CD11B + PIEZO-1 + MACROPHAGES AND USES THEREOF

Abstract

The present invention relates to isolated mammalian CD11b+/PIEZO-1+ macrophage cells, neutrophil cells, and/or an isolated exosome as secreted by said cells, pharmaceutical compositions comprising said cells and/or exosomes, as well as uses thereof.

Claims

1. An isolated mammalian CD11b+/PIEZO-1+ macrophage cell, neutrophil cell, and/or an isolated exosome as secreted by said cell.

2. A pharmaceutical composition comprising the CD11b+/PIEZO-1+ macrophage cell, neutrophil cell, and/or the isolated exosome according to claim 1, together with a pharmaceutically acceptable carrier and/or auxiliary agent.

3. The pharmaceutical composition according to claim 2, further comprising a matrix material selected from alginate, hyaluronic acid, agarose, poloxamers, polyethylene hydrogels, and a modified primary hydroxyl group containing polysaccharide comprising repeating disaccharide units, wherein in at least part of the disaccharide units the primary hydroxyl group is replaced by a functional group selected from halide groups and groups comprising sulfur or phosphorus atoms.

4. A method for producing a pharmaceutical composition according to claim 2, comprising the steps of: a) providing a biological sample derived from a mammal comprising CD11b+ macrophages and/or exosomes as secreted by said cells, b) purifying and/or isolating CD11b+ PIEZO-1+ macrophages or neutrophil cells from said sample based on the expression of CD11b+ and PIEZO-1+, and/or purifying and/or isolating exosomes as secreted by said cell based on their content of CD11b+ and PIEZO-1+, and c) formulating said purified and/or isolated CD11b+ PIEZO-1+ macrophages and/or exosomes into a suitable pharmaceutical composition.

5. The method according to claim 4, wherein said purifying and/or isolating comprises the use of beads or cell sorting.

6. A method for producing a pharmaceutical composition according to claim 2, comprising the steps of: a) providing a biological sample derived from a mammal comprising CD11b+ macrophages, neutrophil cells, or precursor monocytes thereof, b) in vitro differentiating said CD11b+ macrophages, neutrophil cells, or precursor monocytes into CD11b+ PIEZO-1+ macrophages or neutrophil cells, and c) formulating said purified and/or isolated CD11b+ PIEZO-1+ macrophages or neutrophil cells into a suitable pharmaceutical composition.

7. A method for diagnosing angiogenesis in a mammal, comprising identifying CD11b+ PIEZO-1+ macrophages, neutrophil cells and/or exosomes as secreted by said cells in a sample from a mammal to be diagnosed, wherein the presence of CD11b+ PIEZO-1+ macrophages, neutrophil cells and/or exosomes as secreted by said cells is indicative for angiogenesis in said mammal, when compared to a control sample.

8. The method according to claim 7, wherein said identifying further comprises detecting the amount of said CD11b+ PIEZO-1+ macrophages and/or neutrophil cells and/or the level of expression and/or the levels of the biological activity of CD11b+ and PIEZO-1+ in said macrophages and/or neutrophil cells in said sample, wherein an increase of the amount of said CD11b+ PIEZO-1+ macrophages and/or neutrophil cells and/or the level of expression and/or the levels of the biological activity of CD11b+ and PIEZO-1+ in said macrophages and/or neutrophil cells, when compared to a control, is indicative for an increased angiogenesis.

9. A method for identifying a compound that modulates the expression and/or the biological activity of PIEZO-1 in a CD11b+ macrophage and/or neutrophil cell, comprising the steps of a) contacting said CD11b+ macrophage and/or neutrophil cell with at least one compound that potentially modulates the expression and/or the biological activity of PIEZO-1 in said CD11b+ macrophage and/or neutrophil cell, and b) identifying a modulation of the expression and/or the biological activity of PIEZO-1 in the presence of said at least one compound, compared to a control CD11b+ macrophage.

10. A method for manufacturing a pharmaceutical composition, comprising the steps of performing a method according to claim 9, and formulating said compound as identified into a pharmaceutical composition.

11. An in vitro method for producing a CD1 b+/PIEZO-1+ macrophage and/or neutrophil cell, comprising the steps of a) providing a biological sample comprising CD11b+ macrophages, neutrophil cells, or precursor monocytes, and b) in vitro differentiating said CD11b+ macrophages or precursor monocytes into CD11b+ PIEZO-1+ macrophages and/or neutrophil cells using one or more suitable signal compounds.

