Effect of HUVEC on human osteoprogenitor cell differentiation needs heterotypic gap junction communication

F. Villars1, B. Guillotin1, T. Amédée2, S. Dutoya1, L. Bordenave1, R. Bareille1, and J. Amédée1

Institut National de la Santé et de la Recherche Médicale 1 U-443 and 2 U-394, 33076 Bordeaux Cedex, France


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Bone development and remodeling depend on complex interactions between bone-forming osteoblasts and other cells present within the bone microenvironment, particularly vascular endothelial cells that may be pivotal members of a complex interactive communication network in bone. Our aim was to investigate the interaction between human umbilical vein endothelial cells (HUVEC) and human bone marrow stromal cells (HBMSC). Cell differentiation analysis performed with different cell culture models revealed that alkaline phosphatase activity and type I collagen synthesis were increased only by the direct contact of HUVEC with HBMSC. This "juxtacrine signaling" could involve a number of different heterotypic connexions that require adhesion molecules or gap junctions. A dye coupling assay with Lucifer yellow demonstrated a functional coupling between HUVEC and HBMSC. Immunocytochemistry revealed that connexin43 (Cx43), a specific gap junction protein, is expressed not only in HBMSC but also in the endothelial cell network and that these two cell types can communicate via a gap junctional channel constituted at least by Cx43. Moreover, functional inhibition of the gap junction by 18alpha -glycyrrhetinic acid treatment or inhibition of Cx43 synthesis with oligodeoxyribonucleotide antisense decreased the effect of HUVEC cocultures on HBMSC differentiation. This stimulation could be mediated by the intercellular diffusion of signaling molecules that permeate the junctional channel.

coupling; intercellular messenger; connexin43; osteoblast; endothelial cells


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ANGIOGENESIS is a tightly regulated process involved in growth, repair, and bone remodeling (9, 11-13, 23, 28, 50, 54, 56). Several studies have shown that there is a reciprocal regulation and functional relationship between endothelial cells and osteoblast-like cells during osteogenesis, in which systemic hormones and paracrine growth factors or cytokines play an active role (4, 24, 53, 55). In a previous study (51), we showed that osteoblast progenitor cells [human bone marrow stromal cells (HBMSC)] behave differently in terms of proliferation and differentiation when cultured in association with endothelial cells [human umbilical vein endothelial cells (HUVEC)] in various coculture models (coculture with or without direct contact, conditioned medium) than when they are cultured alone. An increase in alkaline phosphatase activity (Al-P) was observed only when HBMSC were cocultured in direct contact with HUVEC. The enhancement of this early osteoblastic marker is not provided when HBMSC and HUVEC are cocultured separately with the use of a semipermeable membrane (51) or when HBMSC are seeded onto a matrix obtained from HUVEC cultures (51).

Therefore, the intercommunication between endothelial cells and osteoblast-like cells not only may require diffusible factors but also may involve junctional communications to form a multicellular network (6, 37). Gap junction communications between osteoblastic cells mediate intracellular exchanges of regulatory ions and small molecules (molecular mass <1 kDa), which allows metabolic cooperation between adjacent cells and control cell differentiation and growth (14, 15, 31, 33, 37, 43, 44, 57). Cell-to-cell communication could be critical to the coordinated cell behavior necessary for bone development and remodeling. Lecanda et al. (31) have demonstrated that gap junction communication modulates transcriptional activity of osteoblast-specific promoters, thus pointing to a fundamental physiological role of intercellular communication for the function of specialized tissues such as bone. The major gap junction protein connexin43 (Cx43) (20) has been identified in osteoblastic (14, 15, 36, 37, 57, 61) and endothelial cells (5, 10, 22, 27, 60). Moreover, heterotypic gap channels may be present between endothelial cells and vascular smooth muscle cells in arteriole and sinusoid walls (8, 16, 48, 58), and some ultrastructural and morphological studies have demonstrated the existence of heterotypic gap junction channels between microvascular cells and osteocytes (41) or other cell types (5). Several studies have shown that many factors regulate the number, localization, nature, and function of gap junctional channels. These factors may be physical (10, 22, 27), physicochemical (14), ionic (15), hormonal, and cellular (5, 14, 37, 38).

In the present study, we focused on cell-to-cell interactions between endothelial cells and osteoprogenitor cells to explain the effect of HUVEC on the HBMSC differentiation observed only when these two cell types are cocultured with direct contact (51). Our results provide new evidence pointing to the function of endothelial mesenchymal cell interactions via gap junctional communication for the development of bone-forming cells.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell Culture

Endothelial cell culture. Endothelial cells were extracted from human umbilical veins essentially as described by Bordenave et al. (3), according to the procedure of Jaffe (26). Cell cultures were maintained in Iscove's modified Dulbecco's medium (IMDM) (GIBCO BRL, Gaithersburg, MD) with 20% (vol/vol) FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 90 µg/ml heparin, 20 µg/ml endothelial cell growth supplement (Sigma-Aldrich Diagnostics, St. Louis, MO). Cell characterization was evaluated either by the assessment of von Willebrand factor (vWF) expression, detected by immunocytochemical reaction with the use of DAKO-EPOS anti-vWF coupled with horseradish peroxidase (HRP) (DAKO DAB chromogene tablets) or by uptake of fluorescent acetylated low-density lipoprotein (DiI-Ac-LDL) by the "scavenger cell pathway" of LDL according to the operative conditions of Voyta et al. (52).

HBMSC culture. Human bone marrow was obtained by aspiration from the femoral diaphysis or iliac bone from patients (age 20-70 yr) undergoing hip prosthesis surgery after trauma. Their informed consent was obtained. Cells were separated into a single suspension by sequential passage through syringes fitted with 16-, 18-, and 21-gauge needles, cultured in IMDM supplemented with 10% (vol/vol) FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin, and incubated in a humidified atmosphere of 95% (vol/vol) air-5% (vol/vol) CO2 at 37°C (42, 49). Dexamethasone at 10-8 M was added to the culture medium during the first 2 wk of culture to induce alkaline phosphatase stimulation and mineralization of adherent cells (1). At the first subculturing, the cells were tested for their intracellular Al-P activity as described previously (2) with the Sigma diagnostic kit (85L-2). HBMSC also were characterized by immunocytochemical detection of intracytoplasmic type I collagen and osteocalcin (Takara Biomedicals). Moreover, additional markers besides the latter (osteopontin, calcium-binding proteins, osteonectin) (49) and their responsiveness to vitamin D3 [1,25(OH)2D3 or dihydroxycholecalciferol], parathyroid hormone, transforming growth factor-beta (TGF-beta ), and bone morphogenetic protein 3 (BMP3) have previously been studied (17, 18).

