopulation of progenitor cells, that they call “fetal myeloid progenitor cells” (FMPCs), and mediates the recruitment of these cells to maternal wound tissue. Moreover, the inventors reported that recruited FMCs/FMPCs improve maternal skin wound healing by organizing blood vessel endothelium and secreting pro-angiogenesis peptides, particularly chemokine CXCL1, to enhance angiogenesis in wound. In particular, the present invention relates to CCR2 agonists for use in the treatment of tissue lesions in a subject in need thereof.

Claims

1. A method of treating a tissue lesion in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a CCR2 agonist, wherein the subject is a female who is pregnant or has been pregnant at least one time, wherein the CCR2 agonist comprises a polypeptide having the identity of SEQ ID NO:3, and wherein the therapeutically effective amount of the CCR2 agonist is sufficient to mediate recruitment of fetal myeloid progenitor cells (FMPCs) from blood circulation to the tissue lesion.

2. The method of claim 1 wherein the CCR2 agonist comprising the polypeptide is an immunoadhesin.

3. The method according to claim 1, wherein the tissue lesion is selected from the group consisting of skin lesion, hepatic lesion, cardiac lesion, lung lesion, neurologic lesion, ocular lesion, stomach lesion, pancreas lesion, spleen lesion, bowel lesion, thyroid lesion, thymus lesion, kidney lesion, artery lesion, vein lesion, bone lesion, bone marrow lesion, muscle lesion, tendon lesion, ligament lesion, reproductive organs lesion and endocrine glands lesion.

4. The method according to claim 1, wherein the tissue lesion is a diabetic foot ulcer resulting from diabetes.

5. The method according to claim 1, wherein the tissue lesion is a leg ulcer resulting from sickle-cell anemia.

6. The method according to claim 1, wherein a conventional treatment of tissue lesions is also administered to the subject.

7. A method of treating a tissue lesion in a female subject in need thereof, wherein the female subject is pregnant or has been pregnant at least one time, comprising the step of administering to the female subject a therapeutically effective amount of a CCR2 agonist comprising a polypeptide having the amino acid sequence identity of SEQ ID NO:3, wherein the therapeutically effective amount of the CCR2 agonist is sufficient to recruit fetal myeloid progenitor cells (FMPCs) from blood circulation to the tissue lesion, and promote neovascularization in the tissue lesion.

8. The method according to claim 7, wherein the tissue lesion is selected from the group consisting of skin lesion, hepatic lesion, cardiac lesion, lung lesion, neurologic lesion, ocular lesion, stomach lesion, pancreas lesion, spleen lesion, bowel lesion, thyroid lesion, thymus lesion, kidney lesion, artery lesion, vein lesion, bone lesion, bone marrow lesion, muscle lesion, tendon lesion, ligament lesion, reproductive organs lesion and endocrine glands lesion.

9. The method according to claim 7, wherein the tissue lesion is a diabetic foot ulcer resulting from diabetes.

10. The method according to claim 7, wherein the tissue lesion is a leg ulcer resulting from sickle-cell anemia.

11. The method according to claim 7, wherein a conventional treatment of tissue lesions is also administered to the subject.

Description

FIGURES

(1) FIG. 1: Skin wound healing processes in virgin and pregnant female mice

(2) (A) Planimetry of wound area at each time point relative to original wound area (n=3). (B) Measurement of neo-epidermal tongues and gaps (n=3). (C) Quantification of Ki67+ cells in epidermal wound edges (EpiD.) and the granulation demal tissues (D.) (n=3). (D) Quantification of the relative vessel surface per 20× field by fluorescence densitometry (n=3). (E) Quantification of the number of vessel type per 20× field (n=3). (F) Quantitative RT-PCR analysis of VEGF-A, VEGFR1, VEGFR2 mRNA expression normalized to mRNA GAPDH level (n=3), (G) Quantitative RT-PCR analysis of VEGF-C, VEGFR3 mRNA expression normalized to mRNA GAPDH level (n=3). T-test Student, * p<0.05; mean±SEM.

(3) FIG. 2: Maternal wound activates FMCs and induces CCR2

(4) Fetal cells quantification in bone marrow (A), blood (B) and skin/wound (C) after maternal skin injury (n=3). (D) PCR array analysis of cytokine and chemokine gene expression in FMCs sorted from maternal bone marrow in mice with or without wound at day 3 (n=6). White columns represent the ligand genes and the black columns represent the receptor genes. Quantitative RT-PCR analysis for CCR2 (E) and its ligand CCL2 (F) mRNA expression normalized to mRNA GAPDH level in normal skin and wound (n=3). (G) Quantitative RT-PCR analysis for CCL2 in sorted leukocytes from day 1 wound. T-test Student, * p<0.05; mean±SEM.

