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Am J Physiol Heart Circ Physiol 294: H2411-H2420, 2008. First published March 7, 2008; doi:10.1152/ajpheart.01217.2007
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Sex Steroids and Gender in Cardiovascular-Renal Physiology and Pathophysiology

Sexual dimorphism in rabbit aortic endothelial function under acute hyperglycemic conditions and gender-specific responses to acute 17β-estradiol

Aditya Goel,1 Der Thor,1 Leigh Anderson,2 and Roshanak Rahimian1

1Department of Physiology and Pharmacology, Thomas J. Long School of Pharmacy and Health Sciences, University of the Pacific, Stockton; and 2Department of Anatomical Sciences, Arthur A. Dugoni School of Dentistry, University of the Pacific, San Francisco, California

Submitted 19 October 2007 ; accepted in final form 27 February 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Epidemiological data suggest that hyperglycemia abrogates the gender-based cardiovascular protection possibly associated with estrogens. This study was designed to investigate 1) whether rabbit aortic rings show gender differences in the development of abnormal endothelium-dependent vasodilation (EDV) under acute hyperglycemic conditions, 2) the potential role of PKC isoforms and superoxide (O2–) in acute hyperglycemia-induced vascular dysfunction, and 3) the effect of acute estrogen administration on hyperglycemia-induced endothelial dysfunction in male and female rabbits. EDV to ACh was determined before and after 3 h of treatment with high glucose (HG) in phenylephrine-precontracted aortic rings from male and female New Zealand White rabbits. Similar experiments were conducted in the presence of inhibitors of PKC-{alpha}, PKC-β, and PKC-{delta} or an O2– scavenger. The effect of acute estrogen administration was evaluated in the presence and absence of HG. Finally, mRNA expression of PKC isoforms was measured by real-time PCR. We found that 1) 3 h of incubation with HG impairs EDV to a greater extent in female than male aorta, 2) inhibition of PKC-β or O2– prevents HG-induced impairment of EDV in female aorta, 3) acute 17β-estradiol aggravates HG-induced endothelial dysfunction in female, but not male, aorta, and 4) PKC-{alpha} and PKC-β expression are significantly higher in female than male aorta. This study reveals the predisposition of female rabbit aorta to vascular injury under hyperglycemic conditions, possibly via activation of PKC-β and O2– production. Furthermore, it suggests that, under hyperglycemic conditions, acute estrogen treatment is detrimental to endothelial function in female rabbits.

cardiovascular disease; superoxide; diabetes; protein kinase C

CARDIOVASCULAR DISEASE (CVD) claims more lives each year than cancer, chronic lower respiratory diseases, accidents, and diabetes mellitus combined (43). Asymptomatic hyperglycemia is associated with an increased risk of death from CVD (10, 12, 18, 25). However, the available evidence suggests that hyperglycemia affects male and female vascular beds differently (21). Clinically, these differences reveal a stronger association between CVD and diabetes in women than men (22, 35). Despite the epidemiological data, the mechanisms underlying the loss of premenopausal female-specific cardiovascular protection in hyperglycemia/diabetes are not clear.

Hyperglycemia brings about several changes in the vascular homeostasis. One of the hallmarks of hyperglycemia-induced vascular disease is endothelial cell dysfunction, characterized by impaired nitric oxide (NO)-dependent vasodilation. Endothelium-dependent vasodilation (EDV) is generally used as a reproducible parameter to probe endothelial function under different pathological conditions. Impaired EDV has been described in diabetes, and the degree of impairment of relaxation was shown to be correlated with glycemic control (48). We recently showed that acute exposure to high glucose (HG) reveals a gender difference in the development of impaired EDV in rat aorta (15), but because different species may react differently to a comparable stimulus (30), it remains to be established whether the above-mentioned sexual dimorphism is species specific or extends to other mammals. Hence, the first objective of the present study was to investigate whether rabbit aortic rings under acute hyperglycemic conditions also show a gender difference in the development of abnormal endothelium- dependent responses.

The second aim of the present study was to explore the potential mechanism(s) by which hyperglycemia impairs vasodilator function in the rabbit. Components of the signaling pathway by which hyperglycemia impairs vascular dilatation are not clearly defined, but PKC isoforms and superoxide (O2–) have been suggested to play important roles. Recent evidence suggests that PKC isoforms, such as PKC-{alpha}, PKC-β, and PKC-{delta}, are upregulated and activated in hyperglycemia and diabetes (9). It has also been shown that activation of PKC isoforms leads to decreased NO production in human (3), mouse (33), rat (8), and rabbit (49) vasculature. Thus experiments were carried out to determine whether inhibition of PKC-{alpha}, PKC-β, or PKC-{delta} activity would affect the vascular impairment caused by acute hyperglycemia in rabbits. Activation of PKC has also been shown to cause generation of reactive oxygen species (ROS), such as O2– (19, 23). Furthermore, there is growing evidence that acute hyperglycemia is accompanied by ROS generation (44). Thus we investigated the role of O2– generation in HG-induced impairment of EDV in rabbit aortic rings.

Finally, we previously reported that administration of 17β-estradiol (E2) to ovariectomized rats improves vascular function in terms of the availability of vasodilating agents, such as NO (37–39). These findings have been substantiated by a number of studies in humans (41, 46) and animals, including rabbits (17). Such a direct effect of estrogen on the vascular wall, along with the reported improvement in the plasma lipid profile (51), may explain why premenopausal women are relatively protected from CVD compared with men. However, premenopausal women with diabetes not only lose this gender-based cardiovascular protection, suggesting that hyperglycemia overcomes some of the beneficial effects of sex steroids, but they also experience a higher relative risk of CVD than men (1, 4, 13, 27, 42, 47). Because the effects of estrogen on vasculature under hyperglycemic conditions are uncertain, our last objective was to evaluate whether acute administration of E2 (100 nM) would affect endothelial function during hyperglycemia in male and female rabbits equally.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Experimental Animals

Adult male and female New Zealand White rabbits (2.5–3 kg body wt; Kralek Farms, Turlock, CA) were euthanized by injection of pentobarbital sodium (80 mg/kg; Sigma, St. Louis, MO). All animal protocols were approved by the Animal Care Committee of the University of the Pacific and complied with the National Institutes of Health guidelines on care for animals used in research.