Coculture of HBMSC and HUVEC with direct contact. HBMSC were plated in 24-multiwell dishes at 104 cells/well in 0.5 ml of IMDM containing 10% (vol/vol) FCS and antibiotics as described above. During the same seeding phase, HUVEC were added to each well at 2 × 104 cells/well in IMDM containing 10% (vol/vol) FCS without growth factors or dexamethasone for direct cocultures.

Quantitative Measurement of Al-P Activity

Intracellular Al-P activity was determined as described by Majeska and Rodan (35) in the different culture conditions. Data are expressed as ratios of nanomoles of inorganic phosphate (Pi) cleaved by the enzyme in 30 min per microgram of protein. The quantitative measurement of cellular proteins was done by using the method of Lowry et al. (34).

Quantitative Measurement of Type I Collagen Synthesis

Collagenous protein synthesis was quantified by L-[5-3H]proline (10 mCi/ml; Amersham, Amersham, UK) incorporation in the culture medium for 24 h. After being washed with 0.1 M phosphate-buffered saline (PBS), pH 7.4, labeled materials were extracted from the cells and immunoprecipitated by using a monoclonal antibody against type I collagen incubated overnight at 4°C. Immunocomplex was trapped by adding 100 µl of washed protein A-agarose bead (Upstate Biotechnology) slurry for 2 h at 4°C. Agarose beads were collected by pulsing in the microcentrifuge and washed three times, and the radioactivity retained was counted with a beta counter (Packard).

Western Blot Analysis

Cells (HBMSC and HUVEC) were grown to confluence, and crude extract proteins were then obtained in 0.1 M ice-cold PBS, pH 7.4, containing protease inhibitors (5 mM EDTA, 0.3 M benzaminidiumchloride, 0.1 mM iodoacetamide, 4.5 mM phenylmethylsulfonyl fluoride, and 0.1 M 6-amino hexanoic acid) before being subjected to Western blot analysis. Proteins (20 µg/lane) were separated by 10% SDS-PAGE in reducing conditions according to Laemmli (30). Samples were transferred onto nitrocellulose membrane, and the free binding capacity of the membrane was blocked with 3% bovine serum albumin (BSA) in 0.1 M PBS, pH 7.4, for 2 h at 37°C. Cx43 was detected with rabbit polyclonal antibodies (Chemicon, Euromedex) diluted 1:1,000 and revealed with anti-rabbit immunoglobulin G (IgG) linked to HRP (Chemicon, Euromedex) diluted 1:20,000. Loading control was performed by using monoclonal antibodies against alpha -tubulin (Chemicon, Euromedex) diluted 1:1,000 and revealed with anti-mouse IgG linked to HRP (Chemicon, Euromedex) diluted 1:20,000. After additional washes with PBS, the membrane was soaked in enhanced chemiluminescence detection reagents (ECL; Amersham). The sheet was then air-dried and exposed to X-ray film.

Immunofluorescence Labeling

Cx43 and vWF were localized using indirect immunofluorescence. HBMSC and HUVEC cultured alone and cocultured HBMSC/HUVEC were fixed in 4% (wt/vol) paraformaldehyde for 20 min at 4°C and rinsed in Tris · HCl [50 mM Trisma base, pH 7.4, buffered with HCl (TB)]. Fixed cells were incubated for 30 min in TB containing 1% (wt/vol) BSA and then for 90 min with a monoclonal antibody against Cx43 (Chemicon, Euromedex) diluted 1:200 in TB and/or with a polyclonal antibody against vWF diluted 1:250 in TB, pH 7.4. Subsequently, cells were washed in TB and incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (Jackson Immunoresearch) diluted 1:100 or with rhodamine-conjugated anti-rabbit IgG (Jackson Immunoresearch) diluted 1:100 for 1 h. Cells were rinsed in TB, and cultures were coverslipped with Vectashield (Vector Laboratories) to prevent bleaching. Cultures were examined with an Axiophot microscope (Zeiss) equipped with the appropriate epifluorescence filter sets. Controls were performed without the primary antibody, and cross-reactivity of the secondary antibodies to mouse and rabbit IgG also were investigated.

Intercellular Dye Transfer and Staining

Coupling was examined in cells bathed in a control saline solution, pH 7.4, containing 124 mM NaCl, 15 mM KCl, 1.3 mM MgSO4, 1.25 mM KH2PO4, 26 mM NaHCO3, 1.8 mM CaCl2, and 11 mM glucose. For dye coupling experiments, we used patch pipettes with a resistance of 5-10 MOmega filled with a solution at pH 7.4 containing 120 mM KCl, 2 mM MgCl2, 1 mM CaCl2, 11 mM glucose, 11 mM EGTA-KOH, and 10 mM HEPES. Lucifer yellow (Sigma-Aldrich Diagnostics) was dissolved in a final concentration of 1% (wt/vol) (47). When these pipettes were applied to the cells, Lucifer yellow spread into the cytoplasm and was visualized under an inverted fluorescent microscope (Nikon Diaphot) with an excitation wavelength of 400-440 nm and an emission wavelength of 480 nm. Dye coupling from the patched cell to adjacent cells was monitored immediately and 5 min later and was confirmed by micrographs. Next, the cells were fixed and analyzed for immunocytological detection of vWF in HUVEC by using DAKO-EPOS anti-vWF coupled with peroxidase (DAKO).