(5) FIG. 3: CCL2 recruits FMCs to maternal wound and improve skin wound healing in pregnant mice

(6) 8 mm wound was performed on female mice pregnant with eGFP+ fetuses and the lesion was injected with CCL2 or PBS immediately after and 2 days after skin excision. (A) FACS analysis demonstrated significantly greater number of eGFP+ cells in wound of pregnant mice injected with CCL2 compare to mice injected with PBS (n=3). (B) Quantification of eGFP+ cells in wound at day 7 of PBS or CCL2 injected pregnant mice (n=3). (C) Quantification of eGFP+ cells in wound sections from pregnant mice with eGFP+ fetuses after PBS or CCL2 injection (n=5). (D) Planimetry of wound area at each time points relative to original wound surface (n=5). (E) Measurement of neo-epidermal tongues and gaps in wound sites (n=5). (F) Quantification of the relative vessel surface per 20× field by fluorescence densitometry (n=4). (G) Quantification of the number of vessel type per 20× field (n=4). (H) Quantification of vWF+ eGFP+ double positive vessel per 4× field (n=4). T-test Student, * p<0.05; mean±SEM.

(7) FIG. 4: CCL2 recruits FMPCs to wound

(8) Peripheral mononuclear blood cells (PBMCs) from female mice pregnant with eGFP.sup.+ fetuses or virgin control mice with or without wound were collected at day 0, 1, 2, 3 and subjected to FACS analysis. (A, B and C) Percentage of CD11b.sup.+ CD34.sup.+ CD31.sup.+ cells. Female mice pregnant with eGFP.sup.+ fetuses were wounded and PBS or CCL2 were injected immediately after and 2 days after skin injury. PBMCs and wounds were collected 7 after for FACS analysis. (D) PBMC and (E) wound tissues were analyzed to determine maternal CD11b.sup.+ CD34.sup.+ CD31.sup.+ myeloid progenitor cells (eGFP.sup.− gate) and fetal CD11b.sup.+ CD34.sup.+ CD31.sup.+ myeloid progenitor cells (eGFP.sup.+ gate) upon CCL2 or PBS administration. (D and E) Percentage of CD11b.sup.+ CD34.sup.+ CD31.sup.+ cells in eGFP± gate (n=4). T-test Student, * p<0.05; mean±SEM.

(9) FIG. 5: FMPCs express high percentage of CCR2 after maternal skin injury

(10) Female mouse pregnant with eGFP.sup.+ fetuses was wounded and eGFP.sup.+ CD11b.sup.+ CD34.sup.+ CD31.sup.+, FMPCs were isolated from blood at wound day 1 or female mouse CaG-eGFP was wounded and eGFP.sup.+ CD11b.sup.+ CD34.sup.+ CD31.sup.+, MPCs were isolated from blood at wound day 1. The recipient mouse was normal virgin female with the same genetic background as the donor mice. 1×10.sup.5 FMPCs or MPCs were transplanted into the day 1 wound of the recipient mouse and the wound was harvested at day 7. (A) Quantification of CCR2.sup.+/eGFP.sup.+ cells in FMPCs or adult MPCs transplantation. (B) Quantification of CCR2.sup.+/eGFP.sup.− in FMPCs or adult MPCs transplantation. (C) Quantification of CCR2.sup.+ cells in CD11b.sup.+ CD34.sup.+ CD31.sup.+ eGFP± gate (n=3).

(11) FIG. 6: FMPCs overexpress CXCL1 in wound

(12) Female mouse pregnant with eGFP.sup.+ fetuses was wounded and eGFP.sup.+ CD11b.sup.+ CD34.sup.+ CD31.sup.+, FMPCs and eGFP.sup.− CD11b.sup.+ CD34.sup.+ CD31.sup.+, maternal MPCs were isolated from wound tissue at day 3. After mRNA extraction, high throughput PCR array analysis was performed. (A) Mouse PCR array analysis of angiogenesis-associated gene expression in FMPCs (right) and MPCs (left) (n=3). (B) Quantitative RT-PCR validation of CXCL1 mRNA expression normalized to mRNA GAPDH level (n=3).

(13) FIG. 7: An 8 mm wound was created in pregnant female mice carrying eGFP.sup.+ fetuses. We injected Ccl2 or PBS into the wound immediately and two days after skin excision. (a) Planimetry of the wound area relative to the initial wound area, at various time points from Ccr2.sup.KO/KO virgin mice (n=5). (b) Planimetry of the wound area relative to the initial wound area, at various time points from Ccr2.sup.KO/KO female mice mated with eGFP.sup.KI Ccr2.sup.KO male mice (n=5). (c) Planimetry of the wound area relative to the initial wound area, at various time points (n=5) from Ccr2.sup.KO/KO female mice mated with eGFP male mice. (d) Quantifications of eGFP.sup.+ cells in sections of wounds from pregnant mice the injections of PBS or Ccl2 (n=3). Student's t-test, * p<0.05; mean±SEM.

(14) FIG. 8: An 8 mm wound was created in 8 months old postpartum female SAD mice that had carried eGFP.sup.+ fetuses, or 8 months old virgin WT female mice. We injected Ccl2 or PBS into the wound immediately and two days after skin excision. (a) Planimetry of the wound area relative to the initial wound area, at various time points from 8 month old postpartum SAD mice (n=3). (b) Planimetry of the wound area relative to the initial wound area, at various time points from 8 months old virgin WT female mice (n=3). Student's t-test, * p<0.05; mean±SEM.