12. A method for the prevention or treatment of insufficient angiogenesis in a mammalian patient, wherein said method comprises administering to the patient a pharmaceutical composition according to claim 2 or a pharmaceutical composition produced by a method comprising identifying a compound that modulates the expression and/or the biological activity of PIEZO-1 in a CD11b+ macrophage and/or neutrophil cell, wherein said identification comprises the steps of a) contacting said CD11b+ macrophage and/or neutrophil cell with at least one compound that potentially modulates the expression and/or the biological activity of PIEZO-1 in said CD11b+ macrophage and/or neutrophil cell, and b) identifying a modulation of the expression and/or the biological activity of PIEZO-1 in the presence of said at least one compound, compared to a control CD11b+ macrophage and formulating said compound as identified into a pharmaceutical composition.

13. A method for the in vitro production/induction of CD11b+ PIEZO-1+ macrophages and/or neutrophil cells wherein said method comprises the use of a pharmaceutical composition produced according to claim 10.

14. A screening tool for identifying a compound that modulates the expression and/or the biological activity of PIEZO-1 in a CD11b+ macrophage, comprising a recombinant CD11b+ macrophage and/or neutrophil cell comprising a genetic construct that allows for detecting the expression and/or the biological activity of PIEZO-1 in said CD11b+ macrophage and/or neutrophil cell.

15. A kit comprising materials and reagents for performing a method according to claim 9.

16. The pharmaceutical composition according to claim 2, wherein the matrix material comprises carboxylated agarose and/or phosphate agarose.

17. The method according to claim 5, wherein said purifying and/or isolating comprises the use of fluorescence activated cell sorting.

18. The method according to claim 11, wherein said biological sample is blood.

19. The method according to claim 11, comprising the use of a signal compound selected from small molecules, cytokines, proteins, and peptides.

Description

[0097] FIG. 1 shows that RGD functionalized carboxylated agarose hydrogels induce angiogenesis. a-d Frozen sections of GC muscles implanted with distinct hydrogel compositions were stained for hematoxylin/eosin and (e-h) immunostained against CD31 (endothelial cells, red), NG2 (pericytes, green), α-SMA (smooth muscle cells, cyan). Quantification of vessel morphology: Vessel diameters (i) and vascular segment length (j) were quantified in the same areas within the hydrogels two weeks post implantation: VLD=vessel length density, is expressed as millimeters of vessel length per square millimeter of area of effect (mm/mm.sup.2); the segment length is expressed as μm of vessel length between 2 consecutive branch points (k). All data sets represent mean values ±SEM; * p<0.05, ** p<0.01 by Kruskal-Wallis test; n=4 independent muscles per each group. Scale bars=1 mm in all HE-stained panels. Scale bars=20 μm in all immunofluorescence-stained panels.

[0098] FIG. 2 shows that soft RGD-functionalized carboxylated agarose supports stable capillaries. a-d Immunofluorescence staining of endothelium (CD31, in light gray), pericytes (NG2, in gray), smooth muscle cell (α-SMA, in dark gray) on frozen sections of leg skeletal muscles of mice injected with distinct hydrogel compositions and sacrificed 7 weeks later. e-h Seven weeks post hydrogel implantation the VLD, the vascular segment length, vessel diameters and perfusion index were quantified in the same areas within the hydrogels: All data sets represent mean values ±SEM; * p<0.05; ** p<0.01, *** p<0.001 and ****p<0.000m by Kruskal-Wallis test; n=4 independent muscles per each group. i-m Endothelial proliferation was assessed by quantifying the percentage of endothelial cells positive for Ki67 by immunofluorescence staining on frozen muscle sections, n=4 independent muscles per group. Scale bars=20 μm in all immunofluorescence-stained panels.

[0099] FIG. 3 shows that RGD functionalized carboxylated agarose recruits myeloid cells. a-f Immunofluorescence staining of endothelial cells (CD31, in green), leukocyte (CD45, in red) and monocytes (CD11b, in green) on cryosections of limb muscles 2 week after injection with 5 kPa and 0.5 kPa hydrogel compositions. Scale bar=20 μm in all panels. c, f Quantification of the number of CD45+ and CD11b+ cells recruited into the implanted hydrogel. All data sets represent mean values ±SEM;** p<0.01, by Mann-Whitney test; n=4 independent muscles per each group.