Measurement of Arterial Tension

The thoracic aorta was excised and placed in oxygenated Krebs solution (in mM: 118.3 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, KH2PO4, 25.0 NaHCO3, and 6.0 glucose) at 37°C. The aorta was cleared of fatty tissue and adhering connective tissue and then cut into 3- to 4-mm-long rings. The rings were suspended horizontally between two stainless steel hooks for measurement of isometric tension in individual organ baths containing 20 ml of Krebs solution at 37°C bubbled with 95% O2-5% CO2. The isometric tension of aortic rings was continuously monitored with a computer- based data acquisition system (PowerLab with Chart version 4.0 software, ADInstruments). The rings were equilibrated for 45 min under a resting tension of 3 g to allow development of a stable basal tone. Stimulation of rings with 80 mM KCl was repeated two or three times every 15 min until contractile responses were stable and uniform. Responsiveness of the rings to the contractile agent phenylephrine (PE, 2 µM) and the endothelium-dependent vasodilator ACh (10 µM) was determined. The tissues that did not relax were discounted and discarded.

Dilator concentration-response curves to ACh. Six rings were selected for the dilator concentration-response curves (CRCs) to ACh. They were washed with Krebs solution to allow relaxation to the basal tone. Each ring was assigned to one of the following experimental protocols

In protocol 1, we investigated the effects of normal glucose (NG) on ACh-induced relaxation. The ring was precontracted with 2 µM PE, which represents a concentration that produces ~80% of the maximal contraction. The first CRC was obtained in Krebs solution containing NG (6 mM) by addition of increasing concentrations of ACh (10–8–10–5 M). The tissue was then washed every 10 min with Krebs solution containing NG to allow relaxation to the basal tone and incubated for an additional 1 h in Krebs solution containing NG. The ring was again precontracted with 2 µM PE, and the second CRC to ACh (10–8–10–5 M) was generated in Krebs solution containing NG. The tissue was washed with Krebs solution to allow relaxation to the basal tone and incubated for 3 h in Krebs solution containing NG before the third CRC to ACh (10–8–10–5 M) was generated after contraction with 2 µM PE. Figure 1 shows a typical trace of experimental protocol 1.


Figure 1
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  		Fig. 1. Representative trace of experimental protocol 1. First concentration-response curve (CRC) to ACh was determined in aortic ring precontracted with 2 µM phenylephrine (PE) in Krebs solution containing normal (6 mM) glucose (NG; A). The same aortic ring was washed (W) with Krebs solution to allow relaxation to basal tone, and 2nd CRC to ACh (10–8–10–5 M) was determined after pretreatment with Krebs solution containing NG (B). The same aortic ring was washed with Krebs solution to allow relaxation to basal tone, and 3rd CRC to ACh was determined after pretreatment with Krebs solution containing NG for 3 h (C).

 
In protocols 2 and 3, we investigated the effects of HG or PMA on ACh-induced relaxation. In the second and third set of rings, the first and second CRCs to ACh in PE-precontracted tissues were obtained in Krebs solution containing NG, as described in protocol 1. After the tissues were washed with Krebs solution every 10 min to allow relaxation to the basal tone, the rings were incubated for 3 h in Krebs solution containing HG (46 mM; protocol 2) or 0.2 µM PMA (Sigma; protocol 3), a PKC activator, and the third CRC to ACh was generated in PE-precontracted aorta. The high concentration of glucose was identical to the concentration used in similar in vitro vascular studies reported previously (15, 49). The use of 0.2 µM PMA was based on our preliminary studies to determine the highest concentration that does not affect the tone (data not shown).

In protocols 4–6, we studied the role of PKC isoforms (PKC-{alpha}, -β, and -{delta}), O2–, and 17β-estradiol in HG- or PMA-induced impairment of ACh relaxation. In our fourth, fifth, and sixth sets of rings, the first CRC to ACh in PE-precontracted tissues was obtained in Krebs solution containing NG, as described in protocol 1. The tissue was then washed with Krebs solution every 10 min to allow relaxation to the basal tone and further incubated for 1 h in NG. During the last 20 min of incubation, the tissues were treated with the PKC-{alpha} inhibitor Ro-32-0432 (9 nM; Sigma), the PKC-β inhibitor LY-379196 (1 µM; a gift of Lilly Research Laboratories, Indianapolis, IN), the PKC-{delta} inhibitor rottlerin (6 µM; Sigma), the membrane-permeant superoxide dismutase mimetic tempol (100 µM; Sigma), or 100 nM E2 (Sigma), and the second CRC to ACh was generated in PE-precontracted rings. Tissues were then washed with Krebs solution to allow relaxation to the basal tone. Aortic rings were again incubated with one of the above-mentioned drugs for 20 min before the addition of NG (protocol 4), HG (protocol 5), or 0.2 µM PMA (protocol 6) to the buffer. Incubation was continued for 3 h, and the third CRC to ACh was generated in PE-precontracted tissues.

Effects of HG on ionomycin-induced relaxation. In a different set of experiments, the effect of HG on relaxation of PE (2 µM)-precontracted male and female rings induced by ionomycin (10–10–10–6 M), a non-receptor-mediated endothelium-dependent vasodilator, was investigated after 3 h of incubation in HG.

PE and ACh were dissolved in double-distilled H2O, E2 was dissolved in ethanol, ionomycin stock solution was prepared in DMSO and then diluted in ethanol, and all other drugs were dissolved in DMSO. In each case, the vehicle had no effect on vascular reactivity.

Real-Time PCR

Rabbit aorta was isolated as described above (see Measurement of Arterial Tension) and cut into ~12-mm segments. Each segment was individually flash frozen in liquid nitrogen and stored at –80°C for later RNA extraction. RNA was extracted from male and female rabbit aortic segments using TRIzol (Invitrogen), as described by Chomczynski and Sacchi (6). First-strand cDNA was synthesized by reverse transcription of 2 µg of total RNA using the Omniscript RT kit (Qiagen), in a total volume of 20 µl, according to the manufacturer's suggestions. The gene fragments were specifically amplified using iQ SYBR Green Supermix (Bio-Rad) and 1 µl of first-strand cDNA with real-time RT-PCR (MyIQ Single-Color Real-Time PCR Detection System, Bio-Rad). Internal variations were normalized using rabbit 18S rRNA, and expression was analyzed as described by Muller et al. (32). The following primers were used for detection of gene expression: 5'-AAACGGCTACCACATCCAAG-3' (forward) and 5'-CCTCCAATGGATCCTCGTTA-3' (reverse) for rabbit 18S rRNA; 5'-GTTAGTGGATACGCAAATCG-3' (forward) and 5'-TGGGAAGTATACCAAATCGT-3' (reverse) for rabbit PKC-{alpha}; 5'-AGAGTAAGCGCATTATCCAA-3' (forward) and 5'-CTTTTACCAGGAACATCAGC-3' (reverse) for rabbit PKC-β; and 5'-ACCAGGGCATCATCTACAGG-3' (forward) and 5'-AGACTTCCCATAGGGCTGGT-3' (reverse) for rabbit PKC-{delta}. Specificity of the primers was verified by electrophoresis of the PCR products on a 2% agarose gel and staining with ethidium bromide.

