Late dual endothelin receptor blockade with bosentan

Life Sciences 118 (2014) 263–267

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Late dual endothelin receptor blockade with bosentan restores impaired cerebrovascular function in diabetes Mohammed Abdelsaid a,c, Handong Ma a,c, Maha Coucha c, Adviye Ergul a,b,c,⁎ a b c

Charlie Norwood Veterans Administration Medical Center, University of Georgia College of Pharmacy, USA Center for Pharmacy and Experimental Therapeutics, University of Georgia College of Pharmacy, USA Department of Physiology, Georgia Regents University, Augusta, GA, USA

a r t i c l e

i n f o

Article history: Received 22 October 2013 Accepted 31 December 2013 Available online 13 January 2014 Keywords: Basilar artery Middle cerebral artery Myogenic tone Endothelial function Endothelin

a b s t r a c t Aims: Up-regulation of the endothelin (ET) system in type-2 diabetes increases contraction and decreases relaxation in basilar artery. We showed that 1) ET-receptor antagonism prevents diabetes-mediated cerebrovascular dysfunction; and 2) glycemic control prevents activation of the ET-system in diabetes. Here, our goal is to determine whether and to what extent glycemic control or ET-receptor antagonism reverses established cerebrovascular dysfunction in diabetes. Main methods: Non-obese type-2 diabetic Goto–Kakizaki rats were administered either vehicle, metformin (300 mg/kg/day) or dual ET-receptor antagonist bosentan (100 mg/kg) for 4-weeks starting at 18-weeks after established cerebrovascular dysfunction (n = 5–6/group). Control group included vehicle-treated agedmatched Wistar rats. Blood glucose and pressure were monitored weekly. At termination, basilar arteries were collected and cumulative dose–response curves to ET-1 (0.1–500 nM), 5-HT (1–1000 nM) and acetylcholine (Ach, 0.1 nM–5 μM) were studied by wire myograph. Middle cerebral artery (MCA) myogenic reactivity and tone were measured using pressurized arteriograph. Key findings: There was no difference in ET-1 and 5-HT-mediated constrictions. Endothelium-dependent relaxation was impaired in diabetes. Bosentan improved sensitivity to Ach as well as the maximum relaxation. Myogenic-tone is decreased over the course of the disease. Both treatments improved the ability of MCAs to develop tone at 80 mm Hg and only bosentan improved the tone at higher pressures. Significance: These results suggest that contractile response is not affected by glycemic control or ET-receptor antagonism. Meanwhile, dual ET-receptor blockade is effective in partially improving endothelium-dependent relaxation and myogenic response in a blood pressure-independent manner even after established cerebrovascular dysfunction and offers therapeutic potential. © 2014 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

Introduction Diabetes increases the risk and severity of cerebrovascular diseases such as ischemic stroke and vascular cognitive impairment (Kodl and Seaquist, 2008; Ergul et al., 2012). Diabetes is also associated with decreased cerebral blood flow, which is increasingly recognized as a major factor contributing to the development and progression of cognitive deficits in this population (Kodl and Seaquist, 2008). Undoubtedly, regulation of cerebrovascular tone is important for maintenance of proper blood flow (Faraci and Heistad, 1990). Myogenic and agonistinduced reactivity of the cerebral vasculature play a key role in controlling cerebral blood flow, and large arteries like basilar artery

⁎ Corresponding author at: Department of Physiology, Georgia Regents University, 1120 15th street CA-2094, Augusta, Georgia 30912, USA. Tel.: + 1 706 721 9103; fax: + 1 706 721 7299. E-mail address: [email protected] (A. Ergul).

