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ENDOCRINE AND DIABETES
Department of Pharmacology, School of Pharmaceutical Sciences, Showa University, Tokyo, Japan
Received March 21, 2006; accepted May 11, 2006.
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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,9-epoxymethano prostaglandin F2; U46619
[GenBank]
) under normal and high-glucose (HG) conditions. U46619
[GenBank]
treatment increased EC layer permeability in a time- and dose-dependent fashion. This response initially disrupted calcium-dependent EC-to-EC connections, namely, vascular endothelial cadherin (VE-CaD). Thereafter, EC force development in association with morphological changes was detected employing a reconstituted EC fiber technique, resulting in paracellular hole formation in the EC layer. Thus, we confirmed that U46619
[GenBank]
-induced enhancement of EC layer permeability involves these sequential steps. Similar trials were performed using a concentration twice that of normal glucose (22.2 mM glucose for 48 h). This treatment significantly enhanced U46619
[GenBank]
-induced EC layer permeability; furthermore, increases in both rate of VE-CaD disruption and EC fiber contraction were evident. Inhibition of calcium-independent protein kinase C and diacylglycerol kinase indicated that the glucose-dependent increase in VE-CaD disruption was mediated by a calcium-independent mechanism. Moreover, EC contraction was regulated by a typical calcium-independent pathway associated with rho kinase and actin stress fiber. Contraction was also enhanced under HG conditions. This investigation revealed that glucose-dependent enhancement of EC layer permeability is related to increases in VE-CaD disruption and EC contraction. Increases in both parameters were mediated by alteration of a calcium-independent pathway.
; Ceriello, 2003). In major diabetic complications, including angioneurosis, retinopathy, and nephropathy, it is generally accepted that dysfunction of microresistant arteries is responsible for perivascular tissue damage (Ostergaard et al., 2005; Yu and Lyons, 2005). Endothelial cells (ECs), which regulate vascular tonus via nitric oxide synthesis (Cohen, 2005), serve as a barrier at the interface of vascular tissue (Lum and Malik, 1994). The increase in EC layer permeability in local resistant artery is thought to be an early manifestation of EC barrier dysfunction and its induced diabetic complications. For example, enhancement of EC layer permeability can lead to edema and proliferative diabetic retinopathy (Qaum et al., 2001). Based on this perspective, evidence corresponding to the relationship between alteration of oxidative stress and/or NO synthesis and complications of diabetic hyperglycemia has accumulated (Prabhakar, 2004; Santilli et al., 2004; Niedowicz and Daleke, 2005). In contrast, studies have indicated that glucose-induced enhancement of EC layer permeability is dependent on protein kinase C (PKC) activity in the absence of mediation of the calcium-NO synthesis pathway (Dang et al., 2005). According to these findings, critical conclusions with respect to alteration of EC function in diabetic hyperglycemia have not been established.
We previously reported that the inflammatory factor thrombin induces enhancement of EC layer permeability (Nobe et al., 2005), which is mediated via a two-step process. Interaction of EC-to-EC adhesion molecules, namely, vascular endothelial cadherin (VE-CaD), was disrupted during the initial stage of thrombin stimulation; cell-to-cell connections were decreased. EC indicated force development (nonmuscle contraction) characterized by morphological changes; cell-to-cell distances increased, resulting in the formation of paracellular holes. We concluded that these sequential steps play a central role in alteration of EC layer permeability. To detect EC force development, a reconstituted EC fiber technique was introduced. This EC fiber was reconstituted in a collagen matrix, which made possible detection of absolute force development in cultured EC. Utility of this EC fiber force measurement system revealed that the thrombin-induced responses involved two distinct pathways, which operated as intracellular signaling pathways. Reduced VE-CaD signals were associated with a calcium-dependent pathway, whereas EC contraction was associated with a calcium-independent pathway. However, alterations of these pathways under high-glucose (HG) conditions, as in diabetes, are poorly understood.
We previously demonstrated that EC-replaced mouse aorta (Nobe et al., 2003) and portal vein (Nobe et al., 2004b) contractions were induced by treatment with the thromboxane A2 (TXA2) analog, U46619.