RT-PCR Analysis

Total RNA was extracted from HBMSC and HUVEC by using the RNeasy Total RNA kit (Qiagen), and 2 µg were used as template for single-strand cDNA synthesis with the Superscript preamplification system (GIBCO BRL) in a 20-µl final volume containing 20 mM Tris · HCl, pH 8.4, 50 mM KCl, 2.5 mM MgCl2, 0.1 mg/ml BSA, 10 mM dithiothreitol (DTT), 0.5 mM each of dATP, dCTP, dGTP, and dTTP, 0.5 µg of oligo(dT)12-18, and 200 units of reverse transcriptase. After incubation for 50 min at 42°C, the reaction was stopped at 70°C for 15 min and kept on ice. Synthesized cDNA were treated with 2 units of RNase H (Escherichia coli) at 37°C for 20 min, and samples were mixed with PCR cocktail containing 20 mM Tris · HCl, pH 8.4, 50 mM KCl, 2.5 mM MgCl2, 0.1 µg/µl BSA, 10 mM DTT, 0.5 mM each of dATP, dCTP, dGTP, and dTTP, 2.5 units of Taq polymerase, and 0.5 µM each of forward and reverse primer (Table 1) in a 50-µl final volume. Amplification was performed in a HYBAID thermocycler under the following conditions: denaturation at 94°C for 5 min, 30 cycles of 94°C for 10 s, hybridization at melting temperature (indicated in Table 1) for 30 s, followed by a final extension step at 72°C for 1 min. To control for the integrity of the various RNA preparations, the expression of beta -actin also was assessed. PCR products were electrophoretically separated on a 1% (wt/vol) agarose gel and visualized by ethidium bromide staining.

                              
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Table 1.   Sequences and melting temperatures of primers used for RT-PCR analysis and expected size of their corresponding PCR products

Gap Junction Channel Inhibition Assay

Assay with 18alpha -glycyrrhetinic acid. HBMSC and HUVEC were plated in 24-multiwell dishes at 104 cells/well and 2 × 104 cells/well, respectively, in IMDM supplemented with 10% (vol/vol) FCS with antibiotics. Four hours after seeding, 18alpha -glycyrrhetinic acid (18alpha -GA; Sigma-Aldrich Diagnostics) was added to the culture medium to obtain a concentration of 100 µM (18alpha -GA stock solution was at 50 mM in DMSO) (48, 58). Thereafter the medium was changed every day and supplemented or not with 18alpha -GA (100 µM) for 1, 3, and 6 days. At each time indicated, the medium was removed and the cells were washed twice with 0.1 M PBS, pH 7.4, and then scraped and sonicated for 30 s at 20 kHz and 8% amplitude (Vibra-Cell; Bioblock-Scientific) in 300 µl of 0.1 M PBS, pH 7.4. They were then frozen at -80°C until quantitative assays of total protein content and Al-P activity were performed.

Assay with oligodeoxyribonucleotide antisense Cx43. HBMSC and HUVEC were plated in 24-multiwell dishes at 104 cells/well and 2 × 104 cells/well, respectively, in IMDM supplemented with 10% (vol/vol) FCS with antibiotics. Four hours after seeding, phosphorothioated sense oligodeoxyribonucleotide (ODN) of Cx43 (TAC CCA CTG ACC TCG CGG) or antisense ODN of Cx43 (GGC GCT CCA GTC ACC CAT) (ODN-Cx43; Genset, Paris, France) were added to the culture medium to obtain a concentration of 10 µM. Thereafter, the medium was changed every day and supplemented or not with sense or antisense ODN-Cx43 (10 µM) for 3 and 6 days (19). At each time, the medium was removed and the cells were washed twice with 0.1 M PBS, pH 7.4, and then scraped, sonicated for 30 s at 20 kHz and 8% amplitude (Vibra-Cell) in 300 µl of 0.1 M PBS, pH 7.4, and frozen at -80°C until quantitative assays of total protein content and Al-P activity were performed.

Statistical Analysis

The data were expressed as means ± SD and were analyzed using the nonparametric U-test of Mann and Whitney (used to compare observed means on a few independent samples).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of HUVEC on HBMSC Differentiation in Coculture With Direct Contact

As shown in Fig. 1 and as previously identified (49), osteoblastic markers were detected in HBMSC obtained from the first subculture. Cytochemical analysis revealed that HBMSC expressed Al-P activity (Fig. 1a), synthesized type I collagen (Fig. 1c), and began to express and synthesize osteocalcin after the first passage, which was then secreted in the matrix (Fig. 1e). The other osteoblastic markers (osteonectin and osteopontin) and their response to 1,25(OH)2D3, BMP3, and TGF-beta have been previously identified (17, 18, 49). Phenotypic characterization of HUVEC revealed that, unlike HBMSC, these cells did not express Al-P (Fig. 1b) or type I collagen (Fig. 1d) but synthesized vWF (Fig. 1f).


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Fig. 1.   Phenotype characterization of human bone marrow stromal cells (HBMSC). a and b: cytochemical analysis of alkaline phosphatase (Al-P) activity in HBMSC (a) and human umbilical vein endothelial cells (HUVEC) (b). c and d: intracytoplasmic type I procollagen detection in HBMSC (c) and HUVEC (d) by immunofluorescence. e: intracytoplasmic osteocalcin detection in HBMSC by using a monoclonal antibody to bovine osteocalcin, which cross-reacts with human antigen (Takara Biochemical). Fixed immunoglobulins were revealed by using goat anti-mouse conjugated to Al-P-labeled polymer goat. f: immunodetection of von Willebrand factor (vWF) in HUVEC by using horseradish peroxidase (HRP) staining systems. Magnification, ×100. Bars, 100 µm.

When Al-P-positive HBMSC and vWF-positive HUVEC (Fig. 2A) were cultured with direct contact for 3, 6, and 9 days, the Al-P activity increased greatly (Fig. 2B). Type I collagen synthesis, which is concomitant to Al-P activity expression in bone cell cultures (45), also was investigated in these cocultures by using [3H]proline incorporation followed by an immunoprecipitation with an antibody against type I collagen. When HBMSC and HUVEC were cultured with direct contact for 3, 6, and 9 days, type I collagen synthesis increased with time of coculture (Fig. 2C), as Al-P enzymatic activity did in the same coculture. In this cell culture model, the endothelial cells could be considered as being "osteoinductive" mediators instead of acting at the level of bone cell maturation.