contribute significantly to regulation of cerebrovascular resistance (Faraci and Heistad, 1990). Thus, vascular dysfunction could contribute to cerebrovascular disease, and indeed, a number of studies have shown that diabetes alters myogenic tone and impairs endotheliumderived relaxation in experimental models (Dumont et al., 2003; Harris et al., 2008; Kelly-Cobbs et al., 2012). We have shown that myogenic tone is increased early in disease but as cerebral blood vessels remodel, they lose their ability to develop tone, and glycemic control started at the onset of diabetes prevents this change in myogenic tone (A. KellyCobbs et al., 2011; A.I. Kelly-Cobbs et al., 2011). Whether and to what extent diabetes-mediated cerebrovascular dysfunction can be reversed if glycemic control is initiated later in the disease is not known. It is well established that the potent vasoconstrictor endothelin-1 (ET-1) and cognate receptors, ETA and ETB (Goto et al., 1996), are activated in both clinical and experimental diabetes (Takahashi et al., 1990; Collier et al., 1992). ETA receptors residing on the smooth muscle cell (SMC) produce vasoconstriction and mediate the proliferative effects of ET-1, while endothelial ETB counteracts these effects. However,

http://dx.doi.org/10.1016/j.lfs.2013.12.231 0024-3205 © 2014 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

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we have shown that 1) there is an upregulation of the VSMC ETB receptors in the cerebrovasculature in diabetes (A. Kelly-Cobbs et al., 2011; A.I. Kelly-Cobbs et al., 2011); and 2) selective ETA, selective ETB or dual ET receptor blockade is vasculoprotective and prevents diabetesmediated cerebrovascular dysfunction (Harris et al., 2005; A. KellyCobbs et al., 2011; A.I. Kelly-Cobbs et al., 2011). We further demonstrated that glycemic control prevents activation of the ET system in diabetes (Sachidanandam et al., 2009), which is associated with improved vascular function (A. Kelly-Cobbs et al., 2011; A.I. KellyCobbs et al., 2011). While these studies provided important evidence with regard to preventive cerebrovascular protective role of ET-1 antagonism, the therapeutic efficacy remained unknown. In this study, we tested the hypothesis that dual ET-1 receptor antagonism reverses established myogenic and endothelial dysfunction in diabetes.

because negative pressure is generated at 0 mm Hg, causing the vessel to collapse. All vessels were exposed to each pressure point for 5 min before readings were recorded. Pressure-diameter curves were obtained, first in the presence of Ca2+ to observe the vessels' contractile properties, and then in Ca2+-free buffer with the addition of 10−7 M papaverine hydrochloride to evaluate the vessels' passive properties. Using the outer diameter (OD) measurements obtained in active conditions (in the presence of Ca2+) and in passive conditions (in the absence of Ca2+), Percent Myogenic Tone (% tone) = 1 − (Active OD / Passive OD) × 100 was calculated over the entire pressure range (20–180 mmHg) and tones at 80 and 120 mm-Hg were reported to represent mid and high end of the pressure curve, respectively.

Materials and methods

Isometric tension exerted by BAs was recorded via a force transducer using the wire-myograph technique (Danish Myo Technologies, Denmark). The myograph chambers were filled with Krebs buffer (NaCl 118.3, NaHCO3 25, KCl 4.7, MgSO4 1.2, KH2PO4 1.2, CaCl2 1.5 and Dextrose 11.1 mM), gassed with 95% O2 and 5% CO2 and maintained at 37 °C. Vessel segments were mounted in the chamber using 40 μmthin wires and adjusted to a baseline tension of 0.4 g. Buffer was then switched to 70 mM KCl containing Krebs buffer in which NaCl concentration was reduced to achieve similar osmolarity and endothelial integrity was checked by relaxation response to 1 μM acetylcholine (Ach). Chambers were washed, refilled with regular Krebs buffer and cumulative dose–response curves to serotonin 5-HT (1–1000 nM) and ET-1 (0.1–500 nM) were generated. The force generated was expressed as % change of KCl. Endothelium-dependent relaxation to Ach (1 nM– 1 μM) was assessed directly after ET-1 dose–response. Sensitivity (EC50) and maximum response (Rmax) values were calculated from the respective dose–response equations.