[GenBank]
These contractions were significantly enhanced under HG conditions; furthermore, intracellular diacylglycerol (DG) level and PKC activity were elevated during this enhancement. Similar changes were also described in other types of tissues (Ramana et al., 2005; Rolo and Palmeira, 2006). We suggested overacceleration of phosphatidylinositol (PI) turnover as a possible mechanism of enhanced vascular contraction. Incorporated glucose is converted to DG via a de novo synthesis pathway under HG conditions; as a result, excess accumulation of DG was believed to be a key step in vascular dysfunction because this situation might lead to activation of PKC. In addition, similar accumulation of DG was observed. Alteration of this vascular smooth muscle contraction might be associated with diabetic complications. We hypothesized that EC function was also influenced under HG conditions in a manner identical to that of smooth muscle dysfunction. However, alteration of EC function due to U46619
[GenBank]
has not been examined. We believe that a thorough understanding of alteration of U46619
[GenBank]
-induced EC function and its intracellular mechanisms are of utmost importance to facilitate the establishment of a new target for care in diabetic complications.
The objective of this investigation was to identify the contribution of extracellular glucose accumulation to U46619 [GenBank] -induced EC layer permeability. Thereafter, the relationships of both VE-CaD response and EC contraction to alteration of EC layer permeability were evaluated as the major mechanisms of diabetic vascular dysfunction.
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Materials and Methods |
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-3-methoxyestra-1,3,5(10)-trien-17-yl]-amino]hexyl]-1H-pyrrole-2,5-dione; U73122
[GenBank]
) and DG kinase inhibitor (6-{2-{4-[(4-fluorophenyl)phenylmethylene]-1-piperidinyl}ethyl]}-7-methyl-5H-thiazolo(3,2-)pyrimidine-5-one; R59022
[GenBank]
) were purchased from Sigma Aldrich Co. (St. Louis, MO). TXA2 receptor antagonist (([1S-[1,2(Z),3,4]]-7-[3[[2-(phenylamino)carbonyl [hydrazino]methyl]-7-oxabicyclo]2.2.1]hept-2-yl]-5-heptenoic acid; SQ29548) was obtained from Cayman Chemical Co. (Ann Arbor, MI) kinase inhibitor. Rho kinase inhibitor ((+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexane carboxamide dihydrochloride, monohydrate; Y27632) and rottlerin were obtained from Wako Pure Chemical Corporation (Osaka, Japan) and Funakoshi Corporation (Tokyo, Japan), respectively. VE-CaD antibody was acquired from Alexis Biochemicals (Tokyo, Japan). DG kinase inhibitor, stemphone, was a gift from Mitsubishi Pharma Corporation (Yokohama, Japan). U73122
[GenBank]
and U46619
[GenBank]
, which were dissolved in dimethyl sulfoxide and ethanol, respectively, served as stock solutions. Concentrations of dimethyl sulfoxide and ethanol in all solutions in the bathing medium were less than 0.05% (these reagents did not affect mechanical responses). The remaining reagents were dissolved in deionized water. All reagent dilutions and reactions were conducted in MOPS-buffered physiological salt solution (PSS) containing 140 mM NaCl, 4.7 mM KCl, 1.2 mM NaH2PO4, 0.02 mM EDTA, 1.2 mM MgSO4, 2.5 mM CaCl2, 11.1 mM glucose, and 20 mM MOPS, pH 7.4, at 37°C. HG conditions were achieved by addition of sufficient glucose to produce a 22.2 mM (twice that of normal conditions) solution in DMEM and PSS at 37°C for 48 h. Cell Culture. ECs were cultured in DMEM supplemented with 10% calf serum. Cells, which were grown on 60-mm dishes in an incubator in an atmosphere of 5% CO2 and 95% air at 37°C, displayed typical cobblestone morphology. Cells were propagated in 0.05% trypsin and phosphate-buffered saline with a split ratio of 1:4 every 3 days. All experimental data were derived from EC obtained from 7 to approximately 20 passages.
