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Cardiovascular Research 2005 67(4):723-735; doi:10.1016/j.cardiores.2005.04.008
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Copyright © 2005, European Society of Cardiology

COX-2 Up-regulation and vascular smooth muscle contractile hyperreactivity in spontaneous diabetic db/db mice

Zhenheng Guoa, Wen Sua, Shannon Allena, Huan Panga, Alan Daughertya,b, Eric Smarta,c and Ming C. Gonga,*

aDepartment of Physiology, University of Kentucky, 509 Wethington Building, 900 South Limestone, Lexington, KY 40536, USA
bInternal Medicine, University of Kentucky, Lexington, KY 40536, USA
cPediatrics, University of Kentucky, Lexington, KY 40536, USA

* Corresponding author. Tel.: +1 859 323 4933x81361; fax: +1 859 257 3646. Email address: mcgong2{at}uky.edu

Received 9 February 2005; revised 21 March 2005; accepted 5 April 2005


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Objective: Abnormalities in vascular constriction and dilatation are associated with early diabetes and contribute to diabetic vascular complications. However, mechanisms underlying such vascular dysfunction remain to be fully elucidated. The current study tests the role of cyclooxygenase-2 (COX-2) in diabetic vascular smooth muscle dysfunction.

Methods: Small mesenteric artery and aorta are isolated from type 2 diabetic db/db and control mice. Isometric contractions in response to serotonin, angiotensin II, phenylephrine and high potassium are determined in small spiral mesenteric arterial or aortic strips. COX-2 mRNA and protein levels are analyzed by using DNA microarray, real-time PCR and immunoblot.

Results: Contractions induced by serotonin, angiotensin II, phenylephrine and high potassium are significantly higher in endothelium-denuded smooth muscle strips isolated from db/db mice than in those isolated from control mice. The contractile hyperreactivity is observed in aortic and third-order branch small mesenteric arterial smooth muscle strips. DNA microarray, real-time PCR and immunoblot analysis show that compared with control mice, COX-2 mRNA and protein are significantly increased in db/db mice aortic smooth muscle. The COX-2 up-regulation is temporally associated with the development of diabetes mellitus and vascular smooth muscle contractile hyperreactivity. Inhibition of COX-2 with NS-398 or SC-58125 partially–but significantly–alleviates agonist-induced but not potassium-induced contractile hyperreactivity. In addition, serum isolated from db/db mice induces COX-2 expression and increases thromboxane A2 production in primary cultured vascular smooth muscle cells (VSMC). SQ-29548, a TP receptor antagonist, diminished the db/db mice vascular smooth muscle contractile hyperreactivity.

Conclusions: COX-2 is up-regulated and contributes at least in part to the vascular smooth muscle contractile hyperreactivity in db/db mice.

KEYWORDS Vascular smooth muscle; Contraction; Type 2 Diabetes; Cyclooxygenase-2


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Sixteen million Americans have type 2 diabetes, and vascular complications are the most common causes of morbidity and mortality in diabetic patients. Among these diabetic vascular abnormalities are alterations in blood vessel constriction and dilatation (vascular reactivity) that have been detected in type 2 diabetic patients [1–6] and type 2 diabetic animal models [7–16]. Such abnormalities in vascular reactivity can alter blood flow and peripheral vascular resistance and thereby contribute to diabetic retinopathy, nephropathy, neuropathy and increased rates of hypertension. Therefore, the vascular reactivity under type 2 diabetic conditions has been the subject of many studies. Endothelium dysfunction has been well-documented and is recognized as an important factor in causing decreased vasodilation and increased vasoconstriction responses [17–21]. In contrast, the dysfunction of vascular smooth muscle and its contribution to the abnormal vascular reactivity under type 2 diabetic conditions is less clear and remains to be fully explored.

Cyclooxygenase (COX) is a rate-limiting enzyme that catalyzes the conversion of free arachidonic acid and O2 into prostaglandin H2, the committed step in the biosynthesis of prostaglandins and thromboxane. Prostaglandins and thromboxane are potent vasoconstrictors and vasodilators that play an important role in regulating vascular function. Of the prostaglandins produced in the vascular wall, prostaglandin I2 (PGI2) relaxes while thromboxane A2 (TxA2), prostaglandin F2{alpha} (PGF2{alpha}) and prostaglandin H2 (PGH2) contract vascular smooth muscle. In addition, COX is the target of commonly used non-steroidal anti-inflammatory drugs such as aspirin and ibuprofen. There are two major isoforms of COX: COX-1 and COX-2 [22]. COX-1 is constitutively expressed in many tissue and cell types. Conversely, COX-2 is not expressed in most tissues under physiological conditions, but its expression can be rapidly and markedly induced [23]. Under physiological conditions, vascular smooth muscle expresses COX-1 whereas COX-2 expression is undetectable. Under type 2 diabetic conditions, it is unknown if COX-2 expression is induced in vascular smooth muscle; if it is induced, the role COX-2 plays in vascular smooth muscle dysfunction remains unknown.

Db/db mice are a well-characterized and extensively used type 2 diabetic mouse model [24]. The syndrome in db/db mice is similar to that found in type 2 diabetic patients. Db/db mice manifest insulin resistance and compensatory hyperinsulinemia starting at 2 weeks of age. They become obese at around 4 weeks and develop hyperglycemia at around 7 weeks [25,26]. The obesity and complex diabetic mellitus in db/db mice is caused by an inability to respond to leptin due to a point mutation in the leptin receptor gene [27–29]. Leptin is an adipocyte-derived hormone that binds to the leptin receptor, resulting in weight loss by reducing appetite and food intake and increasing energy expenditure.

In the present study, isometric contraction measurements were combined with microarray, real-time PCR and immunoblot analysis to investigate vascular smooth muscle dysfunction under type 2 diabetic conditions. Specifically, we tested the hypothesis that COX-2 was induced in vascular smooth muscle under type 2 diabetic conditions and that the up-regulated COX-2 contributed to diabetic vascular smooth muscle dysfunction. To selectively investigate the role of vascular smooth muscle, endothelium-denuded vascular smooth muscle preparations were isolated from db/db and control mice. Our results show that COX-2 plays an important role in vascular smooth muscle hyperreactivity in db/db mice.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 Acknowledgments
 References
 
2.1. Animals and materials
Female C57BL/KsJ db/db mice (db/db –/–) and age/gender-matched non-diabetic (db/db +/?) C57BL/KsJ control mice were purchased from Jackson Lab (Bar Harbor, ME). The mice were housed at an animal care facility at the Medical Center of the University of Kentucky that is accredited by the American Association for Accreditation of Laboratory Animal Care. Male Sprague–Dawley rats were purchased from Harlan (Indianapolis, IN). All animal protocols were approved by the Institutional Animal Care and Use Committee. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication NO. 85-23, revised 1996). The mice were fed a standard diet and RO water ad libitum until they were used at 4, 8 or 12 weeks of age. Other chemicals and reagents were purchased from Sigma (St. Louis, MO) or Fisher (Pittsburgh, PA).