Animals All experiments were performed using male Wistar rats (Harlan; Indianapolis, ID) and age-matched diabetic GK rats (In-house bred, derived from the Tampa colony or purchased from the Tampa colony, Taconic; Hudson, NY). We have chosen this non-obese model of type 2 diabetes because diabetes-induced cerebrovascular alterations can be studied in the absence of confounding comorbidities such as hypertension or hyperlipidemia. The animals were housed at the Georgia Regents University animal care facility that is approved by the American Association for Accreditation of Laboratory Animal Care. All protocols were approved by the institutional animal care and use committee. Animals were fed standard rat chow and tap water ad libitum. Starting at 18 weeks of age after the development of diabetesinduced cerebrovascular dysfunction, the following groups (n = 5–6/ group) were treated for 4 weeks: 1) GK vehicle, 2) GK + metformin (300 mg/kg/day in drinking water which we showed to achieve effective glycemic control in previous studies (Sachidanandam et al., 2009; Elgebaly et al., 2010)), 3) GK + bosentan (100 mg/kg/day by oral gavage), and 4) control Wistar vehicle. In additional groups (n = 5/ group) of 10 and 18 week-old diabetic GK rats, myogenic reactivity was measured to investigate the impact of disease duration on tone development. Body weights and blood glucose measurements were taken biweekly. Blood glucose measurements were taken from tail vein samples using a commercially available glucometer (Freestyle, Abbott Diabetes Care, Inc.; Alameda, CA). Mean arterial pressure (MAP, mm Hg) was measured using the tail-cuff method. All animals were anesthetized with pentobarbital sodium (Fatal-Plus, Vortech Pharmaceuticals Ltd.; Dearborn, MI), exsanguinated via cardiac puncture, and decapitated to extract the brain. Middle cerebral arteries (MCA) and basilar arteries (BA) were isolated for functional studies with pressurized arteriograph and myograph, respectively. Myogenic function studies MCAs were quickly excised and used within 45 min of isolation to ensure viability of the vessels. A pressure arteriograph system (Living Systems; Burlington, VT) was used to evaluate MCA myogenic reactivity and tone. For these studies, MCA segments approximately 200–250 μm in diameter and proximal to the junction between the MCA and the inferior cerebral vein were used exclusively. Vessels were first mounted onto glass cannulas in an arteriograph chamber and HEPES bicarbonate buffer (in mM: 130 NaCl, 4 KCl, 1.2 MgSO4, 4 NaHCO3, 10 HEPES, 1.18 KH2PO4, 5.5 glucose, 1.8 CaCl2) was circulated and maintained at 37 ± 0.5 °C. MCA segments were then pressurized at 60 mm Hg for 1 h to generate spontaneous tone. A video dimension analyzer connected to the arteriograph system was used to measure media thickness (MT) and lumen diameter (LD) at pressures ranging from 5 to 180 mm-Hg, in 20 mm-Hg increments. The first measurement was taken at 5 mm Hg

Endothelial function studies

Statistical analysis Results are given as mean ± SEM. For EC50 and Rmax values, analysis of variance (ANOVA) was performed with a post-hoc Tukey test. A repeated measures ANOVA was used to determine group differences (Diabetic vs. Control) across the ET-1 or Ach concentrations. Post-hoc group comparisons at each concentration used a Tukey's adjustment for the multiple comparisons. Graphpad Prism 5.0 was used for all statistical tests performed. Results Effect of bosentan and metformin on metabolic profile Body weight was slightly lower and blood glucose higher in diabetic animals as compared to control. Treatment with metformin did not affect body weight or blood pressure but bosentan increased blood pressure in diabetic animals (Table 1). Effect of bosentan and metformin on contractile response ET-1 mediated constriction is a dose dependent manner (Fig. 1A) but there was no difference among the groups with respect to maximal contraction or sensitivity to ET-1 (Fig. 1B and C). KCl and HT-induced contractions were also similar in all groups (data not shown). Effect of bosentan and metformin on relaxation response Endothelium-dependent relaxation to Ach was significantly impaired; i.e. maximum relaxation was less and EC50 was higher indicating reduced sensitivity, in diabetic animals as compared to age-matched control animals (Fig. 2A). While it did not reach statistical significance, only bosentan increased relaxation (Rmax) despite elevated blood pressure


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Table 1 Animal characteristics (*P b 0.05).

