Kamakshi Sachidanandam

Glycemic control prevents microvascular remodeling and increased tone in Type 2 diabetes: link to endothelin-1

Medial thickening and vascular hypertrophy of resistance arteries can lead to cardiovascular complications associated with diabetes. While previous studies have established a role of type 1 diabetes in vascular remodeling, we recently extended these observations to type 2 diabetes and reported increased collagen deposition due to alterations in matrix metalloproteinase expression and activity in mesenteric resistance arteries. These studies also showed that remodeling response was mediated by endothelin-1 (ET-1) via activation of ET(A) receptors, whereas blockade of ET(B) receptors exacerbated the remodeling. However, the effectiveness of glycemic control strategies in preventing these vascular changes, including activation of the ET system still remained unclear. Also, very little is known about whether and to what extent reorganization of the extracellular matrix (ECM) affects vascular compliance and vasomotor tone. Accordingly, this study assessed structural remodeling of mesenteric microvessels, vascular compliance, and myogenic tone, as well as the role of matrix metalloproteinases (MMP) in mediating these processes. Spontaneously diabetic, non-obese Goto-Kakizaki (GK) rats, a model for type 2 diabetes, and normoglycemic Wistar rats were used for the studies. A subset of GK rats were administered metformin to achieve euglycemia. Glycemic control normalized the increased media-to-lumen ratios (M/L) and myogenic tone seen in diabetes, as well as normalizing plasma ET-1 levels and mesenteric ET(A) receptor expression. There was increased collagen synthesis in diabetes paralleled by decreased collagenase MMP-13 activity, while glycemic control attenuated the process. These findings and our previous study taken together suggest that hyperglycemia-mediated activation of ET-1 and ET(A) receptors alter vascular structure and mechanics in type 2 diabetes.

Effect of chronic endothelin receptor antagonism on cerebrovascular function in type 2 diabetes

Diabetes increases the risk of stroke and contributes to poor clinical outcomes in this patient population. Myogenic tone of the cerebral vasculature, including basilar arteries, plays a key role in controlling cerebral blood flow. Increased myogenic tone is ameliorated with ET receptor antagonism in Type 1 diabetes. However, the role of endothelin-1 (ET-1) and its receptors in cerebrovascular dysfunction in Type 2 diabetes, a common comorbidity in stroke patients, remains poorly elucidated. Therefore, we hypothesized that 1) cerebrovascular dysfunction occurs in the Goto-Kakizaki (GK) model of Type 2 diabetes, and 2) pharmacological antagonism of ETA receptors ameliorates, while ETB receptor blockade augments vascular dysfunction. GK or control rats were treated with antagonists to either ETA (atrasentan, 5 mg.kg(-1).day(-1)) or ETB (A-192621, 15 or 30 mg.kg(-1).day(-1)) receptors for 4 wk and vascular function of basilar arteries was assessed using a wire myograph. GK rats exhibited increased sensitivity to ET-1. ET(A) receptor antagonism caused a rightward shift, indicating decreased sensitivity in diabetes, while it increased sensitivity to ET-1 in control rats. Endothelium-dependent relaxation was impaired in diabetes. ETA receptor blockade restored relaxation to control values in the GK animals with no significant effect in Wistar rats and ETB blockade with 30 mg.kg(-1).day(-1) A-192621 caused paradoxical constriction in diabetes. These studies demonstrate that cerebrovascular dysfunction occurs and may contribute to altered regulation of myogenic tone and cerebral blood flow in diabetes. While ETA receptors mediate vascular dysfunction, ETB receptors display differential effects. These results underscore the importance of ETA/ETB receptor balance and interactions in cerebrovascular dysfunction in diabetes.