2.2. Measurement of blood glucose and plasma insulin
Blood glucose was determined by using a One Touch Ultra Blood Glucose Meter with a glucose test strip (LifeScan, Inc., Milpitas, CA). The mice were then anesthetized and blood was collected. Serum was separated by centrifugation and stored at –20 °C until use. Serum insulin was measured using an ELISA kit (Crystal Chem Inc., Chicago, IL) according to the manufacturer's instructions.

2.3. Isometric tension measurement
After removing connective tissue, mouse thoracic aortas and the third order branch of mesenteric arteries were cut into small spiral strips (about 3 mm in length and 1.6 mm in width for aortic strips, or 400 µm in width for mesenteric arterial strips). Endothelium was denuded and verified by the losses of acetylcholine-induced relaxation. Isometric contractions were measured as described previously [30].

2.4. Sectioning of vascular smooth muscle tissue
The muscle strips were immediately stored in 10% formalin after the completion of the isometric contraction measurements, embedded in O.C.T. Compound (Tissue Tek, Sakura Finetek, U.S.A., Inc. Torrance, CA) and cut into 8 µm thick cross-sections. The sections were stained with hematoxylin (Biomeda Corp., Foster City, CA). Images of the cross-sections were captured on a Spot Camera (Diagnostic Instruments) and medium smooth muscle areas were measured using Image-Pro Software (Media Cybernetics, Silver Springs, MD).

2.5. Immunoblot analysis
COX-2 and smooth muscle {alpha}-actin were detected by immunoblot as described previously (anti-COX-2: BD Biosciences, San Diego, CA; anti-{alpha}-actin: Sigma, St. Louis, MO) [31].

2.6. DNA microarray
Thoracic and abdominal aortas were harvested from 12- to 13-week-old db/db and control mice and immediately placed in RNAlater solution. After carefully removing the adventitia and endothelium, RNA was extracted using an RNeasy mini-kit (Qiagen, Valencia, CA). RNA samples from three mice were pooled to obtain a sufficient quantity for one DNA array. The reverse transcription, biotin-labeling, fragmentation and hybridization to Affymetrix MGU-74A chip were carried out by the University of Kentucky Microarray Core Facility according to the Expression Analysis Technical Manual (Affymetrix, Santa Clara, CA). A total of 9 db/db mice and 9 control mice were used. Three pairs of DNA arrays were run.

2.7. Real-time PCR
Total RNA was isolated from aortas as described above. Genomic DNA contamination was removed by DNase DNA-freeTM kit (Ambion, Austin, TX). The cDNA was synthesized by a SuperScriptTM kit (Invitrogen, Carlsbad, CA) using random hexamers. The real-time PCR primers used were: 5'-AAG GCA GAG GCA GTT GGA TCT-3' (forward), 5'-CAT GGC TGG CCT AGA ACT CAC T-3' (reverse) for mouse COX-1; 5'-CCA GCA CTT CAC CCA TCA GTT-3' (forward), 5'-ACC CAG GTC CTC GCT TAT GA-3' (reverse) for mouse COX-2; 5'-AGA AAC GGC TAC CAC ATC CAA-3' (forward), 5'-GGG TCG GGA GTG GGT AAT TT-3' (reverse) for mouse 18S rRNA. The real-time PCR reactions were performed using a QuantiTectTM SYBR Green PCR kit (Qiagen, Valencia, CA) in an ABI Prism 7700 Sequence Detection System. The specificity of the PCR was verified by dissociation curve analysis, agarose gel electrophoresis, and DNA sequencing. COX-1 and COX-2 mRNAs were normalized to the 18S rRNA and relatively quantified by standard curve analysis. Two controls, one with no template and one with no reverse transcription, were included in each real-time PCR run to ensure no contamination of genomic DNA.

2.8. Primary cultured rat vascular smooth muscle cells (VSMC)
The procedure for isolation and culture of primary VSMC from male Sprague–Dawley rats' thoracic aorta was adapted from a well-established method [32]. The smooth muscle identity was verified by expression of smooth muscle maker proteins: smooth muscle {alpha}-actin, myosin-heavy chains, calponin, and caldesmon (all antibodies were purchased from Sigma, St. Louis, MO) using immunocytochemistry. To test the effect of serum on COX-2 protein expression and on thromboxane A2 production, post-confluent primary cultured cells were starved for 96 h. The starvation medium was replaced for 24 h with medium containing 10% serum isolated from db/db or from control mice. Then the COX-2 protein was analyzed by immunoblot. The TxB2, stable metabolites of TxA2, were assayed using a commercially available ELISA kit (Neogene Corp., Lexington, KY) according to the manufacturer's instructions.

2.9. Statistical analysis
Each experiment was repeated a minimum of 3 times. Data were expressed as mean ± SEM. Statistical analysis was performed by using an unpaired t-test.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 Acknowledgments
 References
 
3.1. Body weight, blood glucose and serum insulin in db/db and control mice
Body weight, non-fasting blood glucose and serum insulin were measured in 4- to 5-, 8- to 9- and 12- to 13-week-old db/db and control mice. As shown in Table 1, at 4 weeks of age, db/db mice were overweight, exhibited severe hyperinsulinemia but did not have hyperglycemia. By 8 weeks of age, db/db mice exhibited obesity and hyperglycemia in addition to hyperinsulinemia. Similar differences remained at 12–13 weeks of age (Table 1). Consistent with literature reports [25,26], these data show that the db/db mice used in the present study manifest obesity, hyperinsulinemia and hyperglycemia.


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  		Table 1 Age-related changes in body weight, blood glucose, and serum insulin in db/db and control (+/?) mice

 
3.2. Contractile responses were significantly enhanced in vascular smooth muscle tissue isolated from db/db mice
To determine if the contractile responses are enhanced in db/db mice vascular smooth muscle tissue, we determined phenylephrine (PE)- and 5-HT-induced contractions in aorta and third-order branch mesenteric artery strips.

As shown in Fig.1A and B, maximal concentrations of 5-HT (1 µM) and Ang II (100 nM) induced markedly higher contractions in aortic strips isolated from 8- to 9-week-old db/db mice than in those isolated from control mice. To test if agonist-induced contractions were selectively enhanced, we determined potassium depolarization-induced contractions. As shown in Fig 1C, potassium (143 mM) also induced higher contractions in strips isolated from db/db mice than in those from control mice. Interestingly, 5-HT-induced contractions remained significantly higher in strips isolated from db/db mice than in those from control mice even when they were normalized to respective potassium depolarization-induced contractions (Fig. 1D).