Body weight (g) Blood glucose (mg/dl) Mean arterial pressure (mm-Hg)

Control

Diabetes

Diabetes + metformin

Diabetes + bosentan

443 ± 14

371 ± 5*

371 ± 6*

377 ± 12*

112 ± 9

228 ± 26*

126 ± 5

199 ± 20*

97.6 ± 4

108 ± 5

105 ± 5

120 ± 5*

(Fig. 2B). The response curve in the metformin group was almost flat and therefore nonlinear regression analysis is not dependable to report an EC50 value. Bosentan, however, improved sensitivity to Ach (Fig. 2C). Effect of treatment and duration of disease on myogenic response Myogenic tone was lower in diabetic rats as compared to agematched control group (Fig. 3A). Treatment with metformin and bosentan restored tone at 80 mm-Hg pressure but only bosentan was effective in improving tone at higher pressure (Fig. 3A). Diabetic animals developed higher pressure shortly after onset of diabetes at

A

Control (C) Diabetes (D) D+Bosentan (D+B) D+Metformin (D+M)

200

Discussion We have shown that dual ET receptor antagonism is an effective preventive vasculoprotection strategy in decreasing hypersensitivity to ET-1 and improving vascular relaxation of basilar arteries in diabetes when treatment is started at the onset of diabetes. The current study was designed to test the hypothesis that dual ET-1 receptor antagonism will be as effective in reversing established myogenic and endothelial dysfunction in diabetes. Our results demonstrate that 1) bosentan treatment is as effective, if not better, as glycemic control with metformin in

A Relaxation (%ET-1)

Contraction (% KCl)

250

10 weeks of age but by 18 weeks myogenic tone decreased and remained lower till 22 weeks (Fig. 3B).

150 100 50 0

60

C D D+B D+M

#

40 20 0 -10

-10

-9

-8

-7

-9

B

300

200

-7

-6

-5

80

Rmax (% ET-1)

B

-8

Ach (Log M)

-6

ET-1(log M)

Rmax (%KCl)

80

60 40 20

100 0 C

D

D+M

D+B

0 C

D

D+M

D+B

C

150

EC50 (nM)

C EC50 (nM)

150

100

*

100

50

ND 50

0 C

0 C

D

D+M

D+B

Fig. 1. Effect of glycemic control and endothelin receptor antagonism on ET-1-induced isometric contractions in basilar arteries in diabetes. (A) Dose–response curves to ET-1 in control (C), diabetes (D), diabetes + bosentan (D + B) and diabetes + metformin (D + M) groups were similar. There was no difference in maximum response (B) or sensitivity (C) to ET-1 among the groups. Contractile response was expressed as % KCl response and results are given as mean ± SEM, n = 5–6/group.

D

D+M

D+B

Fig. 2. Effect of glycemic control and endothelin receptor antagonism on endotheliumdependent vasorelaxation in basilar arteries in diabetes. (A) Dose–response curves to Ach in ET-1-preconstricted vessels showed severely impaired vasorelaxation in diabetic animals. (B) Maximum relaxation to Ach (Rmax) was significantly lower in diabetic animals. While metformin had no effect on Rmax, bosentan improved it. (C) Sensitivity to Ach was decreased in diabetes as indicated by greater EC50 values. Bosentan improved sensitivity. Since the metformin dose–response curve was flat, EC50 was not determined (ND) for this group. Results are given as mean ± SEM, n = 5–6/group. #p b 0.001, *p b 0.05 vs. C, **p b 0.05 vs. D + M.