Preparation of Three Dimensionally Reconstituted Endothelial Cell Fibers. EC fibers were prepared according to Nobe et al. (2005). Rat tail collagen (type I) solution was neutralized with 0.1 N NaOH in an ice bath. Dispersed cells were suspended in a solution containing 1 x 107 cells/ml and 0.5 mg/ml collagen in DMEM. A cell suspension (2 ml) was poured into a specially designed mold (0.8- x 5- x 0.5-cm deep), which was cut into a layer of silicone rubber in a 60-mm dish and placed in a CO2 incubator at 37°C. EC fiber preparations were incubated for 3 days. In a manner identical to that of the monolayer EC culture, DMEM was exchanged daily.
Measurement of Isometric Force Development in EC Fibers. EC fibers were cut into 5-mm sections and mounted between stainless wire posts with cyanoacrylate glue. One post was fixed, whereas the opposite post was connected to a silicone strain gauge force transducer (model AME801; SensoNor, Horton, Norway). EC fibers were mounted isometrically under resting tension of 20 ± 1.0 µN at 37°C; fibers were bathed in PSS.
Measurement of EC Layer Permeability. EC layer permeability was measured as described previously (Imai-Sasaki et al., 1995; Nobe et al., 2005). Results of BSA permeability of the EC layer were calculated as: total amount of BSA (micrograms)/area of EC layer (centimeters squared).
Measurement of Intracellular Free Ca2+ Concentration ([Ca2+]i). ECs were grown on cover glasses (diameter, 25 mm) placed inside 35-mm dishes. [Ca2+]i was measured as described previously (Nobe et al., 2005).
VE-Cadherin Immunostaining. VE-cadherin immunostaining was conducted as described previously(Sandoval et al., 2001).
Labeling of EC Structures and Digital Imaging. To determine the mechanism(s) via which cytoskeletal molecules are affected by stimulators, ECs were grown to the 3rd day of post-subconfluence on a cover glass inside 35-mm dishes. Following rinsing of the culture medium with PSS, ECs were treated under various conditions at 37°C. The reaction was terminated, and fluorescent staining was performed as described previously (Nobe et al., 2005). A digital imaging microscope was used to view the stained samples [MRC600 confocal microscope (Carl Zeiss, Oberkochen, Germany) equipped with a 60x, 1.4 numerical aperture oil immersion objective]. Fluorescent images of the samples excited at 488 nm (Alexa Fluor 488-phalloidin) and 568 nm (SYTO-17) and emitting at 504 to approximately 540 and 585 to approximately 700 nm, respectively, were collected. Digital images were qualified and/or merged with Confocal Assistant (Carl Zeiss) and Photoshop (Adobe Systems, San Jose, CA) software.
Statistics and Data Analysis. Data presented in the text and the illustrations are expressed as mean ± S.E.M. The permeability value at each condition was assessed in terms of statistical significance for comparison with appropriate control data employing the analysis of variance techniques. Paired data were used when appropriate. p < 0.01 was considered significant. Analysis of variance statistical comparisons were performed with the Y-Stat program. For western blot and fluorescent imaging, similar patterns of changes were detected in nearly all samples (>six trials).
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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To elucidate the mechanisms, effects of a PKC inhibitor (rottlerin) and a DG kinase inhibitor (stemphone) on U46619 [GenBank] -induced VE-CaD depression were also examined. In normal PSS, U46619 [GenBank] -induced depression of VE-CaD signals was apparently reduced by pretreatment with rottlerin and stemphone. In excess of 50% of the original signals remained in the presence of these inhibitors. Similar effects were detected under HG conditions. Advanced VE-CaD depression reached the normal level in the presence of these inhibitors.