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Fig. 2.   Effect of HUVEC cocultures onto HBMSC differentiation. A: both cytochemical detection of enzymatic intracellular Al-P activity in HBMSC and immunodetection of vWF in HUVEC cultured in direct contact. Magnification, ×100. Bar, 100 µm. B: time course of HUVEC effect on Al-P activity of HBMSC cultured alone (control) and HBMSC cocultured with HUVEC with direct contact (HBMSC + HUVEC). Enzymatic activity was quantified according to MATERIALS AND METHODS. Results are expressed as nM Pi cleaved per µg protein in 30 min. Each point represents a mean of 6 determinations, and data are expressed as means ± SD of 4 experiments. * P < 0.05. C: time course of HUVEC effect on collagenous proteins and type I collagen synthesis of HBMSC cultured alone (control) and HBMSC cocultured with HUVEC with direct contact (HBMSC + HUVEC). Collagen synthesis was monitored by using [3H]proline incorporation as described in MATERIALS AND METHODS. Results are expressed as cpm per µg protein. Each point represents a mean of 4 determinations, and data are expressed as means ± SD. * P < 0.05.

We next explored the effect of cell seeding density on osteoblastic phenotype potential (Fig. 3). The optimal effect of HUVEC on HBMSC Al-P activity was obtained at 3 days, for a HUVEC seeding density of 5-10 × 103 cells/cm2 cocultured with HBMSC, themselves seeded at 5 × 103 cells/cm2. This effect was maintained at days 6 and for the same HBMSC seeding density (5 × 103 cells/cm2) and also was maintained for higher HUVEC seeding densities such as 15 × 103 cells/cm2 when Al-P activity was normalized for protein content.


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Fig. 3.   Effect of cell density of HUVEC onto HBMSC differentiation. Time course of effect of HUVEC seeding densities on Al-P activity of HBMSC. HBMSC were seeded at 5,000 cells/cm2 and cultured alone (control) or in coculture with HUVEC in direct contact at different cell densities (1,250, 2,500, 5,000, 10,000, and 20,000 HUVEC/cm2). Enzymatic activity was quantified according to MATERIALS AND METHODS. Results are expressed as nM Pi cleaved per µg protein in 30 min. Each point represents a mean of 4 determinations, and data are expressed as means ± SD of 3 experiments. * P < 0.05; ** P < 0.01.

Functional Coupling Exists Between HUVEC and HBMSC

Because no effect of HUVEC was noted with a semipermeable membrane (51), we investigated the gap junctional communication between HUVEC and HBMSC. Lucifer yellow dye transfer confirmed that HBMSC were coupled homotypically (Fig. 4, a and b), as were HUVEC (Fig. 4, c and d). Interestingly, in coculture (Fig. 4, e-p), immunostaining of vWF in HUVEC (Fig. 4, g, j, m, and p) performed a few minutes after the dye transfer experiment shows that HBMSC and HUVEC also were coupled heterotypically (Fig. 4, f, i, l, and o).


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Fig. 4.   Representative photographs illustrating Lucifer yellow dye transfer in HBMSC and/or HUVEC. Typical phase views show spatial organization of cells for HBMSC (a), HUVEC (c), and HBMSC cocultured with HUVEC with direct contact (e, h, k, and n). Lucifer yellow dye transfer in cells shows efficient homotypic gap junctional channel established between HBMSC (b) or HUVEC (d) and heterotypic gap junctional communication between HBMSC and HUVEC (f, i, l, and o). Dye coupling from the patched cell to adjacent cells was monitored 5 min after dye injection. Immunocytological detection of vWF in fixed HUVEC cocultured with HBMSC (g, j, m, and p) was performed a few minutes after the Lucifer yellow dye transfer experiment. This immunocytological labeling revealed which type of cells (HUVEC or/and HBMSC) were affected by dye transfer around the injected cell. Immunocytological detection was done by using DAKO-EPOS anti-vWF coupled with peroxidase (DAKO A/S Denmark) as described in MATERIALS AND METHODS. Asterisks indicate the injected cell; B, bone cell; E, endothelial cell.

In bone cells, the predominant gap junction protein is Cx43, which plays a fundamental role in osteoblast differentiation and also has been formally identified in endothelial cells (22). This cellular intercommunication between HUVEC and HBMSC was strongly suggested by the expression of Cx43 mRNA in both cell types (Fig. 5A). Moreover, both Western blot with anti-Cx43 (Fig. 5B) and fluorescence staining of Cx43 performed on confluent HBMSC (Fig. 6a) or confluent HUVEC (Fig. 6b) demonstrated that these cells synthesized these proteins. In both cell types (Fig. 6, a and b) under indirect immunofluorescence, Cx43 appeared mainly in bright, punctate regions located where cells were close together, probably corresponding to gap junctions. Double staining of Cx43 (Fig. 6, c, f, and i) and vWF (Fig. 6, d, g, and j) were performed on the coculture (Fig. 6, e, h, and k), and immunolabeling of Cx43 was identified in neighboring cells either both negative to vWF or both positive to vWF. In some areas of the coculture, a specific labeling of Cx43 between a vWF-positive cell and a vWF-negative cell was observed, which suggested gap junctional coupling between HBMSC and HUVEC (Fig. 6, c-k).


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Fig. 5.   Expression of connexin43 (Cx43) in HUVEC and HBMSC. A: detection of mRNA expression for Cx43 by RT-PCR in HBMSC and HUVEC. Total RNA was isolated from HBMSC and HUVEC after they had reached confluence and was reverse transcribed. Products of PCR amplification were subjected to electrophoresis on a 1% agarose gel and detected by staining with ethidium bromide. Results are representative of 3 experiments. HBMSC and HUVEC express Cx43 (lanes 1 and 2, respectively). beta -Actin (lane 4, HBMSC; lane 5, HUVEC) was used as housekeeping gene. Lanes 3 and 6: 1-kb DNA ladder (GIBCO). B: immunodetection of Cx43 by Western blot analysis (lane 1, HBMSC; lane 2, HUVEC). Immunoblots also were revealed by anti-alpha -tubulin (lane 1', HBMSC; lane 2', HUVEC). Lanes 3 and 3': 10-kDa protein ladder (GIBCO).