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A 80 mmHg

Myogenic tone %

20

120 mmHg

15

10

5

0 C

D

D+M D+B

C

D

D+M D+B

B Myogenic tone %

40

80 mmHg

120 mmHg

30

##

20

10

## #

#

D 22 22 w w D D D+ 22 w M D +B

D

w

w

18

10

D 22 22 w w D D D+ 22 w M D +B

w 18

10

w

D

0

Fig. 3. Effect of glycemic control and endothelin receptor antagonism on pressure-induced myogenic tone of isolated MCAs. (A) Myogenic tone at 80 and 120 mm-Hg was lower in diabetic animals and bosentan improved tone development. (B) MCA myogenic tone was decreased during the disease progression in diabetes and only bosentan improved tone at higher pressure points. Mean ± SEM, n = 5–6, *p b 0.05 vs. control, **p b 0.05 vs. D, #p b 0.05 vs. 10 w, ##p b 0.05 vs. 22w D.

restoring impaired vasorelaxation and improving myogenic tone of larger cerebral arteries, 2) this protective effect is independent of changes in blood pressure, and 3) the gradual decline in myogenic tone of middle cerebral arteries in early diabetes does not further progress in the disease process. Accumulating evidence suggests that cerebrovascular disease and alterations in cerebral blood flow (CBF) are important for stroke and neurodegenerative diseases such as dementia and Alzheimer's disease in patients with diabetes (Kodl and Seaquist, 2008; Ergul et al., 2012). CBF is tightly regulated to meet the constant energy demands and proper function of the brain (Iadecola and Nedergaard, 2007). Cerebrovascular autoregulation is essential for maintaining constant CBF despite changes in cerebral perfusion pressure and this is achieved by the regulation of vascular tone by myogenic, neuronal, and liganddependent mechanisms (Faraci and Heistad, 1998). Large arteries like BAs and MCAs contribute significantly to cerebrovascular resistance (Faraci and Heistad, 1990; Palomares and Cipolla, 2011). Thus, a number of studies including our own investigated the impact of diabetes on vascular function in these vascular beds and reported enhanced constriction, reduced vasodilatation and/or impaired myogenic reactivity leading to increased tone and reduced CBF (Zimmermann et al., 1997; Jarajapu et al., 2008; Ergul et al., 2012; Kelly-Cobbs et al., 2012). We have shown that myogenic tone is increased and is associated with reduced CBF in early diabetes (10 weeks) as compared to nondiabetic counterparts but as disease progresses MCAs remodel and develop less tone by 18 weeks of age (A. Kelly-Cobbs et al., 2011; A.I. KellyCobbs et al., 2011; Kelly-Cobbs et al., 2012). Glycemic control with metformin prevented this decrease in MCA tone when the treatment was started at the onset of diabetes. In the current study, we used both BAs and MCAs to assess agonist-induced (contractile and dilatory function) and pressure-induced (myogenic function) vasoreactivity. The

