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VASCULAR BIOLOGY
1Division of Endocrinology and Metabolism, 2Division of Pulmonary and Critical Care Medicine, Department of Medicine, University of California, San Diego, La Jolla, California
Submitted 20 September 2007 ; accepted in final form 5 May 2008
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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gap junction; endothelium-dependent relaxation; microvascular rarefaction; coronary vascular complications; Sp1
The endothelium regulates vascular tone by producing and releasing nitric oxide (NO) and prostacyclin and by hyperpolarizing smooth muscle cell (SMC). ECs can evoke SMC hyperpolarization by releasing endothelium-derived hyperpolarizing factor (EDHF) and directly by intercellular communication through gap junctions. Gap junctions are cell membrane channels made of connexin (Cx) proteins, and 20 Cx genes have been identified in the mouse genome (37). Cx37, Cx40, and Cx43 (and Cx45 in some case) are described in the vascular wall, and Cx40 is highly expressed in ECs (45, 50). The Cx40-knockout mice are hypertensive (11), whereas polymorphisms in the promoter of Cx40 gene have been shown to associate with increased risk of hypertension in patients (21). Furthermore, in hypertensive rats, Cx40 protein expression is decreased in ECs (28, 41) [but not in the kidney (24) and the cardiomyocyte (2)]. These data suggest that Cx40 in ECs may play an important role in the development of hypertension; however, the role of Cx40 in coronary endothelial dysfunction in diabetes is unexplored.
Insufficient growth and rarefaction of capillaries, followed by endothelial dysfunction, may represent one of the most critical mechanisms involved in heart damage. It has been reported that capillary density in the heart is progressively decreased in Type 1 and Type 2 diabetic animal models, as well as in human diabetic patients (5, 23, 25, 47, 55). Decreased density of capillaries is due to loss of existing capillary network and attenuated regeneration of new capillaries. Increasing capillary density in the heart, which improves the capacity to transport oxygen to cardiac tissues with a high tissue perfusion and a short diffusion distance for oxygen, is therefore another potential therapeutic strategy for coronary vascular disease.
The present study was designed to investigate the role of Cx40 on vascular complication in the diabetic heart. To that end, we determined the protein expression level of Cxs in coronary endothelial cells and gap junction intercellular communication (GJIC) contribution on endothelial function in diabetes.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Preparation of diabetic mice. Six-week-old male NIH Swiss mice were purchased from Harlan Sprague Dawley (Indianapolis, IN), and mice in the diabetic group received a single injection of streptozotocin (160 mg/kg iv, dissolved in citrate buffer). All data were obtained from mice 10–12 wk after injection. Plasma glucose levels were 7.9 ± 0.4 mM in control mice and 36.5 ± 1.6 mM in diabetic mice. This study was conducted in accordance with the guidelines established by Committee on Animal Research at the University of California, San Diego.
Isolation of coronary vascular endothelial cells. Mouse coronary ECs were isolated using a modification of previously described methods (56). Briefly, dissected heart tissues were minced and incubated with M199 containing 1 mg/ml collagenase II and 0.6 U/ml dispase II, for 1 h at 37°C. The digested material was filtered through sterile 40-µm nylon mesh and washed in 2% fetal calf serum-M199. Subsequently, the cells were incubated with Dynabeads (Invitrogen), which were prepared as follows: beads coated with sheep anti-rat IgG were incubated with purified rat anti-mouse CD31 monoclonal antibody (1 µg/ml) at 4°C overnight and then washed with PBS containing 0.1% BSA and 2 mM EDTA. Cell suspension was incubated with beads for 1 h at 4°C, and then beads attached to endothelial cells were captured by Dynal magnet (Invitrogen). Purity of the EC population in cells isolated from hearts was tested by DiI-acLDL uptake and Bandeiraea Simplicifolia lectin-FITC (BS-l, Sigma Aldrich) staining in mouse coronary ECs. Cultured human coronary smooth muscle cells (HCSMC) were used as negative control. Mouse coronary ECs were seeded on a cover glass in a four-well chamber. After 3 days of incubation, cells were stained with Dil-acLDL (10 µg/ml, Invitrogen) and incubated for 4 h at 37°C. At the last 30 min, Hoechst (1 µg/ml, Invitrogen) was added to the cells to stain nuclei. Cells were washed with PBS, fixed with 4% paraformaldehyde, and incubated with BS-l for 30 min. The percentage of positive cells that exhibit both acLDL and BS-l signal was over 91% in mouse coronary EC cultures (the experiments were reproduced five times).
