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Cardiovascular Research Advance Access originally published online on April 29, 2008
Cardiovascular Research 2008 79(3):509-518; doi:10.1093/cvr/cvn112
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Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2008. For permissions please email: journals.permissions@oxfordjournals.org

Activation of endothelial cells to pathological status by down-regulation of connexin43

Hsueh-Hsiao Wang1, Chang-I Kung1, Yuen-Yi Tseng1, Yi-Chun Lin1, Chi-Hau Chen1, Cheng-Ho Tsai2,3,4 and Hung-I Yeh2,3,4,*

1 Department of Medical Research, Mackay Memorial Hospital, Taipei, Taiwan
2 Department of Internal Medicine, Mackay Memorial Hospital, 92, Sector 2, Chung San North Road, Taipei 10449, Taiwan
3 Mackay Medicine, Nursing and Management College, Taipei, Taiwan
4 Taipei Medical University, Taipei, Taiwan

* Corresponding author. Tel: +886 2 25433535 (ext. 2456); fax: +886 2 25433642. E-mail address: hiyeh{at}ms1.mmh.org.tw

Received 25 November 2007; revised 20 March 2008; accepted 18 April 2008

Time for primary review: 23 days


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 
Aims: We investigated the effects of connexin43 (Cx43) down-regulation on endothelial function.

Methods and results: We used two different sequences of Cx43-specific small interference RNA (siRNA) to reduce de novo synthesis of Cx43 in human aortic endothelial cells and then examined the expression profiles, proliferation activity and viability, and angiogenic potential. The involvement of mitogen-activated protein kinase signalling pathways was analysed. In parallel, the effect of inhibition of gap-junctional communication by connexin-mimetic peptides was evaluated. During the down-regulation of Cx43 by the siRNA, the cells exhibited impaired gap-junctional communication, proliferation, viability, and angiogenic potential. In addition, plasminogen activator inhibitor-1 (PAI-1) and von Willebrand factor were up-regulated. Furthermore, c-jun N-terminal kinase (JNK) and its downstream target c-jun were activated, while caspase-3, p38, and extracellular signal-regulated kinase remained unchanged. Inhibition of JNK by SP600125 blocked the siRNA-induced increased expression of PAI-1 and partially recovered the impaired angiogenic potential. Short-term inhibition of Cx43 channels by connexin-mimetic peptides did not activate JNK.

Conclusion: Down-regulation of Cx43 inhibits gap-junctional communication and activates endothelial cells to pathological status, as characterized by up-regulation of coagulatory molecules and impairment of proliferation, viability, and angiogenesis. The processes are associated with activation of JNK signalling pathways and rectified by inhibition of the activation. These results suggest that inadequate expression of Cx43 per se impairs endothelial function by the activation of stress-activated protein kinase.

KEYWORDS Angiogenesis; Connexins; Coagulatory factors; Gap junctions; c-jun N-terminal kinase


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 
The luminal side of the vascular wall is covered by a monolayer of endothelial cells, which possess numerous functions for regulation of vascular tone, haemostasis, and inflammation. Impairment of the functions of this single layer is critical in the initiation of a variety of vascular disorders, including atherosclerosis.1 Maintenance of the functional integrity of vascular endothelium requires coordination of activity between individual cells, in which communication between neighbouring cells through gap junctions plays an essential role. Gap junctions are cell membrane protein channels made of paired hemichannels named connexons. For construction of a channel, cells from either sides of the channel equally contribute a connexon, which is a hexameric assembly of connexins.2 In mammals, endothelial cells mainly express connexin43 (Cx43), Cx40 and Cx37,3,4 of which Cx43 is, by far, predominant in cultured endothelial cells.5,6