[0100] FIG. 4 shows that the soft RGD functionalized carboxylated agarose microenvironment recruits CD11b±/Piezo-1+ cells. (a-h) Immunofluorescence staining of monocytes (CD11b, in red) and of PIEZO-1±cells (in light blue) on cryosections of limb muscles 2 week after injection with 5 kPa and 0.5 kPa hydrogel compositions. Scale bar=20 μm in all panels. (i) Quantification of the number of CD11b±/Piezo1+ cells in sites within the implanted gels. (j-n) Circulating Piezo-1+/CD11b+ monocytes were identified in both mouse and human blood by FACS. (o) Fold enrichment of recruited CD11b±/Piezo1+ monocytes in sites within the implanted gels compared with circulating CD11b±/Piezo1+ monocytes. (p) Quantification of CD11b+/Piezo1+ cells in sites within the implanted gels (% of total CD11b+ cells), see (i), above (TOP), and Quantification of Piezo1+ monocytes and Piezo1+ neutrophils in sites within the implanted gels (% of total CD11b+ cells) (BOTTOM). All data sets represent mean values ±SEM; **** p<0.0001, by parametric unpaired t-test; n=3 mice and n=3 human donors.

EXAMPLES

Materials and Methods

Gels Preparation and Characterization

[0101] Native agarose (1 g) (Merck, Darmstadt, Germany) was transferred into a 3-necked round bottom flask equipped with a mechanical stirrer and pH-meter (WTW, Weilheim, Germany), and dissolved in deionized water at a concentration of 1% w/v by heating to 90° C. The flask was cooled down to 0° C., using an ice bath, under vigorous mechanical stirring in order to prevent gelation of the agarose, and the reactor was charged with 99% (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) (20.6 mg, 0.16 mmol), NaBr (0.1 g, 0.9 mmol), and NaOCl (2.5 mL, 15% solution) all obtained from Sigma Aldrich (Steinheim, Germany). As the reaction occurs, the solution becomes acidic. The pH of the solution was maintained at 10.8 by dropwise addition of NaOH (0.1 M) (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) throughout the duration of the reaction. The degree of carboxylation was back calculated by using the volumes of NaOH (0.1 M) solution added during the reaction. The reaction was quenched by the addition of NaBH.sub.4 (0.1 g) (Sigma-Aldrich Chemie GmbH, Steinheim, Germany), following which the solution was acidified to pH 8 (0.1 mHCl) and stirred for 1 h. The modified agarose was then precipitated by the sequential addition of NaCl (12 g, 0.2 mol) and ethanol (500 mL) (technical grade). The product was collected by vacuum filtration using a fritted glass funnel and then washed using ethanol (500 mL). The ethanol, catalyst, and salts were removed by extensive dialysis against water for 2 days with replacement of the water every 12 h. The modified agarose was then freeze-dried on a Beta 2-8 LD (Martin Christ Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany) overnight to yield a white solid. The degree of carboxylation was verified by the appearance of peaks associated with aliphatic carboxylic acid groups via FTIR (KBr) (vc=0: 1750 cm-1) (Bruker Optics, Ettlingen, Germany) and NMR300 Mhz (13C: 180 ppm) (Bruker BioSpin, Rheinstetten, Germany).

Carboxylated Agarose Mechanical Properties

[0102] Rheology experiments were performed with a Physica MCR 301 (Anton Paar, Wundschuh, Austria) equipped with a Peltier cell to control the temperature and the experiment was performed with a plate geometry PPR25 (Anton Paar, 250 Wundschuh, Austria). Samples in deionized water were prepared by heating at 90° C. for 10 min until a clear solution was obtained. The liquid was then poured on the rheometer plate and the following sequence was used to determine the shear modulus: cool down from 80° C. to 5° C. in 30 min, 30 min equilibration at 5° C. to allow the gel to form, followed by heating to 37° C. and equilibration for 30 min prior to measuring G′ and G″ by increasing the rotation frequency from 0.01 rad/s up to 10 rad/s with a 1% deformation. The G′ of the gel was determined at 1 Hz shear frequency.

Carboxylated Agarose RGD Functionalization

[0103] Functionalization of CA with the RGDSP (Peptides International, Louisville, Ky., USA) peptide was performed using 1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide (EDC) (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) coupling chemistry. CA was sterilized overnight in 70% ethanol, and the ethanol was re-moved by extensive dialysis against water.

[0104] The sterile CA was freeze-dried overnight to yield a white solid. CA (30 mg, 0.25 μmol) was dissolved in MES sterile buffer (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) and an excess EDC (210 mg, 1.3 mmol) was added and the solution stirred for 30 min. Following this the peptide was added (500 μg, 0.66 μmol for the CA-60 gels and 1 mg, 1.33 μmol for the gels) and the solution stirred for an additional 2 h at room temperature. Unreacted reagents were removed by dialysis against water. RGD incorporation was verified using elemental analysis Vario EL (Elementar Analysen systeme GmbH, Langenselbold, Germany) equipped with a thermal conductivity detector and an adsorption column for CO.sub.2 at 110° C. and H.sub.2O at 150° C. All samples were accurately weighed to 3 mg before measurements and the percentage of nitrogen was used to calculate the peptide attachment. It was found that 11.6±0.9% of the repeat units where functionalized on both CA-28 and CA-60.