Figure 1
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  		Fig. 1 Enhanced contractile responses in the absence of an increase in medium smooth muscle area in the endothelium-denuded aortic smooth muscle strips isolated from db/db mice. The thoracic aortas isolated from 8- to 9-week-old female db/db and control mice were cut into spiral strips, denuded of endothelium and stimulated with 1 µM 5-HT, 100 nM Ang II or 154 mM potassium. Contractions were expressed as absolute force (mg, A, B, and C) or normalized to potassium-induced contractions (%, D). After completion of the contractile study, the muscle strips were fixed and the medium layer thickness (E) and cross-sectional areas (F) were determined. N = 5–7. *p<0.05, **p<0.01, ***p<0.0001.

 
To test a possible role for increased smooth muscle mass in the enhanced contractile response, we examined the medium thickness and medium cross-sectional area in the same aortic strips in which the isometric contractions were measured. As shown in Fig. 1E and F, no significant difference in the medium thickness and cross-sectional area were observed between strips isolated from db/db and control mice.

To determine if the observed aortic smooth muscle contractile hyperreactivity was also present in small arteries, we isolated third-order branch mesenteric arteries from 12- to 13-week-old db/db and control mice. As shown in Fig. 2A, the phenylephrine concentration–response curve shifted to the left in strips isolated from db/db mice compared with those isolated from control mice. Potassium also induced higher contractions in strips isolated from db/db mice than in those from control mice (31 ± 2.2 mg vs 24 ± 2.0 mg, p<0.05). The phenylephrine-induced contractions remain significantly different when normalized to their respective high potassium-induced contractions (Fig. 2B). Similar to aorta, no difference was detected in the medium thickness and cross-sectional areas between mesenteric arterial strips isolated from db/db and control mice (Fig. 2C and D).


Figure 2
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  		Fig. 2 Increased phenylephrine (PE)-induced contractions in the absence of an increase in medium smooth muscle area in third-order branch mesenteric arterial strips isolated from db/db mice. The third-order branch mesenteric arterials isolated from 12- to 13-week-old female db/db and control mice were cut into spiral strips and denuded of endothelium. Cumulative PE concentration–response were expressed as absolute force (mg, A) or normalized to potassium-induced contractions (%, B). Then the muscle strips were fixed and the medium thickness (C) and cross-sectional area (D) were determined. N = 7 for db/db and 4 for control mice. *p<0.05, **p<0.01.

 
In our initial experiments, we have determined both phenylephrine- and 5-HT-induced contractions in aorta and in mesenteric artery strips. Similar contractile hyperreactivity in response to phenylephrine and to 5-HT were observed in aortic as well as in mesenteric artery strips. The phenylephrine-induced contractions in aortic and the 5-HT-induced contractions in mesenteric artery strips are small in amplitude. Therefore, we used 5-HT in aorta and phenylephrine in mesenteric artery in subsequent experiments.

3.3. COX-2 mRNA and protein were up-regulated in vascular smooth muscle in db/db mice
To elucidate the molecular mechanisms underlying the observed vascular smooth muscle contractile hyperreactivity, the gene expression patterns in aortic smooth muscle tissue isolated from 12- to 13-week-old db/db and control mice were determined and compared using DNA microarray analyses (Affymetrix). In more than 12,000 genes checked, 103 genes showed an over 2-fold up- or down-regulation that was statistically significant. The expression levels of known contraction-related genes are listed in Table 2. Among them, COX-2 and the potassium inwardly-rectifying channel are significantly increased, whereas the expression of sarcoendoplasmic reticulum Ca2+ ATPase (SERCA3b) is significantly decreased. Since the potassium inwardly-rectifying channel mediates smooth muscle relaxation [33,34], the increased expression of potassium inwardly-rectifying channel in db/db mice may be a compensatory response. COX-2, a key enzyme in the synthesis of prostanoids and thromboxane, was up-regulated 3-fold in db/db mice aorta. In contrast, COX-1 showed a tendency to decrease (0.7-fold of the control level, Table 2 and Fig. 3A). Interestingly, genes downstream of COX-2 such as thromboxane A2 synthase and thromboxane-prostanoid receptor (TP receptor) were not significantly different between db/db mice and control mice (Table 2).


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  		Table 2 Expression levels of contraction-related genes in db/db and control mouse thoracic aorta

 

Figure 3
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  		Fig. 3 COX-2 is up-regulated at the mRNA (A and B) and protein (C) level in db/db mice vascular smooth muscle. Total RNA was extracted from aortas isolated from 12- to 13-week-old db/db and control mice. DNA microarray hybridization signal intensities are presented in A and real-time PCR qualifications of the COX-2/COX-1 mRNA were normalized to 18S rRNA and presented in B. A representative immunoblot of COX-2 protein is presented in C. N = 5–7. *p<0.05.

 
To confirm that COX-2 mRNA was indeed selectively up-regulated, COX-2 and COX-1 mRNA were also quantified by using real-time PCR. Consistent with the microarray results, COX-2 mRNA was significantly increased whereas the COX-1 mRNA was not significantly different between the aortas from db/db and control mice (Fig. 3B). Furthermore, to determine if the increased COX-2 mRNA resulted in COX-2 protein increase, we immunoblotted the COX-2 protein in aortas isolated from db/db and control mice. In the aortas isolated from control mice, COX-2 protein was hardly detectable (Fig. 3C). Conversely, the COX-2 protein was markedly induced and readily detectable in the aortas from db/db mice.

3.4. The vascular smooth muscle contractile hyperreactivity in db/db mice was temporally associated with type 2 diabetic mellitus and COX-2 up-regulation
To test a possible causative relationship between diabetes mellitus and vascular smooth muscle hyperreactivity, we first investigated the time course of vascular smooth muscle hyperreactivity. As shown in Fig. 4A, at 4–5 weeks of age, when the db/db mice were pre-diabetic, 5-HT induced contractions only showed a small increase in aortas isolated from db/db mice. At 8–9 or 12–13 weeks of age, when the db/db mice exhibit severe obesity, hyperglycemia and hyperinsulinemia, dramatic differences in 5-HT-induced contractions were detected between strips isolated from db/db and control mice (Fig 4C and E). The EC50s were –7.7 ± 0.27 (n = 10) in 8- to 9-week-old db/db mice and –7.1 ± 0.25 (n = 10, p<0.0001) in control mice. The maximal responses were 96.0 ± 19.6 mg (n = 10) in 8- to 9-week-old db/db mice and 28.9 ± 10.74 mg (n = 10, p<0.0001) in control mice. Interestingly, 5-HT-induced maximal contractions increased with the age of db/db mice but the maximal contractions decreased as the age in control mice increased (Fig. 4A and C); this suggests that the aging of the mice did not account for the observed vascular smooth muscle hyperreactivity. In addition, we tested if leptin directly affects vascular smooth muscle contractility as the leptin receptor is expressed in smooth muscle. Acute leptin treatment (50 nM, 30 min) neither causes vascular smooth muscle contraction nor affects phenylephrine-induced contractions in mesenteric artery strips isolated from normal control mice (data not shown).