Western blot analysis. Isolated ECs were lysed and centrifuged at 16,000 g for 10 min at 4°C. Supernatants were used as a sample protein. Samples were separated through a SDS-polyacrylamide gel and transferred to nitrocellular membranes. Blots were incubated with a primary antibody, anti-Cx37, Cx40, Cx43 antibodies (1:1,000), anti-Sp1 antibody (1:2,000), or anti-actin antibody (1:4,000), and followed by secondary antibody application. The immunoblots were detected with the ECL Western blotting detection reagents (Perkin-Elmer, Norton, OH). Band intensity was normalized to actin controls and is expressed in arbitrary units.
Detection of GJIC. Isolated ECs were cultured in M199 supplemented with 20% FCS, 100 µg/ml endothelial cell growth supplement (ECGS), 100 IU/ml penicillin, 100 µg/ml streptomycin, 50 mg/l D-valine, and 16 U/ml of heparin. Cells were plated on the coverslips coated with 5% gelatin, cultured for 3–5 days, and used to determine GJIC activity. Cells on 25-mm coverslips were placed in a recording cell chamber on the stage of an inverted Nikon microscope (Eclipse/TE 200) with the TE-FM epifluorescence attachment. Excitation (428 nm) was provided by a mercury lamp. Fluorescence emission (536 nm) was collected using a x40 Nikon Plan Fluor objective (0.75 numerical aperture) and an intensifier. The fluorescence images based on the fluorescence signals emitted from the cells were acquired at 1 Hz using an Image Intensifier Tube/Philips 1381 system (Stanford Photonics, Palo Alto, CA) and stored on an IBM-compatible computer for later analysis. Dye transfer through GJIC was studied using an electrode in the whole cell mode to deliver a known concentration (2 mM) of Lucifer Yellow (LY, Invitrogen) (49). GJIC activity was assessed by counting the number of surrounding cells showing LY fluorescence 30 min after loading LY into a cell in the middle of a group of cells.
Isometric tension measurement of coronary arterial ring.
The heart was isolated and placed in Krebs-Henseleit solution for dissection. Second- or third-order small coronary arteries (CAs) were cleaned of any adherent connective tissue and cardiomyocytes and cut into 1- to 1.5-mm segments. Rings were placed with tissue hooks onto force-displacement transducer, set at a resting tension of 0.1 g, and allowed to equilibrate for 60 min with intermittent washes every 20 min. After equilibration, each CA ring was contracted by treatment with PGF2
(1–5 µM). Functional vessels were confirmed by demonstrating the ring's ability to relax to 100 µM sodium nitroprusside (SNP). CAs in which 100% relaxation of the PGF2 response occurred were regarded as intact and functional vessels.
Electrophysiological measurements.
Isolated coronary ECs from control and diabetic heart were used for electrophysiological measurement. Whole cell K+ currents (IK) were recorded from ECs with an Axopatch 1D amplifier and a DigiData 1200 interface (Axon Instruments, Sunnyvale, CA) using conventional patch-clamp techniques. Briefly, a coverslip plated with cells was mounted on a Plexiglas perfusion chamber on a Nikon inverted microscope, and cells on the coverslip were bathed in Ca2+-free physiological salt solution (PSS) containing (in mM) 141 NaCl, 4.7 KCl, 3 MgCl2, 10 HEPES, 1 EGTA, and 10 glucose (pH 7.4). The pipette (intracellular) solution contained (in mM) 135 KCl, 4 MgCl2, 10 HEPES, 10 EGTA, and 5 Na2ATP (pH 7.2). All experiments were performed at room temperature (22–24°C). Patch pipettes (2–3 M
) were fabricated on an electrode puller (Sutter Instrument, Novato, CA) using borosilicate glass tubes and fire polished on a microforge (Narishige Scientific Instruments, Tokyo, Japan). Command voltage protocols and data acquisition were performed using pCLAMP-8 software (Axon Instruments). With the use of the 2- to 3-M pipettes, the series resistance was at a range of 4–9 M when the whole cell configuration was formed. Series resistance compensation was performed in most of the whole cell experiments. Leak and capacitative currents were subtracted using the P/4 protocol in pCLAMP software.
Analysis of capillary densities in left ventricular myocardium.
