Chemicals and reagents
All the fluorenylmethyloxycarbonyl (Fmoc) amino acids and Fmoc-Lys (Boc)-Wang Resin were purchased from AAPPTec (Louisville, KY, USA). Dichloromethane (DCM), acetic acid, acetic anhydride, thioanisole, phenol, hydroxybenzotriazole (HOBt), N,N-diisopropylethylamine (DIEA), N-trityl-1,2-ethanediamine, phenol, thioanisol, dimethylformamide (DMF), N,N′-diisopropylcarbodiimide (DIC), trifluoroacetic acid (TFA), iodine, methyl tert-butyl ether (MTBE), O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU), and dexamethasone (DEX) were purchased from Sigma-Aldrich (St Louis, MO). Hematoxylin and eosin (H&E) stains were purchased from MilliporeSigma (St Louis, MO). Rabbit anti-pANXA2 (phospho-Tyr24) antibody was purchased from Signalway Antibody (College Park, MD). AlexaFluor 594-conjugated donkey anti-rabbit antibody was purchased from Thermo Fisher Scientific (Waltham, MA).
Synthesis of LS301
LS301 (cypate-cyclic (DCys-Gly-Arg-Asp-Ser-Pro-Cys)-Lys-OH) was synthesized as previously reported . Briefly, the linear GRD peptide, H-DCys (Acm)-Gly-Arg (Pbf)-Asp (tBu)-Ser (tBu)-Pro-Cys (Acm)-Lys (Boc)-OH, was prepared via a CEM Liberty Blue microwave peptide synthesizer (Matthews, NC, USA) on the Fmoc-Lys (Boc)-wang resin. The resin (0.1 mmol) was swelled in DCM for 1 h before use. Fmoc-amino acids (0.5 mmol, 5 eq), coupling reagent (HBTU, 0.5 mmol, 5 eq), and DIEA (1 mmol, 10 eq) were added to the resin and the mixture was reacted for 15 min under microwave irradiation (100W, 90°C). The resin was washed three times with DMF. Deprotection of Fmoc group was carried out by treatment of 20% piperidine/DMF for 5 min under microwave irradiation (100W, 90 °C). The peptidyl resin was washed and the peptide cyclized through the disulfide bridge with iodine (1.2 eq) in DMF for 90 min. Subsequently, cypate (3 eq) was conjugated to the cyclic peptide on solid support in the presence of DIC (5 eq) in DMF to afford the LS301 peptidyl resin. The resin was then treated with a cleavage cocktail of TFA: thioanisol: phenol: water (85:5:5:5, v/v/v/v) for 90 min at room temperature. The cleaved peptide product was concentrated in vacuo before purification by reverse-phase HPLC (Gilson, Middleton, WI, USA). Analytical HPLC was used to determine product purity (> 95%) and the compound identity was confirmed by electrospray ionization mass spectrometry on a Shimadzu LCMS-2020 Mass Spectrometer (Columbia, MD) with peaks observed at 1470 (M+1) and 735 (M+2/2).
Male 5–7-week-old C57BL/6J mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and housed in designated animal facilities. Mice were fed ad libitum and inspected regularly. All animal experiments were performed in compliance with guidelines and protocols approved by the Division of Comparative Medicine at Washington University in St. Louis. The animal protocol is subjected to annual review and approval by The Animal Studies Committee of Washington University.
Arthritis mouse models
The K/BxN mice with spontaneous arthritis (F1) [38,39,40] were maintained in the laboratory of Dr. Christine Pham (Department of Internal Medicine, Washington University School of Medicine). To establish serum transfer arthritis (STA), male 6–8 weeks old C57BL/6J mice (The Jackson Lab, Bar Harbor, ME, USA) were injected intraperitoneally with 150–175 μL of serum derived from F1 mice (8–9 weeks old), with day 0 denoting the day of serum transfer/disease induction. Clinical manifestation of arthritis in each paw was assessed daily on a scale of 0—3 with 0 = no swelling or erythema, 1 = slight swelling or erythema, 2 = moderate erythema and swelling in multiple digits or entire paw, and 3 = pronounced erythema and swelling of an entire paw, with a maximum score of 12 per mouse as previously described . Ankle thickness of two hind paws was measured using calipers. Animals were monitored for signs of distress during arthritis induction including their ability to move around the cage and access food/water.
In vivo imaging
Animals were shaved and excess hair removed using commercially available hair removal cream. Mice were anesthetized with isoflurane for injection and imaging procedures. LS301 stock (or LS301-methotrexate or LS301-methylpresdnisolone conjugate stock) in dimethyl sulfoxide solution was diluted in phosphate-buffered saline to a final concentration of 60 μM and injected via tail vein into mice. In vivo near-infrared fluorescence using 785 nm excitation and 820 nm emission filters was assessed pre-injection, post-injection, and/or at indicated time points post-injection with a Pearl Small Animal Imaging System (LICOR Biotechnology, Lincoln, NE). Regions of interest (ROIs) for fluorescence quantitation were drawn and analyzed using the Pearl Small Animal Imaging System software.