current study further expanded our previous observations and showed that decreased tone persists up to 22 weeks and it can be partially restored to control levels even if blood glucose is regulated after the onset of myogenic dysfunction. Endothelial dysfunction is a prominent feature of cardiovascular diseases and also plays an important role in both micro and macrovascular complications of diabetes (Jansson, 2007; Versari et al., 2009). In early stages, the imbalance of increased vasoconstrictors like ET-1 and decreased bioavailability of vasodilator nitric oxide (NO) due to hyperglycemia-driven oxidative stress results in impaired vasorelaxation (Kalani, 2008). As the disease progresses, the prolonged loss of protective effects of NO and the activation of the ET system lead to structural alterations, thrombosis and plaque development in the vessel wall (Kalani, 2008; Ergul, 2011). Increasing evidence suggests that ET-1 is involved in the pathology of cerebrovascular disease (Khan and Chakrabarti, 2003; Ergul, 2011). Contractile responses to ET-1 are augmented in the rat and rabbit basilar arteries of type 1 diabetic rats, respectively (Alabadi et al., 2004; Matsumoto et al., 2004), and augmented myogenic tone is decreased after ET receptor antagonism in type 1 diabetes (Dumont et al., 2003). We have shown that early in the disease basilar arteries from diabetic GK rats exhibit increased sensitivity to ET-1 (Harris et al., 2008). Endothelium-dependent relaxation was also impaired in this study. In the current study, we used older animals and did not observe a difference in the contractile response to ET-1 between control and diabetic animals; however, endothelial dysfunction remained to be a prominent feature of cerebrovascular dysfunction observed in this model and even further suppressed. Given the potential involvement of ET-1 in cerebral complications of diabetes, in a series of studies we investigated the relative role(s) of ET receptors in diabetic cerebrovascular dysfunction. While selective ETA receptor blockade restored relaxation to control values in the GK animals and selective ETB blockade caused paradoxical constriction in diabetes (Harris et al., 2008) suggesting that there may be an upregulation of vascular smooth muscle ETB receptors in diabetes. We next showed that dual antagonism to be as effective as selective ETA blockade in prevention of basilar artery dysfunction in diabetes and suggested that when blocked simultaneously with the ETA receptor, the ETB receptor antagonism is protective by improving cerebrovascular endothelial dysfunction in diabetes (Li et al., 2011). Building on this foundation, the current study evaluated the efficacy of dual receptor blockade with bosentan in reversing established endothelial and myogenic dysfunction in later stages of diabetes. Bosentan not only improved sensitivity to Ach as also seen with metformin but it also improved maximum relaxation which was different than complete lack of effect of metformin treatment on this end point, which was intriguing. Given that hyperglycemia is a potent stimulant for ET-1 synthesis, one would predict that with glycemic control ET-1 levels would be normalized as we have shown with early glycemic control (Sachidanandam et al., 2009). In this study, we did not measure circulating and tissue ET-1 levels. The lack of effect of metformin on maximum relaxation may be due to failure of suppressing ET-1 levels and needs to be confirmed. Bosentan also improved myogenic tone, which was quite pronounced at higher end of the pressure-response curve. ET-1, being the most potent vasoconstrictor, bosentan-mediated improvement of tone may sound counterintuitive. Myogenic reactivity is the inherent ability of vascular smooth muscle to respond to changes in pressure the vessels are exposed to and plays a critical role in certain vascular beds including cerebral, coronary and renal circulation to regulate blood flow. While enhanced tone is detrimental, lack of tone development is also detrimental as the vessels lose their ability to regulate lumen diameter and hence blood flow. Myogenic reactivity in response to changes in pressure the vessels are exposed to requires proper actin cytoskeleton arrangement. We have shown that oxidative stress, peroxynitrite in particular, nitrates filamentous F-actin and impairs myogenic tone development (Kelly-Cobbs et al., 2012). ET-1, a potent pro-oxidant, may be acting through this mechanism which needs to be further


confirmed by studying total nitrotyrosine formation and actin nitration in bosentan-treated animals. It also has to be noted that the autoregulatory range for middle cerebral arteries is 80 to 120 mm Hg, after which vessels show forced dilation. The finding that bosentan improves tone even at higher end of this range in diabetic animals suggests that bosentan provides protection at higher pressures. Interestingly, blood pressure was increased in bosentan-treated animals. We observed this effect in our previous studies which employed younger rats and used both tail-cuff and telemetry approaches to measure blood pressure (A. Kelly-Cobbs et al., 2011; A.I. Kelly-Cobbs et al., 2011; Li et al., 2011). While we do not have an explanation for this finding as bosentan has been reported to lower blood pressure in other studies involving hypertensive animal models, blood pressure observed is similar to what we have reported with selective ETB blockade (A. Kelly-Cobbs et al., 2011; A.I. Kelly-Cobbs et al., 2011). It is also possible that the renal ET system is altered in diabetic rats contributing to elevated blood pressure with treatment. Nevertheless, the important conclusion is that dual receptor blockade restores cerebrovascular function independent of changes in blood pressure in diabetes. There are several limitations of the current study that need to be recognized. First, we did not study mechanisms underlying cerebrovascular endothelial and myogenic function in diabetes and whether treatment with metformin or bosentan affects different signaling pathways. Second, our goal was to determine whether established vascular dysfunction in diabetes can be reversed and how this compares to age-matched control animals. As such we did not include control animals treated with metformin or bosentan. Third, we did not study the impact of glycemic control on local and circulating ET-1 levels and cerebrovascular ET receptor distribution to determine whether the protective effects of metformin are mediated via modulation of the ET system. Nevertheless, numerous studies have shown that glycemic control is an effective strategy in prevention and reduction of microvascular complications of diabetes, however, the impact of glycemic control on cerebrovascular complications remains unknown (Stettler et al., 2006; Akalin et al., 2009). Finally, there is evidence that ETB receptors may not be involved in mediating vasoconstriction of human pial arteries (Pierre and Davenport, 1999). Whether this applies to entire cerebral circulation or limited to vial vessels in humans is not known but it is an important point to be considered in the evaluation of the therapeutic potential of ET receptor antagonists. Taken together with our previous studies, the findings of the current study suggest that glycemic control offers preventive and some therapeutic potential for cerebrovascular dysfunction in diabetes. Late ET receptor antagonism, however, is more effective in improving endothelial and especially myogenic function in the cerebrovasculature in experimental diabetes. The impact of chronic regulation of cerebrovascular function by these approaches on cerebrovascular complications including cognitive impairment and stroke warrants future research. Conflict of interest statement We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.