Cx43 has been known to possess distinct properties. Apart from forming gap junction channels for exchange of ions and small molecules between neighbouring cells, connexon hemichannels made of Cx43 also exist in the cell membrane and work as pathways connecting the cytosol and extracellular space.7,8 In addition, the C-terminal tail of Cx43 is able to interact with other molecules to exert channel-independent effects,9,10 such as those implicated in the modification of transcription and cell-cycle regulation.11 Furthermore, Cx43 is known to exist in mitochondria and is involved in cardioprotection against ischaemia12 and regulation of apoptosis.13

Animal studies showed that Cx43 is down-regulated in endothelial cells exposed to risk factors for atherosclerosis, such as ageing,14 hypertension,15 and diabetes.16 Endothelial cell-specific knockout of Cx43 in mice was reported to cause hypotension.17 In view of the versatile functions of Cx43, we suspected that underexpression of Cx43 per se may increase cell stress and activate endothelial cells to pathological status. To clarify this possibility, in the present study we examined the phenotypic features of human aortic endothelial cells (HAEC), in which the expression of Cx43 is repressed by small interference RNA (siRNA). We also checked the pathway of c-jun N-terminal kinase (JNK), a stress-activated protein kinase involved in the regulation of several aspects of endothelial activity.18 In parallel, the effect of gap 26 and gap 27, connexin-mimetic peptides known to block Cx43 channels,19 were also examined.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 
2.1 Cell culture, small interference RNA transfection, and gap peptide treatment
HAEC (Cascade Biologics, Portland, OR, USA) were maintained in medium 200 with low serum growth supplement (named complete medium; from Cascade Biologics). The culture was seeded in 1% gelatin-coated plastic-ware and maintained in the incubator at 37°C under a humidified 95% air and 5% CO2 atmosphere. To conduct transfection experiments, HAEC were grown in complete medium 200 and serially passaged until they reached passage 8. All the following experiments, unless mentioned, were conducted using a seeding density of 10 000/cm2. Chemically synthetic siRNAs from two different regions of Cx43 used are as follows: Cx43 transmembrane domain I Cx43siRNA1 (5'-GGTGTGGCTGTCAGTACTT-3')20 and C-terminal domain Cx43siRNA3 (5'-GCTGGTTACTGGCGACAGA-3'). Nonsense siRNA was used as negative control (5'-TTCTCCGAACGTGTCACGT-3'). These chemically synthesized and HPLC-purified siRNAs (Dharmacon, Lafayette, CO, USA) were used in all further experiments. The siRNAs were transfected into HAEC using LipofectAMINE 2000 reagent according to the manufacturer's instructions. After 5.5 h of transfection and periods of recovery, according to the experimental protocols, cells were processed for western blotting, reverse transcription-polymerase chain reaction (RT-PCR), and immunocytochemical examination, as described in Supplementary materials and methods. For the connexin-mimetic peptides, the synthetic oligopeptides corresponded to Cx43 extracellular loop I (Gap 26, VCYDKSFPISHVR) and extracellular loop II (Gap 27, SRPTEKTIFII) were used. The scrambled peptides (scrambled Gap 26, PSDVFRSCVKHYI; scrambled Gap 27, FKTIRTISIEP) were treated as negative controls.19 All peptides were synthesized to a purity >95% (MDBio, Taipei, Taiwan) and treated at concentrations of 300 µmol/L. After 1.5 h of treatment and periods of recovery, according to the experimental designs, cells were applied to dye transfer analysis and western blotting. HeLa cells expressing either Cx37 or Cx40, kindly donated by Professor Klaus Willecke (University of Bonn, Germany)21 were used in western blotting experiment to confirm the signals of Cx37 or Cx40.