(15) FIG. 9: Postpartum female mice that had carried eGFP.sup.+ fetuses received an hepatectomia. We injected Ccl2 or PBS into the damaged liver lobe. Mice were analyzed 7 days after the surgery. (a) Quantifications of eGFP.sup.+ cells in sections of liver from postpartum mice that received the injections of PBS or Ccl2 (n=3). Student's t-test, * p<0.05; mean±SEM.

(16) FIG. 10: An 8 mm wound was created in virgin mice, pregnant mice or postpartum mice treated with clobetasol. We injected Ccl2 or PBS into the lesion immediately and two days after skin excision. Sirius red staining and quantification of collagen density (a) in virgin mice (n=3) (b) pregnant mice (n=3) and (c) postpartum mice treated with clobetasol (n=3). Scale bars: 50 μm. Quantitative RT-PCR analysis of Col1a, Col3a and TGFb mRNA levels normalized against GAPDH mRNA levels in the wound on day 7 in (d) virgin mice (n=3) (e) pregnant mice (n=3) and (f) postpartum mice treated with clobetasol (n=3).

EXAMPLE 1

(17) Material & Methods

(18) Mice: Male enhanced green fluorescence protein (eGFP) transgenic mice were obtained from Riken Laboratories (CD57BL/6-Tg (CAG-EGFP)lObs/J) and mated to Wild-type (WT) females on background C57BL/6 of 6-8 weeks old obtained from Harlan (Harlan). All mice care were in compliance with ethical rules of Université Pierre et Marie Curie (UPMC) animal care regulations.

(19) Flow cytometry: Back skin was shaved and wounds were harvested, incubated overnight at 4° C. in 0.05% trypsin-EDTA (Invitrogen) for mechanical separation of the epidermis. The tissue were digested in Collagenase IV for 60 min at 37° C., vortexed every 10 min and filtered using a 100 μm cell strainer, followed by 40 μm cell strainer (BD Pharmingen) to obtain a single cell suspension. Blood were draw from the heart of the mice and the peripheral mononuclear blood cells were separated from erythrocytes and platelets using Ficoll 1.088 method (Health Care). After collected the ring, the cells suspension was washed with PBS (Life Technologies) and then filtered using a 40 μm cell strainer (BD Pharmingen). Antibodies used for cytometry were CD34-eFluor660 (1:100; eBioscience), CD11b-PERCP-Cy5.5 (1:100; eBioscience), CD31-PE-Cy7 (1:100; eBioscience), CCR2 (1:100; Santa Cruz) crossed with an anti-goat-alexa 555 (1:1, 1000 Invitrogen). Flow cytometry data was acquired using a BD LSRII (BD Pharmingen) and sorting were performed on a MoFlo cell sorter (Beckman Coulter), and subsequently analyzed with FlowJo software (Treestar, San Carlos, Calif.).

(20) Immunostaining: We performed 5 μm cryosections from frozen tissue. After permeabilization with cold acetone, sections were blocked with 2% bovine serum albumin (BSA) (Sigma-Aldrich). Primary antibodies used including: rat anti-mouse CD31 (1:40; BD Biosciences), rabbit anti-mouse Lyve-1 (1:200; Abcam), rat anti-mouse F4/80 (1:250; Abcam), rat anti-mouse GR-1 (1:250; eBiosciences) rabbit anti-mouse Ki67 (1:200; Abcam), goat anti-mouse CCR2 (1:200; Santa Cruz biotechnology), goat anti-mouse CCL2 (1:200; Santa Cruz), rabbit anti-mouse Von Willebrand Factor (1:800; Abcam). For immunofluorescence, we used secondary antibodies goat anti-rabbit IgG labeled with Cy3 or Alexa 488, donkey anti-rat IgG labeled with Alexa 488 or Cy3 and rabbit anti-goat Alexa 555 (1:1000; Invitrogen). Slides were counterstained with 0.3 μg/ml DAPI (Sigma-Aldrich).

(21) Microscopy, scoring and measurements: We used Nikon Eclipse 90i fluorescent microscope equipped with Nikon DS-Fil C digital camera (Nikon, Tokyo, Japan). For cell scoring, photographs of 3 different fields were taken, and labeled cells were counted by fluorescence densitometry and reported as percentage of total nuclei. Mean percentage of labeled cells was calculated for each specimen. Measurements were done using ImageJ software (NIH, Bethesda, Md.).

(22) RNA extraction and quantitative PCR: Total RNA was extracted from cells or tissues using Trizol reagent as per the manufacturer's (Invitrogen) instructions and then reverse-transcribed with iScript cDNA synthesis kit (Bio-Rad). The resulting cDNA was used for PCR with the SYBR-Green Master PCR Mix (Roche). PCR and data collection were performed on a LightCycler 480 (Roche). The expression levels of samples were normalized to the housekeeping gene β-Actin.