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Fig. 6.   Immunocytochemistry of Cx43 and vWF in HBMSC, HUVEC, and coculture. Immunostaining of Cx43 and vWF were performed as described in MATERIALS AND METHODS by using indirect immunofluorescence. Cx43 and vWF were revealed by using FITC-conjugated goat anti-mouse IgG or rhodamine-conjugated anti-rabbit IgG, respectively. a: Cx43 in HBMSC. b: Cx43 in HUVEC. c-k: double staining of Cx43 and vWF in coculture (c, f, and i: Cx43 labeling; d, g, and j: vWF labeling; e, h, and k: typical phase view showing spatial organization of cocultured cells). Arrows show Cx43 labeling between neighboring cells; B, bone cell; E, endothelial cell.

HUVEC Effect on Osteoblast Differentiation is Mediated by Gap Junctions

Because Cx43 was able to constitute gap junctional communication between HUVEC and HBMSC, we further investigated the function of these gap junctions in this signaling pathway.

Functional gap junction inhibition. Glycyrrhetinic acid (GA) is a lipophilic aglycone isolated from licorice root, with a steroidal structure that can occur in alpha - and beta -isoforms. Dye transfer studies have shown that 18alpha -GA reversibly interrupts gap junctional communication in cultured cells at micromolar concentrations. This effect remains specific for 18alpha -GA even at concentrations up to 100 µM (48, 58). At this concentration, 18alpha -GA inhibits communication in HBMSC and HUVEC, as shown by using Lucifer yellow dye transfer (data not shown), and this reduced communication significantly decreased the HUVEC effect on HBMSC Al-P activity by ~76% at day 3 after the coculture and by ~82% at day 6 (Fig. 7). Concerning HBMSC cultured alone, 18alpha -GA induced a weak but significant decrease in enzymatic activity after day 3 of treatment but not at day 6. As also demonstrated by Schiller et al. (44), disturbance of gap junctions in osteoblastic cells by 18alpha -GA results in decreased Al-P activity.


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Fig. 7.   Functional coupling inhibition assay between HBMSC and HUVEC. Time course of effect of 18alpha -glycyrrhetinic acid [18alpha -GA; 100 µM in Iscove's modified Dulbecco's medium (IMDM) with 10% (vol/vol) FCS] on Al-P activity in HBMSC cultured alone (control) or cocultured with HUVEC with direct contact (HBMSC + HUVEC). Results are expressed as nM Pi cleaved per µg protein in 30 min. Each bar represents the mean of 4 determinations, and data are expressed as means ± SD of a representative of 3 experiments. * P < 0.05; # no significant difference.

Gap junction synthesis inhibition. To directly inhibit gap junctional channels composed of Cx43, we used antisense ODN-Cx43. Addition of antisense-ODN-Cx43 at 10 µM inhibited protein synthesis, as demonstrated by immunocytochemistry performed on HBMSC (data not shown), and decreased the HUVEC effect on HBMSC-Al-P activity (Fig. 8) by ~82% at day 3 after the direct contact and by ~79% at day 6 compared with untreated cells. Both antisense ODN-Cx43 and sense ODN-Cx43 did not exert any significant effect on HBMSC cultured alone. Moreover, the effect of sense ODN-Cx43 on HBMSC Al-P activity in coculture is not significant compared with that in untreated cells. The decrease observed in HBMSC Al-P activity between sense and antisense treatment remains significant at day 6 (P < 0.001).


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Fig. 8.   Synthesis inhibition assay of Cx43 protein in HBMSC and HUVEC. Time course of effect of sense or antisense oligodeoxyribonucleotide [ODN; 10 µM in IMDM with 10% (vol/vol) FCS] to Cx43 treatment on Al-P activity in HBMSC cultured alone or cocultured with HUVEC in direct contact for 6 days. Results are expressed as nM Pi cleaved per µg protein in 30 min. Each bar represents the mean of 12 determinations, and data are expressed as means ± SD. * P < 0.01; ** P < 0.001; # no significant difference.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Gap junctions provide a fundamental regulatory mechanism controlling cell proliferation and gene expression in tissues whose specialized function requires the synchronization of multicellular activity (31, 33, 63). Coordination of gene expression during osteoblast differentiation and bone remodeling represents an excellent model for the physiological role of gap junctional intercellular communication (31). Palazzini et al. (41) showed that the cells belonging to the osteogenic lineage form a continuous protoplasmic network that extends from the osteocytes to the vascular endothelium, passing through osteoblasts and stromal cells in the perimedullary spaces.

The occurrence of gap junctions in the cytoplasmic network forms a functional syncytium-like structure, suggesting that the activity of the cells pertaining to the osteogenic lineage might be regulated not only by diffusion of systemic or paracrine factors through the intercellular fluids but also by signals transmitted through the cytoplasmic network (31, 41, 59). Therefore, inadequate control of intercellular communication among osteoblasts or a reduction in the number of coupling cells that may occur in pathological conditions could contribute to the impaired synthesis of new bone and reduced trabecular wall thickness, which is typical of osteoporotic bone (31). This is strongly supported by a typical feature of osteoporosis in which a reduction in the number of sinusoids and arterial capillaries in the bone marrow (4) is associated with a bone formation deficiency.

Gap junction channels are aqueous intercellular channels that allow the cell-to-cell diffusion of small molecules and ions. Each gap junction pore is formed by the juxtaposition of two hemichannels in adjacent cells, composed of a hexameric array of transmembrane proteins called connexins. While Cx43, Cx40, and Cx37 have been formally identified in endothelial cells (60), only Cx43 and Cx45 have already been reported in osteoprogenitor cells. These connexins have different molecular permeabilities (46) and differing abilities to interact with each other, e.g., gap junction pores formed by Cx45, which are less expressed in osteoblast-like cells, are less permeable than Cx43 pores to negatively charged dyes such as Lucifer yellow, carboxyfluorescein, or calcein. Lecanda et al. (31) hypothesized that the type of gap junctional communication provided by Cx43 allows the full development of mature osteoblastic phenotypes, because they showed the correlation between permeability to negatively charges, which in osteoblasts is mediated by Cx43 gap junctions, and osteocalcin and bone sialoprotein gene transcription. Li et al. (33) recently demonstrated that Cx45 in rat osteosarcoma cells may contribute to altered gene expression as in osteocalcin or Al-P. These results were confirmed by Furlan et al. (21).