After 10 wk of STZ injection, the mice were used for an analysis of myocardial capillary vasculature similar to previously described methods (56) with modifications. The ventricle was dissected, embedded in OCT compound (Sakura Finetek, Torrance, CA), frozen in 2-methylbutane precooled with liquid nitrogen, and then kept at –80°C until sectioned. Sections (6 µm) were fixed in 4% formaldehyde for 5 min, blocked with 5% BSA for 30 min, and incubated with Bandeiraea Simplicifolia lectin-FITC (BS-l) for 30 min. BS-l is used to probe the terminal -galactosyl saccharides associated with endothelial cells surface of arterioles and venules as well as capillaries. Subepicardial regions of the left ventricular (LV) free wall on the section were photographed in sequence by a CCD camera connected to a fluorescence microscope with a x20 objective lens. For every experimental condition, at least two sections from each sample were examined, and at least eight microscopic fields were investigated. Capillary count was analyzed with ImageJ 1.33 (National Institutes of Health, Bethesda, MD). The average capillary numerical density NA/mm2 (number of capillaries per mm2) was calculated for each heart.
Determination of Cxs localization in a CA.
Vertical section of the CA (6 µm) was fixed with ice-cold acetone and stained with BS-1 and -smooth muscle action (SMA), makers for ECs and SMCs, respectively. The same heart but a different section was fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton-X, and stained with Cx37 or Cx40 (1:200). FITC-labeled secondary antibody (1:2,000) was applied to the section for visualization.
Assay for in vitro capillary network formation. Four-well Permanox cell culture slides were coated with Matrigel (150 µl/well) and allowed to solidify for 1 h at 37°C. Rat vascular ECs (4 x 104 cells/well, Cell Applications, San Diego, CA) were seeded on the Matrigel. For high-glucose (HG) treatment, 30 mM glucose was added to the medium (the final glucose concentration was 35 mM). In a control group of cells, equimolar mannitol was added to exclude the potential effect of changes in osmolarity (NG: glucose concentration, 5 mM). After 24 h, 10 microscopic fields selected at random were photographed. Each cell was outlined manually, and the total tube lengths (L, mm/mm2) and the density of capillary network D/mm2 (D = number of cross section of cells and grid; grid width = 10 pixels in 640 x 480 pixel image) were analyzed by MATLAB software (The Math Works, Natick, MA).
Trasfection of Cx40 into human coronary ECs for capillary network formation assay. Human coronary ECs were transfected with an empty adenovirus (Control-Adv) or adenovirus containing the mouse Cx40 gene (Cx40-Adv) (200 pfu/cell). Three days after infection, cells (104 cells/well) were seeded on four-well Permanox slides coated with Matrigel, and capillary network formation assay was performed as described above.
Statistical analysis. Values are expressed as means ± SE. Responses were evaluated with one-way ANOVA, with a Dunn multiple-range test. Statistical comparison between dose-response curves was made by two-way ANOVA with the Bonferroni correction performed post hoc to correct for multiple comparisons. Differences were considered to be statistically significant when P < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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-induced contraction) between control (132.2 ± 6.6%) and diabetic (135.2 ± 5.9%, P = 0.20) mice. However, ACh-induced relaxation in CAs from diabetic mice was significantly attenuated compared with CAs from control mice (Fig. 3A).
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In addition, 20-min treatment of the CA rings isolated from control mice with 18β-glycyrrhetinic acid (18β-GA, 50 µM), a nonspecific gap junction inhibitor (1, 46), markedly attenuated ACh-induced vasodilatation (Fig. 4). Pretreatment of CA rings for 1 h with 300 µM 40GAP27, a selective inhibitor of Cx40, had no effect on ACh-induced vasorelaxation in control CA but strongly inhibited the relaxation in diabetic CA (Fig. 5). Only after the concentration of 40GAP27 was increased to 600 µM, it finally exhibited the inhibitory effect on the relaxation in control CA, indicating that diabetic CA forms much less gap junction with Cx40. These data suggest that GJIC is required for endothelium-dependent coronary vasodilatation, and attenuated vasodilatation observed in diabetic CA results in reduced GJIC function by a decrease in Cx40 expression in coronary ECs. An alternative explanation for the change in sensitivity of ACh-mediated EDR in diabetic CAs to 40GAP27 is that the gap junction-mediated EDR is altered in diabetes to be more dependent on Cx40.