Data Analysis

The recorded decrease in the force of contraction (residual contraction) was calculated as percentage of the maximum contraction obtained with PE immediately before addition of each concentration of ACh. Relaxation of the precontracted aorta is expressed as the percent residual contraction left at each dose: residual contraction (%) = (maximum PE contraction as 100%) – [extent of relaxation (%) obtained at a particular drug concentration]. mRNA expression was normalized to expression of 18S rRNA levels. Values are reported as means ± SE. Data were analyzed by paired t-test for comparisons of two group means in a pre-/posttest format unless otherwise stated. P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Contractile Responses to PE

In rabbit aorta, PE (2 µM)-induced contraction was not significantly affected by incubation of tissues with HG in male or female animals (n = 9 in each group). PE-induced contraction in male rabbit aortic rings was 3.04 ± 0.51 and 2.64 ± 0.67 g in NG and HG, respectively. PE-induced contraction in female rabbit aortic rings was 2.72 ± 0.49 and 2.53 ± 0.49 g in NG and HG, respectively.

Relaxation Responses to ACh

ACh (10–8–10–5 M) concentration dependently relaxed PE-precontracted aortic rings. No significant differences in response to ACh occurred among the first, second, and third CRCs generated in Krebs solution containing NG (data not shown; see Emax values in NG, Table 1).


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  		Table 1. Emax to ACh in experimental groups

 
To investigate whether there is a gender difference in EDV in PE-precontracted rabbit aortic rings exposed to HG, the differences in the ACh relaxation response between the second CRC (generated in Krebs solution containing NG) and the third CRC (generated in Krebs solution containing HG) in male and female aortic rings were compared. Figure 2, A and B, shows the effect of HG pretreatment on ACh-induced relaxation of PE-precontracted aortic rings from male and female rabbits, respectively. A 3-h incubation in HG attenuated ACh-induced relaxation (3rd CRC) in male and female animals (Fig. 2, A and B; P < 0.05 vs. 2nd CRC, n = 9 in each group). However, the extent of HG-induced EDV impairment was significantly greater in female than male rabbit aorta. In male rabbits, relaxation of aorta was significantly decreased by acute exposure to HG only at the maximum concentration of ACh (10–5 M). In female rabbits, HG leads to a significant decrease in ACh (10–6–10–5 M)-induced relaxation of the aorta. Figure 2, C and D, shows the effect of pretreatment with PMA, a diacylglycerol analog, on ACh-induced relaxation of PE-precontracted aortic rings. Acute treatment of aortic rings with Krebs solution containing PMA (0.2 µM) for 3 h significantly attenuated ACh-induced relaxation of aortic rings from male and female animals (P < 0.05 vs. 2nd CRC, n = 9 in each group). Similarly, the extent of PMA-induced EDV impairment was greater in female than male aortic rings. Neither HG nor PMA changed the sensitivity to ACh as assessed by logEC50 in either gender (data not shown).


Figure 2
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  		Fig. 2. Effect of high (46 mM) glucose (HG) and activation of PKC by PMA (0.2 µm) on relaxation response to cumulative concentrations of ACh in intact aortic rings from male (A and C) and female (B and D) rabbits precontracted with 2 µM PE. Rings were incubated with Krebs solution containing NG (1st and 2nd responses) or Krebs solution containing HG or PMA (3rd response) for 3 h before CRC to ACh was determined. In males, 3rd CRC at 10–5 M ACh was reduced compared with 2nd CRC. In females, 3rd CRC at 10–6–10–5 M ACh was reduced compared with 2nd CRC. Values are means ± SE. *P < 0.05 (paired t-test).

 
Effect of PKC inhibition on ACh-induced relaxation. Pretreatment of female aortic rings with the PKC-{alpha} inhibitor Ro-32-0432 (9 nM) or the PKC-β inhibitor LY-379196 (1 µM) for 20 min in NG or 3 h in NG (protocol 4) did not significantly affect EDV, as indicated by no significant change of the relaxation response in the first, second, and third CRCs (Table 1B).

On the other hand, pretreatment with Ro-32-0432 or LY-379196 for 20 min before the addition of HG or PMA to the buffer for 3 h appeared to prevent the impairment of ACh-mediated relaxation induced by HG (Fig. 3, A and B) or PMA (Fig. 4, A and B), as indicated by no significant difference between the maximum dilator response (Emax) values of the first, second, and third CRCs (in protocol 5 or 6) to ACh (Table 1B). However, when the decrease in Emax under the HG or PMA conditions in the presence (3rd CRC in protocol 5 or 6) and absence (3rd CRC in protocol 2 or 3) of Ro-32-0432 or LY-379196 was compared, the extent of decrease in Emax was significant in the presence of LY-379196, but not Ro-32-0432 (P < 0.05, unpaired t-test; Fig. 5).


Figure 3
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  		Fig. 3. Effect of Ro-32-0432 (A), LY-379196 (B), rottlerin (C), or tempol (D) on HG-induced impairment of ACh responses in female rabbit aortic rings. Responses to ACh were determined in rings incubated with Krebs solution containing NG as 1st response, Krebs solution containing various inhibitors in NG for 20 min as 2nd response, and Krebs solution containing the same inhibitors in HG for 3 h as 3rd response. Ro-32-0432 (9 nM), LY-379196 (1 µM), or tempol (100 µM) prevented abnormal ACh relaxation of female rabbit aorta incubated in HG. Rottlerin (6 µM) did not prevent HG-induced impairment of responses to ACh. Pretreatment with rottlerin before the 2nd response in NG significantly decreased ACh response compared with the 1st response. Values are means ± SE. *#P < 0.05 (paired t-test).