(23) Surgical wounds: Mice were anesthetized by inhalation of 4.9% isoflurane at 300 ml/min ambient air flow. After depilation, four 6 mm or one 8 mm surgical wounds were generated using punch biopsy devices. All tissues above the panniculus carnosus were excised. Wounds were left uncovered until they were harvested. Standardized pictures of the wounds were taken on different time points using a Sony Cybershot 10.1 megapixels DSC-W180 digital camera (Sony, Tokyo, Japan). Wound tissues were harvested either snap-frozen in liquid nitrogen or stored at −80° C.

(24) Corticoïds treatment: 4 days after delivery, mice were treated with topical application on shaved dorsal skin of 200 μL of clobetasol (Dermoval) during 12 days.

(25) Chemokine/Cells injection: After generated 8 mm wound, 100 μL of CCL2 (Clinisciences, Nanterre, France) was injected at day 0 and day 2 in the fourth cardinal points of the wound bed at a concentration of 0.5 ng.Math.μL.sup.−1. Or 10 000 cells were injected after FACS sorting resuspended into PBS using the same procedure at day 1.

(26) PCR Array for Cytokines and Chemokines

(27) The change in cytokine and chemokine expression was measured using RT.sup.2 Profiler PCR Array for cytokines and chemokines system (Qiagen, Hilden, Germany). Total RNA was extracted from FACS isolated eGFP+ cells from bone marrow. Expression analysis of 86 cytokine and chemokine genes was performed Lightcycler 1536 system (Roche Diagnostics, Mannheim, Germany) according to the manufacturer's instructions. Data analysis was performed using the RT.sup.2 Profiler PCR Array Data Analysis Template (Qiagen, Hilden, Germany). Data normalization was performed using five housekeeping genes (Actb, B2m, Gapdh, Gusb, Hsp90ab1) and the relative expression levels were calculated with 2.sup.−ΔΔCt.

(28) PCR Arrays were performed in AP-HP Hopital La pitié Salpétrière ICM, using a LightCycler 1536 (Roche) with RT.sup.2 Profiler PCR Arrays.

(29) Statistical analysis: Statistical analysis was performed with the statistical software Graphpad Prism. Results were reported as mean±SEM. A single comparison between two groups was performed with an unpaired, two-tailed Student's t-test. Statistical significance of difference was defined when p-value was <0.05.

(30) Results

(31) Pregnancy Improve Skin Wound Healing by Enhancing Vascular Angiogenesis

(32) To evaluate whether pregnancy affects skin wound healing, we performed excisional wounds on dorsal skin of pregnant mice at gestation day 15.5 and their age matched respective virgin littermate. Wound closure was significantly accelerated in pregnant mice (mean wound surface on day 5: pregnant vs virgin=8.39% vs 13.67%; p=0.025) (FIG. 1A). Re-epithelialization, as measured by the length of the neo-epidermis covering the granulation tissue, was quicker in pregnant mice (FIG. 1B). Consistently, we also discovered increased number of Ki67.sup.+ proliferating cells in both epidermis and dermis area of pregnant mice (FIG. 1C).

(33) Inflammation and neovascularization are two crucial factors of wound healing process. Here we found CD31.sup.+ blood vessel density within the wound bed increased in pregnant compared to virgin mice, while there were no difference of LYVE1.sup.+ lymphatic vessel density between both groups (FIGS. 1D and 1E). VEGF-A binds to cell surface receptors VEGFR1 and VEFGR2 and mediates vascular angiogenesis, while VEGF-C acts on receptor VEGFR3 and promotes lymphatic angiogenesis. We discovered gene expression of VEGF-A, VEGFR1 and VEFGR2 were significantly elevated in wound sites of pregnant mice (FIG. 1F), however, VEGF-C and VEGFR3 levels were not altered (FIG. 1G). These results suggest that vascular, but not lymphatic angiogenesis, increased in pregnant wound.

(34) Immunofluorescence analysis revealed no difference between pregnant and virgin mice in wound sites for infiltrating GR-1.sup.+ neutrophils, and F4/80.sup.+ macrophages, thus suggest inflammation appears to be unchanged in pregnant wound (Data not shown).

(35) Taken together, these results provided initial indication that pregnancy improves maternal skin wound healing by enhancing vascular angiogenesis.

(36) Maternal Skin Wound Activates FMCs by Inducing CCR2

(37) Our laboratory previously reported that FMCs actively participates in maternal wound healing and VEGF-A plays a crucial role in recruiting these cells. To further investigate the role of fetal cells in maternal wound healing, we mated virgin C57BL/6 females with heterozygous enhanced green fluorescence protein (eGFP) males, and performed excisional wounds on dorsal skin at 15.5 day of pregnancy (Data not shown). The FMCs were identified by their expression of eGFP and quantified by fluorescence-activated cell sorting (FACS) from maternal organs. We found that fetal cell dramatically increased 1 days after wound in bone marrow (FIG. 2A), blood (FIG. 2B) and wound (FIG. 2C), and restored to normal level at day 3, thus indicates that fetal cells activation is an early and transient process.