Among the connexins expressed by endothelial cells and cells of osteogenic lineage, Cx43 is most closely associated with the initiation of osteogenesis and bone formation. Minkoff et al. (39) suggested that expression of Cx43 appears to precede the overt expression of the osteogenic phenotype (38, 63).

Our results demonstrate for the first time that human endothelial cells and osteoprogenitor cells arising from human bone marrow stromal cells do couple. Moreover, our data demonstrate 1) that this heterotypic gap junctional coupling contributes to the regulation of gene expression in osteoblastic cells and 2) that lack of coupling, including osteoblast to osteoblast but also endothelial cells to osteoblastic cells through gap junctions composed of the Cx43 expressed in both cells, is sufficient to alter osteoblastic marker expression such as Al-P in HBMSC and its increase occurring in direct contact with HUVEC. Together, our data suggest that Cx43 expression in HUVEC and HBMSC contributes to osteoblastic differentiation.

The nature of the mechanism that links gap junctional communication to gene expression remains elusive, but it certainly depends on the type of signals that permeate the junctional channel. This stimulation could be mediated by the intercellular diffusion of signaling molecules such as inositol phosphates as well as propagation of calcium waves (29, 32), which require gap junctional communication. Other second messengers also could be partly involved in the relationship between our endothelial and osteoblastic cell culture models. Inoue et al. (25) reported that the cyclic nucleotides cAMP and cGMP significantly and reciprocally regulate osteocalcin synthesis and AL-P activity and modulate the formation of mineralized nodules by osteoblast-like cells in culture (7). HUVEC could act as a donor of nitric oxide, which is well known to enhance the level of mRNA osteocalcin in osteoblastic cells (40, 62) and the rate of production of intracellular cGMP by activation of soluble guanylate cyclase. The identification of the signal transduction pathways involved in these cocultures is now underway.


    ACKNOWLEDGEMENTS

We thank Dr. R. Cooke for help in preparing the manuscript. We thank Dr. L. Jaubert (Bel Air Maternity Hospital, Bordeaux, France) for collecting umbilical cords and Dr. A. Durandeau (Pellegrin Hospital, Bordeaux, France) for the bone marrow samples.


    FOOTNOTES

This work was supported by a grant from the Fondation pour la Recherche Medicale and Pole Genie Biologique et Medical Aquitaine.

Address for reprint requests and other correspondence: F. Villars, INSERM U-443, Université Bordeaux 2 Victor Segalen, 146 rue Léo Saignat, 33076 Bordeaux Cedex, France (E-mail: joelle.amedee{at}bordeaux.inserm.fr).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajpcell.00310.2001

Received 6 July 2001; accepted in final form 19 November 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Abe, Y, Aida Y, Abe T, Hirofuji T, Anan H, and Maeda K. Development of mineralized modules in fetal rat mandibular osteogenic precursor cells: requirement for dexamethasone but not for beta -glycerophosphate. Calcif Tissue Int 66: 66-69, 2000[ISI][Medline].

2.   Ackerman, GA. Substituted naphthol AS phosphate derivatives for the localization of leukocyte alkaline phosphatase activity. Lab Invest 11: 563-567, 1962[ISI].

3.   Bordenave, L, Baquey C, Bareille R, Lefebvre F, Lauroua C, Guerin C, Rouais F, More N, Vergnes C, and Anderson JM. Endothelial cell compatibility testing of three different Pellethanes. J Biomed Mater Res 27: 1367-1381, 1993[ISI][Medline].

4.   Burkhardt, R, Kettner G, Bohm W, Schmidmeier M, Schlag R, Frisch B, Mallmann B, Eisenmenger W, and Gilg TH. Changes in trabecular bone, hematopoiesis and bone marrow vessels in aplastic anemia, primary osteoporosis, and old age : a comparative histomosphometric study. Bone 8: 157-164, 1987[ISI][Medline].

5.   Cai, J, Jiang WG, and Mansel RE. Gap junctional communication and the tyrosine phosphorylation of connexin 43 in interaction between breast cancer and endothelial cells. Int J Mol Med 1: 273-278, 1998[ISI][Medline].

6.   Collin-Osdoby, P. Role of vascular endothelial cells in bone biology. J Cell Biochem 55: 304-309, 1994[ISI][Medline].

7.   Conget, PA, and Minguell JJ. Phenotypical and functional properties of human bone marrow mesenchymal progenitor cells. J Cell Physiol 181: 67-73, 1999[ISI][Medline].

8.   Daniel, EE. Vascular gap junctional communication and heptanol. Hypertension 30: 306, 1997[ISI][Medline].

9.   Decker, B, Bartels H, and Decker S. Relationship between endothelial cells, pericytes, and osteoblasts during bone formation in the sheep femur following implantation of tricalciumphosphate-ceramic. Anat Rec 242: 310-320, 1995[ISI][Medline].

10.   DePaola, N, Davies PF, Pritchard WF, Florez L, Harbeck N, and Polacek DC. Spatial and temporal regulation of gap junction connexin43 in vascular endothelial cells exposed to controlled disturbed flows in vitro. Proc Natl Acad Sci USA 96: 3154-3159, 1999[Abstract/Free Full Text].

11.   Diaz-Flores, L, Gutierrez R, Lopez-Alonzo A, Gonzales R, and Varela H. Pericytes as supplementary source of osteoblasts in periosteal osteogenesis. Clin Orthop 275: 280-286, 1992[Medline].

12.   Doherty, MJ, Ashton BA, Walsh S, Beresford JN, Grant ME, and Canfield AE. Vascular pericytes express osteogenic potential in vitro and in vivo. J Bone Miner Res 13: 828-838, 1998[ISI][Medline].

13.   Doherty, MJ, and Canfield AE. Gene expression during vascular pericyte differentiation. Crit Rev Eukaryot Gene Expr 1: 1-17, 1999.

14.   Donahue, HJ, McLeod KJ, Rubin CT, Andersen J, Grine EA, Hertzberg EL, and Brink PR. Cell-to-cell communication in osteoblastic networks: cell line-dependent hormonal regulation of gap junction function. J Bone Miner Res 10: 881-889, 1995[ISI][Medline].

