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First published online 12 September 2006
doi: 10.1242/jcs.03179

Journal of Cell Science 119, 4117-4126 (2006)
Published by The Company of Biologists 2006
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A novel endothelial-specific heat shock protein HspA12B is required in both zebrafish development and endothelial functions in vitro

Guang Hu1,*, Jian Tang1,*, Bo Zhang1,*, Yanfeng Lin1,*, Jun-ichi Hanai1,*, Jenna Galloway2, Victoria Bedell3, Nathan Bahary4, Zhihua Han5, Ramani Ramchandran3, Bernard Thisse6, Christine Thisse6, Leonard I. Zon2 and Vikas P. Sukhatme1,{ddagger}

1 Renal Division, Center for Study of the Tumor Microenvironment and Center for Vascular Biology Research, Department of Medicine, Beth Israel Deaconess Medical Center, Boston, MA 02215, USA
2 Division of Hematology/Oncology, Children's Hospital, Department of Medicine, Boston, MA 02215, USA
3 National Cancer Institute, National Institutes of Health, Rockville, MD 20850, USA
4 Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, W1257 BSTWR, Pittsburgh, PA 15261, USA
5 Department of Biochemistry and Molecular Biology, East Tennessee State University, Johnson City, TN 37614, USA
6 Institut de Biologie Moleculaire et Cellulaire, CNRS, INSERM, Universite Louis Pasteur, C. U. de Strasbourg, France


Figure 1
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  		Fig. 1. HspA12B whole-mount in situ hybridization at different zebrafish developmental stages. (A-G) Middle somite stages. (A-C) Dorsal views of head, trunk and tail, respectively, showing staining in ventral hematopoietic and vasculogenic mesoderm. (D,E) Lateral (D) and enlarged lateral view (E) of head region showing expression in rhombomeres. (F) Lateral view showing expression in first somite. (G) Dorsal view of ear showing staining in the anterior part of the otic vesicle. (H-J) Embryos at 24 hpf, dorsal view of head region (H), lateral view of the whole embryo (I), JB4 cross section in the truck region of embryos at 24 hpf (J). Arrows point to arterial and venous structures. NT, neural tube; NO, notochord. (K-N) Embryos at 36 hpf. Enlarged lateral view of the head (K), front view showing staining in the heart (arrow) (L), dorsal view of the head region (M); arrow indicates duct of Cuvier. Lateral view of trunk (N) showing staining in both axial vessels and ISVs. (O) Lateral view of an embryo at 48 hpf.

 

Figure 2
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  		Fig. 2. Knockdown of HspA12B by MOs during zebrafish development. (A) In vitro transcription and translation in the absence of MOATG or in the presence of various concentrations of MOATG or MM, demonstrating the efficiency of MOATG in blocking the translation of HspA12B. (B) RT-PCR of zebrafish HspA12B from zebrafish embryos injected with various amounts of MOs3rd. (C-E) Embryos at 48 hpf injected with (C) 1.0 mM MM, (D) 1.0 mM or (E) 2.0 mM MOATG. (F) Percentage of normal circulation in morphants injected with 1.0 mM MOATG or MM at 26 hpf. Data are presented as the mean ± s.e.m. of three or more independent experiments; the difference between MM and MOATG was significant (P<0.01) as accessed by two tailed Student's t-test.

 

Figure 3
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  		Fig. 3. Vascular phenotypes in morphants defined by microangiography and AP staining. (A,B) Angiogram of fish injected with 1 mM MM (A) or MOATG (B) at 48 hpf. (C-J) AP staining and statistical analysis of morphants treated with various concentrations of MM or MOATG. (C-E) SIVs. (F-H) Pectoral fin vessels. (I,J) Statistical analysis. (A,C,F) Embryos injected with 1.0 mM MM. (D,G) Embryos injected with 0.5 mM MOATG. (B,E,H) Embryos injected with 1.0 mM MOATG.

 

Figure 4
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  		Fig. 4. Impaired angiogenic processes revealed by knockdown of HspA12B in the [Tg(fli1:EGFP)y1] zebrafish line. (A,C,E,G,I) Morphants injected with 1 mM MM. (B,D,F,H,J) Morphants injected with 1 mM MOATG. (A,B,G,H) Morphants at 27 hpf. (C,D) Morphants at 36 hpf. (E,F) Morphants at 54 hpf. (I,J) Morphants at 24 hpf. In panels G-J, the rostral is to the left.

