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Midkine, a heparin-binding growth factor, promotes growth and glycosaminoglycan synthesis of endothelial cells through its action on smooth muscle cells in an artificial blood vessel model

¹ Department of Oral and Maxillofacial Surgery, Nagoya Daini Red Cross Hospital, 2-9 Myoken-cho, Showa-ku, Nagoya 466-8650, Japan
² Department of Biochemistry, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan
³ Department of Tissue Engineering Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan
⁴ Department of Oral and Maxillofacial Surgery, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan

View larger version (116K): [in a new window]   Fig. 1. Morphologies of BVMs cultured with or without MK. BVMs were cultured in DMEM-FCS for 1 day (24 hours) (A,C,E,G) or 3 days (72 hours) (B,D,F,H) with (C-H) or without (A,B) 100 ng/ml of MK. For antibody inhibition studies, anti-MK antibody (90 µg/ml) (E,F) or anti-IL-8 antibody (30 µg/ml) (G,H) was added. Then, BVMs were embedded in paraffin, cut into 5 µm sections and stained with hematoxylin-eosin. (A,C) HUVECs were attached and formed a monolayer on the HASMC-populated collagen gel on Day 1. (B) HUVECs were attached and formed a monolayer and secreted extracellular matrix on the HASMC-populated collagen gel on Day 3. (D) HUVECs were stratificated on the HASMC-populated collagen gel on Day 3. The extracellular matrix was secreted in large quantities. Bar, 100 µm.

View larger version (109K): [in a new window]   Fig. 2. Anti-PCNA immunostaining of BVMs. (A,B,E,F) Control staining without the first antibody on Day 1 (A,E) and on Day 3 (B,F). (C,D,G,H) Anti-PCNA immunostaining on Day 1 (C,G) and Day 3 (D,H). BVMS were either untreated (A,B,C,D) or cultured with 100 ng/ml of MK (E,F,G,H) as in Fig. 1.

View larger version (20K): [in a new window]   Fig. 3. [35S]-labeled glycosaminoglycan synthesis by HUVEC treated with or without MK. Measurement of glycosaminoglycan synthesis was performed 24-48 hours and 48-72 hours after the start of culture. The number of samples in each experiment was six. *P<0.05; **P<0.01.

View larger version (36K): [in a new window]   Fig. 4. HUVEC proliferation assay. (A) HUVEC proliferation assay in the co-culture system (Transwell 24-well culture plate). (B) HUVEC proliferation assay with HASMC-conditioned medium treated with or without MK. The number of samples in both A and B was five, and the experiments were repeated twice with reproducible results. *P<0.05; **P<0.001.

View larger version (69K): [in a new window]   Fig. 5. Morphologies of BVMs cultured in DMEM with or without IL-8 and MK. BVMs were cultured for 1 or 2 days, with IL-8 (100, 500, 1000 ng/ml), MK (100 ng/ml) or without factors (Control). For antibody inhibition, 30 µg/ml of anti-IL-8 antibody was added. Bar, 100 µm.

View larger version (32K): [in a new window]   Fig. 6. Effects of treatment with 100 ng/ml MK for 24 hours on expression of PTP, LRP and PG-M/versican. (A) Proteins were separated by SDS-PAGE, and PTP and LRP were detected by western blotting with the respective antibody. The intensity of bands was normalized to that of actin, and each band was shown as the ratio of the value in the treated cells to that in the non-treated cells. (B) Expression of versican revealed by RT-PCR. The intensity of bands was normalized to that of GAPDH and was shown as the ratio of the value in the treated cells to that in the non-treated cells.