Interleukin-1β, lipocalin 2 and nitric oxide synthase 2 are mechano-responsive mediators of mouse and human endothelial cell-osteoblast crosstalk

Endothelial cells are spatially close to osteoblasts and regulate osteogenesis. Moreover, they are sensitive to mechanical stimuli, therefore we hypothesized that they are implicated in the regulation of bone metabolism during unloading. Conditioned media from endothelial cells (EC-CM) subjected to simulated microgravity (0.08g and 0.008g) increased osteoblast proliferation and decreased their differentiation compared to unit gravity (1g) EC-CM. Microgravity-EC-CM increased the expression of osteoblast Rankl and subsequent osteoclastogenesis, and induced the osteoblast de-differentiating factor, Lipocalin 2 (Lcn2), whose downregulation recovered osteoblast activity, decreased Rankl expression and reduced osteoclastogenesis. Microgravity-EC-CM enhanced osteoblast NO-Synthase2 (NOS2) and CycloOXygenase2 (COX2) expression. Inhibition of NOS2 or NO signaling reduced osteoblast proliferation and rescued their differentiation. Nuclear translocation of the Lcn2/NOS2 transcription factor, NF-κB, occurred in microgravity-EC-CM-treated osteoblasts and in microgravity-treated endothelial cells, alongside high expression of the NF-κB activator, IL-1β. IL-1β depletion and NF-κB inhibition reduced osteoblast proliferation and rescued differentiation. Lcn2 and NOS2 were incremented in ex vivo calvarias cultured in microgravity-EC-CM, and in vivo tibias and calvarias injected with microgravity-EC-CM. Furthermore, tibias of botulin A toxin-treated and tail-suspended mice, which featured unloading and decreased bone mass, showed higher expression of IL-1β, Lcn2 and Nos2, suggesting their pathophysiologic involvement in endothelial cell-osteoblast crosstalk.

using fine forceps under a stereoscopic microscope. The proximal portion of the aorta was inserted with 24-gauge cannula and ligated with a silk thread and the inside of the lumen was washed with serum free DMEM. The other side of the aorta was filled with type II collagenase solution and incubated for 45 minutes at 37ºC. Then the aorta was flushed with 5 ml of DMEM containing 20% FBS to release the endothelial cells, the supernatant was centrifuged at 1200 rpm for 5 minutes and the endothelial cells were collected. Finally, the endothelial cells were seeded on matrigel coated dishes containing culture medium [20% FBS, 100 U/ml penicillin-G, 100 µg/ml of streptomycin, 2 mM of L-glutamine, 1X sodium pyruvate, 25 mM HEPES (pH 7.0 -7.6)] supplemented with 5 ng VEGF. After one week, endothelial cells were confluent and ready for the experimental procedures.

Human endothelial cell line
The human endothelial cell line, EA.hy926, was obtained from ATCC (CRL-2922) and was cultured in type 1 collagen-coated dish containing DMEM supplemented with 10% FBS, 1% penicillin/streptomycin in a humidified atmosphere of 5% CO2 at 37°C.

Primary mouse osteoblast cultures
Calvarias from 7 day-old C57BL/6J wild type and LCN2 knockout mice were removed, cleaned thoroughly and digested three times with 1 mg/mL clostridium histolyticum type IV collagenase and 0.25% trypsin, for 20 minutes at 37°C, with gentle agitation. Cells from the second and third digestions were plated and grown in standard conditions, in DMEM containing 10% FBS and 1% penicillin/streptomycin. These cells expressed the osteoblast markers ALP, Runx2, Osterix, Coll 1 chain-a, and osteocalcin.

Primary human osteoblast cultures
Primary human osteoblasts were isolated from bone fragments collected from patients after traumatic bone fractures, under their informed consent and approval by the University of L'Aquila Ethics Board. The bone fragments were thoroughly cleaned from fat and soft tissues, chopped in small pieces and processed for osteoblast isolation as described above.

Total bone marrow cell culture
Bone marrow cells of the long bones of 7-day-old wild type and LCN2 KO mice (C57BL/6J background) were flushed out, diluted 1:1 in Hank's balanced salt solution and centrifuged at 400g for 30 minutes. Then cells were re-suspended in DMEM containing 10% FBS and plated in culture dishes at a density of 10 6 cells/cm 2 . After 3 hours, cell cultures were rinsed to remove non-adherent cells and cultured with 1g-, 0.08g or 0.008g-EC-CM for 7 days.

Purified osteoclast cultures
Mouse primary osteoclast precursors were isolated from 7 day-old CD1 mice. The bone marrow from the bone cavity of the long bones was flushed out and diluted 1:1 in Hank's balanced salt solution, layered over Histopaque 1077 solution and centrifuged at 400g for 30 minutes. Cells were washed twice with Hank's solution, resuspended in DMEM supplemented with 10% FBS and plated in culture dishes at a density of 10 6 cells/cm 2 . After 3 hours, cultures were rinsed to remove non-adherent cells and maintained for 7 days in 1gor 0.008g EC-CM supplemented with 50ng/mL rhM-CSF.