 

Figure 4
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  		Fig. 4. Effect of Ro-32-0432 (A), LY-379196 (B), rottlerin (C), and tempol (D) on PMA-induced impairment of responses to ACh in female rabbit aortic rings. Responses to ACh were determined in rings incubated with Krebs solution containing NG as 1st response, Krebs solution containing various inhibitors in NG for 20 min as 2nd response, and Krebs solution containing the same inhibitors in 0.2 µM PMA for 3 h as 3rd response. Ro-32-0432 (9 nM), LY-379196 (1 µM), or tempol (100 µM) restored abnormal ACh relaxation of female rabbit aorta incubated in PMA. Rottlerin did not prevent PMA-induced impairment of responses to ACh. Pretreatment with rottlerin before 2nd response in NG significantly decreased ACh response compared with 1st response. Values are means ± SE. *#P < 0.05 (paired t-test).

 

Figure 5
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  		Fig. 5. Comparison of decrease in maximum dilator response (Emax) to ACh (10–5 M) in female rabbit aortic rings after 3 h of incubation in Krebs solution containing HG or PMA (0.2 µM) in the presence and absence of Ro-32-0432 (Ro), LY-379196 (LY), rottlerin (Rot), or tempol (Tem). Decreases in Emax are calculated as percent change of Emax in 3rd CRC to ACh normalized to 2nd CRC. Extent of decrease in Emax was significantly less in female aortic rings incubated with LY-379196 or tempol in HG or PMA for 3 h than in those incubated in HG alone and PMA alone, respectively. Values are means ± SE. *#P < 0.05 (unpaired t-test).

 
In contrast to the effect of PKC-{alpha} and PKC-β inhibitors, incubation of aortic rings with the PKC-{delta} inhibitor rottlerin (6 µM) for 20 min before the second CRC (in NG) significantly decreased the EDV compared with the first CRC to ACh (P < 0.05, n = 10; Figs. 3C and 4C). Emax in the presence of rottlerin was reduced by 18.27 ± 2.32% in the second CRC compared with the first CRC (Table 1B). Moreover, pretreatment with rottlerin for 20 min before 3 h of incubation in HG or PMA did not prevent further impairment of EDV (Figs. 3C and 4C). The extent of decrease in Emax in the presence of rottlerin + HG or rottlerin + PMA was comparable to that observed after incubation of female aortic rings in HG or PMA alone (Fig. 5).

Effect of O2– scavenging on ACh-induced relaxation. Pretreatment of female rabbit aortic rings with 100 µM tempol, an O2– scavenger, for 20 min before 3 h of incubation in Krebs solution containing HG (Fig. 3D) or PMA (Fig. 4D) prevented HG- or PMA-induced impairment of EDV; the extent of decrease in Emax in the presence of tempol + HG or tempol + PMA was significantly less than in HG or PMA alone (Fig. 5).

Furthermore, Figs. 3D and 4D show that pretreatment of tissues for 20 min with tempol (100 µM) alone did not alter the ACh-induced relaxation compared with control, as also indicated by no significant differences between Emax from the first and second CRCs to ACh (Table 1B).

Effect of acute administration of E2 on HG- or PMA-induced blunting of endothelium-dependent relaxation. To examine the effect of acute administration of estrogen on endothelial function under hyperglycemic conditions, aortic rings from male and female rabbits were incubated with 100 nM E2 for 20 min before addition of HG or PMA (3rd CRC) to the buffer. Furthermore, to investigate the effect of E2 alone on EDV, in some tissues the second and third CRCs to ACh were generated after pretreatment with E2 in NG.

Incubation of aortic rings with 100 nM E2 in NG did not alter the ACh-induced relaxation compared with control (1st CRC) in either gender (Fig. 6), as also indicated by no significant differences between Emax values of the first, second (in E2 + NG), and third (in E2 + NG) CRCs to ACh (Table 1, A and B). However, pretreatment of tissues with E2 not only failed to prevent HG- or PMA-induced impairment in endothelium from male and female rabbits, but it also significantly enhanced the extent of impairment in tissues from female animals (P < 0.05 vs. HG alone, unpaired t-test). In female rabbit aorta, Emax to ACh was significantly reduced by 24.32 ± 4.63% and 40.93 ± 3.34% in HG and E2 + HG, respectively, compared with NG (2nd CRC; Fig. 7). Similarly, the extent of PMA-induced impairment in female aortic rings was significantly greater in the presence than absence of E2; Emax to ACh was significantly reduced by 30.03 ± 4.39% and 47.87 ± 4.29% in PMA and E2 + PMA, respectively, compared with NG (2nd CRC; Fig. 7).


Figure 6
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  		Fig. 6. Acute effect of 17β-estradiol (E2) on HG- and PMA-induced impairment of ACh responses in aortic rings from male (A and C) and female (B and D) rabbits. Aortic rings were incubated with Krebs solution containing NG (1st response), Krebs solution containing E2 (100 nM) for 20 min in NG (2nd response), or Krebs solution containing E2 in HG or 0.2 µM PMA (3rd response) for 3 h before CRC to ACh was determined. There was no significant difference between 1st and 2nd CRC to ACh in any group. In males, 3rd CRC at 10–5 M ACh was reduced compared with 2nd CRC. In females, 3rd CRC at 10–6–10–5 M ACh was reduced compared with 2nd CRC. Values are means ± SE. *P < 0.05 (paired t-test).

 

Figure 7
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  		Fig. 7. Decrease in Emax to ACh (10–5 M) in female rabbit aortic rings after 3 h of incubation in Krebs solution containing HG or PMA (0.2 µM) in the presence and absence of E2. Decreases in Emax are calculated as percent change of Emax in 3rd CRC to ACh normalized to 2nd CRC. Extent of decrease in Emax was significantly greater in female aortic rings incubated with E2 + HG or E2 + PMA (0.2 µM) for 3 h than in those incubated with HG alone and PMA alone, respectively. Values are means ± SE. *#P < 0.05 (unpaired t-test).

 
Effect of HG on Ionomycin-Induced Relaxation

Aortic relaxation responses to ionomycin (10–10–10–6 M), a non-receptor-mediated endothelium-dependent vasodilator, were not significantly different between the first and second CRCs in male or female animals. However, incubation of tissues from females, but not males, for 3 h in Krebs solution containing HG significantly decreased (P < 0.05, n = 3) the maximum relaxation response to 10–6 M ionomycin; the maximum relaxation was significantly reduced by 23.32 ± 7.85% in HG compared with NG (data not shown).