Isometric force development of EC accompanied by morphological changes, i.e., paracellular hole formation, was determined using three dimensionally reconstituted EC fibers (Fig. 3). EC fibers were preincubated in normal and HG-DMEM for 48 h in a manner consistent with that of the aforementioned experiments; subsequently, 5-mm sections were mounted on a specially designed isometric force measurement system. The length was increased to match the original fiber length in the mold. Following stress relaxation, the force attained a baseline level of 20.30 ± 0.27 µN (n = 5). Treatment with 10 µM U46619 [GenBank] induced sustained force development in normal glucose-PSS (Fig. 3). This response was time-dependent. Pretreatment of EC fibers with HG-DMEM slightly increased the nonstimulated resting level (29.30 ± 1.86 µN; n = 5). Although U46619 [GenBank] treatment led to an increase in sustained force development, the increase was rapid, and the response was large. Maximal force responses, including baseline, to U46619 [GenBank] under normal and HG conditions after the 60-min period were 45.00 ± 1.80 and 63.80 ± 3.14 µN, respectively (n = 5). Significant enhancement of U46619 [GenBank] -induced force development under HG conditions was observed from 3 min of stimulation. To confirm alteration of EC fiber contractility in HG-PSS, increased force value in the initial 5 min of U46619 [GenBank] stimulation was calculated as the "initial rate" (Fig. 3B, inset). This initial rate was significantly elevated from 2.49 ± 0.15 to 4.81 ± 0.32 µN/min (n = 5).
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These HG-induced alterations of EC responses were dependent on the extracellular glucose concentration and preincubation period. In preliminary trials, each condition of extracellular glucose concentration (11.1-33.3 mM) or preincubation period (1-96 h) was challenged in terms of permeability and contraction assays (data not shown). Based on consideration of stability and reproducibility of effects, HG conditions (22.2 mM glucose for 48 h) were adopted as a chronic HG model in this study.
Intracellular Signaling Mechanisms of HG-Induced Enhancement of EC Responses. To establish the intracellular mechanisms governing U46619 [GenBank] -induced EC responses, several key factors of cellular functions were investigated. A general intracellular factor, [Ca2+]i, was measured with the calcium indicator, Fura-2, loaded into the EC (Fig. 4A). Pretreatment of EC with HG-DMEM did not affect resting [Ca2+]i. Averages of [Ca2+]i in randomly selected nonstimulated 30 cells under normal and HG conditions were 41.17 ± 1.11 and 43.33 ± 2.20 nM, respectively. In normal glucose-PSS, treatment of EC with 10 µM U46619 [GenBank] led to a rapid increase in [Ca2+]i. The maximal peak level, which was 153.5 ± 8.17 nM, was attained only 42 s after the stimulation. This response returned to the resting level during the 4th min of stimulation. A similar transient increase in [Ca2+]i was observed in EC pretreated with HG-DMEM-pretreated EC. Patterns of changes and the maximal level (163.00 ± 3.59 nM) also overlapped with the response in normal PSS. ECs were pretreated with the intracellular calcium inhibitor, BAPTA/AM, for 30 min prior to U46619 [GenBank] treatment (Fig. 4A, inset). The transient increases in [Ca2+]i under normal and HG conditions were suppressed (34.83 ± 1.70 and 39.67 ± 2.54 nM, respectively) without affecting resting levels. Differences between normal and HG-PSS could not be detected in intracellular calcium measurements.
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The roles of rho and the rho kinase pathway in U46619 [GenBank] -induced EC fiber contraction were examined to discern alteration of sustained function following completion of intracellular calcium events under HG conditions (Fig. 5). The rho kinase inhibitor, Y27632 (1 µM), markedly reduced U46619 [GenBank] -induced force development under normal and HG conditions (Fig. 5, A and B). The maximal force developments in the presence of Y27632 were 22.50 ± 0.83 and 23.80 ± 1.13 µN (n = 5), respectively. Differences between normal and HG conditions were suppressed. Although the initial rate of the response was also diminished, significant differences between normal and HG-PSS remained (Fig. 5C). As a downstream of the Rho-Rho kinase pathway, actin stress fiber (ASF) formation was measured (Fig. 5D). ASF reconstitution was induced by treatment with 10 µM U46619 [GenBank] in a time-dependent fashion. The pattern of the time course was similar to the response in EC fiber force development. This response was suppressed upon pretreatment with 1 µM Y27632. Under HG conditions, enhanced U46619 [GenBank] -induced ASF formation was apparent; however, it was inhibited in the presence of Y27632. Paracellular hole formation was also reduced following Y27632 treatment in normal and HG-PSS.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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; Kobayashi et al., 2005). This importance is based mainly on the low concentration (0.1-30 nM) of the TXA2 analog, U46619