15.   Donahue, HJ, Li Z, Zhou Z, and Yellowley CE. Differentiation of human fetal osteoblastic cells and gap junctional intercellular communication. Am J Physiol Cell Physiol 278: C315-C322, 2000[Abstract/Free Full Text].

16.   Dora, KA, Martin PEM, Chaytor AT, Evans WH, Garland C, and Griffith TM. Role of heterocellular gap junctional communication in endothelium-dependent smooth muscle hyperpolarization : inhibition by a connexin-mimetic peptide. Biochem Biophys Res Commun 254: 27-31, 1999[ISI][Medline].

17.   Faucheux, C, Bareille R, Amédée J, and Triffitt JA. Effect of 1,25(OH)2D3 on bone morphogenetic protein-3 mRNA expression. J Cell Biochem 73: 11-19, 1999[ISI][Medline].

18.   Faucheux, C, Ulysse F, Bareille R, Reddi AH, and Amédée J. Opposing actions of BMP3 and TGF beta 1 in human bone marrow stromal cell growth and differentiation. Biochem Biophys Res Commun 241: 787-793, 1997[ISI][Medline].

19.   Fibbi, G, Caldini R, Chevanne M, Pucci M, Schiavone N, Morbidelli L, Parenti A, Granger HJ, Del Rosso M, and Ziche M. Urokinase-dependent angiogenesis in vitro and diacylglycerol production are blocked by antisense oligonucleotides against the urokinase receptor. Lab Invest 78: 1109-1119, 1998[ISI][Medline].

20.   Fishman, GI, Spray DC, and Leinwand LA. Molecular characterization and functional expression of the human cardiac gap junction channel. J Cell Biol 111: 589-598, 1990[Abstract].

21.   Furlan, F, Lecanda F, Sheikh S, Weitzmann L, and Civitelli R. Reduced osteogenic potential and support of osteoclastogenesis by connexin43 deficient osteoblasts. J Bone Miner Res 15, Suppl1: S140, 2000[ISI].

22.   Gabriels, JE, and Paul DL. Connexin43 is highly localized to sites of disturbed flow in rat aortic endothelium but connexin37 and connexin40 are more uniformly distributed. Circ Res 83: 636-643, 1998[Abstract/Free Full Text].

23.   Guenther, HL, Fleisch H, and Sorgente N. Endothelial cells in culture synthesize a potent bone cell active mitogen. Endocrinology 119: 193-201, 1986[Abstract]..

24.   Hunter, WL, Arsenault AL, and Hodsman AB. Rearrangement of the metaphyseal vasculature of the rat growth plate in rickets and rachitic reversal: a model of vascular arrest and angiogenesis reviewed. Anat Rec 229: 453-461, 1991[ISI][Medline].

25.   Inoue, A, Hiruma Y, Hirose S, Yamaguchi A, and Hagiwara H. Reciprocal regulation by cyclic nucleotides of the differentiation of rat osteoblast-like cells and mineralisation of nodules. Biochem Biophys Res Commun 215: 1104-1110, 1995[ISI][Medline].

26.   Jaffe, EA. Culture of human endothelial cells. Transplant Proc 12, Suppl: 49-53, 1980[ISI][Medline].

27.   Jansson, K, Bengtsson L, and Haegerstrand A. Time-course for in vitro development of basement membrane, gap junctions, and repair by adult endothelial cells seeded on precoated ePTFE. Eur J Vasc Endovasc Surg 16: 334-341, 1998[ISI][Medline].

28.   Jones, AR, Clark CC, and Brighton CT. Microvessel endothelial cells and pericytes increase proliferation and repress osteoblast phenotype markers in rat calvarial bone cell cultures. J Orthop Res 13: 553-561, 1995[ISI][Medline].

29.   Jørgensen, NR, Geist ST, Civitelli R, and Steinberg TH. ATP and gap junction-dependent intracellular calcium signaling in osteoblastic cells. J Cell Biol 139: 497-506, 1997[Abstract/Free Full Text].

30.   Laemmli, UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685, 1970[ISI][Medline].

31.   Lecanda, F, Towler DA, Ziambaras K, Cheng SL, Koval M, Steinberg TH, and Civitelli R. Gap junctional communication modulates gene expression in osteoblastic cells. Mol Biol Cell 9: 2249-2258, 1998[Abstract/Free Full Text].

32.   Leybaert, L, Paemeleire K, Strahonja A, and Sanderson MJ. Inositol-trisphosphate-dependent intercellular calcium signaling in and between astrocytes and endothelial cells. Glia 24: 398-407, 1998[ISI][Medline].

33.   Li, Z, Zhou Z, Yellowley CE, and Donahue HJ. Inhibiting gap junctional intercellular communication alters expression of differentiation markers in osteoblastic cells. Bone 25: 661-666, 1999[ISI][Medline].

34.   Lowry, OH, Rosebrough NJ, Farr AL, and Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265-275, 1951[Free Full Text].

35.   Majeska, RJ, and Rodan GA. The effect of 1,25(OH)2D3 on alkaline phosphatase in osteoblastic osteosarcoma cells. J Biol Chem 257: 3362-3365, 1982[Abstract/Free Full Text].

36.   Mason, DJ, Hillam RA, and Skerry TM. Constitutive in vivo mRNA expression by osteocytes of beta-actin, osteocalcin, connexin-43, IGF-I, c-fos and c-jun, but not TNF-alpha nor tartrate-resistant acid phosphatase. J Bone Miner Res 11: 350-357, 1996[ISI][Medline].

37.   Massas, R, and Benayahu D. Parathyroid hormone effect on cell-to-cell communication in stromal and osteoblastic cells. J Cell Biochem 69: 81-86, 1998[ISI][Medline].

38.   Massas, R, Korenstein R, and Benayahu D. Estrogen modulation of osteoblastic cell-to-cell communication. J Cell Biochem 69: 282-290, 1998[ISI][Medline].

39.   Minkoff, R, Rundus VR, Parker SR, Hertzberg EL, Laing JG, and Beyer EC. Gap junction proteins exhibit early and specific expression during intramembranous bone formation in the developing chick mandible. Anat Embryol (Berl) 190: 231-241, 1994[ISI][Medline].