Analysis of mRNA Expression of PKC Isoforms

Real-time PCR analysis revealed that the levels of mRNA expression for PKC-{alpha} and PKC-β were significantly higher in aortas from female than male rabbits (P < 0.05, unpaired t-test, n = 4–6 in each group; Fig. 8). However, there was no significant difference between male and female PKC-{delta} mRNA expression.


Figure 8
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  		Fig. 8. Real-time PCR analysis of PKC-{alpha}, PKC-β, and PKC-{delta} mRNA expression in male and female rabbit aorta. mRNA expression levels of PKC-{alpha}, PKC-β, or PKC-{delta} in aorta from male and female rabbits were normalized to 18S rRNA. Each bar represents mean ± SE of 4–6 animals. *P < 0.05 vs. male (unpaired t-test).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
We set out to study the influence of gender on rabbit aortic responses to acute hyperglycemia and E2. We observed that acute exposure to HG reveals a gender difference in the development of EDV impairment in rabbit aorta. This impairment was restored by PKC-{alpha} and PKC-β inhibition or O2– scavenging. We also observed higher PKC-{alpha} and PKC-β mRNA expression in female than male rabbit aorta. In addition, hyperglycemia-induced impairment of EDV is further aggravated by acute incubation of tissues with E2, but only in female rabbits.

Several lines of evidence suggest that endothelial dysfunction represents early steps in the development of vascular complications in diabetes, and hyperglycemia is the central initiating factor for many types of vascular complications in diabetes. In agreement with previous reports demonstrating compromised EDV in different vascular beds under hyperglycemic conditions (3, 8, 14, 20, 45, 49), we observed that HG impairs EDV in rabbit aorta. Moreover, in accordance with our recent report on rat aorta (15), we showed a gender-based difference in the vascular response under acute hyperglycemic conditions in rabbits. Incubation of aorta for 3 h with Krebs buffer containing HG impaired EDV to a greater extent in aorta from female than male rabbits. Gomes et al. (16) reported that 3 h of incubation in HG impaired EDV in male and female rabbit aortic rings. However, they performed a pooled data analysis and made no attempt to determine whether there was a gender effect in their study. Others have shown that a longer exposure to HG induces EDV impairment in male rabbits (50). We obtained similar findings by incubating male aorta with HG for 6 h (data not shown). Thus, in male rabbits, longer exposure to HG would lead to impairment of the vascular response similar to that observed in female rabbits. This is in agreement with our recent report showing a predilection toward earlier impairment of EDV in acute hyperglycemia in female than male rat aorta (15). These results may, in part, also correlate with the findings of prospective population-based studies on humans that suggest an association of asymptomatic hyperglycemia with increased risk of CVD mortality in both genders, but a greater degree of association in women than men (21, 24). Nevertheless, it is important to note that the gender differences observed under acute hyperglycemic conditions were significant only at high, probably nonphysiological, concentrations of ACh. Further studies are required to more clearly establish the relevance of the present findings.

Furthermore, similar to the findings for ACh, aortic relaxation to ionomycin, a calcium ionophore, but not to sodium nitroprusside, an NO donor, was affected by HG to a greater extent in females than males, suggesting that attenuation of ACh-mediated relaxation by HG is a phenomenon of endothelial dysfunction (data not shown). A change in osmolarity of the incubating solution does not seem to play a role in the acute hyperglycemia-induced impairment of EDV, since incubation of tissues with a high concentration of mannitol (46 mM) did not affect EDV (data not shown).

Previous studies have shown that exposure to HG upregulates PKC activity and increases generation of intracellular ROS (23, 34). In our experiments, hyperglycemia-induced impairment of EDV in female rabbit aorta was completely prevented by prior incubation of tissues with the selective PKC-β inhibitor LY-379196, which would support a role of PKC-β in the alteration of EDV by HG. In support of this hypothesis, we demonstrated a greater impairment of ACh-induced relaxation of female aortic rings exposed to Krebs solution containing PMA, a PKC activator. Also consistent with our working hypothesis are the data demonstrating that attenuation of EDV by PMA is completely prevented after treatment of tissues with a selective PKC-β inhibitor. The enhanced activity of PKC-β in female rabbit aortas exposed to HG might be explained in part by an elevated constitutive level of PKC-β expression. Accordingly, we demonstrated higher PKC-β mRNA expression in female than male rabbit aorta. In addition to the effect of PKC-β inhibition, addition of a PKC-{alpha} inhibitor, Ro-32-0432, also seemed to prevent the HG- or PMA-induced impairment of EDV in female aortic rings. Activation of PKC-{alpha} and PKC-β under hyperglycemic conditions has been reported in monocytes, leading to release of interleukin-6 (11). However, in the present study, we cannot rule out the possibility of a partial inhibition of PKC-β by the PKC-{alpha} inhibitor Ro-32-0432. This argument is supported by the comparison of decreased Emax values under hyperglycemic conditions in the presence and absence of PKC-{alpha} or PKC-β inhibitors. In contrast to the effect of LY-379196, the extent of the decrease in the Emax values in the presence of Ro-32-0432 was not significantly different from that observed after incubation of female aortic rings in HG alone. This suggests that activation of PKC-{alpha} probably does not play a role in the alteration of EDV by HG. Nevertheless, our mRNA expression study clearly indicates a gender difference in the mRNA level of PKC-{alpha} in rabbit aorta.

There is also conflicting data regarding PKC-{delta} and its action on the vasculature. PKC-{delta} activation has been linked to activation of vasodilatory mechanisms (2). Conversely, a parallel increase in PKC-{delta} activation and O2– generation under hyperglycemic conditions has been reported (40). In the present study, inhibition of PKC-{delta} by rottlerin not only failed to prevent the HG- or PMA-induced impairment of EDV in female aortic ring, but it also induced EDV impairment under normoglycemic conditions. Interestingly, the extent of HG-induced impairment of EDV after PKC-{delta} inhibition was the same as that observed under hyperglycemic conditions alone.

There is growing evidence that an acute increase of glycemia is accompanied by ROS generation (44). Therefore, we assessed the role of O2– in mediating the greater hyperglycemia-induced impairment of EDV in female rabbit aorta. Prior incubation with an O2– scavenger, tempol, completely prevented HG- or PMA-induced impairment of EDV in female rabbit aorta. This suggests that in female rabbit aortic rings the decrease in relaxation is due to an augmentation of O2– production under hyperglycemic conditions.