(38) In order to identify novel targets, especially chemokines, which are activated by maternal skin wound in FMCs, we performed PCR array analysis on sorted fetal cells from bone marrow with or without skin injury. CCR2 was the most significantly upregulated chemokine receptor after injury (FIG. 2D), thus indicated that maternal wound may induce CCR2 in FMCs.

(39) Moreover, eGFP.sup.+ FMCs were detected in wound tissue and a considerable portion of these cells expressed CCR2 (Data not shown). Furthermore, in peripheral blood, only 1.5% of eGFP.sup.+ FMCs expressing functional CCR2 in unwounded mice. However, 90% of these cells expressed CCR2 one day after skin injury, while 20% of them still expressing CCR2 at day 3 (Data not shown). Collectively, these results suggest that early post-wound activation of CCR2 signaling in fetal cells could serve to trigger FMCs from circulation blood to wound site.

(40) Overexpression of CCL2/CCR2 During Early Skin Wound Healing is not Affected by Pregnancy

(41) To further determine CCR2 chemotactic cues in wound and which ligand of CCR2 is triggering the movement of FMCs to injured skin, we measured the expression of CCR2 and its two major ligands CCL2 and CCL8 at skin wound bed of pregnant and virgin mice. CCR2 mRNA dramatically increased one day after injury and significantly decreased at day 3 from the level of day 1 (FIG. 2E), therefore suggests that CCR2 overexpression in injured skin is an early and transient event. Correspondingly, protein level of CCR2 follows the same pattern as its mRNA (Data no shown). Interestingly, one of the CCR2 ligand, CCL2 level is in parallel to CCR2 expression, with strong increased at day 1 and dropped at day 3 (FIG. 2F). Meanwhile, the other CCR2 ligand, CCL8 expression only have slight increase at day 1 and significantly increased at day 3 (Data not shown). These data indicate that CCL2/CCR2 signaling is specifically overexpressed early during wound healing. In addition, the mRNA level of CCR2, CCL2 and CCL8 in wound tissue have no difference between virgin and pregnant mice (FIG. 2E, 2F and supplemental figure not shown) indicate that pregnancy does not affect this signaling pathway.

(42) To further identify the specific cell types secreting CCL2, we sorted CD45.sup.+ leucocytes from normal skin and day 1 wound, and discovered robust overexpression of CCL2 in wound, thus suggest immunocytes' secretion of CCL2 during early wound healing process (FIG. 2G and supplemental figure not shown). Co-immunofluorescent analysis demonstrated that F4/80.sup.+ monocytes, not GR-1.sup.+ neutrophils, expressed CCL2 (data not shown), therefore suggest resident monocytes secret initially CCL2. In addition, CCL2 were also detected in CD31.sup.+ endothelial cells (data not shown). Moreover pregnancy does not affected early inflammation (data not shown) and the secretion of CCL2 (FIG. 2G).

(43) Collectively, these data suggested that monocytes and endothelial cells secrete CCL2 during initial stage of skin wound healing, and not affected by pregnancy.

(44) CCL2 Recruits FMCs to Maternal Wound

(45) FMCs are able to infiltrate into the maternal wound and participate in the healing process. Due to the high percentage of FMCs expressing CCR2 on their surface and overexpression of CCL2 in wound tissue during early stage of healing, we speculated that CCL2 may mediates the early recruitment of FMCs to wound. To attest this hypothesis, we injected recombinant CCL2 mouse protein or PBS as control into the wound bed at day 0 and day 2 after excisional wounds. We initially quantified eGFP.sup.+ cells number in blood and skin wound of pregnant mice injected with CCL2 or PBS using FACS analysis at day 7. In blood, the eGFP.sup.+ cell number wasn't different between the mice injected with CCL2 and PBS (data not shown). Interestingly, skin wound tissue injected with CCL2 contained more than double number of eGFP.sup.+ cells than PBS (FIGS. 3A and B). In order to confirm this finding, we performed immunofluorescence analysis on skin wound granulation tissue at day 7. In accordance with our FACS results, the number of eGFP.sup.+ cells was almost three times more in the sections of CCL2 injected mice in compare with PBS injected mice (FIG. 3C). Together, the results suggest CCL2-CCR2 pathway mediated signals mediates the recruitment of FMCs to wound sites.