Figure 4
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  		Fig. 4 Age-dependent contractile hyperreactivity (A, C, E) and COX-2 mRNA up-regulation (B, D, F) in db/db mice aortic smooth muscle tissue. Cumulative 5-HT concentration-response curves (A, C, E) and real-time PCR quantification of COX-2 mRNA (B, D, F) were carried out in two separate sets of endothelium-denuded aortic strips isolated from different ages of db/db and control mice. A & B: 4–5 weeks; C & D: 8–9 weeks; E & F: 12–13 weeks. n = 5–7. *p<0.05, **p<0.01, ***p<0.0001.

 
To test a possible association between COX-2 up-regulation and vascular smooth muscle hyperreactivity, we determined the time course of COX-2 mRNA up-regulation and compared it with the time course of diabetes mellitus and vascular smooth muscle contractile hyperreactivity. As shown in Fig. 4 and Table 1, at 8–9 and 12–13 weeks of age, the COX-2 mRNA expression level in db/db mice aorta showed a marked increase (Fig. 4D and F) when diabetic mellitus were severe (Table 1) and the vascular smooth muscle contractile hyperreactivity was dramatic (Fig. 4C and E). In contrast, at 4–5 weeks of age, COX-2 mRNA levels were comparable in db/db and control mice aortas when db/db mice were pre-diabetic (Table 1) and the vascular smooth muscle contractile responses were only slightly increased in response to low concentration of 5-HT (Fig. 4A).

3.5. Inhibition of COX-2 activity alleviated vascular smooth muscle contractile hyperreactivity
To further test a possible causative relationship between the COX-2 up-regulation and vascular smooth muscle hyperreactivity, we investigated the effect of COX-2 inhibition on 5-HT-induced vascular smooth muscle contractile hyperreactivity. We first determined the effects of indomethacin, a potent inhibitor of COX-1 and COX-2, on the contractile hyperreactivity. As shown in Fig. 5C, indomethacin significantly inhibited the 5-HT-induced contractions. To further investigate the contribution of COX-2 to the contractile hyperreactivity, we used two structurally different selective COX-2 inhibitors, NS-398 and SC-58125. The IC50 of NS-398 and SC-58125 for ovine or murine COX-2 are over 200- to 1400-fold lower than the IC50 for COX-1 [35,36] and have been used to selectively inhibit COX-2 in a system similar to the current study [37–39]. Thirty min pre-incubation with 3 µM NS-398 (Fig. 5A) or 10 µM SC-58125 (Fig. 5B) significantly alleviated the contractile hyperreactivity to 5-HT in aortic strips isolated from db/db mice without significantly affecting the contractions in strips isolated from control mice. Interestingly, neither NS-398 nor SC-58125 significantly inhibited K+-induced contractions (NS-398: 12 ± 5.2 vs. 8 ± 3.5 in control and 41 ± 7.6 vs. 36 ± 8.5 in db/db mice; SC-58125: 14 ± 7.1 vs. 14 ± 9.8 in control and 41 ± 5.1 vs. 40 ± 5.1 in db/db mice).


Figure 5
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  		Fig. 5 Inhibition of COX-2 diminishes the vascular smooth muscle contractile hyperreactivity in response to 5-HT. Cumulative 5-HT concentration–response curves were determined in endothelium-denuded aortic strips isolated from 12- to 13-week-old db/db or control mice. Then the strips were incubated with 3 µM NS398 (A) or 10 µM SC58125 (B) or 10 µM indomethacin (C) for 30 min, and the cumulative 5-HT concentration–response curves were obtained again in the presence of the inhibitors. n = 5–7. *p<0.05, **p<0.01, ***p<0.0001.

 
To determine if COX-2 up-regulation is selectively involved in 5-HT-induced contractile hyperreactivity in db/db mice, we determined the effects of the COX-2 inhibitor NS-398 on Ang II- and PE-induced contractions. As shown in Fig. 6, NS-398 (3 µM, 30 min pre-incubation) significantly inhibited Ang II- and PE-induced hyperreactivity in db/db mice aorta and mesenteric artery strips, respectively.


Figure 6
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  		Fig. 6 Inhibition of COX-2 diminishes the vascular smooth muscle contractile hyperreactivity in response to angiotensin II and phenylephrine. (A) control and db/db mice aortic strips were treated with 3 µM NS-398 or vehicle for 30 min. Then 100 nM angiotensin II (Ang II)-induced contractions were obtained. (B) 10 µM phenylephrine-induced contractions were determined in endothelium-denuded third order branch mesenteric artery strips isolated from 12- to 13-week-old db/db or control mice. Then the strips were incubated with 3 µM NS-398 for 30 min, and the phenylephrine-induced contractions were obtained again in the presence of the NS-398. n = 4–6. *p<0.05, **p<0.01.

 
3.6. COX-2 up-regulation in primary cultured vascular smooth muscle cells (VSMC) by diabetic serum increased TxA2 production
In the whole animal, multiple factors may up-regulate COX-2. To test if diabetic serum can directly act on smooth muscle cells to up-regulate COX-2, we incubated primary cultured VSMC with serum isolated from 12 to 13-week-old db/db or control mice. Serum isolated from diabetic db/db mice induced much higher COX-2 protein expression than serum isolated from control mice in VSMC (Fig. 7A).


Figure 7
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  		Fig. 7 Serum isolated from db/db mice induces COX-2 protein expression (A) and increases thromboxane A2 (TxA2) production (B). Post-confluent primary cultured rat aortic smooth muscle cells were starved for 96 h and then incubated with medium containing 10% serum isolated from diabetic db/db or from control mice. (A) The COX-2 protein expression was analyzed by immunoblot. (B) The medium TxB2 was assayed using an ELISA kit according the manufacturer's instructions. (C) U-46619, a TP receptor agonist, potentiates 5-HT-induced contraction in aortic smooth muscle strips. The thoracic aortic strips isolated from control mice were stimulated with 1 nM U46619 or 10 nM 5-HT or both. n = 4–8. ***p<0.001.

 
To test if the COX-2 up-regulation is associated with TxA2 production increase, we measured TxB2 (a stable metabolite of TxA2) in cells after incubation with diabetic serum. Accompanied with the COX-2 up-regulation (Fig. 7A), diabetic serum dramatically increased TxB2 production (Fig. 7B).

To directly demonstrate that TxA2 can potentiate agonist-induced smooth muscle contractions, we determined the contractions induced by low concentrations of U-46619 alone (1 nM, a TP receptor agonist), 5-HT alone (10 nM) and U-46619 plus 5-HT. As shown in Fig. 7C, 1 nM U-46619 induced a small contraction but it significantly enhanced 5-HT-induced contractions.