2.2 Analysis of gap-junctional communication function
For scrape loading dye transfer analysis, cells in 35 mm culture dish were scraped with a surgical blade followed by the addition of Lucifer yellow/Rhodamine dextran (Invitrogen, Carlsbad, CA, USA) mixture and incubated at room temperature for 2 min. Cells were then carefully washed and replaced with fresh medium. After 5 min incubation, cells were fixed with 1% paraformaldehyde and examined. The distance between the middle scrape line stained with Rhodamine dextran and the bilateral edges of Lucifer yellow transfer was measured and compared between experiments. At least three independent experiments with each dish scraped three lines were conducted for each siRNA. For microinjection, sharp micropipette were loaded with a dye mixture of 4.6% Lucifer yellow (w/v) and 10% Rhodamine dextran B 10 000 (w/v). Cells were impaled using a micromanipulator (Leica, Wetzlar, Germany). Three minutes after dye injection, the fluorescence was recorded. At least 10 cells in each dish were randomly injected and three independent experiments were performed. Cells positive for Lucifer yellow excluded Rhodamine dextran were counted. All these images were viewed by a microscope (DM IRBE; Leica) with a 4 x 0.1 or 20 x 0.4 objective (Leica) and acquired by the charge-coupled device camera.

2.3 Analysis of angiogenic potential
After 5.5 h of transfection, PD98059 or SP600125 (Calbiochem, San Diego, CA, USA) were added in selected experiments. After 2 h, the cells were washed and supplemented with complete M200 medium and incubated for another 16.5 h. In experiments without the addition of PD98059 or SP600125, the cells were incubated for 18.5 h after transfection (i.e. cells were kept for a total of 24 h since the start of transfection, regardless of the addition of PD98059 or SP600125). The cells were then trypsinized and resuspended with medium 200 containing 0.5% fetal bovine serum (FBS) at a density of 3 x 104 cells/mL. From the cell suspension, 1 mL was plated onto 24-well plate containing 200 µL solidified Matrigel (Becton Dickinson, Franklin Lakes, NJ, USA) per well. After 24 h, formation of tube structures was evaluated. The images were acquired and the tube length was measured using QWIN image analysis software (Leica) as an indicator of angiogenic potential. For each type of treatment, the cumulative tube length in five randomly selected microscopic fields (40x) derived from three independent experiments was calculated, as previously described.22

2.4 Cell viability and proliferation analysis
Cell viability was measured using the 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay to reflect the dehydrogenase activity of mitochondria. HAEC were seeded onto 24-well plates. After the transfection and 38.5 h of recovery, medium was replaced with MTT-containing medium (1.2 mmol/L) and cells were incubated for 4 h at 37°C. The cells were lysed with isopropanol–HCl solution and subject to absorbance measurement at 540 nm with 630 nm reference. Cell proliferation was determined using BrdU labelling kit (Calbiochem) to show the cells in the stage of DNA synthesis. At least three independent experiments were conducted for each group of cells.

2.5 Data analysis
Data are expressed as mean ± SD. Analysis was conducted using one-way analysis of variance or Student's t-test. P-value <0.05 is considered statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Supplementary material
 Funding
 References
 
3.1 Connexin43-specific small interfering RNA attenuated the expression of connexin43 transcripts, proteins, and gap junctions, and inhibited the function of gap-junctional communication in human aortic endothelial cells
Cells were well in contact in all experiments (see Supplementary material, Figure S1). After 6 h of transfection with 25 nmol/L or more Alexa Fluor 488-labelled Cx43siRNA1, virtually all HAEC took up the siRNA1, and the uptake appeared in a dose-dependent manner (Figure 1A, a–c). Cells treated with liposome only (Figure 1A, d) or Alexa Fluor 488-labelled Cx43siRNA1 without liposome (Figure 1A, e) displayed none or less fluorescence. Consistently, delivery of each of Cx43siRNA1 and Cx43siRNA3 decreased the Cx43 mRNA expression levels in a dose-dependent manner (Figure 1B). Analysis showed that, for both Cx43siRNA1 and Cx43siRNA3, the maximal reduction of the transcripts was more than 60% (Figure 1B). Accordingly, Cx43 protein was also down-regulated in a dose-dependent manner (Figure 1C). The expression of Cx43 was not affected by the nonsense siRNA (Figure 1B and C).