(46) CCL2 Administration Improves Wound Healing by Enhancing Neovascularization in Pregnant but not in Virgin Mice

(47) FMCs participate in inflammation and angiogenesis during maternal wound healing. Here, we also demonstrated that CCL2 recruits FMCs to wound (FIG. 3A-C). Therefore, we further analyzed the maternal wound healing process upon CCL2. For the mice injected with CCL2, injured surfaces reduced to less than 16.8% (FIG. 3D), meanwhile for mice injected with PBS, wound areas remained over 28.4% of the original lesion at day 7 after skin excision (p=0.024) (FIG. 3D). Concomitantly, neo-epidermal tongue measurements on tissue sections, showed increased re-epithelialization after CCL2 injections (FIG. 3E). Moreover, proliferation as measured by the number of Ki67.sup.+ cells, was elevated in both epidermis and dermis area of CCL2 injected mice (data not shown). Neovascularization as measured by CD31.sup.+ blood vessel density as well as VEGF-A, VEGFR1 and VEGFR2 gene expression were significantly elevated in wound site of CCL2 injected mice (FIGS. 3F and 3G and data not shown). On the contrary, lymphatic angiogenesis was not altered upon CCL2 administration (FIGS. 3F and 3G, and data not shown). In addition, inflammation in the wound, as measured by the number of GR-1.sup.+ and F4/80.sup.+ cells in granulation tissue demonstrated no difference between CCL2 and PBS injected mice (data not shown). Nassar et al. reported that FMCs form blood vessel in maternal wound. Consistently, we also identified Von Willebrand Factor (vWF) positive blood vessels largely, at least partially, comprised of eGFP.sup.+ FMCs, and the number of these fetal microchimeric vessels was significantly higher in CCL2 injected compared to PBS injected mice (FIG. 3H). We concluded from these data that CCL2 improve healing by promoting maternal neovascularization and formed fetal origin vessels.

(48) To further determine whether CCL2 improves skin healing through the effect of FMCs or directly affect wound closure, we analyzed the wound healing upon CCL2 administration in virgin mice. Strikingly, for virgin mice, CCL2 injection did not change the healing for all paradigms compared to PBS injection: wound surface, neo-epidermal tongue, proliferation, inflammation, angiogenesis and lymphangiogenesis (data not shown). Therefore the effect of CCL2 on improving wound healing is limited to pregnant, not virgin mice, thus extended our argument that CCL2-recruited FMCs contributed to promoting maternal neovascularization and improving wound healing.

(49) Overall, these data suggested that CCL2's effect on healing is through fetal cells.

(50) A Specific Subpopulation of FMCs Respond to CCL2 in Maternal Wound Healing

(51) Maternal wound recruits fetal myeloid progenitors (FMPCs) and fetal endothelial progenitors (FEPCs). Here, we further investigated the dynamics of MPCs and EPCs in peripheral blood during wound healing. In virgin control, the number of MPCs, defined as CD11b.sup.+ CD34.sup.+ CD31.sup.+, maintained at the same level as unwound condition during the first two days, and significantly decreased at day 3 after wound (FIG. 4A). In pregnant mice, the number of maternal MPCs, defined as eGFP.sup.− CD11b.sup.+ CD34.sup.+ CD31.sup.+, displayed the same pattern as the virgin MPCs (FIG. 4B). On the contrary, the number of FMPCs, defined as eGFP.sup.+ CD11b.sup.+ CD34.sup.+ CD31.sup.+, markedly elevated at day 1 post wound compares to unwound condition. For day 2 and day 3, although reduced from day 1, FMPC number still maintained the same level as unwound (FIG. 4C). EPCs are well recognize as a crucial factor for neovascularization in wound healing process. Here we also studied the pattern of maternal (eGFP.sup.−) and fetal (eGFP.sup.+) EPCs defined as CD11b.sup.− CD34.sup.+ CD31.sup.+. Both populations displayed a delayed and abrupt increase at day 3 after skin lesion (data not shown). Collectively, these observations demonstrated that: unlike maternal MPCs, FMPCs elevated during early healing process, while maternal and fetal EPCs share parallel pattern. Thus, FMPCs are specifically activated after wound.

(52) We further investigated this fetal specific population of MPCs response to CCL2 administration. The number of FMPCs was significantly decreased in peripheral blood (FIG. 4D) and markedly increased in the wound tissue of mice injected with CCL2 (FIG. 4E). Meanwhile, the number of maternal MPCs was not change upon CCL2 injection in both blood and wound tissue (FIGS. 4D and 4E). Furthermore, both maternal and fetal EPCs numbers detected in blood and wound were same between CCL2 and PBS injection (data not shown). Collectively, these results suggest that CCL2 specifically recruits FMPCs from blood to wound during the healing process.

(53) FMPCs Organize Blood Vessel Endothelium in Wound

(54) To assess the cellular lineage of FMPCs in vivo, we isolated this population from the peripheral blood of mice pregnant with eGFP.sup.+ fetuses at wound day 1. As control eGFP.sup.+ adult MPCs were isolated from peripheral blood of virgin heterozygous eGFP+ female mouse. The cells were injected into the day 1 wound site of a eGFP.sup.− wild type BL/6 virgin mouse. Immunofluorescence analysis was utilized to observe the wound tissue section of the recipient mice at wound day 7. eGFP.sup.+ FMPCs displayed vWF.sup.+ endothelial phenotype. We even discovered blood vessels largely comprise of eGFP.sup.+ vWF.sup.+ FMPCs, with confocal microscopy confirming the intimal position of eGFP.sup.+ cells in vessel (data not shown). eGFP.sup.+ FMPCs also expressed marker of smooth muscle cell (α-SMA), but not marker of macrophage (F4/80) (data not shown). On the contrary, eGFP.sup.+ adult MPCs did not express endothelial marker vWF and myofibroblast marker α-SMA (data not shown). These results suggest that FMPCs differentiate into endothelial and myofibroblast lineage in wound.