Gels Implantation In Vivo

[0105] Gels were implanted into 10-15 week old immune-deficient SCID CB.17 mice (Charles River Laboratories, Sulzfeld, Germany). Animals were treated in accordance with Swiss Federal guidelines for animal welfare, and the study protocol was approved by the Veterinary Office of the Canton of Basel-Stadt (Basel, Switzerland; Permit 2071). Gels were pre-loaded in 1 ml syringes and kept on ice. Fifty μl of cold PBS (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) followed by 50 μ1 of cold gels were implanted in both the medialis and lateralis portions of Gastrocnemius (GC) leg muscle, using a syringe with a 291/2G needle (Becton Dickinson, Allschwil, Switzerland).

Tissue Staining and Microscopy

[0106] For the studies performed on frozen tissue sections, mice were anesthetized and the tissues were fixed by vascular perfusion of 1% paraformaldehyde in PBS pH 7.4 for 4 minutes under 120 mm/Hg of pressure. GC muscles were harvested, post fixed in 0.5% paraformaldehyde in PBS for 2 hours, cryo-protected in 30% sucrose in PBS overnight at 4° C., embedded in OCT compound (CellPath, Newtown, Powys, UK), frozen in freezing isopentane and cryosectioned. Tissue sections (30 μm) were stained with Hematoxylin and eosin (H&E) and in addition, the gel biocompatibility was examined with Masson trichrome staining (Réactifs RAL, Martillac, France), performed according to manufacturer's instructions. For immunofluorescent staining of neighboring 30 μm thick longitudinal cryosections, the sections were blocked with PBS 0.1% triton supplemented with 5% normal goat or donkey serum and 2% BSA (all reagents from Sigma-Aldrich Chemie GmbH, Steinheim, Germany) for 1 hour. The slides were then incubated for 1.5 hour at room temperature with the following primary antibodies and dilutions: rat anti-mouse PECAM-1 (clone MEC 13.3, BD Biosciences, Basel, Switzerland) at 1:100 or hamster monoclonal 300 anti-mouse CD31 (clone 2H.sub.8, Millipore, Merck, Germany) at 1:200; mouse anti-mouse/human a-SMA (clone 1A4, MP Biomedicals, Basel, Switzerland) at 1:400, anti-mouse NG2 (Chemicon International, Hampshire, UK) at 1:200; rabbit anti-Ki67 (Abcam, Cambridge, UK) at moo, rat monoclonal anti-CD11b (clone M1/70, Abcam, Cambridge, UK) at moo, rat anti-mouse CD45 (PE conjugated, clone 30 F11, BD Biosciences, Basel, Switzerland) at 1:400. Negative controls lacking primary antibody were always performed. Sections were rinsed in PBS 0.1% triton and then incubated for 1.5 hour at room temperature with fluorescently labeled secondary antibodies (Invitrogen, Basel, Switzerland) diluted at 1:200. The slides were then rinsed and mounted.

[0107] Piezo1 staining immunohistochemistry experiments were performed on Ventana Discovery Ultra instrument (Roche Diagnostics, Manheim, Germany) by using the procedure RUO Discovery Universal instead. Cryosections were fixed for 12 minutes with 4% paraformaldehyde followed by 1 hour incubation at 37° C. with rat anti-CD11b (1:100) and 32 minutes incubation at 37° C. with a fluorescently labeled anti rat secondary antibody used at 1:200 (Invitrogen, Basel, Switzerland). Next, after an antibody denaturation step, sections were pre-treated for 16 minutes with Cell Conditioning Solution (CC1) (Roche Diagnostics, Mannheim). Rabbit anti Piezo1 (Proteintech, Manchester, UK) diluted at 1:500 was then incubated for 1 hour at 37° C. and detected with the secondary antibody (ImmPRESS reagent kit peroxidase anti-rabbit Ig MP-7401, Vector) applied manually (200 μl) for 32 minutes.