Figure 1
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  		Figure 1 Uptake of Cx43siRNA reduces connexin43 expression, as confirmed using confocal microscopy (A), semi-quantitative reverse transcriptase-polymerase chain reaction (B), and western blotting (C) (see text for details). (A) The concentration of Alexa Fluor 488-labelled Cx43siRNA1 was denoted at upper border of each image and the blue fluorescence is the nucleus counterstained with bisbenzimide. Control, cells without any treatment. NS, nonsense control; siRNA1, Cx43siRNA1; siRNA3, Cx43siRNA3. Bar, 50 µm. *P < 0.05; **P < 0.01 compared with nonsense control.

 
Immunoconfocal microscopy showed that, after 48 h of transfection, cells treated with liposomes only or nonsense in liposomes had abundant Cx43 gap junctions in cell borders (Figure 2A, a and b), similar to those without any treatment (Figure 2A, c). Cx43 gap junctions were markedly reduced in cells transfected with Cx43siRNA1 or Cx43siRNA3 using liposomes (Figure 2A, d and e).


Figure 2
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  		Figure 2 Connexin43-specific siRNA down-regulates Cx43 gap junctions (A) and attenuates the gap-junctional communication function (B and C) (see text for details). Note that (A) shows immunoconfocal images, in which gap junctions are seen as red labels mainly distributed at cell borders. (B) Scrape loading/dye transfer experiments demonstrated that cells treated with liposome-only control (a) and nonsense control (b) exhibited the same spread pattern of Lucifer yellow, seen as green fluorescence. However, those with Cx43siRNA1 (c) and Cx43siRNA3 (d) showed attenuated dye transfer. The red fluorescence of Rhodamine dextran was used to mark the line of scrape. Analysis of the distance between the front of dye transfer and the scrape line is shown in the histogram. (C) Microinjection showed similar patterns of dye diffusion in the control groups (a and b) but the diffusion was almost abolished in human aortic endothelial cells (HAEC) treated with Cx43siRNA1 (c) or Cx43siRNA3 (d). Analysis showed that <10% of cells was capable of dye transfer in HAEC treated with Cx43-specific siRNA, as indicated in the histogram. Note that injected cells appeared yellow because of overlapping of green labels (Lucifer yellow) and red labels (Rhodamine dextran). Data are expressed as relative percentage of the number of cells positive for green fluorescence. Abbreviations are the same as used in Figure 1. #P < 0.005 compared with liposome-only control. Bars, 50 µm in (A) and (C); 200 µm in (B).

 
The effect of Cx43 silencing on gap-junctional communication function was evaluated using scrape loading/dye transfer (Figure 2B) and microinjection (Figure 2C), in which cells after 48 h of transfection showed that both Cx43siRNA1 and Cx43siRNA3 drastically inhibited the communication.

3.2 Down-regulation of connexin43 by Cx43-specific small interfering RNA altered the expression profile of human aortic endothelial cells
To evaluate the endothelial gene profile affected by Cx43 down-regulation, HAEC were harvested for 24 and 48 h post-transfection with the siRNA. Multiple genes including gap-junctional components (Cx37, Cx40, and Cx43), growth-related factors [Flt-1, KDR, and vascular endothelial growth factor (VEGF)], angiogenesis factors (Ang1 and Ang2), coagulatory factors [plasminogen activator inhibitor-1 (PAI-1) and von Willebrand factor (vWF)], vasodilatory enzyme endothelial nitric oxide synthase (eNOS), adheren junctional protein vascular endothelial cadherin (VE-Cad), and cytokine (IL-8) were examined. At the time point of 24 h when the down-regulation of Cx43 transcripts was apparent (decrement, Cx43siRNA1, 61%; Cx43siRNA3, 52%; both P < 0.005), only PAI-1 was significantly up-regulated in cells transfected with Cx43siRNA3 (77% increment, P < 0.01). At 48 h post-transfection, PAI-1 was up-regulated by both Cx43siRNA1 and Cx43siRNA3 (increment, 53% and 81%, respectively; both P < 0.05). In addition, the siRNA also lead to the enhanced expression of other transcripts, including Cx37 and Cx40. Analysis of data of transcripts were summarized in Figure 3.