(55) FMPCs Form Proliferative Cluster and Express High Percentage of CCR2

(56) Unlike adult MPCs, which maintain isolated single-cell form, FMPCs formed proliferative cluster and stained positive for Ki67 (data not shown). Interestingly, immunofluorescence analysis demonstrated that FMPCs detected in skin wound site, expressed high percentage of CCR2 (data not shown), lead us to speculate that CCL2/CCR2 interactions guide the recruitment of FMPCs into wound tissue. Indeed, in situ, almost all of FMPCs, but none of the adult MPCs expressed CCR2 (FIG. 5A). Meanwhile, the percentage of CCR2 expressing non eGFP cells was almost identical between FMPCs and adult MPCs injected samples (FIG. 5B). Furthermore, FACS analysis was performed on blood of mice pregnant with eGFP.sup.+ fetuses at wound day 1. CCR2 was expressed by about 90% of FMPCs and by only 0.3% of maternal MPCs (FIG. 5C). Collectively, these results suggest that CCR2 mediate the recruitment of FMPCs from blood to wound site.

(57) FMPCs Overexpressed CXCL1 in Wound

(58) To investigate the potential paracrine effects of FMPCs on angiogenesis, we sorted eGFP.sup.+ FMPCs and eGFP.sup.− maternal MPCs from the wounded skin tissue of mice pregnant with eGFP.sup.+ fetuses at day 3. Angiogenesis stage of skin wound healing occurs at day 2-3, angiogenetic factors secretion as well as recruitment of EPCs reach maximum at day 3-4. Here, we studied the gene expression profile of angiogenesis factors at day 3 using mouse angiogenesis PCR array analysis, and the prevalent transcripts were plotted in FIG. 6A. The most upregulated genes include CXCL1, Sphk and TGF-β2, with chemokine CXCL1 being the highest increased transcript. On the other hand, certain angiogenesis inhibitor genes were downregulated, namely Thbs2 and Bai1. FIG. 6B shows quantitative RT-PCR analysis validation of CXCL1 expression, which again demonstrated almost six fold higher CXCL1 mRNA level in FMPCs than in maternal MPCs. Finally, we performed immunofluorescence analysis on sections of wounds injected with eGFP+ FMPCs or eGFP+ adult MPCs, and discovered only FMPCs overexpressed CXCL1 (data not shown). These results suggest FMPCs secret pro-angiogenesis factors, especially CXCL1, to enhance maternal angiogenesis.

(59) CCL2 Improves Delayed Wound Healing in Postpartum Mice (Data not Shown)

(60) Fetal cells have been reported transferred into maternal circulation during pregnancy and engrafted in maternal bone marrow postpartum, even throughout life. Corticoids can delay skin repair, therefore clobetasol administration has been recognized as chronic wound healing model in mice. Here, to evaluate the effect of CCL2 in a of delay wound healing model for postpartum situation, we mated virgin females with heterozygous eGFP transgenic males. 2 weeks after delivery, females mice which gave birth to eGFP.sup.+ pups received daily topical application of dermoval (clobetasol cream) on the lower back for 14 days to induce skin atrophy. Then excisional skin injury was performed on dermoval treated area, and CCL2 or PBS was injected at day 0 and day 2 post wound. Pre-wound clobetasol application effectively prolong the normal healing process with wound surface at day 7 maintained at 100% of original lesion in compare to normal healing process for 30% of original wound. Meanwhile, CCL2 injection improved wound closure with unhealed area of 40% compared to PBS injection which were still at 100% of the initial surface. Neo-epidermal tongues of CCL2 injected wounds were significantly longer than PBS injected wounds. CCL2 also recruited more than double of fetal cells to wound bed compared to PBS, as demonstrated by FACS analysis. There was also a significant increase in epidermal and dermal cell proliferation, blood vessel angiogenesis and VEGF-A, VEGFR1 and VEGFR2 gene expression in wounds injected with CCL2 compared to PBS. Meanwhile, lymphogenesis as measured by LYVE1.sup.+ lymphatic vessel density as well as VEGF-C, VEGFR3 gene expression, and inflammation, as measured by the number of GR1.sup.+ and F4/80.sup.+ cells in granulation tissue demonstrated no difference. Therefore, CCL2 recruit FMCs and improve delayed wound healing in postpartum mice.