Finally, the present study is, to our knowledge, the first to assess the role of gender differences in the effect of acute administration of E2 on vascular function under hyperglycemic conditions. However, one study in the literature did examine the influence of chronic E2 on vascular function in an obese insulin-resistant male rat (5). In the present study, short-term administration of the natural estrogen (E2) did not have an effect on the ACh-induced vascular response in the control (NG) condition in males or females. These data suggest that acute effects of sex hormones are unlikely to directly cause a change in a vasodilatory parameter under normal conditions in males or females. In favor of this theory, it has been shown that, in rabbit coronary artery (26) and rat femoral artery (29), relaxation by E2 in males and females is similar. In our experiments, acute addition of E2 had no effect on HG- or PMA-induced EDV impairment in male aortic rings. However, the extent of hyperglycemia-induced EDV impairment in female aortic rings was enhanced by acute administration of E2. These results are in accordance with observations of Collins et al. (7), who showed that acute E2 modulates ACh-induced coronary artery responses of female, but not male, atherosclerotic coronary arteries in vivo. In the study of Collins et al., acute exposure to E2 converted the vascular responses of atherosclerotic coronary arteries to ACh from constriction to dilatation in females. In contrast, our study showed an adverse effect of acute estrogen on ACh responses under hyperglycemic conditions in females. Nevertheless, both studies underscore the selective sensitivity of female vasculature to estrogen.

Despite an apparent absence of an acute effect of estrogen in male tissues in our study, Brooks-Asplund and co-workers (5) showed that chronic estradiol treatment partially restored the impaired ACh-mediated vasodilation in obese male rats by reducing EC50. Contrary to its influence on EC50, however, estrogen also significantly reduced Emax to ACh in obese male rats. The reason for the lack of an estrogen effect in male aorta under acute hyperglycemic conditions in the present study is unclear, but our data argue against a nonspecific effect of estrogen. Our present findings could also indicate that, in acute hyperglycemia, estrogen is working via a mechanism that is more sensitive in female than male arteries. Consistent with these results is the observation that estradiol increased NO in female guinea pigs after 5 days of treatment, whereas a similar increase occurred in males only after 10 days of estrogen exposure (52). Although we did not attempt to elucidate the underlying mechanisms responsible for preferential interaction of E2 within the female vessel, the observation that, in female rabbits, acute exposure to estrogen leads to a greater impairment of vascular responses under hyperglycemic conditions may correlate with the findings of prospective population-based studies on humans that suggest an association of asymptomatic hyperglycemia with a higher risk of CVD in women than men. Lastly, the greater impairment of vascular responses by HG in the presence of estrogen could possibly be explained by increased PKC expression or activity. Several studies have shown that estrogen upregulates various isoforms of PKC mRNA expression in pituitary (31), breast cancer (28), and ovarian granulosa (36) cells. Along similar lines, we showed a higher basal expression level of PKC-β in female than male rabbit aorta. Moreover, this theory is further supported in the present study by observations of a greater extent of PMA-induced endothelial dysfunction in female rabbit aorta in the presence of estrogen. However, our study would not reveal the molecular effector and substrate(s) for the PKC isoforms or whether HG or estrogen alters the expression of PKC. Clearly, further studies are needed to elucidate the nature of the possible interaction between estrogen and PKC.

In summary, the present study reveals a predisposition of female rabbit aorta toward vascular injury under acute hyperglycemic conditions, possibly via activation of PKC-β and O2– generation. Moreover, it verifies that acute administration of estrogen causes a gender-specific endothelial dysfunction under acute hyperglycemic conditions in rabbit aorta. Our study sheds light on a mechanism that may possibly explain why premenopausal diabetic women lose their gender-based cardiovascular protection and suggests that estrogen might actually be detrimental to the cardiovascular system in diabetic women.


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REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grant R15 HL-71520 and National Institute for Dental and Craniofacial Research Grant DE-016587.


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   FOOTNOTES
 

Address for reprint requests and other correspondence: R. Rahimian, Thomas J. Long School of Pharmacy & Health Sciences, Univ. of the Pacific, 3601 Pacific Ave., Stockton, CA 95211 (e-mail: rrahimian{at}pacific.edu)

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.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