Microvascular Versus Macrovascular Dysfunction in Type 2 Diabetes: Differences in Contractile Responses to Endothelin-1:

Vascular dysfunction characterized by a hyperreactivity to vasoconstrictors and/or impaired vascular relaxation contributes to increased incidence of cardiovascular disease in diabetes. Endothelin (ET)-1, a potent vasoconstrictor, is chronically elevated in diabetes. However, the role of ET-1 in resistance versus larger vessel function in mild diabetes remains unknown. Accordingly, this study investigated vascular function of third-order mesenteric arteries and basilar arteries in control Wistar and Goto-Kakizaki (GK) rats, a model of mild Type 2 diabetes. Six weeks after the onset of diabetes, contractile responses to 0.1–100 nM ET-1 and relaxation responses to 1 nM–10 μM acetylcholine (ACh) in vessels preconstricted (baseline + 60%) with serotonin (5-HT) were assessed by myograph studies in the presence or absence of a nitric oxide synthase (NOS) inhibitor, N-nitro-l-arginine (L-NNA). Maximum contractile response to ET-1 was augmented in mesenteric vessels (155 ± 18% in GK vs. 81 ± 6% in control; n = 5–7) but not in the basilar artery (134 ± 29% in GK vs. 107 ± 17% in control; n = 4 per group). However, vascular relaxation was impaired in the basilar arteries (22 ± 4% in GK vs. 53 ± 7% in control; n = 4 per group) but not in mesenteric arteries of GK rats. Inhibition of NOS decreased the relaxation response of basilar arteries to 15 ± 8% and 42 ± 5% in GK and control rats, respectively; whereas, in resistance vessels, corresponding values were 56 ± 7% and 89 ± 3% (vs. 109 ± 2% and 112 ± 3% without NOS blockade), indicating the involvement of different vasorelaxation-promoting pathways in these vascular beds. These findings provide evidence that the ET system is activated even under mild hyperglycemia and that it contributes to the hyperreactivity of resistance vessels, therefore, the ET system may play an important role in elevated blood pressure in Type 2 diabetes.

Evidence for Vasculoprotective Effects of ETB Receptors in Resistance Artery Remodeling in Diabetes

OBJECTIVE—Vascular remodeling, characterized by extracellular matrix deposition and increased media-to-lumen (M/l) ratio, contributes to the development of microvascular complications in diabetes. Matrix metalloproteinases (MMPs) play an important role in the regulation of extracellular matrix (ECM) turnover and vascular remodeling. Vasoactive factor endothelin (ET)-1 not only causes potent vasoconstriction but also exerts profibrotic and proliferative effects that change vessel architecture, which makes it a likely candidate for a key role in vascular complications of diabetes. Thus, this study investigated the regulation of MMP activity of resistance arteries under mild-to-moderate diabetes conditions, as seen in type 2 diabetes, and the relative role of ET receptors in this process. RESEARCH DESIGN AND METHODS—Vessel structure, MMP activity, and ECM proteins were assessed in control Wistar and diabetic Goto-Kakizaki (GK) rats treated with vehicle, ETA receptor antagonist atrasentan (5 mg · kg−1 · day−1), or ETB receptor antagonist A-192621 (15 mg · kg−1 · day−1) for 4 weeks. RESULTS—M/l ratio was increased in diabetes. Atrasentan prevented this increase, whereas A-192621 caused further thickening of the medial layer. Increased MMP-2 activity in diabetes was prevented by atrasentan treatment. Collagenase activity was significantly decreased in diabetes, and while ETA antagonism improved enzyme activity, ETB blockade further reduced collagenase levels. Accordingly, collagen deposition was augmented in GK rats, which was reversed by atrasentan but exacerbated with A-192621. CONCLUSIONS—ET-1 contributes to the remodeling of mesenteric resistance arteries in diabetes via activation of ETA receptors, and ETB receptors provide vasculoprotective effects.

Effect of chronic and selective endothelin receptor antagonism on microvascular function in Type 2 diabetes

Vascular dysfunction, which presents either as an increased response to vasoconstrictors or an impaired relaxation to dilator agents, results in worsened cardiovascular outcomes in diabetes. We have established that the mesenteric circulation in Type 2 diabetes is hyperreactive to the potent vasoconstrictor endothelin-1 (ET-1) and displays increased nitric oxide-dependent vasodilation. The current study examined the individual and/or the relative roles of the ET receptors governing vascular function in the Goto-Kakizaki rat, a mildly hyperglycemic, normotensive, and nonobese model of Type 2 diabetes. Diabetic and control rats received an antagonist to either the ET type A (ETA; atrasentan; 5 mg x kg(-1) x day(-1)) or type B (ET(B); A-192621; 15 or 30 mg x kg(-1) x day(-1)) receptors for 4 wk. Third-order mesenteric arteries were isolated, and vascular function was assessed with a wire myograph. Maximum response to ET-1 was increased in diabetes and attenuated by ETA antagonism. ETB blockade with 15 mg/kg A-192621 augmented vasoconstriction in controls, whereas it had no further effect on ET-1 hyperreactivity in diabetes. The higher dose of A-192621 showed an ETA-like effect and decreased vasoconstriction in diabetes. Maximum relaxation to acetylcholine (ACh) was similar across groups and treatments. ETB antagonism at either dose had no effect on vasorelaxation in control rats, whereas in diabetes the dose-response curve to ACh was shifted to the right, indicating a decreased relaxation at 15 mg/kg A-192621. These results suggest that ETA receptor blockade attenuates vascular dysfunction and that ETB receptor antagonism exhibits differential effects depending on the dose of the antagonists and the disease state.