40.   Otsuka, E, Hirano K, Matsushita S, Inhoue A, Hirose S, Yamaguchi A, and Hagiwara H. Effects of nitric oxide from exogenous nitric oxide donors on osteoblastic metabolism. Eur J Pharmacol 349: 345-350, 1998[ISI][Medline].

41.   Palazzini, S, Palumbo C, Ferretti M, and Marotti G. Stromal cell structure and relationships in perimedullary spaces of chick embryo shaft bones. Anat Embryol (Berl) 197: 349-357, 1998[ISI][Medline].

42.   Rickard, DJ, Kassem M, Hefferan TE, Sarkar G, Spelsberg TC, and Riggs BL. Isolation and characterization of osteoblast precursor cells from human bone marrow. J Bone Miner Res 11: 312-324, 1996[ISI][Medline].

43.   Schiller, PC, D'ippolito G, Balkan W, Roos BA, and Howard GA. Gap-junctional communication mediates parathyroid hormone stimulation of mineralization in osteoblastic cultures. Bone 28: 38-44, 2001[ISI][Medline].

44.   Schiller, PC, D'ippolito G, Balkan W, Roos BA, and Howard GA. Gap-junctional communication is required for the maturation process of osteoblastic cells in culture. Bone 28: 362-369, 2001[ISI][Medline].

45.   Stein, GS, Lian JB, and Owen TA. Relationship of cell growth to the regulation of tissue specific gene expression during osteoblast differentiation. FASEB J 4: 3111-3123, 1990[Abstract].

46.   Steinberg, TH, Civitelli R, Geist ST, Robertson AJ, Hick E, Veenstra RD, Wang HT, Warlow PM, Westphale EM, Laing JG, and Beyer EC. Connexin43 and connexin45 form gap junctions with different molecular permeabilities in osteoblastic cells. EMBO J 13: 744-750, 1994[Abstract].

47.   Stewart, WW. Lucifer dyes-highly fluorescent dyes for biological tracing. Nature 292: 17-21, 1981[ISI][Medline].

48.   Taylor, HJ, Chaytor AT, Evans WH, and Griffith TM. Inhibition of the gap junctional component of endothelium-dependent relaxations in rabbit iliac artery by 18-alpha glycyrrhetinic acid. Br J Pharmacol 125: 1-3, 1998[Abstract].

49.   Vilamitjana-Amédée, J, Bareille R, Rouais F, Caplan AI, and Harmand MF. Human bone marrow stromal cells express and osteoblastic phenotype in culture. In Vitro Cell Dev Biol Anim 29A:: 699-707, 1993[ISI].

50.   Villanueva, JE, and Nimni ME. Promotion of calvarial cell osteogenesis by endothelial cells. J Bone Miner Res 5: 733-739, 1990[ISI][Medline].

51.   Villars, F, Bareille R, Bordenave L, and Amédée J. Effect of human endothelial cells on human bone marrow stromal cell phenotype: role of VEGF? J Cell Biochem 15: 672-685, 2000.

52.   Voyta, JC, Via DP, Butterfield CE, and Zetter BR. Identification and isolation of endothelial cells based on their increased uptake of acetylated-low density lipoprotein. J Cell Biol 99: 2034-2040, 1984[Abstract].

53.   Wang, DS, Miura M, Demura H, and Sato K. Anabolic effects of 1,25-dihydroxyvitamin D3 on osteoblast are enhanced by vascular endothelial growth factor produced by osteoblasts and by growth factors produced by endothelial cells. Endocrinology 138: 2953-2962, 1997[Abstract/Free Full Text].

54.   Wang, DS, Yamazaki K, Nohtomi K, Shizume K, Ohsumi K, Demura H, Sato K, and Shibuya M. Increase of vascular endothelial growth factor mRNA expression by 1,25-dihydroxyvitamin D3 in human osteoblast-like cells. J Bone Miner Res 11: 472-479, 1996[ISI][Medline].

55.   Winet, H. The role of the microvasculature in normal and perturbed bone healing as revealed by intravital microscopy. Bone 19, Suppl: 39S-57S, 1996[Medline].

56.   Winet, H, Bao JY, and Moffat R. A control model for tibial cortex neovascularization in the bone chamber. J Bone Miner Res 5: 19-30, 1990[ISI][Medline].

57.   Yamaguchi, DT, Huang JT, and Ma D. Regulation of gap junction intercellular communication by pH in MC3T3-E1 osteoblastic cells. J Bone Miner Res 10: 1891-1899, 1995[ISI][Medline].

58.   Yamamoto, Y, Fukuta H, Nakahira Y, and Suzuki H. Blockade by 18-beta -glycyrrhetinic acid of intercellular electrical coupling in guinea-pig arterioles. J Physiol (Lond) 511: 501-508, 1998[Abstract/Free Full Text].

59.   Yamazaki, K, and Eyden BP. A study of intercellular relationship between trabecular bone and marrow stromal cells in the murine femoral metaphysis. Anat Embryol (Berl) 192: 9-20, 1995[ISI][Medline].

60.   Yeh, HI, Rothery S, Dupont E, Coppen SR, and Severs NJ. Individual gap junction plaques contain multiple connexins in arterial endothelium. Circ Res 83: 1248-1263, 1998[Abstract/Free Full Text].

61.   Yellowley, CE, Li Z, Zhou Z, Jacobs CR, and Donahue HJ. Functional gap junctions between osteocytic and osteoblastic cells. J Bone Miner Res 15: 209-217, 2000[ISI][Medline].

62.   Zaman, G, Pitsillides AA, Rawlinson SCF, Suswillo RFL, Mosley JR, Cheng MZ, Platts LAM, Hukkanen M, Polak JM, and Lanyon LE. Mechanical strain stimulates nitric oxide production by rapid activation of endothelial nitric oxide synthase in osteocytes. J Bone Miner Res 14: 1123-1131, 1999[ISI][Medline].

63.   Zhang, D, Weinbaum S, and Cowin SC. Electrical signal transmission in a bone cell network : the influence of a discrete gap junction. Ann Biomed Eng 26: 644-659, 1998[ISI][Medline].


Am J Physiol Cell Physiol 282(4):C775-C785
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