Treatment response monitoring studies
Experiments were performed in a blinded fashion, where the technician responsible for clinical assessment of paw score and ankle measurements was blinded to treatment groups. For studies on disease remission, mice with STA were injected with 6 nmol of intravenous LS301 on day 4 post-disease induction and imaged at 18h post LS301 injection using the Pearl Small Animal Imaging System as described above. Clinical paw scores and ankle measurements were obtained daily. On day 23 post-disease induction when the clinical paw scores of mice were near the baseline, mice were imaged again with 6 nmol intravenous LS301. Regions of interest (ROI) were quantitated, encompassing mouse upper extremities (all structures including and distal to the wrist) and lower extremities (all structures including and distal to the ankle), applied universally to all images using the Pearl software. Total extremity fluorescence (quantitated from ROIs) per mouse, averaged among n = 3 mice, was compared between groups. For studies on response to DEX treatment, mice with STA were injected with 6 nmol of intravenous LS301 on day 3 post disease induction and imaged at 18h post LS301 injection using the Pearl Small Animal Imaging System with λ = 785 nm (excitation)/820 nm (emission). Mice then received intraperitoneal DEX (10 mg/kg/dose) daily over a 6-day period. Clinical paw scores and ankle measurements were obtained daily. On day 9 (day of final DEX treatment), mice were imaged again with 6 nmol intravenous LS301. Regions of interest (ROI) were quantitated and analyzed as described above.
H&E staining, immunohistochemical staining for pANXA2, and microscopic analysis were performed as previously described . Tissues of interest were harvested and frozen at -80°C in Optimal Cutting Temperature (OCT) media. Frozen sections were cut at 10 μm thickness, and slides were stored at – 40 °C. Consecutive sections were subjected to H&E and immunohistochemical analysis as follows. H&E staining was performed by the Musculoskeletal Histology and Morphometry Core, Washington University School of Medicine. Briefly, frozen sections were fixed for 10 min in 4% paraformaldehyde solution (Sigma, St. Louis, MO, USA) and stained with Harris hematoxylin for 90 s and with eosin (Sigma, St. Louis, MO) for 15 s, and then washed with tap water for 5 min. Some sections were stained with Safranin O and Fast Green counterstain (Musculoskeletal Histology and Morphometry Core, Washington University School of Medicine, St Louis, MO. For immunohistochemistry, slides were blocked with appropriate serum for 35 min or with 5% non-fat milk PBS (pH 7.4) overnight at 4 °C and incubated with primary antibody overnight at 4 °C or 1h at 37 °C. For pANXA2 studies, tissue sections were incubated with 1: 250 rabbit anti-pANXA2 (phospho-Tyr24) antibody (Signalway Antibody, College Park, MD). After washing twice with PBS, the tissue sections were incubated with 1:1000 AlexaFluor 594-conjugated donkey anti-rabbit antibody (Thermo Fisher Scientific, Waltham, MA) for 1 h at 25 °C respectively. Slides were washed again and stained with DAPI nuclear stain for 5 min (Thermo Fisher Scientific, Waltham, MA) for 45 min at 37 °C. After final washes, a coverslip with aqueous fluorescence-saving mounting media was applied prior to imaging. Slides were viewed using an Olympus B61 epifluorescence microscope (Olympus Corp., Tokyo, Japan) with filters/channels as follows: DAPI (Ex/Em = 330–385/420 nm), FITC (Ex/Em = 460–500/510–560 nm), Texas Red (Ex/Em = 542–582/604–644 nm), cypate (Ex/Em = 750–800/818–873 nm), using exposure times 1 to 30 s and sensitivity settings ISO200-ISO1600, with the same parameters used for control and treatment groups. ImageJ software (National Institutes of Health, Bethesda, MD, USA) was used for image processing.
Tissues were homogenized using an ultrasonic processor in RIPA buffer (20mM Tris-HCl, pH 7.5, 150mM NaCl, 1mM Na2EDTA, 1mM EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5mM sodium pyrophosphate, 1mM b-glycerophosphate, 1mM Na3VO4, 1 μg/ml leupeptin, 1mM PMSF). The tissue lysates were clarified by centrifugation. The protein was denatured in SDS gel-loading buffer (100mM Tris-HCl, 200mM DTT, 4% SDS, 0.2% bromophenol blue, and 20% glycerol) at 95 °C for 10 min and then separated on 12% SDS-polyacrylamide gels (50 μg of the tissue protein per sample). After electrophoresis, proteins were transferred to PVDF membrane using an EC140 Mini Blot Module (Thermo EC, Holbrook, NY) apparatus. The membrane was blocked for 1 h at room temperature in PBS containing 5% nonfat dry milk (w/v), 0.1% (v/v) Tween-20 (PBS-T), followed by incubation with Annexin A2 rabbit mAb (1:2000; Cat. 8235, Cell Signaling Tech.) or p-Annexin A2 mouse mAb (1:500; sc-135753, Lot# J2920; Santa Cruz) in PBS-T containing 3% nonfat dry milk (w/v) at 4°C overnight. After washing three times for 10 min each in PBS-T, the membrane was incubated for 1 h with diluted polyclonal goat anti-rabbit IgG or polyclonal goat anti-mouse IgG conjugated to horseradish peroxidase in PBS-T containing 3% nonfat dry milk (w/v). The membrane was then washed three times for 10 min each in PBS-T and developed using the chemiluminescence ECL kit (Pierce) according to the manufacturer’s instructions.
Differences between sample means were analyzed by two-tailed unpaired t-test (Microsoft Excel) with p < 0.05 as the threshold for statistical significance. Correlations between fluorescence measurements, clinical paw scores, and change in ankle thickness were analyzed using Pearson correlation (Microsoft Excel). For semiquantitative analysis of fluorescence, 3 mice per group allows 80% power to detect an effect size of 1.67 by 2-sided 2-sample t-test at alpha=5% .