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deact, Fig. 6E) were also comparable between control and diabetic ECs. These data suggest that attenuated EDH-mediated vascular relaxation in diabetes is not due to changes in K+ channel activity.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Coronary blood flow and coronary vascular resistance are tightly controlled by vascular tone and vascular density. EC serves as a major player in regulation of vascular tone and density; endothelial dysfunction is thus considered a risk factor of cardiovascular complications in many diseases, including diabetes. Diabetic CAs exhibit attenuated endothelium-dependent relaxation (EDR) (Fig. 3A). To confirm that the inhibited EDR in diabetic CAs involves the contribution of a non-NO/prostacyclin mediator of EDH, we conducted a series of pharmacological experiments using the combination of Apa and ChTX in the presence of L-NAME and Indo. When NOS and COX were blocked, ACh-induced coronary vasodilatation was almost abolished in diabetic mice, whereas ACh still caused dose-dependent relaxation in control mice (Fig. 3B). The difference of ACh-induced coronary vasodilatation between control and diabetic mice, in the presence of L-NAME and Indo, was likely due to the difference of the EDR independent of NO and prostacyclin. Inhibition of calcium-dependent K (KCa) channels with ChTX + Apa, in the presence of L-NAME and Indo, abolished ACh-induced relaxation in control CAs and eliminated the difference of ACh-induced coronary vasodilatation between control and diabetic mice (Fig. 3C). In the absence of L-NAME and Indo, the magnitude of ACh-induced relaxation in CAs treated with ChTX + apamin (Fig. 3D) or TEA alone (Fig. 3E) was almost the same between control and diabetic mice. These data suggest that ACh-induced EDH is attenuated in diabetic CAs.
There is accumulating evidence showing that EDH is impaired in diabetic animal models and diabetic patients, although some studies suggest that EDH is enhanced in diabetes (8, 10, 20, 22, 33, 36, 48). These inconsistent results imply that the changes in vascular relaxation mediated by EDH in diabetes may vary depending on the tissues studied, the diabetic period, and the type of diabetic models. Inhibition of direct cell-to cell coupling by Cx-mimetic peptides (e.g., Gap26 and Gap27) and GA derivatives attenuate EDH and vasodilatation (14, 46). Inhibition of GJIC with 18β-GA significantly decreased EDR in control CAs (Fig. 4), whereas we were unable to evaluate the effect of 18β-GA in diabetic CAs since vehicle treatment (0.25% EtOH) interfered with EDR in diabetic CAs. The mechanism that induces inhibitory effect by EtOH on EDR in diabetic CAs is not clear and needs further study.
Cx mimetic peptides are effective in diminishing GJIC by simulating crucial extracellular loop sequences and interacting prematurely with unpaired connexons in the plasma membrane, thus disrupting the docking of the complementary hemichannels and reducing the number of operational gap junctions (17). The density of functional Cxs on the surface membrane can be influenced by modulation of transport along cytoskeletal elements, and a gap junction's resident time in the surface membrane is likely to be short because of the rapid turnover of Cxs (1–5 h) (18, 30). Thus Cx mimetic peptides are usually exposed to the tissues at least for 1 h to block not only the Cxs in the surface membrane but also renewed Cxs from cytosol. In contrast, some reports demonstrated different inhibitory mechanisms by Cx mimetic peptides in different cell types. In A7r5 cells, Martin et al. (34) reported that Cx-mimetic peptides (e.g., 37,43GAP26 and 37,43GAP27) had no effect on Cx trafficking or the de novo formation of gap plaques. Furthermore, in coupled cell pairs under cell culture condition, Cx-mimetic peptides are also capable of blocking electric coupling within 5 min (35). These observations indicate that the Cx-mimetic peptide might exert the inhibitory effect on gap junction via multiple mechanisms. As shown in Fig. 5, 300 µM 40GAP27 completely inhibited EDR in diabetic CAs but not in control CAs. Twice higher concentration of 40GAP27 was needed to partially inhibit EDR in control CAs. These data suggest that the total amount of Cx40 in diabetic CAs is much less than that in control CAs, which is in line with the result of Western blot analysis of Cx40 in coronary ECs (Fig. 1B). When taken together, the data from current study demonstrate that EDR is attenuated in diabetic CAs because of downregulated expression (and/or decreased function) of Cx40.
Although our data indicate that Cx40 is an important gap junction peptide required for normal coronary vascular function, it has to be notified that multiple gap junction peptides may be required to completely block endothelium-dependent hyperpolarization (6). More study, using combinations of different Cx mimetic peptides, is needed to further reveal the identity of the gap junction proteins involved in coronary vascular dysfunction in diabetic mice. Furthermore, it has to be emphasized that changes in Cx expression and function may not be the sole contributors to endothelial dysfunction, and multiple mechanisms contribute to the inhibited coronary vasodilatation in diabetic mice. In addition to the impaired GJIC by decreased Cx40 expression, we believe that the inhibited endothelium-dependent hyperpolarization in diabetic CA may also involve other mechanisms: for example, 1) dysfunctional coupling of Cx peptides and their spatial co-association with K+ channels (12, 42), and 2) changes in expression and activity K+ channels in the vascular smooth muscle cells (13, 26).