  1. Aviles-Santa L, Salinas K, Adams-Huet B, Raskin P. Insulin therapy, glycemic control, and cardiovascular risk factors in young Latin Americans with type 2 diabetes mellitus. J Investig Med 54: 20–31, 2006.[CrossRef][Web of Science][Medline]
  2. Barman SA, Zhu S, White RE. PKC activates BKCa channels in rat pulmonary arterial smooth muscle via cGMP-dependent protein kinase. Am J Physiol Lung Cell Mol Physiol 286: L1275–L1281, 2004.[Abstract/Free Full Text]
  3. Beckman JA, Goldfine AB, Gordon MB, Garrett LA, Creager MA. Inhibition of protein kinase Cβ prevents impaired endothelium-dependent vasodilation caused by hyperglycemia in humans. Circ Res 90: 107–111, 2002.[Abstract/Free Full Text]
  4. Brandenburg SL, Reusch JE, Felder KK, Nelson-Wong E, Lindenfeld J, Manco-Johnson M, Regensteiner JG. Impaired fibrinolysis in premenopausal women and age-matched men with type 2 diabetes mellitus: a pilot study. J Investig Med 50: 110–115, 2002.[CrossRef][Web of Science][Medline]
  5. Brooks-Asplund EM, Shoukas AA, Kim SY, Burke SA, Berkowitz DE. Estrogen has opposing effects on vascular reactivity in obese, insulin-resistant male Zucker rats. J Appl Physiol 92: 2035–2044, 2002.[Abstract/Free Full Text]
  6. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156–159, 1987.[Web of Science][Medline]
  7. Collins P, Rosano GM, Sarrel PM, Ulrich L, Adamopoulos S, Beale CM, McNeill JG, Poole-Wilson PA. 17β-Estradiol attenuates acetylcholine-induced coronary arterial constriction in women but not men with coronary heart disease. Circulation 92: 24–30, 1995.[Abstract/Free Full Text]
  8. Cotter MA, Jack AM, Cameron NE. Effects of the protein kinase Cβ inhibitor LY333531 on neural and vascular function in rats with streptozotocin-induced diabetes. Clin Sci (Lond) 103: 311–321, 2002.[Medline]
  9. Das Evcimen N, King GL. The role of protein kinase C activation and the vascular complications of diabetes. Pharmacol Res 55: 498–510, 2007.[CrossRef][Web of Science][Medline]
  10. DECODE Study Group. Glucose tolerance and cardiovascular mortality: comparison of fasting and 2-hour diagnostic criteria. Arch Intern Med 161: 397–405, 2001.[Abstract/Free Full Text]
  11. Devaraj S, Venugopal SK, Singh U, Jialal I. Hyperglycemia induces monocytic release of interleukin-6 via induction of protein kinase C{alpha} and -β. Diabetes 54: 85–91, 2005.[Abstract/Free Full Text]
  12. De Vegt F, Dekker JM, Ruhe HG, Stehouwer CD, Nijpels G, Bouter LM, Heine RJ. Hyperglycaemia is associated with all-cause and cardiovascular mortality in the Hoorn population: the Hoorn Study. Diabetologia 42: 926–931, 1999.[CrossRef][Web of Science][Medline]
  13. Di Carli MF, Afonso L, Campisi R, Ramappa P, Bianco-Batlles D, Grunberger G, Schelbert HR. Coronary vascular dysfunction in premenopausal women with diabetes mellitus. Am Heart J 144: 711–718, 2002.[Web of Science][Medline]
  14. Giugliano D, Marfella R, Coppola L, Verrazzo G, Acampora R, Giunta R, Nappo F, Lucarelli C, D'Onofrio F. Vascular effects of acute hyperglycemia in humans are reversed by L-arginine. Evidence for reduced availability of nitric oxide during hyperglycemia. Circulation 95: 1783–1790, 1997.[Abstract/Free Full Text]
  15. Goel A, Zhang Y, Anderson L, Rahimian R. Gender difference in rat aorta vasodilation after acute exposure to high glucose: involvement of protein kinase Cβ and superoxide but not of Rho kinase. Cardiovasc Res 76: 351–360, 2007.[Abstract/Free Full Text]
  16. Gomes MB, Affonso FS, Cailleaux S, Almeida AL, Pinto LF, Tibirica E. Glucose levels observed in daily clinical practice induce endothelial dysfunction in the rabbit macro- and microcirculation. Fundam Clin Pharmacol 18: 339–346, 2004.[CrossRef][Web of Science][Medline]
  17. Hayashi T, Fukuto JM, Ignarro LJ, Chaudhuri G. Basal release of nitric oxide from aortic rings is greater in female rabbits than in male rabbits: implications for atherosclerosis. Proc Natl Acad Sci USA 89: 11259–11263, 1992.[Abstract/Free Full Text]
  18. Heine RJ, Balkau B, Ceriello A, Del Prato S, Horton ES, Taskinen MR. What does postprandial hyperglycaemia mean? Diabet Med 21: 208–213, 2004.[CrossRef][Web of Science][Medline]
  19. Higashi M, Shimokawa H, Hattori T, Hiroki J, Mukai Y, Morikawa K, Ichiki T, Takahashi S, Takeshita A. Long-term inhibition of Rho-kinase suppresses angiotensin II-induced cardiovascular hypertrophy in rats in vivo: effect on endothelial NAD(P)H oxidase system. Circ Res 93: 767–775, 2003.[Abstract/Free Full Text]
  20. Houben AJ, Schaper NC, de Haan CH, Huvers FC, Slaaf DW, de Leeuw PW, Nieuwenhuijzen Kruseman AC. The effects of 7-hour local hyperglycaemia on forearm macro- and microcirculatory blood flow and vascular reactivity in healthy man. Diabetologia 37: 750–756, 1994.[CrossRef][Web of Science][Medline]
  21. Hu G. Gender difference in all-cause and cardiovascular mortality related to hyperglycaemia and newly-diagnosed diabetes. Diabetologia 46: 608–617, 2003.[Web of Science][Medline]
  22. Hu G, Jousilahti P, Qiao Q, Katoh S, Tuomilehto J. Sex differences in cardiovascular and total mortality among diabetic and non-diabetic individuals with or without history of myocardial infarction. Diabetologia 48: 856–861, 2005.[CrossRef][Web of Science][Medline]
  23. Inoguchi T, Li P, Umeda F, Yu HY, Kakimoto M, Imamura M, Aoki T, Etoh T, Hashimoto T, Naruse M, Sano H, Utsumi H, Nawata H. High glucose level and free fatty acid stimulate reactive oxygen species production through protein kinase C-dependent activation of NAD(P)H oxidase in cultured vascular cells. Diabetes 49: 1939–1945, 2000.[Abstract]
  24. Janghorbani M, Jones RB, Gilmour WH, Hedley AJ, Zhianpour M. A prospective population based study of gender differential in mortality from cardiovascular disease and "all causes" in asymptomatic hyperglycaemics. J Clin Epidemiol 47: 397–405, 1994.[CrossRef][Web of Science][Medline]
  25. Jarrett RJ, Shipley MJ. Type 2 (non-insulin-dependent) diabetes mellitus and cardiovascular disease—putative association via common antecedents: further evidence from the Whitehall Study. Diabetologia 31: 737–740, 1988.[CrossRef][Web of Science][Medline]
  26. Jiang CW, Sarrel PM, Lindsay DC, Poole-Wilson PA, Collins P. Endothelium-independent relaxation of rabbit coronary artery by 17β-oestradiol in vitro. Br J Pharmacol 104: 1033–1037, 1991.[Web of Science][Medline]