[0108] Discovery Rhodamine (Roche Diagnostics, Mannheim) applied for 12 minutes was used for the detection. To study vessel perfusion, 100 μg of biotinylated Lycopersicon esculentum (tomato) lectin (Vector Laboratories, Burlingame, Calif.) was dissolved in 100 μl, which binds the luminal surface of all blood vessels, and injected intravenously through the femoral vein. After four minutes the thoracic cavity was opened and the tissues were fixed by perfusing the animal with 1% paraldehyde and leg muscle were collected and processed as described above. Fluorescently labeled Streptavidin (eBioscience, Vienna, Austria) at 1:200 was used to visualize the perfused vasculature. Frozen sections were mounted with Faramount Aqueous Mounting Medium (Dako, Agilent Technologies, Basel, Switzerland), and fluorescence images were taken with 40× objectives on a Carl Zeiss LSM710 3-laser scanning confocal microscope (Carl Zeiss, Feldbach, Switzerland) or with a 20× objective on an Olympus BX61 microscope (Olympus, Volketswil, Switzerland). All Image analysis were performed with either Cell Sense software (Olympus, Volketswil, Switzerland) or Imaris 7.6.5 software (Bitplane, Zürich, Switzerland) on fluorescence images acquired with a 20× objective on an Olympus BX61 microscope or with a 40× objective on a Carl Zeiss LSM710 3-laser scanning confocal microscope.

Histological Analysis

[0109] The quantification of vessel length density (VLD) and vessel perfusion was performed on sections of leg muscles harvested after intravascular staining with biotinylated lectin and fluorescently labeled streptavidin, as described above. After co-staining with a fluorescent anti-CD31 antibody, VLD was measured on 6-10 randomly acquired fields per leg and 4 muscles per group (n=4) by tracing the total length of vessels in the fields and dividing it by the area of the fields. The total lengths of lectin-positive and CD31-positive vascular structures in each field were traced independently and the vessel perfusion index was calculated as the ratio between the two values. Vascular segment length was also measured in all representative fields per muscles tracing the total length of vessels and dividing it for the number+1 of branching points. Vessel diameters were measured by overlaying a captured microscopic image with a square grid. Squares were chosen at random, and the diameter of each vessel (if any) in the center of selected squares was measured. Two to five hundred total vessel diameter measurements were obtained from 4 muscles 350 per each group (n=4). KI67+ endothelial cells (ECs) were quantified from the total number of ECs (260-890 total ECs were counted per condition at 7 weeks post gel implantation) in vascular structures visible in each of 3-5 fields, in each area of effect. 10 areas with a clear angiogenic effect were analyzed per group. The quantification of leukocytes (CD45) and monocyte (CD11b) were performed on 7 random areas per muscle (n=4) per group by counting them and normalizing to the absolute number of CD45+ and CD11b+ cells with area. (9000-1400 total CD45+ cells and 5000-6000 total CD11+ cells were counted per condition at 2 weeks post gel implantation). The quantification of Piezo1+/CD11b+ cells was performed on 7-10 random areas per muscle (n=4) after immunostaining for CD11b (700-800 total CD11+ cells were counted per condition at 2 weeks post gel implantation).

[0110] All Image measurements were performed with both Cell Sense software (Olympus Volketswil, Switzerland) and Imaris 7.6.5 software on fluorescence images acquired with a 20× objective on an Olympus BX61 microscope or with a 40× objective on a Carl Zeiss LSM710 3-laser scanning confocal microscope.

Blood Cell Analysis by FACS

[0111] Peripheral Blood Mononuclear Cells (PBMCs) were isolated from 3 immune-deficient SCID CB.17 mice (Charles River Laboratories, Sulzfeld, Germany) and 3 human healthy donor using a density gradient technique (Histopaque-1077, Sigma-Aldrich Chemie GmbH, Steinheim, Germany). Briefly, after centrifugation, carefully aspirated PBMCs from the Ficoll-plasma interface were stained for APC-anti-human CD11b (clone ICRF44, BD Biosciences, Basel, Switzerland) at 1:100 or rat monoclonal anti-CD11b at 1:100 and anti-human Piezo1 (Abcam, Cambridge, UK) at 1:500. Fluorescently labeled secondary antibody to detect Piezo1 (Invitrogen, Basel, Switzerland) was used at 1:200. Samples were acquired by LSR Fortessa (BD Biosciences, Basel, Switzerland), 375 and data analyzed by FlowJo software (Tree Star, Ashland, Oreg., USA).

Statistical Analysis

[0112] Data are presented as mean±standard error. The significance of differences was assessed with the GraphPad Prism 7.03 software (GraphPad Software). The normal distribution of all data sets was tested and, depending on the results, multiple comparisons were performed with the parametric 1-way analysis of variance (ANOVA) followed by the Sidak test for multiple comparisons, or with the non-parametric Kruskal-Wallis test followed by Dunn's post-test, while single comparisons were analyzed with the non-parametric Mann-Whitney test or the parametric unpaired t-test.

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