In addition to regulation of vascular tone, ECs also play an important role in revascularization in the capillary network. The data shown in Fig. 7 demonstrate that capillary density in LV myocardium was significantly decreased in diabetic mice compared with that in control mice. Decreased density of capillaries is due to loss of existing capillary network and to attenuated regeneration of new capillaries. EC apoptosis and occlusion of arteriole and capillaries both contribute to microvascular rarefaction. Indeed, we observed apoptosis of capillary ECs in the LV myocardium of diabetic mice; the apoptotic rate in capillary ECs was higher in diabetic mice (5.8 ± 0.7%; P < 0.05) than that in control mice (2.6 ± 0.6%). Our results are consistent with the observations by other investigators (5, 55).
Regeneration of new EC monolayer and formation of new vessels attribute to the migration and proliferation of neighboring mature ECs and to the mobilization of circulating endothelial progenitor cells in sites of vascular wall where ECs are injured and/or lost. The results from our study show that ECs treated with HG had significantly impaired ability to form capillary network in vitro (Fig. 8, A–C), implying that hyperglycemia may attenuate ECs to form new capillary network, subsequently decreasing capillary density. In addition, 18β-GA significantly attenuated capillary network formation (Fig. 8, E–G), which indicates that GJIC is required for EC-derived capillary network formation. Interestingly, we also found that Cx40 protein expression was decreased by HG treatment during network formation (Fig. 8D). Furthermore, we demonstrated that overexpression of Cx40 gene in ECs restored capillary network formation that was attenuated by hyperglycemia (Fig. 9). These data support the contention that hyperglycemia downregulates Cx40 protein expression, which makes ECs dysfunctional to form new capillary network and subsequently decreases capillary density in the heart.
Participation of gap junctions in vascular development and control of cell proliferation are reported but in a highly specific manner. In cultured ECs, wound-induced migration is associated with an increased Cx43 expression and a downregulated Cx37 expression, but with no change in Cx40 expression (29). Thromboxane A2-induced reduction of endothelial tube formation is mediated by impairment of GJIC (1). After mechanical injury in SMCs, Cx43 expression is upregulated in the neointima of rat carotid artery, whereas no change of Cx43 is observed in rabbit iliac artery (40, 54). This evidence suggests that vascular Cxs seems to be essential to the coordination of cell proliferation, migration, and angiogenesis.
Mouse Cx40 gene contains a number of potential transcription factor binding sites (e.g., for AP-2, Sp1, and p53) (37, 43). It has been shown that Sp1 positively regulates Cx40 gene transcription in mice, rats, and humans (4, 16, 32). We demonstrate here that Sp1 expression was decreased in coronary ECs isolated from diabetic mice and in ECs treated with HG for 24 h compared with that in respective controls (Fig. 10). These data suggest that reduction of Cx40 expression by hyperglycemia might be caused by downregulation of Sp1 protein expression and/or attenuation of Sp1 function. Functional change of Sp1 by O-GlcNacylation are well studied compared with the change in protein expression of Sp1 in the diabetic state (7, 15, 38, 51), although those data reached inconsistent results. Yang et al. (51) showed that O-GlcNAcylation of Sp1 represses Sp1-mediated transcription. In contrast, Du et al. (15) demonstrated that O-GlcNAcylated Sp1 activates transcription of responsive genes. Our laboratory showed that HG treatment increases Sp1 O-GlcNAcylation and decreases SERCA promoter activity in cardiomyocytes (7). Further study is needed to investigate whether O-GlcNAcylation of the Sp1, which regulates Cx40 gene transcription, is involved in downregulating Cx40 expression and subsequently causing coronary endothelial dysfunction in diabetes.
In conclusion, the results of this study demonstrate that hyperglycemia may contribute to the development of diabetic coronary vascular complications by selectively downregulating the Cx40 protein expression in coronary arterial ECs.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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8); N, sodium nitroprusside (SNP); W, wash. The concentration of 300 µM 40GAP27 exhibits no inhibitory effect on EDR, whereas 600 µM 40GAP27 starts to affect EDR in control CAs. However, 300 µM 40GAP27 is sufficient to inhibit EDR in diabetic CAs. B: dose-response curves of ACh-induced vasodilatation in control (n = 5) and diabetic (n = 5) CA rings treated with 300 µM scramble peptide and 300 µM 40GAP27. Data are expressed as means ± SE. *P < 0.05 vs. scramble peptide.