  27. Kannel WB. Metabolic risk factors for coronary heart disease in women: perspective from the Framingham Study. Am Heart J 114: 413–419, 1987.[CrossRef][Web of Science][Medline]
  28. Karp G, Maissel A, Livneh E. Hormonal regulation of PKC: estrogen up-regulates PKC{varepsilon} expression in estrogen-responsive breast cancer cells. Cancer Lett 246: 173–181, 2006.[CrossRef][Web of Science][Medline]
  29. Kitazawa T, Hamada E, Kitazawa K, Gaznabi AK. Non-genomic mechanism of 17β-oestradiol-induced inhibition of contraction in mammalian vascular smooth muscle. J Physiol 499: 497–511, 1997.[Abstract/Free Full Text]
  30. Mackert JL, Parsons AA, Wahl M, Schilling L. Mediation of endothelium-dependent relaxation: different response patterns in rat and rabbit basilar artery. Neurol Res 19: 521–526, 1997.[Web of Science][Medline]
  31. Maeda T, Lloyd RV. Protein kinase C activity and messenger RNA modulation by estrogen in normal and neoplastic rat pituitary tissue. Lab Invest 68: 472–480, 1993.[Web of Science][Medline]
  32. Muller PY, Janovjak H, Miserez AR, Dobbie Z. Processing of gene expression data generated by quantitative real-time RT-PCR. Biotechniques 32: 1372–1379, 2002.[Web of Science][Medline]
  33. Nangle MR, Cotter MA, Cameron NE. Protein kinase Cβ inhibition and aorta and corpus cavernosum function in streptozotocin-diabetic mice. Eur J Pharmacol 475: 99–106, 2003.[CrossRef][Web of Science][Medline]
  34. Nishikawa T, Edelstein D, Du XL, Yamagishi S, Matsumura T, Kaneda Y, Yorek MA, Beebe D, Oates PJ, Hammes HP, Giardino I, Brownlee M. Normalizing mitochondrial superoxide production blocks three pathways of hyperglycaemic damage. Nature 404: 787–790, 2000.[CrossRef][Medline]
  35. Onat A, Hergenc G, Keles I, Dogan Y, Turkmen S, Sansoy V. Sex difference in development of diabetes and cardiovascular disease on the way from obesity and metabolic syndrome. Metabolism 54: 800–808, 2005.[CrossRef][Web of Science][Medline]
  36. Peters CA, Cutler RE, Maizels ET, Robertson MC, Shiu RP, Fields P, Hunzicker-Dunn M. Regulation of PKC{delta} expression by estrogen and rat placental lactogen-1 in luteinized rat ovarian granulosa cells. Mol Cell Endocrinol 162: 181–191, 2000.[CrossRef][Web of Science][Medline]
  37. Rahimian R, Dube GP, Toma W, Dos Santos N, McManus BM, van Breemen C. Raloxifene enhances nitric oxide release in rat aorta via increasing endothelial nitric oxide mRNA expression. Eur J Pharmacol 434: 141–149, 2002.[CrossRef][Web of Science][Medline]
  38. Rahimian R, Laher I, Dube G, van Breemen C. Estrogen and selective estrogen receptor modulator LY117018 enhance release of nitric oxide in rat aorta. J Pharmacol Exp Ther 283: 116–122, 1997.[Abstract/Free Full Text]
  39. Rahimian R, Van Breemen C, Karkan D, Dube G, Laher I. Estrogen augments cyclopiazonic acid-mediated, endothelium-dependent vasodilation. Eur J Pharmacol 327: 143–149, 1997.[CrossRef][Web of Science][Medline]
  40. Ramana KV, Friedrich B, Tammali R, West MB, Bhatnagar A, Srivastava SK. Requirement of aldose reductase for the hyperglycemic activation of protein kinase C and formation of diacylglycerol in vascular smooth muscle cells. Diabetes 54: 818–829, 2005.[Abstract/Free Full Text]
  41. Reis SE, Gloth ST, Blumenthal RS, Resar JR, Zacur HA, Gerstenblith G, Brinker JA. Ethinyl estradiol acutely attenuates abnormal coronary vasomotor responses to acetylcholine in postmenopausal women. Circulation 89: 52–60, 1994.[Abstract/Free Full Text]
  42. Ren J, Ceylan-Isik AF. Diabetic cardiomyopathy: do women differ from men? Endocrine 25: 73–83, 2004.[CrossRef][Web of Science][Medline]
  43. Rosamond W, Flegal K, Friday G, Furie K, Go A, Greenlund K, Haase N, Ho M, Howard V, Kissela B, Kittner S, Lloyd-Jones D, McDermott M, Meigs J, Moy C, Nichol G, O'Donnell CJ, Roger V, Rumsfeld J, Sorlie P, Steinberger J, Thom T, Wasserthiel-Smoller S, Hong Y. Heart disease and stroke statistics—2007 update: a report from the American Heart Association Statistics Committee and Stroke Statistics Subcommittee. Circulation 115: e69–e171, 2007.[Free Full Text]
  44. Sercombe R, Vicaut E, Oudart N, Sercombe C, Girard P. Acetylcholine-induced relaxation of rabbit basilar artery in vitro is rapidly reduced by reactive oxygen species in acute hyperglycemia: role of NADPH oxidase. J Cardiovasc Pharmacol 44: 507–516, 2004.[CrossRef][Web of Science][Medline]
  45. Sobrevia L, Mann GE. Dysfunction of the endothelial nitric oxide signalling pathway in diabetes and hyperglycaemia. Exp Physiol 82: 423–452, 1997.[Abstract]
  46. Stampfer MJ, Colditz GA, Willett WC, Manson JE, Rosner B, Speizer FE, Hennekens CH. Postmenopausal estrogen therapy and cardiovascular disease. Ten-year follow-up from the Nurses' Health Study. N Engl J Med 325: 756–762, 1991.[Abstract]
  47. Steinberg HO, Paradisi G, Cronin J, Crowde K, Hempfling A, Hook G, Baron AD. Type II diabetes abrogates sex differences in endothelial function in premenopausal women. Circulation 101: 2040–2046, 2000.[Abstract/Free Full Text]
  48. Stettler C, Allemann S, Juni P, Cull CA, Holman RR, Egger M, Krahenbuhl S, Diem P. Glycemic control and macrovascular disease in types 1 and 2 diabetes mellitus: meta-analysis of randomized trials. Am Heart J 152: 27–38, 2006.[CrossRef][Web of Science][Medline]
  49. Tesfamariam B, Brown ML, Cohen RA. Elevated glucose impairs endothelium-dependent relaxation by activating protein kinase C. J Clin Invest 87: 1643–1648, 1991.[Web of Science][Medline]
  50. Tesfamariam B, Brown ML, Deykin D, Cohen RA. Elevated glucose promotes generation of endothelium-derived vasoconstrictor prostanoids in rabbit aorta. J Clin Invest 85: 929–932, 1990.[Web of Science][Medline]
  51. Walsh BW, Schiff I, Rosner B, Greenberg L, Ravnikar V, Sacks FM. Effects of postmenopausal estrogen replacement on the concentrations and metabolism of plasma lipoproteins. N Engl J Med 325: 1196–1204, 1991.[Abstract]
  52. Weiner CP, Lizasoain I, Baylis SA, Knowles RG, Charles IG, Moncada S. Induction of calcium-dependent nitric oxide synthases by sex hormones. Proc Natl Acad Sci USA 91: 5212–5216, 1994.[Abstract/Free Full Text]




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