Figure 3
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  		Figure 3 Down-regulation of connexin43 alters the mRNA expression profiles of HAEC. Note that 25 nmol/L of various synthetic small interfering RNA was used (see text for details). Abbreviations are the same as used in Figure 1. *P < 0.05; **P < 0.01; #P < 0.005 compared with nonsense control.

 
Western blots showed that cells transfected with Cx43siRNA1 or Cx43siRNA3 exhibited Cx43 silencing for at least 96 h (Figure 4A and B). However, the maximal reduction occurred at different time points (maximal decrement, Cx43siRNA1, 98% at 48 h; Cx43siRNA3, 89% at 24 h; Figure 4C). Regarding PAI-1, the expression levels gradually escalated and reached a maximum at 48 h in cells transfected with Cx43siRNA3 (increment 187% compared with nonsense control) and at 96 h for those with Cx43siRNA1 (increment 98%; Figure 4D). Similarly, vWF protein was increased (increment at 48 h post-transfection, 181% with Cx43siRNA1; at 72 h, 63% with Cx43siRNA3; both P < 0.05). In contrast, the nonsense siRNA exerted no significant effect on the expression of Cx43, PAI-1, and vWF protein for up to 96 h post-transfection (Figure 4E). For eNOS and VE-Cad, consistent with the results of RT-PCR, no significant change was observed. Although at the mRNA levels Cx37 and Cx40 were up-regulated (Figure 3), no obvious difference was detected by western blotting in all cell preparations (see Supplementary material, Figure S2). Immunoconfocal microscopy also confirmed that Cx43 silencing had no effect on the expression of VE-Cad, Neural cadherin (N-Cad), ZO-1, Cx37, and Cx40 (see Supplementary material, Figure S3).


Figure 4
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  		Figure 4 Time-course of down-regulation of connexin43 (Cx43) and up-regulation of plasminogen-activator inhibitor-1 (PAI-1) protein in human aortic endothelial cells treated with Cx43-specific small interfering RNA (siRNA) (see text for details). Note that in (A–E 25 nmol/L of various synthetic siRNA were used, and the incubation time for nonsense siRNA is 48 h. Other time-points of cells treated with nonsense siRNA are shown in (E). Abbreviations are the same as used in Figure 1. *P < 0.05; **P < 0.01; #P < 0.005 compared with nonsense control.

 
3.3 Connexin43 silencing impaired the proliferation activity, viability, and angiogenic potential of human aortic endothelial cells
Analysis of the activity of cell proliferation using BrdU incorporation showed that Cx43-specific siRNA retarded the proliferation of HAEC (Figure 5A). For each of the Cx43siRNA1 and Cx43siRNA3, the maximal reduction was more than 50%, compared with liposome-only control. The viability, determined by the MTT assay, at 48 h post-transfection of HAEC was reduced by more than 35% for each siRNA at 40 nmol/L (P < 0.05 compared with the liposome-only control; see Supplementary material, Figure S4).


Figure 5
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  		Figure 5 Analysis of the activity of proliferation and angiogenesis in human aortic endothelial cells (HAEC) treated with connexin43-specific small interfering RNA, as evaluated by BrdU incorporation assay (A) and tube formation assay (B) (see text for details). Note that tube formation capability of HAEC is not affected by SP600125 (B, g). *P < 0.05; **P < 0.01 compared with nonsense control. Bar, 200 µm.