EXAMPLE 2

(61) Results

(62) The Mobilization of FMCs to Maternal Tissues Through Ccl2 is Mediated by Ccr2 Receptor on Fetal Cells

(63) Once we showed that Ccl2 was able to recruit FMCs to injected maternal tissue, it was important to demonstrate whether the fetal cell signaling was dependent on Ccr2. To answer this question, we analyzed virgin female Ccr2.sup.KO/KO mice, female Ccr2.sup.KO/KO mice mated with eGFP.sup.+ males and female Ccr2.sup.KO/KO mice mated with eGFP.sup.KI Ccr2.sup.KO males (FIG. 7 a,b). When Ccr2.sup.KO/KO female mice bear Ccr2.sup.KO/KO fetuses, the Ccl2 injections do not modify wound healing at any day (FIG. 7b). In contrast, when Ccr2.sup.KO/KO female mice bear Ccr2.sup.WT/KO fetuses, Ccl2 decreases wounded area by 49.08, 28.96 and 57.58% at days 2, 5 and 7 respectively (FIG. 7c). Interestingly, this ratio is similar to the ratio we found with Ccl2 in WT mice. In addition, only Ccr2.sup.KO/KO mice bearing Ccr2.sup.WT/KO fetuses displayed an increase in fetal cell infiltrate in granulation tissue upon Ccl2 local injections (FIG. 7d). Finally, the virgin Ccr2.sup.KO/KO mice did not show any change when treated with Ccl2 (FIG. 7a). Therefore, all these data demonstrate that Ccl2 enhances wound healing through Ccr2-dependent fetal cell recruitment to wound bed.

(64) Low Doses (“Physiological”) Doses of Ccl2 Improves Delayed Wound Healing in Post Parous Old “Sickle Cell” SAD Mice

(65) Sickle cell anemia may be complicated by prolonged ulcers. Nguyen V T el al., have recently developed a murine model of such chronic wounds, by performing wounds on old SAD mice (Nguyen V T et al., J Invest Dermatol, 2016, 136(2):497-506). We have therefore mated young female SAD mice with eGFP.sup.+ WT males. Then the postpartum female SAD were allowed to age until 8 months, a time when these mice have a delayed wound healing. We have afterwards performed 8 mm wounds in such post-partum old SAD mice (n=3) or same age virgin old SAD females. At days 0 and 2, 50 ng of Ccl2 was injected in wounds of these mice as previously done. Analysis of wound closure showed that Ccl2 induced a significant improvement of healing kinetics in post-partum treated mice at days 2, 5 and 7 (FIG. 8a). Interestingly, and as observed previously for the two other models, in virgin SAD wounds; Ccl2 did not modify healing kinetics (FIG. 8b). These results indicate that in a murine model of sickle cell anemia, Ccl2 treatment of post-partum mice induces an intense mobilization of FMCs that results in a 73% reduction in wound area.

(66) Ccl2 Improves FMCs Recruitment after Liver Injury

(67) In order to determine whether “natural stem therapy” with recruitment of fetal cells may allow improvement of other maternal tissues than the skin, we studied liver fate after hepatectomy. Median lobe hepatectomy was performed in C57/Bl6 post-partum females previously mated with eGFP C57/Bl6 males. Ccl2 50 ng was injected in the hepatectomy site in 3 animals while PBS was injected in 3 others. Mice were sacrificed 7 days later. Livers were harvested. Cryosections allowed the analysis of eGFP fetal cells. The percentage of such cells was 3.1% under PBS injection, while it reached 8.5% in Ccl2 treated livers (p<0.018) (FIG. 9a). This result shows that in liver repair, Ccl2 leads also to the recruitment of fetal cells.

(68) Low Doses of Ccl2 in Pregnant and Post-Partum Mice Improves Wound Healing without Fibrosis

(69) Finally, an important question remains the difference in the mechanisms observed with the low doses of Ccl2 used. Indeed, when Ccl2 is given at higher dosages in virgin animals in other settings than wound healing, recruitment of monocytes with risks of fibrosis was noted. We have therefore studied skin and liver post Ccl2 injections in wounds. In pregnant and post-partum clobetasol pretreated mice, Ccl2 wound injections lead to a reduced dermal fibrosis at day 7 post wounding as assessed through Sirius red staining (FIG. 10a-c). Collagen and 3 mRNAs levels analyzed through RT-PCR, in dermal day 7 wounds showed that Ccl2 led to a reduction of these messengers. In contrast, Ccl2 in wounds of virgin mice induced an increase of dermal fibrosis (FIG. 10d-f) as well as an increase in Collagen 1 and 3 mRNA levels. These results clearly show that low physiological doses of Ccl2 injected in wounds do not induce adult mono-macrophage recruitment with fibrosis, but rather FMPCs infiltration.

(70) In summary, our results indicate that in the absence of any deficiency in the CCR2/CCL2 axis, CCL2 given early, at low doses in wounds are able to improve angiogenesis through the mobilization of certain fetal cell population CD34+CD11b+CD31+ (FMPCs). In our hands, these cells (or fetal cells) express CCR2 on their surface with levels 100 times higher than their adult equivalents.

(71) Our result agrees with (a) the absence of increase of F480 macrophages at various time points in CCL2 treated post-partum mice, and the absence of the profibrotic effect reported with high CCL2 (b) previous studies showing no improvement of a normal skin wound repair when high doses of CCL2 are injected in virgin mice (Dipietro et al., Wound Repair and Regeneration, 2001).

REFERENCES

(72) Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.