3.7. TP receptor mediates part of the vascular smooth muscle contractile hyperreactivity in db/db mice
To test the possibility that increased production of the contractile prostaglandins TxA2/PGH2 by COX-2 up-regulation mediates the contractile hyperreactivity, we determined the effects of SQ-29548, a TP receptor antagonist, on 5-HT- and PE-induced contractions. As shown in Fig. 8, SQ-29548 (10 µM, 30 min pre-incubation) partially but significantly inhibited 5-HT- and phenylephrine-induced contractile hyperreactivity in db/db mice aortic and mesenteric artery strips.


Figure 8
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  		Fig. 8 Blocking TP receptor by SQ-29548 diminishes 5-HT- and phenylephrine-induced vascular smooth muscle contractile hyperreactivity in db/db mice. Aortic (A) or third order branch mesenteric artery strips (B) isolated from control or db/db mice were stimulated by 30 nM 5-HT or 10 µM PE. Then the strips were incubated with 10 µM SQ-29548 for 30 min and the 5-HT and PE responses were obtained again in the presence of SQ-29548. n = 4–7. **p<0.01, ***p<0.0001.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
Acknowledgments
 References
 
4.1. Vascular smooth muscle hyperreactivity in type 2 diabetes
Increased vascular contractile responses to various agonists or transmural pressure have been reported in thoracic aorta [10–12,14] and mesenteric arteries [13,40] isolated from db/db mice when compared with those isolated from control mice. In addition to endothelium dysfunction, vascular smooth muscle dysfunction has been implicated in vascular hyperreactivity. In endothelium-intact vascular preparations, the contractile responses after blockage of the endothelium-derived nitric oxide (NO) production remain significantly higher in tissue isolated from db/db mice than in that from control mice [14]. This suggests that the basal eNOS activity is not altered in db/db mice. Furthermore, the ACh-induced endothelium-dependent relaxation is only slightly attenuated in tissue isolated from db/db mice compared with that from control mice [12]. Such a small difference in endothelium-dependent relaxation could not account for the dramatic increases in vascular contractile responses. By using endothelium-denuded vascular smooth muscle preparations, the present study provides clear evidence that compared to control mice, vascular smooth muscle isolated from db/db mice is indeed hyperreactive in response to agonist and high-potassium stimulations. However, our results do not exclude the possibility that long-term endothelium dysfunction under diabetic conditions can alter gene expression patterns in vascular smooth muscle and consequently cause contractile hyperreactivity.

Multiple mechanisms including alterations in receptors, signaling mechanisms, channels, contractile proteins and smooth muscle mass may contribute to the observed enhancement of agonist- and K+-induced contractions in arterial smooth muscle tissue isolated from db/db mice. K+-depolarization initiates smooth muscle contraction mainly by increasing cytoplasmic Ca2+ whereas agonists induce smooth muscle contraction by increasing cytoplasmic Ca2+ and by increasing the sensitivity of the contractile apparatus to Ca2+ (Ca2+-sensitization). The enhancements of both K+-induced and agonist-induced contractions, even when the agonist-induced contractions were normalized to their respective K+-induced contractions, suggest dysfunction in mechanisms regulating intracellular Ca2+ as well as Ca2+-sensitization in db/db mice. For example, the significant decrease in the sarcoendoplasmic reticulum Ca2+ATPase mRNA (SERCA 3b) detected by microarray, if paralleled by a decrease in protein function, may prolong the cytoplasmic free Ca2+ increase and contribute to high K+- and agonist-induced contractile hyperreactivity. Further, different agonists may initiate contraction by activating divergent signaling pathways, and activation of two or more receptors may induce synergistic force output. For example, 5-HT and U-46619 caused synergistic contractions (Fig. 6C). And finally, autocrine and/or paracrine mechanisms also are altered and can contribute to the observed hyperreactivity (see below).

4.2. Involvement of COX-2 in the vascular hyperreactivity in db/db mice
COX activity may contribute to the cause underlying vascular hyperreactivity in db/db mice as indomethacin (an inhibitor of COX-1 and COX-2) [12,40] and SQ29548 (a TP receptor antagonist) [40] significantly alleviate the hyperreactivity in endothelium-intact preparations. However, the specific isoform of COX involved (COX-1 vs. COX-2) and the source of COX (endothelium vs. smooth muscle cells) remains unclear. Furthermore, the underlying mechanisms (transcriptional and/or post-translational) responsible for COX activity up-regulation in db/db mice remain unclear.

Our data provide several lines of evidence suggesting that transcriptional up-regulation of COX-2 in vascular smooth muscle contributes to the vascular smooth muscle hyperreactivity in db/db mice. First, the combination of DNA microarray, real-time PCR and immunoblot analysis clearly shows that COX-2 (but not COX-1) is up-regulated at the mRNA and protein level in vascular smooth muscle tissue isolated from db/db mice (Figs. 3 and 4GoGo). Second, the time course of COX-2 mRNA up-regulation mostly coincides with the appearances of vascular smooth muscle hyperreactivity (Fig. 4) and diabetic mellitus (Table 1) in db/db mice. Third, selective inhibition of COX-2 activity with NS398 and SC58125 significantly alleviates the agonist-induced vascular smooth muscle contractile hyperreactivity in strips isolated from db/db mice, whereas this type of selective inhibition does not significantly affect the contractions in strips isolated from control mice (Fig. 5). However, the inhibition is incomplete, suggesting mechanisms in addition to COX-2 up-regulation contribute to the contractile hyperreactivity. Taken together, these data suggest that the selective COX-2 up-regulation in aortic vascular smooth muscle tissue is partially responsible for the vascular smooth muscle contractile hyperreactivity in db/db mice.

In addition to COX-2 up-regulation, other mechanisms also are likely involved in the observed vascular smooth muscle contractile hyperreactivity. Two pieces of evidence support this notion. First, the 5-HT-induced contractile responses in tissue isolated from db/db mice remain significantly higher than in that isolated from ontrol mice after COX-2 inhibition (Fig. 5). Secondly, at 4–5 weeks of age, the small but significant increase in low concentrations of 5-HT-induced contractions (Fig. 4A) is not associated with the COX-2 mRNA increase (Fig. 4B).