 
In addition, tube formation assay to evaluate angiogenesis capacity showed that, instead of regular polygonal structure formed in the control cells, Cx43 silencing resulted in irregular, incomplete polygonal structure as well as reduced tubular length (Figure 5B). Analysis of the length of tube after 48 h of transfection demonstrated that both Cx43siRNA1 and Cx43siRNA3 reduced the length by more than 40% (Figure 5B).

3.4 Connexin43 down-regulation induced the activation of c-jun N-terminal kinase without alteration of caspase-3 level
As JNK is one of the upstream regulators of PAI-1, the signal pathway of JNK was studied. As shown in Figure 6, JNK phosphorylation was activated after 6 h of transfection with Cx43siRNA1 and such a change is accompanied by an increase of c-jun and its phosphorylation (Figure 6A). Changes were similar in Cx43siRNA1 and Cx43siRNA3 (data not shown). Real-time PCR showed that Cx43-specific siRNA almost completely abolished Cx43 transcripts in the period of 3–6 h post-transfection (maximal decrement, Cx43siRNA1, 93% at 6 h; Cx43siRNA3, 98% at 3 h; see Supplementary material, Figure S5), which was parallel to the time point of JNK activation. In contrast, during the period of Cx43 silencing no change of procaspase 3 was seen and caspase 3, an indicator of apoptosis, was undetectable (Figure 6A). Similarly, western blots analysis using antibodies specific to p38, phosphorylated p38, extracellular signal-regulated kinase (ERK), and pERK demonstrated that there was little difference between nonsense and Cx43siRNA on the activation of p38 and ERK (Figure 6B). These data indicate that the reduced proliferation and viability of HAEC were not contributed by the process of apoptosis.


Figure 6
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  		Figure 6 Effect of connexin43 (Cx43) silencing on JNK, c-jun, procaspase 3, caspase 3, ERK, and p38 (A and B) and effect of Cx43 silencing plus SP600125 on plasminogen-activator inhibitor-1 expression (C) (see text for details). *Jurkat cell lysate was used as positive control for caspase 3.

 
3.5 Blockade of c-jun N-terminal kinase activation suppressed the increased expression of plasminogen activator inhibitor-1 and partially recovered the attenuated angiogenic potential induced by Cx43-specific small interfering RNA
Western blots showed that SP600125, an inhibitor of JNK, attenuated the increased expression of PAI-1 induced by the siRNA (Figure 6C). To further explore the effect of JNK activation on angiogenic potential, SP600125 was added to the culture medium while PD98059, an inhibitor of MEK was used as a negative control. Compared with the impaired tube formation in cells transfected with the Cx43-specific siRNA (Figure 5B, b), cells treated with the siRNA plus SP600125 showed partial recovery in tube formation (Figure 5B, e), while PD98059 had no effect (Figure 5B, f). In contrast, MTT assay showed that co-treatment with the siRNA plus SP600125 did not improve the reduced viability seen in cells treated with the siRNA alone (see Supplementary material, Figure S4).

3.6 Short-term blockade of connexin43 channels by connexin-mimetic peptides did not reduce expression of Cx43 proteins or activate c-jun N-terminal kinase
To determine the contribution of inhibited gap-junctional communication to the activation of JNK, we treated HAEC with gap 26 or gap 27 for 90 min. Scrape loading/dye transfer assay showed that the communication was inhibited by each of the peptides (see Supplementary material, Figure S6A). However, western blots demonstrated no reduction of Cx43 proteins or activation of JNK (see Supplementary material, Figure S6B). Cells treated for 2 h or longer with either scrambled peptides, gap 26, or gap 27 lost the cobble stone-like appearance, indicating the peptides possessed toxicity, which is not specific to the sequence (see Supplementary material, Figure S6C).