[GenBank]
, necessary to induce significant contraction in diabetic models (Pfister et al., 2004). We also documented 10 nM U46619
[GenBank]
-induced submaximal contractions in mouse aorta (Nobe et al., 2003), portal vein (Nobe et al., 2004a), and porcine coronary artery (Nobe and Paul, 2001). However, this range of U46619
[GenBank]
concentrations did not markedly influence EC layer permeability (Teixeira et al., 1995; Nobe et al., 2005). Although these results indicated that the physiological concentration of TXA2 might not affect EC function, the effect under abnormal conditions is unknown. In actuality, these findings revealed that some types of prostanoids, including TXA2, are produced locally in excess in instances of inflammatory diseases (Tonshoff et al., 1992). Diabetic hyperglycemia and inflammatory conditions were presumed in this investigation; therefore, higher concentrations (10 nM-30 µM) of U46619
[GenBank]
were introduced. TXA2 receptor-mediated time- and dose-dependent increases in EC layer permeability were detected within this range (Fig. 1). Moreover, previous reports demonstrated that similar levels of TXA2 induced enhancement in EC layer permeability in other types of EC (Chang and Ohara, 1993), which was consistent with the response obtained in this study. During U46619
[GenBank]
treatment, cell-to-cell connector, e.g., VE-CaD, signals were reduced (Fig. 2), and significant increases in EC fiber contraction (Fig. 3) were observed. These responses were similar to those of thrombin stimulation (Nobe et al., 2005), which suggested that thrombin and U46619
[GenBank]
induced both disruption of VE-CaD signals and EC contraction, which were mediated by common mechanisms in EC layer permeability. Contraction of EC in U46619
[GenBank]
-induced enhancement of EC layer permeability was identified in this investigation; however, it appears that this phenomenon preferentially accompanies hyperglycemia rather than solely elevating TXA2 in diabetes.
Based on this perspective, alteration of U46619
[GenBank]
-induced EC responses under HG conditions was considered to evaluate EC dysfunction in diabetic complications. Under HG conditions, ECs were preincubated with medium containing 22.2 mM glucose (DMEM and MOPS-PSS) for 48 h in accordance with our previous experiments involving vascular smooth muscle tissue (Nobe et al., 2004a,b). We previously confirmed that HG medium treatment induced neither hyperosmolar nor nonselective actions (data not shown). Moreover, HG-induced responses in this investigation were submaximum; higher concentration (>22.2 mM) and long-term treatment (>48 h) were not dependent on increases in EC response (data not shown). Reports appearing in the literature indicate that viability and cellular functions of rat aortic ECs were maintained under similar HG conditions (Lee et al., 2004). These results suggested that similar HG treatment of EC led to enhancement of nonstimulated basal EC layer permeability; however, meaningful differences were not detected in the current study. Although several possibilities exist regarding these differences (EC species, culture conditions, etc.), a critical cause remains unknown.
Under HG conditions, U466619-induced EC layer permeability was enhanced (Fig. 1). EC layer permeability was not derived from enhancement of TXA2 receptor sensitivity (Fig. 1B, inset). Similar enhancement was not evident under high-sucrose conditions (11.1 mM sucrose added to normal medium for 48 h); alteration of EC layer permeability was caused by an increase in extracellular glucose concentration. In our preliminary measurements, enhanced EC layer permeability in HG medium was recovered upon restoration of EC to normal glucose medium, although it was slow and incomplete (data not shown). This recovery rate appears to be dependent on glucose concentration and pretreatment period. This finding may suggest that sustained elevation of blood glucose level leads to a long-term EC disorder.