MTT proliferation assay
Primary human or mouse osteoblasts (10000/well) were cultured in 96-well plates with 1g-, 0.08g or 0.008g-EC-CM. 48 hours later 20 µL of 5mg/mL of MTT solution was added to each well. After 3 hours, the solution was removed and 200 µL of DMSO was added and the color change was measured at 595 nm using a spectrophotometer.

Alkaline phosphatase activity assay
Primary human osteoblasts, or mouse osteoblasts from wild type or LCN2 KO mice, were cultured with 1g-, 0.08g or 0.008g-EC-CM for 48 hours. Cells were then fixed in 4% of paraformaldehyde for 15 minutes and washed twice with PBS. Analysis of ALP activity was evaluated cytochemically by the Sigma-Aldrich kit n. 85, according to the manufacturer's instruction. Quantitative analysis was performed by scanning densitometry using the Molecular Analyst software for the model 670 scanning densitometer (Bio-Rad Laboratories, Hercules, CA, USA) to obtain arbitrary density units.

Conventional and real----time RT----PCR
Total RNA was isolated from the endothelial cells, osteoblasts, calvarias and tibias using the TRIzol method. Two µg of RNA was reverse transcribed using the Moloney murine leukemia virus reverse transcriptase, and the equivalent of 0.1 µg was used for PCR reactions. Realtime PCR was performed employing the Brilliant SYBR Green QPCR master mix and conventional PCR by the green taq master mix. PCR conditions and primer pairs are listed in Supporting Table 2. Results were expressed as fold increase for real-time RT-PCR or shown by electrophoresis of PCR products in 2% agarose gel plus ethidium bromide for conventional RT-PCR. Results were normalized versus the housekeeping gene Gapdh.

Western blotting
For total protein extraction, osteoblasts or endothelial cells were lysed in RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) containing protease inhibitors, then proteins were resolved by 10% SDS-PAGE and transferred to nitrocellulose membranes. Blots were probed with the primary antibody for 1 hour at room temperature, washed and incubated with the appropriate HRP-conjugated secondary antibody for 1 hour at room temperature. Protein bands were revealed by enhanced ECL.

Mineralization assay
Mouse primary osteoblasts were cultured with 1g-and 0.008g-EC-CM along with the supplementation of 10 mM β-glycerophosphate and 50 mg/ml of ascorbic acid for 21 days.
Analysis of mineralization was performed by the Von Kossa staining.

Von Kossa staining
Osteoblast cells grown in 48 wells/plate were fixed in 4% of paraformaldehyde for 15 minutes and washed twice with distilled water. Cells were then incubated under UV light for 1 hour with 300 µL of 5% silver nitrate (AgNO3). The solution was removed and dishes washed twice with distilled water. Then 300 µL of 5% sodium thiosulfate (Na2S2O3) was added and dishes incubated for 2 minutes. The solution was removed, dishes were washed with distilled water and images were taken.

Immunofluorescence
Mouse primary osteoblasts or primary endothelial cells on microbeads were fixed with 4% paraformaldehyde and incubated for 1 hour at room temperature with specific primary

TRAcP activity assay
Osteoclasts, total bone cells or whole calvarial bones were treated with 1g-, 0.08g or 0.008g EC-CM. After 7 days, cells were fixed in 4% paraformaldehyde for 15 min and extensively washed with the PBS. TRAcP activity was analyzed cytochemically using the Sigma-Aldrich kit no 386 according to the manufacturer's instruction.

Micro-CT analysis
Calvaria images were acquired in a SkyScan 1174 micro-CT scanner, with a voxel size of 6 μm (X-ray voltage 50kV). The Skyscan Nrecon software was used for image reconstruction by employing a modified Feldkamp algorithm. Beam hardening correction and Fourier transform-based ring artefact reduction were applied to the reconstructed images. Area of the bone fraction was calculated for the calvarial bone selected regions of interest 2 using the Image J software.

Bone histomorphometry of calvarial bones
Mice were subjected to subcutaneous injection of 1g-, or 0.008g-EC-CM in the area above the calvarial bones, and to intraperitoneal double injection of calcein, 7 and 2 days before sacrifice, performed after 7 days from EC-CM injection.  To recover from anesthesia mice were placed in warm, clean, dry, quiet environment away from other animals. Commercially-available surgical heating pad were used to warm up animals. Bedding material was replaced with toweling material to avoid bedding to stick to eyes or be inhaled while animals are recovering from anesthesia.
All the operation on animals were done in the morning and finished before noon and animals monitored for further 5 hours.