Acknowledgments Adviye Ergul is a research pharmacologist at the Charlie Norwood Veterans Affairs Medical Center in Augusta, Georgia. This work was supported in part by VA Merit Award (BX000347) and NIH award (NS054688) to Adviye Ergul. The authors declare no conflict of interest. The contents do not represent the views of the Department of Veterans Affairs or the United States Government.

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References Akalin S, Berntorp K, Ceriello A, Das AK, Kilpatrick ES, Koblik T, et al. Intensive glucose therapy and clinical implications of recent data: a consensus statement from the Global Task Force on Glycaemic Control. Int J Clin Pract 2009;63(10):1421–5. Alabadi JA, Miranda FJ, Llorens S, Centeno JM, Marrachelli VG, Alborch E. Mechanisms underlying diabetes enhancement of endothelin-1-induced contraction in rabbit basilar artery. Eur J Pharmacol 2004;486(3):289–96. Collier A, Leach JP, McLellan A, Jardine A, Morton JJ, Small M. Plasma endothelin-like immunoreactivity levels in IDDM patients with microalbuminuria. Diabetes Care 1992;15:1038–40. Dumont AS, Dumont RJ, McNeill JH, Kassell NF, Sutherland GR, Verma S. Chronic endothelin antagonism restores cerebrovascular function in diabetes. Neurosurgery 2003;52(3):653–60. [discussion 659–660]. Elgebaly MM, Prakash R, Li W, Ogbi S, Johnson MH, Mezzetti EM, et al. Vascular protection in diabetic stroke: role of matrix metalloprotease-dependent vascular remodeling. J Cereb Blood Flow Metab 2010;30(12):1928–38. Ergul A. Endothelin-1 and diabetic complications: focus on the vasculature. Pharmacol Res 2011;63(6):477–82. Ergul A, Kelly-Cobbs A, Abdalla M, Fagan SC. Cerebrovascular complications of diabetes: focus on stroke. Endocr Metab Immune Disord Drug Targets 2012;12(2):148–58. Faraci FM, Heistad DD. Regulation of large cerebral arteries and cerebral microvascular pressure. Circ Res 1990;66(1):8–17. Faraci FM, Heistad DD. Regulation of the cerebral circulation: role of endothelium and potassium channels. Physiol Rev 1998;78(1):53–97. Goto K, Hama H, Kasuya Y. Molecular pharmacology and pathophysiological significance of endothelin. Jpn J Pharmacol 1996;72:261–90. Harris AK, Hutchinson JR, Sachidanandam K, Johnson MH, Dorrance AM, Stepp DW, et al. Type 2 diabetes causes remodeling of cerebrovasculature via differential regulation of matrix metalloproteinases and collagen synthesis: role of endothelin-1. Diabetes 2005;54(9):2638–44. Harris AK, Elgebaly MM, Li W, Sachidanandam K, Ergul A. Effect of chronic endothelin receptor antagonism on cerebrovascular function in type 2 diabetes. Am J Physiol 2008;294(4):R1213–9. Iadecola C, Nedergaard M. Glial regulation of the cerebral microvasculature. Nat Neurosci 2007;10(11):1369–76. Jansson PA. Endothelial dysfunction in insulin resistance and type 2 diabetes. J Intern Med 2007;262(2):173–83. Jarajapu