To elucidate the mechanism(s) governing EC layer alteration under HG conditions, both VE-CaD signal (Fig. 2) and EC contraction (Fig. 3) were examined; moreover, alterations of both parameters were observed. These findings indicated that these alterations are directly attributable to enhancement of EC layer permeability. However, questions regarding intracellular mechanisms associated with extracellular glucose level abound. A major contractile factor, [Ca2+]i, was measured during U46619 [GenBank] stimulation in normal and HG mediums. A transient increase in intracellular calcium response was detected; however, the response was not dependent on extracellular glucose concentration (Fig. 4A). Inhibition of the transient increase in [Ca2+]i by BAPTA/AM did not suppress the EC layer response. It is noteworthy that significant differences between the two glucose conditions remained in the presence of BAPTA/AM (Fig. 4B). We hypothesized that extracellular glucose influenced a point downstream of the calcium signaling pathway or the calcium-independent signaling pathway. Downstream of the calcium signaling pathway, the association of PKC and DG kinase activities with EC function was assessed consequent to overactivation of PKC and DG kinase, which leads to dysfunction of vascular smooth muscle cells mediated by overacceleration of PI turnover. Each treatment with PKC and DG kinase inhibitor suppressed reduction of U46619 [GenBank] -induced EC layer permeability as well as differences between normal and HG mediums (Fig. 4B). As a result of these treatments, differences of VE-CaD signals under normal and HG conditions were also diminished (Fig. 2). These findings suggested that elevated extracellular glucose accelerated PI turnover mediated by PKC-DG kinase activities; additionally, it induced enhancement of the disruption rate of VE-CaD signals (Fig. 6, A and B).
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Interestingly, inhibitors of PKC (rottlerin) and DG kinase (stemphone) possess selectivity for calcium-independent isoforms (De Witt et al., 2001; Nobe et al., 2004a). We hypothesized that the elevated response of EC under HG conditions is mediated by the calcium-independent PKC-DG kinase pathway. We previously reported that incorporated glucose under HG conditions is converted to a non-natural species of DG via a de novo synthesis pathway in rat and mouse vascular smooth muscle cells (Nobe et al., 2004a). As a preliminary measurement, we also measured a [14C]DG formation from extracellular [14C]glucose as similar as our previous report (Nobe et al., 2004b). Treatment of the EC with the [14C]glucose-contained HG-PSS significantly enhanced the [14C]DG formation (262% of normal glucose). This enhancement was maintained in the U46619
[GenBank]
stimulation (296% of normal glucose). These results indicated a possibility that the DG formation under HG condition was increased without mediating phospholipase C. Depending on the DG formation from extracellular glucose, total PKC activity was also increased (data not shown). Other investigators documented similar findings (Marignani et al., 1996; Deacon et al., 2002). In EC, data suggested that the elevation in extracellular glucose induces accumulation of the non-natural DG species as well as PKC activation; furthermore, this phenomenon might contribute to the rapid disruption of VE-CaD signals. Several groups reported that -catenin, a regulatory factor of VE-CaD, is phosphorylated by PKC (Berk et al., 1995; Sandoval et al., 2001), which leads to an uncoupling of VE-CaD connections (Fig. 6, A and B); in contrast, other researchers noted that activation of PKC is not involved in VE-CaD regulation (Vouret-Craviari et al., 1998). Regulatory mechanisms of this point remain incomplete; however, the current results are consistent with those of the former investigations.
In our preliminary trials employing the nonselective PKC inhibitor, calphostin C (1 µM pretreatment for 10 min), and the calcium-dependent PKC inhibitor, Gö6976 (1 µM pretreatment for 10 min), U46619 [GenBank] -induced VE-CaD disruption was detected exclusively following calphostin C application (data not shown). These results supported the possibility that increased disruption of VE-CaD under HG conditions is mediated by a calcium-independent PKC-DG kinase pathway. Transient elevation of [Ca2+]i induced by U46619 [GenBank] contributed to activation of PI turnover; however, the [Ca2+]i increase may not participate in extracellular glucose-dependent overactivation of the PKC-DG kinase pathway. The initial rate of U46619 [GenBank] -induced EC contraction served as a marker of initial EC response. Moreover, this rate was enhanced under HG conditions (Fig. 3B, inset); additionally, this enhancement remained despite the presence of a rho kinase inhibitor (Fig. 5C). These findings were also suggestive of the importance of regulation of VE-CaD disruption by the calcium-independent PKC-DG kinase pathway.