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Supplementary material
 Funding
 References
 
In this study, we demonstrated that reduced Cx43 expression in endothelial cells not only led to attenuation of gap-junctional communication function, but also affected the activities of endothelial cells in several aspects, including inhibition of proliferation and survival, reduction of angiogenic potential, and enhancement of the expression of coagulatory factors PAI-1 and vWF. In addition, we also showed that during the reduction of Cx43, the JNK pathway and its downstream molecule, c-jun, is activated. Furthermore, inhibition of JNK by SP600125 was able to attenuate the increased expression of PAI-1 and partially recover the attenuated angiogenic potential, but had no effect on the reduced viability of endothelial cells owing to the decrease of Cx43. In contrast, inhibition of gap-junctional communication by connexin-mimetic peptides did not reduce Cx43 protein or activate JNK. All these novel findings indicate that expression of Cx43 is important in the maintenance of normal endothelial function.

Our findings in the present study were based on the use of two distinct segments of siRNA, the sequence of each corresponding to different domains of human Cx43 coding sequence. The effect of Cx43siRNA1 on Cx43 silencing is consistent with a previous report using the same sequence.20 Although the action of both segments on Cx43 silencing varied slightly in time course and efficacy, the phenotypic changes of endothelial cells, as mentioned above, behaved in the same direction. In contrast, the effects of these two segments of siRNA on Cx43 expression were not seen in cells treated with control nonsense siRNA. On the other hand, treatment with either segment of the siRNA did not change the protein levels of other endothelial connexins, Cx37 and Cx40, although the transcripts were increased. Taken together, the changes in the expression level of endothelial Cx43 in the present study are specific to the action of the siRNA, which does not affect the expression of Cx37 and Cx40 proteins.

We in the present study explored the signalling pathways of mitogen-activated protein kinase (MAPK), a family of central signalling molecules responding to a variety of stress,23 because we thought that MAPK may be activated owing to the reduction of intracellular Cx43, which possessed multiple functions, as mentioned before. The findings in the present study that JNK and c-jun were activated and PAI-1 was increased in expression as well as that SP600125 abolished the increased expression are consistent with a previous report, which showed that activation of JNK induced the expression of PAI-124 and that JNK inhibitor reduced the c-jun-dependent transcription activity of PAI-1 promoter.25 Regarding JNK and angiogenesis, in the present study inhibition of JNK by SP600125 rescued the angiogenic potential of HAEC, indicating that activation of JNK attenuates angiogenic potential. This finding is different from a recent report showing that inhibition of JNK reduced endothelial migration,26 which is required for angiogenesis. On the other hand, the partial rather than complete recovery of angiogenic potential by SP600125 suggests that signalling pathways other than JNK are involved in the modulation of the potential during the period of Cx43 reduction. In addition, the impaired angiogenic potential after the knockdown of Cx43 was partially recovered by SP600125, but the reduced viability after the knockdown was not changed by SP600125, indicating that the impairment of angiogenic potential is not entirely contributed by the reduction of viability. On the other hand, a short-term inhibition of the Cx43 channels by the connexin-mimetic peptides neither activate JNK, nor reduce Cx43 protein, suggesting that either the activation required a longer period of time of inhibition, reduction of Cx43 protein is necessary to activate JNK, or the activation is not attributed to the channel-dependent properties of Cx43. With respect to PAI-1 and angiogenesis, the finding of the present study that enhancement of PAI-1 expression is associated with reduced angiogenesis is consistent with a recent report demonstrating that PAI-1 of endothelial origin inhibited angiogenesis,27 although pharmacological inhibition of PAI-1 was reported to attenuate tumour angiogenesis.28 Apart from JNK and PAI-1, in the present study the reduced angiogenesis capacity resulting from the reduction of Cx43 is contradictory to the experiments examining human breast cancer cell lines, which showed that silencing of Cx43 decreased the expression of anti-angiogenesis molecule20 (i.e. reduction of Cx43 favoured tumour angiogenesis), while overexpression of Cx43 reduced tumour angiogenesis.29 These findings indicate that in HAEC and breast cancer cell lines, modulation of angiogenesis by Cx43 expression levels is operated in an opposite direction. Administration of Cx43 siRNA at wound sites has been shown to enhance keratinocyte proliferation,30 opposite to the inhibited proliferation of HAEC seen in the present study. However, administration of Cx43 siRNA at wound sites may affect the expression of Cx43 in several types of cells; therefore, the response of keratinocytes may not merely be attributed to the down-regulation of their Cx43.