4.3. COX-2 up-regulation in vascular smooth muscle cells (VSMC) of db/db mice
In VSMC, COX-2 can be rapidly and markedly up-regulated by various stimulations, including serum [41–43], interleukin-1β [44–46], EGF [41,43], Ang II [47–49] and thrombin [41]. In addition, high glucose [44,50] and Advanced Glycation End Products (RAGE) [51] have been shown to up-regulate or potentiate interleukin-1β-induced up-regulation of COX-2. At 8 weeks of age, when db/db mice demonstrate vascular smooth muscle contractile hyperreactivity, changes are observed in multiple serum components. Among these changes are increases in glucose, insulin, triglyceride, total cholesterol, LDL and vLDL; decreases in HDL, and altered cytokine and growth factor levels [52]. Indeed, several of the potential COX-2 up-regulators including high glucose, increased TNF{alpha}[53] and other cytokines [54], activation of the rennin–angiotensin system [55], increased EGF [56,57], and increased RAGE [58,59] are detected in diabetic patients and in diabetic animal models. That supports the in vivo significance of the COX-2 up-regulation in vascular dysfunction in diabetes. It is therefore conceivable that high glucose in combination with these multiple changes in diabetic serum up-regulates COX-2. Indeed, we find that serum isolated from db/db mice is much more potent than the serum isolated from control mice in inducing COX-2 protein expression in VSMC (Fig. 6A). However, further studies are required to clarify the exact contribution of each serum component to COX-2 up-regulation in db/db mice.

4.4. Mechanisms mediate up-regulated COX-2-induced vascular smooth muscle hyperreactivity
We (Fig. 6B) and others [45,47] show that COX-2 up-regulation is associated with significantly increased production of the potent vasoconstrictor TxA2 in VSMC. This suggests that the increased production of contractile prostanoids such as TxA2, PGF2{alpha} and PGH2 is one potential mechanism mediating COX-2-induced vascular smooth muscle hyperreactivity. By autocrine and paracrine mechanisms, the vasoconstrictive prostanoids can enhance the vascular smooth muscle contractions in response to stimuli (Fig. 6C). Indeed, blockade of the TP receptor by SQ-29548 diminished the vascular smooth muscle hyperreactivity (Fig. 8). Up-regulated COX-2 may also cause vascular smooth muscle hyperreactivity by increasing oxidative stress [60] or decreasing NO production by down-regulated iNOS [45,61].

Although COX-1 and COX-2 exhibit similar biochemical activity in converting arachidonate to PGH2 in vitro [62,63], the ultimate function and prostanoids they produce in vivo may be different due to differential regulation of COX-1 and COX-2, tissue distribution, cellular localization and availability of the down-stream prostanoid synthases [64,65]. Therefore, it is conceivable that functionally more contractile prostanoids are produced by the up-regulated COX-2 under diabetic conditions than what COX-1 produces under physiological conditions.

4.5. Possible pathological significance and perspective
Type 2 diabetes is one of the most serious long-term health problems in the United States, with enormous social and economic consequences. Cardiovascular disease is a major complication and the leading cause of premature death among people with diabetes. The present study shows that transcriptional up-regulation of COX-2 in vascular smooth muscle is partially responsible for vascular hyperreactivity in db/db mice. As abnormal vascular smooth muscle responses may contribute to the etiology of diabetic vascular complications, our study raises the possibility that the selective inhibition of vascular smooth muscle COX-2 activity may help prevent vascular complications in type 2 diabetes.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
References
 
We thank the Cardiovascular Research Group for invaluable advice and assistance. This work is supported by NIH HL67284 and by a Career Development Award (1-04-CD-04) from American Diabetes Association to M.G.