We previously noted that disruption of VE-CaD connections was insufficient for the thrombin-induced increase in EC layer permeability (Nobe et al., 2005). It was suggested that EC contraction also contributed to the increase in EC layer permeability consequent to paracellular holes. Rho and the rho kinase-mediated calcium-independent signaling pathway were involved; furthermore, rho and the rho kinase pathway may contribute to U46619
[GenBank]
-induced EC contraction. In U46619
[GenBank]
-induced EC contractions, initial rate and maximal force developments were significantly enhanced under HG conditions (Fig. 3). These responses were followed by ASF formation (Fig. 5D). Pretreatment with a rho kinase inhibitor markedly reduced maximal contraction and ASF formation without suppressing differences in initial rates between normal and HG conditions (Fig. 5). These findings revealed that rho/rho kinase-mediated ASF rearrangements are correlated with U46619
[GenBank]
-induced EC contraction (Fig. 6C). Enhancement of this contraction under HG conditions is derived from overactivation of this pathway. Inhibition of paracellular hole formation by Y27632 also provided evidence regarding the importance of this pathway (Fig. 5D). In addition, inhibition of EC layer permeability by Y27632 was also confirmed under same condition of Fig. 5D (data not shown). Alteration of EC layer permeability under HG conditions might entail an increase in VE-CaD signal disruption in the initial period as well as a secondary step consisting of overcontraction of EC.
The current investigation demonstrated that dysfunction of the EC layer under HG conditions, as in diabetes, is associated with both the rapid, extracellular glucose-dependent disruption of VE-CaD connections during the initial period as well as with the ensuing rho kinase-ASF-mediated EC overcontraction. In the initial phase, accumulation of the glucose-derived non-natural DG species stimulated calcium-independent PKC and DG kinase isoforms, which might lead to an increase in VE-CaD disruption. In the contraction phase, the rho-rho kinase pathway was also activated by elevation of extracellular glucose level, which results in paracellular hole formation via overcontraction of EC. This sequential process contributes to enhancement of EC layer permeability under HG conditions.
Despite the findings of the present study, important and difficult questions remain in terms of detailed regulatory mechanism(s) governing VE-CaD disruption by activated PKC in HG medium and direct or indirect correlation of extracellular glucose level with the rho-rho kinase pathway. Although these points require additional examination, this investigation confirmed that elevation of extracellular glucose level in diabetes influences vascular smooth muscle function as well as EC layer function. These dysfunctions in major components of vascular tissue might lead to diabetic complications mediated by excessive vascular permeability. To treat EC dysfunction, normalization of this process appears to be essential. Artificial regulation of these steps might be a suitable therapeutic target for diabetic vascular dysfunction.
In conclusion, exposure of the EC layer to TXA2 under diabetic-like HG conditions induces both suppression of cell-to-cell connections via VE-CaD and enhancement of paracellular hole formation due to EC overcontraction. These extracellular glucose-dependent responses may induce diabetic complications via enhanced EC layer permeability.
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Acknowledgements |
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: EC, endothelial cell; PKC, protein kinase C; VE-CaD, vascular endothelial cadherin; HG, high glucose; TXA2, thromboxane A2; U46619
[GenBank]
, 9,11-dideoxy-11,9-epoxymethano prostaglandin F2; DG, diacylglycerol; PI, phosphatidylinositol; DMEM, Dulbecco's modified Eagle's medium; AM, acetoxymethyl ester; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; MOPS, 4-morpholinepropanesulfonic acid; PSS, physiological salt solution; BSA, bovine serum albumin; FITC, fluorescein isothiocyanate; ASF, actin stress fiber; Gö-6976, 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole; U73122
[GenBank]
, 1-[6-[[17-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione; SQ29548, [1S-[1,2(Z),3,4]]-7-[3[[2-[(phenylamino)carbonyl[hydrazino]methyl]-7-oxabicyclo]2.2.1]hept-2-yl]-5-heptenoic acid; R59022
[GenBank]
, 6-{2-{4-[(4-fluorophenyl)phenylmethylene]-1-piperidinyl}ethyl}-7-methyl-5H-thiazolo(3,2-)pyrimidine-5-one; Y27632, ((+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexane carboxamide dihydrochloride, monohydrate.
Address correspondence to: Dr. Koji Nobe, Department of Pharmacology, School of Pharmaceutical Sciences, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-0555, Japan. E-mail: kojinobe{at}pharm.showa-u.ac.jp
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, p < 0.01 versus U46619 response.