In the present study although the mRNA expression levels of Flt-1, KDR, VEGF, Ang1, and Ang2, which are intimately linked to endothelial proliferation and/or angiogenesis, remained stationary, BrdU uptake, MTT assay, and tube formation assay showed depressed proliferation, viability, and angiogenesis. One possibility for this discrepancy is that the changes of these molecules may occur at the post-transcriptional level, behaving in a manner similar to vWF, which is increased at the protein level but remains stationary at the mRNA level. Moreover, in parallel to the changes of proliferation, viability, and angiogenic capacity suggestive of endothelial damage or dysfunction, the increase of endothelial content of vWF in the presence of endothelial damage or dysfunction may enhance thrombosis and therefore accelerate the process of atherothrombosis. vWF had been reportedly increased in the presence of the major risk factors for atherosclerosis and cardiovascular disease, in which damage or dysfunction of endothelial cells exists.31–33

Previous studies investigating the expression of endothelial gap junctions in healthy and diseased state had shown that the expression is altered in the presence of cardiovascular risk factors related to atherosclerosis, such as ageing,14 hypertension,15 diabetes,16,34 hyperlipidaemia,35 exposure to nicotine,6 arsenic trioxide,36 and mechanical injury.37 Interestingly, all of the above studies showed that endothelial gap junctions are reduced, suggesting that down-regulation of gap junctions is a common phenomenon in endothelial cells exposed to the atherogenic factors. Therefore, reduction of endothelial gap junctions may serve as a marker of impaired endothelial function. With the use of Cx43-specific siRNA we in the present study unequivocally showed that reduction of endothelial Cx43 protein per se impairs a variety of functions of endothelial cells. Such in vitro findings implicate that Cx43 is potentially a target for further in vivo investigation of endothelial pathology.

Our findings in the present study strengthen the role of connexins in atherosclerosis, in which multiple connexins exert their actions by regulation of the activities of endothelial cells, smooth muscle cells, and monocytes/macrophages at different stages of the vascular disorder.38 As gap junctions are located at cell borders, whether down-regulation of Cx43 gap junctions is associated with a redistribution of other junctional proteins, such as junctional adhesion molecules, requires further studies.39 Another limitation of the present study is that it is difficult to determine whether the channel-dependent or independent properties of Cx43, or both, are involved in the endothelial changes seen during the knockdown of Cx43. Experiments examining replacement of native Cx43 by mutant Cx43 that loses communication at the junction without changes in total Cx43 protein expression levels may give clues.

In conclusion, transfection with different synthetic siRNA specific to Cx43 results in similar inhibition of Cx43 expression and gap-junctional communication, which activates endothelial cells to pathological status, as characterized by enhanced expression of coagulatory molecules and inhibition of proliferation, viability, and angiogenesis. The processes are associated with activation of JNK signalling pathways and rectified by inhibition of the activation. In contrast, a short-term blockade of Cx43 channels without reduction of the protein did not activate JNK. These results suggest that inadequate expression of Cx43 per se alters the phenotypic features of endothelial cells by the activation of stress-activated protein kinase.


   Supplementary material
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
Funding
 References
 
Supplementary material is available at Cardiovascular Research online.

Conflict of interest: none declared.


   Funding
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
References
 
National Science Council, Taiwan (NSC 95-2314-B-195-006) and Medical Research Department of the Mackay Memorial Hospital, Taiwan (MMH-E-95003).


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 

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Silencing TRPM7 promotes growth/proliferation and nitric oxide production of vascular endothelial cells via the ERK pathway
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