YP, Guberski DL, Grant MB, Knot HJ. Myogenic tone and reactivity of cerebral arteries in type II diabetic BBZDR/Wor rat. Eur J Pharmacol 2008;579(1–3):298–307. Kalani M. The importance of endothelin-1 for microvascular dysfunction in diabetes. Vasc Health Risk Manag 2008;4(5):1061–8. Kelly-Cobbs A, Elgebaly MM, Li W, Ergul A. Pressure-independent cerebrovascular remodelling and changes in myogenic reactivity in diabetic Goto-Kakizaki rat in response to glycaemic control. Acta Physiol (Oxf) 2011a;203(1):245–51. Kelly-Cobbs AI, Harris AK, Elgebaly MM, Li W, Sachidanandam K, Portik-Dobos V, et al. Endothelial endothelin B receptor-mediated prevention of cerebrovascular remodeling is attenuated in diabetes because of up-regulation of smooth muscle endothelin receptors. J Pharmacol Exp Ther 2011b;337(1):9–15. Kelly-Cobbs AI, Prakash R, Coucha M, Knight RA, Li W, Ogbi SN, et al. Cerebral myogenic reactivity and blood flow in type 2 diabetic rats: role of peroxynitrite in hypoxia-mediated loss of myogenic tone. J Pharmacol Exp Ther 2012;342(2): 407–15. Khan ZA, Chakrabarti S. Endothelins in chronic diabetic complications. Can J Physiol Pharmacol 2003;81(6):622–34. Kodl CT, Seaquist ER. Cognitive dysfunction and diabetes mellitus. Endocr Rev 2008;29(4):494–511. Li W, Sachidanandam K, Ergul A. Comparison of selective versus dual endothelin receptor antagonism on cerebrovascular dysfunction in diabetes. Neurol Res 2011;33(2): 185–91. Matsumoto T, Yoshiyama S, Kobayashi T, Kamata K. Mechanisms underlying enhanced contractile response to endothelin-1 in diabetic rat basilar artery. Peptides 2004; 25(11):1985–94. Palomares SM, Cipolla MJ. Vascular protection following cerebral ischemia and reperfusion. J Neurol Neurophysiol 2011. [doi:pii: S1- 004]. Pierre LN, Davenport AP. Blockade and reversal of endothelin-induced constriction in pial arteries from human brain. Stroke 1999;30(3):638–43. Sachidanandam K, Hutchinson JR, Elgebaly MM, Mezzetti EM, Dorrance AM, Motamed K, et al. Glycemic control prevents microvascular remodeling and increased tone in type 2 diabetes: link to endothelin-1. Am J Physiol 2009;296:R952–9. Stettler C, Allemann S, Juni P, Cull CA, Holman RR, Egger M, et al. Glycemic control and macrovascular disease in types 1 and 2 diabetes mellitus: meta-analysis of randomized trials. Am Heart J 2006;152(1):27–38. Takahashi K, Ghatei MA, Lam HC, O'Halloran DJ, Bloom SR. Elevated plasma endothelin in patients with diabetes mellitus. Diabetologia 1990;33:306–10. Versari D, Daghini E, Virdis A, Ghiadoni L, Taddei S. Endothelial dysfunction as a target for prevention of cardiovascular disease. Diabetes Care 2009;32(Suppl. 2):S314–21. Zimmermann PA, Knot HJ, Stevenson AS, Nelson MT. Increased myogenic tone and diminished responsiveness to ATP-sensitive K+ channel openers in cerebral arteries from diabetic rats. Circ Res 1997;81(6):996–1004.