   Notes
 
Time for primary review 21 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 

  1. McVeigh G.E., Brennan G.M., Johnston G.D., McDermott B.J., McGrath L.T., Henry W.R., et al. Impaired endothelium-dependent and independent vasodilation in patients with type 2 (non-insulin-dependent) diabetes mellitus. Diabetologia (1992) 35:771–776.[Web of Science][Medline]
  2. Williams S.B., Cusco J.A., Roddy M.A., Johnstone M.T., Creager M.A. Impaired nitric oxide-mediated vasodilation in patients with non-insulin-dependent diabetes mellitus. J Am Coll Cardiol (1996) 27:567–574.[Abstract]
  3. Caballero A.E., Arora S., Saouaf R., Lim S.C., Smakowski P., Park J.Y., et al. Microvascular and macrovascular reactivity is reduced in subjects at risk for type 2 diabetes. Diabetes (1999) 48:1856–1862.[Abstract]
  4. Dandona P., James I.M., Newbury P.A., Woollard M.L., Beckett A.G. Cerebral blood flow in diabetes mellitus: evidence of abnormal cerebrovascular reactivity. Br Med J (1978) 2:325–326.[Abstract/Free Full Text]
  5. Menon R.K., Grace A.A., Burgoyne W., Fonseca V.A., James I.M., Dandona P. Muscle blood flow in diabetes mellitus. Evidence of abnormality after exercise. Diabetes Care (1992) 15:693–695.[Abstract]
  6. Dandona P., Aljada A., Chaudhuri A. Vascular reactivity and thiazolidinediones. Am J Med (2003) 115(Suppl_8A):81S–86S.[Web of Science]
  7. Harker C.T., O'Donnell M.P., Kasiske B.L., Keane W.F., Katz S.A. The renin–angiotensin system in the type II diabetic obese Zucker rat. J Am Soc Nephrol (1993) 4:1354–1361.[Abstract]
  8. Stepp D.W., Frisbee J.C. Augmented adrenergic vasoconstriction in hypertensive diabetic obese Zucker rats. Am J Physiol Heart Circ Physiol (2002) 282:H816–H820.[Abstract/Free Full Text]
  9. Zemel M.B., Reddy S., Sowers J.R. Insulin attenuation of vasoconstrictor responses to phenylephrine in Zucker lean and obese rats. Am J Hypertens (1991) 4:537–539.[Web of Science][Medline]
  10. Kamata K., Kojima S. Characteristics of contractile responses of aorta to norepinephrine in db/db mice. Res Commun Mol Pathol Pharmacol (1997) 96:319–328.[Web of Science][Medline]
  11. Kanie N., Kamata K. Contractile responses in spontaneously diabetic mice: II. Effect of cholestyramine on enhanced contractile response of aorta to norepinephrine in C57BL/KsJ (db/db) mice. Gen Pharmacol (2002) 35:319–323.[Web of Science]
  12. Kanie N., Kamata K. Contractile responses in spontaneously diabetic mice: I. Involvement of superoxide anion in enhanced contractile response of aorta to norepinephrine in C57BL/KsJ (db/db) mice. Gen Pharmacol (2002) 35:311–318.[Web of Science]
  13. Pannirselvam M., Verma S., Anderson T.J., Triggle C.R. Cellular basis of endothelial dysfunction in small mesenteric arteries from spontaneously diabetic (db/db –/–) mice: role of decreased tetrahydrobiopterin bioavailability. Br J Pharmacol (2002) 136:255–263.[CrossRef][Web of Science][Medline]
  14. Piercy V., Taylor S.G. A comparison of spasmogenic and relaxant responses in aortae from C57/BL/KsJ diabetic mice with those from their non-diabetic litter mates. Pharmacology (1998) 56:267–275.[CrossRef][Web of Science][Medline]
  15. Traupe T., Lang M., Goettsch W., Munter K., Morawietz H., Vetter W., et al. Obesity increases prostanoid-mediated vasoconstriction and vascular thromboxane receptor gene expression. J Hypertens (2002) 20:2239–2245.[CrossRef][Web of Science][Medline]
  16. Okon E.B., Szado T., Laher I., McManus B., van Breemen C. Augmented contractile response of vascular smooth muscle in a diabetic mouse model. J Vasc Res (2003) 40:520–530.[CrossRef][Web of Science][Medline]
  17. Feener E.P., King G.L. Endothelial dysfunction in diabetes mellitus: role in cardiovascular disease. Heart Fail Monit (2001) 1:74–82.[Medline]
  18. King G.L., Shiba T., Oliver J., Inoguchi T., Bursell S.E. Cellular and molecular abnormalities in the vascular endothelium of diabetes mellitus. Annu Rev Med (1994) 45:179–188.[CrossRef][Web of Science][Medline]
  19. Caballero A.E. Endothelial dysfunction in obesity and insulin resistance: a road to diabetes and heart disease. Obes Res (2003) 11:1278–1289.[Web of Science][Medline]
  20. Yki-Jarvinen H. Insulin resistance and endothelial dysfunction. Best Pract Res Clin Endocrinol Metab (2003) 17:411–430.[CrossRef][Medline]
  21. Hsueh W.A., Quinones M.J. Role of endothelial dysfunction in insulin resistance. Am J Cardiol (2003) 92:10J–17J.[Web of Science][Medline]
  22. Feng L., Sun W., Xia Y., Tang W.W., Chanmugam P., Soyoola E., et al. Cloning two isoforms of rat cyclooxygenase: differential regulation of their expression. Arch Biochem Biophys (1993) 307:361–368.[CrossRef][Web of Science][Medline]
  23. Davidge S.T. Prostaglandin H synthase and vascular function. Circ Res (2001) 89:650–660.[Abstract/Free Full Text]
  24. Bell R.H. Jr., Hye R.J. Animal models of diabetes mellitus: physiology and pathology. J Surg Res (1983) 35:433–460.[CrossRef][Web of Science][Medline]
  25. Leiter E.H., Coleman D.L., Hummel K.P. The influence of genetic background on the expression of mutations at the diabetes locus in the mouse: III. Effect of H-2 haplotype and sex. Diabetes (1981) 30:1029–1034.[Abstract]
  26. Leiter E.H., Coleman D.L., Eisenstein A.B., Strack I. Dietary control of pathogenesis in C57BL/KsJ db/db diabetes mice. Metabolism (1981) 30:554–562.[CrossRef][Web of Science][Medline]
  27. Chua S.C. Jr., Chung W.K., Wu-Peng X.S., Zhang Y., Liu S.M., Tartaglia L., et al. Phenotypes of mouse diabetes and rat fatty due to mutations in the OB (leptin) receptor. Science (1996) 271:994–996.[Abstract]
  28. Lee G.H., Proenca R., Montez J.M., Carroll K.M., Darvishzadeh J.G., Lee J.I., et al. Abnormal splicing of the leptin receptor in diabetic mice. Nature (1996) 379:632–635.[CrossRef][Medline]
  29. White D.W., Wang D.W., Chua S.C. Jr., Morgenstern J.P., Leibel R.L., Baumann H., et al. Constitutive and impaired signaling of leptin receptors containing the Gln->Pro extracellular domain fatty mutation. Proc Natl Acad Sci U S A (1997) 94:10657–10662.[Abstract/Free Full Text]
  30. Gong M.C., Cohen P., Kitazawa T., Ikebe M., Masuo M., Somlyo A.P., et al. Myosin light chain phosphatase activities and the effects of phosphatase inhibitors in tonic and phasic smooth muscle. J Biol Chem (1992) 267:14662–14668.[Abstract/Free Full Text]
  31. Guo Z., Su W., Ma Z., Smith G.M., Gong M.C. Ca2+-independent phospholipase A2 is required for agonist-induced Ca2+ sensitization of contraction in vascular smooth muscle. J Biol Chem (2003) 278:1856–1863.[Abstract/Free Full Text]
  32. Pauly R.R., Bilato C., Cheng L., Monticone R., Crow M.T. Vascular smooth muscle cell cultures. Methods Cell Biol (1997) 52:133–154.[Web of Science][Medline]
  33. Nelson M.T., Quayle J.M. Physiological roles and properties of potassium channels in arterial smooth muscle. Am J Physiol (1995) 268:C799–C822.[Web of Science][Medline]
  34. Zaritsky J.J., Eckman D.M., Wellman G.C., Nelson M.T., Schwarz T.L. Targeted disruption of Kir2.1 and Kir2.2 genes reveals the essential role of the inwardly rectifying K(+) current in K(+)-mediated vasodilation. Circ Res (2000) 87:160–166.[Abstract/Free Full Text]