 

Figure 5
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  		Fig. 5. Expression profile of human HspA12A and HspA12B in human cell lines. (A) Detection of HspA12B mRNA in human cell lines by northern blot. Signal is evident only in the HUVEC lane (upper panel), 28S and 18S staining is shown as equal loading control (lower panel). (B) Comparison of HspA12A and HspA12B mRNA levels in cell lines using real-time PCR. Data from two independent experiments were normalized with the expression levels in HUVECs and are presented as the mean ± s.e.m.; the difference of expression levels between HUVECs and other cell lines were all significant (P<0.05) as tested by two tailed Student's t-test.

 

Figure 6
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  		Fig. 6. Knockdown and overexpression of HspA12B. (A) HEK 293 cells cultured in six-well plate were transfected with 0.1 µg pCS2+-HspA12B-Flag and various siRNAs at 62.5 nM final concentration. Cell lysates were collected after 48 hours and the expression level of HspA12B-Flag was checked by western blot using anti-FLAG antibody. P, pool of si1, si2, si3 and si4. (B) GAPDH loading control for (A). (C) HUVECs were transfected with si1 and si3 at 62.5 nM final concentration in six-well plates and endogenous HspA12B expression was examined after 48 hours by western blot using Ab4112 antibody; Lane 4 was loaded with lysate from HUVECs infected with HspA12B-C-FLAG adenovirus. (D) GAPDH loading control for (C).

 

Figure 7
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  		Fig. 7. Effect of HspA12B on VEGF-induced HUVEC migration. HUVECs were infected with empty control or HspA12B adenoviruses and migration assay was done in DMEM supplemented with 0.5% serum or DMEM supplemented with 0.5% serum and VEGF (10 ng/ml) in the lower chamber. HUVECs were transfected with NC, si1 and si3. Data from three experiments are shown as the mean ± s.d. All comparison were tested for statistic significance using two-tailed Student's t-test. *P<0.05.

 

Figure 8
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  		Fig. 8. Wound healing assays using HUVECs. (A,B) HUVECs infected with empty control and sense adenoviruses were grown to confluence. Wounds were made using 200 µl pipette tips in the cell lawn 48 hours after infection and media was changed to EBM-2 supplemented with 1% serum after the injury. Pictures were taken at 0 and 48 hours. (C,D) HUVECs infected with the same adenoviruses were grown to confluence. After making the wound, EBM-2 supplemented with 1% serum and VEGF (10 ng/ml) was added. Pictures were taken at 0 and 30 hours post injury. (E,F) HUVECs transfected with NC, si1 and si3 were grown to confluence and wounds were made in the cell monolayer 48 hours after transfection. EBM-2 supplemented with 1% serum and VEGF (10 ng/ml) was added. Pictures were taken at 0 and 24 hours post injury. Representative pictures were shown from at least three independent experiments.

 

Figure 9
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  		Fig. 9. HUVEC tube formation assay. HUVECs were transfected with NC, si1 and si3. After 48 hours, cells were trypsinized and resuspended in EBM-2 supplemented with 2% serum, and 4x104 cells were added to each well of a 48-well tissue culture plate with 100 µl solidified growth-factor-reduced Matrigel. Pictures were taken at 24 hours. Representative pictures from five independent experiments are shown. Quantitative data are shown as the mean ± s.e.m. All difference between groups were statistically significant (P<0.05), tested by two tailed Student's t-test.

 

Figure 10
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  		Fig. 10. Western blot analysis of Akt phosphorylation in cells transfected with siRNAs, and HUVEC migration rescue experiment with myr-Akt. (A) HUVECs in six-well plates were transfected with NC, si1 and si3 on day 0, split on day 1, serum-starved with EBM-2 supplemented with 0.5% serum on day 2 and stimulated with EBM-2 supplemented with VEGF (5 ng/ml) and 0.5% serum or with EGM-2 MV for 30 minutes on day 3. (B) HUVECs in 10-cm plates were transfected with NC, si1 and si3 on day 0, split onto 2 plates in the early morning of day 1 and infected with myr-Akt adenovirus in the late afternoon. EGM-2 MV was changed 6 hours later, EBM-2 supplemented with 2.5% serum was changed in the evening of day 2 and migration assay was done on day 3 by stimulating with DMEM supplemented with VEGF (10 ng/ml) and 0.5% serum. Data from three experiments are shown as the mean ± s.d. All indicated P values for the comparisons were obtained using two tailed Student's t-test.

 

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© The Company of Biologists Ltd 2006