  35. Johnson J.L., Wimsatt J., Buckel S.D., Dyer R.D., Maddipati K.R. Purification and characterization of prostaglandin H synthase-2 from sheep placental cotyledons. Arch Biochem Biophys (1995) 324:26–34.[CrossRef][Web of Science][Medline]
  36. Seibert K., Zhang Y., Leahy K., Hauser S., Masferrer J., Perkins W., et al. Pharmacological and biochemical demonstration of the role of cyclooxygenase 2 in inflammation and pain. Proc Natl Acad Sci U S A (1994) 91:12013–12017.[Abstract/Free Full Text]
  37. Errasti A.E., Rey-Ares V., Daray F.M., Rogines-Velo M.P., Sardi S.P., Paz C., et al. Human umbilical vein: involvement of cyclooxygenase-2 pathway in bradykinin B1 receptor-sensitized responses. Naunyn Schmiedebergs Arch Pharmacol (2001) 364:149–156.[CrossRef][Web of Science][Medline]
  38. Hernanz R., Alonso M.J., Briones A.M., Vila E., Simonsen U., Salaices M. Mechanisms involved in the early increase of serotonin contraction evoked by endotoxin in rat middle cerebral arteries. Br J Pharmacol (2003) 140:671–680.[CrossRef][Web of Science][Medline]
  39. Potenza M.A., Botrugno O.A., De Salvia M.A., Lerro G., Nacci C., Marasciulo F.L., et al. Endothelial COX-1 and -2 differentially affect reactivity of MVB in portal hypertensive rats. Am J Physiol Gastrointest Liver Physiol (2002) 283:G587–G594.[Abstract/Free Full Text]
  40. Lagaud G.J., Masih-Khan E., Kai S., van Breemen C., Dube G.P. Influence of type II diabetes on arterial tone and endothelial function in murine mesenteric resistance arteries. J Vasc Res (2001) 38:578–589.[CrossRef][Web of Science][Medline]
  41. Rimarachin J.A., Jacobson J.A., Szabo P., Maclouf J., Creminon C., Weksler B.B. Regulation of cyclooxygenase-2 expression in aortic smooth muscle cells. Arterioscler Thromb (1994) 14:1021–1031.[Abstract/Free Full Text]
  42. Rich G., Yoder E.J., Prokuski L., Moore S.A. Prostaglandin production in cultured cerebral microvascular smooth muscle is serum dependent. Am J Physiol (1996) 270:C1379–C1387.[Web of Science][Medline]
  43. Rich G., Yoder E.J., Moore S.A. Regulation of prostaglandin H synthase-2 expression in cerebromicrovascular smooth muscle by serum and epidermal growth factor. J Cell Physiol (1998) 176:495–505.[CrossRef][Web of Science][Medline]
  44. Lee S.H., Woo H.G., Baik E.J., Moon C.H. High glucose enhances IL-1beta-induced cyclooxygenase-2 expression in rat vascular smooth muscle cells. Life Sci (2000) 68:57–67.[CrossRef][Web of Science][Medline]
  45. Shiokoshi T., Ohsaki Y., Kawabe J., Fujino T., Kikuchi K. Downregulation of nitric oxide accumulation by cyclooxygenase-2 induction and thromboxane A2 production in interleukin-1beta-stimulated rat aortic smooth muscle cells. J Hypertens (2002) 20:455–461.[CrossRef][Web of Science][Medline]
  46. Bartlett S.R., Sawdy R., Mann G.E. Induction of cyclooxygenase-2 expression in human myometrial smooth muscle cells by interleukin-1beta: involvement of p38 mitogen-activated protein kinase. J Physiol (1999) 520(Pt 2):399–406.[Abstract/Free Full Text]
  47. Young W., Mahboubi K., Haider A., Li I., Ferreri N.R. Cyclooxygenase-2 is required for tumor necrosis factor-alpha- and angiotensin II-mediated proliferation of vascular smooth muscle cells. Circ Res (2000) 86:906–914.[Abstract/Free Full Text]
  48. Ohnaka K., Numaguchi K., Yamakawa T., Inagami T. Induction of cyclooxygenase-2 by angiotensin II in cultured rat vascular smooth muscle cells. Hypertension (2000) 35:68–75.[Abstract/Free Full Text]
  49. Hu Z.W., Kerb R., Shi X.Y., Wei-Lavery T., Hoffman B.B. Angiotensin II increases expression of cyclooxygenase-2: implications for the function of vascular smooth muscle cells. J Pharmacol Exp Ther (2002) 303:563–573.[Abstract/Free Full Text]
  50. Cosentino F., Eto M., De Paolis P., van der Loo B., Bachschmid M., Ullrich V., et al. High glucose causes upregulation of cyclooxygenase-2 and alters prostanoid profile in human endothelial cells: role of protein kinase C and reactive oxygen species. Circulation (2003) 107:1017–1023.[Abstract/Free Full Text]
  51. Shanmugam N., Kim Y.S., Lanting L., Natarajan R. Regulation of cyclooxygenase-2 expression in monocytes by ligation of the receptor for advanced glycation end products. J Biol Chem (2003) 278:34834–34844.[Abstract/Free Full Text]
  52. Cooper M.E., Bonnet F., Oldfield M., Jandeleit-Dahm K. Mechanisms of diabetic vasculopathy: an overview. Am J Hypertens (2001) 14:475–486.[CrossRef][Web of Science][Medline]
  53. Lu H., Raptis M., Black E., Stan M., Amar S., Graves D.T. Influence of diabetes on the exacerbation of an inflammatory response in cardiovascular tissue. Endocrinology (2004) 145:4934–4939.[CrossRef][Web of Science][Medline]
  54. Pradhan A.D., Ridker P.M. Do atherosclerosis and type 2 diabetes share a common inflammatory basis? Eur Heart J (2002) 23:831–834.[Free Full Text]
  55. Leiter L.A., Lewanczuk R.Z. Of the renin–angiotensin system and reactive oxygen species Type 2 diabetes and angiotensin II inhibition. Am J Hypertens (2005) 18:121–128.[CrossRef][Web of Science][Medline]
  56. Gilbert R.E., Cox A., McNally P.G., Wu L.L., Dziadek M., Cooper M.E., et al. Increased epidermal growth factor in experimental diabetes related kidney growth in rats. Diabetologia (1997) 40:778–785.[CrossRef][Web of Science][Medline]
  57. Gilbert R.E., Rumble J.R., Cao Z., Cox A.J., van Eeden P., Allen T.J., et al. Endothelin receptor antagonism ameliorates mast cell infiltration, vascular hypertrophy, and epidermal growth factor expression in experimental diabetes. Circ Res (2000) 86:158–165.[Abstract/Free Full Text]
  58. Flyvbjerg A., Denner L., Schrijvers B.F., Tilton R.G., Mogensen T.H., Paludan S.R., et al. Long-term renal effects of a neutralizing RAGE antibody in obese type 2 diabetic mice. Diabetes (2004) 53:166–172.[Abstract/Free Full Text]
  59. Ziyadeh F.N., Cohen M.P., Guo J., Jin Y. RAGE mRNA expression in the diabetic mouse kidney. Mol Cell Biochem (1997) 170:147–152.[CrossRef][Web of Science][Medline]
  60. Kontos H.A. Oxygen radicals from arachidonate metabolism in abnormal vascular responses. Am Rev Respir Dis (1987) 136:474–477.[Web of Science][Medline]
  61. Yamada T., Fujino T., Yuhki K., Hara A., Karibe H., Takahata O., et al. Thromboxane A2 regulates vascular tone via its inhibitory effect on the expression of inducible nitric oxide synthase. Circulation (2003) 108:2381–2386.[Abstract/Free Full Text]
  62. Smith W.L., Garavito R.M., DeWitt D.L. Prostaglandin endoperoxide H synthases (cyclooxygenases)-1 and -2. J Biol Chem (1996) 271:33157–33160.[Free Full Text]
  63. Fitzpatrick F.A., Soberman R. Regulated formation of eicosanoids. J Clin Invest (2001) 107:1347–1351.[Web of Science][Medline]
  64. Garavito R.M., Mulichak A.M. The structure of mammalian cyclooxygenases. Annu Rev Biophys Biomol Struct (2003) 32:183–206.[CrossRef][Web of Science][Medline]
  65. Fitzpatrick F.A. Cyclooxygenase enzymes: regulation and function. Curr Pharm Des (2004) 10:577–588.[CrossRef][Web of Science][Medline]

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