Differential effects of diet-induced dyslipidemia and hyperglycemia on mesenteric resistance artery structure and function in type 2 diabetes.

Type 2 diabetes and dyslipidemia oftentimes present in combination. However, the relative roles of diabetes and diet-induced dyslipidemia in mediating changes in vascular structure, mechanics, and function are poorly understood. Our hypothesis was that addition of a high-fat diet would exacerbate small artery remodeling, compliance, and vascular dysfunction in type 2 diabetes. Vascular remodeling indices [media/lumen (M/L) ratio, collagen abundance and turnover, and matrix metalloproteinase dynamics], mechanical properties (vessel stiffness), and reactivity to pressure and vasoactive factors were measured in third-order mesenteric arteries in control Wistar and type 2 diabetic Goto-Kakizaki (GK) rats fed either a regular or high-fat diet. M/L ratios, total collagen, and myogenic tone were increased in diabetes. Addition of the high-fat diet altered collagen patterns (mature versus new collagen) in favor of matrix accumulation. Addition of a high-fat diet caused increased constriction to endothelin-1 (0.1–100 nM), showed impaired vasorelaxation to both acetylcholine (0.1 nM–1 μM) and sodium nitroprusside (0.1 nM–1 μM), and increased cardiovascular risk factors in diabetes. These results suggest that moderate elevations in blood glucose, as seen in our lean GK model of type 2 diabetes, promote resistance artery remodeling resulting in increased medial thickness, whereas addition of a high-fat diet contributes to diabetic vascular disease predominantly by impairing vascular reactivity in the time frame used for this study. Although differential in their vascular effects, both hyperglycemia and diet-induced dyslipidemia need to be targeted for effective prevention and treatment of diabetic vascular disease.

Endothelial Endothelin B Receptor-Mediated Prevention of Cerebrovascular Remodeling Is Attenuated in Diabetes Because of Up-Regulation of Smooth Muscle Endothelin Receptors

Structure and function of the cerebrovasculature is critical for ischemic stroke outcome. We showed that diabetes causes cerebrovascular remodeling by activation of the endothelin A (ETA) receptors. The goal of this study was to test the hypotheses that vasculoprotective endothelial ETB receptors are decreased and pharmacological inhibition of the ETB receptor augments vascular remodeling of middle cerebral arteries (MCAs) in type 2 diabetes. MCA structure, matrix metalloprotease (MMP) activity, and matrix proteins as well as ETA and ETB receptor profiles were assessed in control Wistar and diabetic Goto-Kakizaki rats treated with vehicle, the ETB receptor antagonist (2R,3R,4S)-4-(1,3-benzodioxol-5-yl)-1-[2-[(2,6-diethylphenyl)amino]-2-oxoethyl]-2-(4-propoxyphenyl)pyrrolidine-3-carboxylic acid (A192621) (30 mg/kg/day), or the dual ET receptor antagonist bosentan (100 mg/kg/day) for 4 weeks. Diabetes increased vascular smooth muscle (VSM) ETA and ETB receptors; the increase was prevented by chronic bosentan treatment. MCA wall thickness was increased in diabetes, and this was associated with increased MMP-2 activity and collagen deposition but reduced MMP-13 activity. Because of up-regulation of VSM ET receptors in diabetes, selective ETB receptor antagonism with A192621 blunts this response, and combined ETA and ETB receptor blockade with bosentan completely prevents this response. On the other hand, A192621 treatment augmented remodeling in control animals, indicating a physiological protective role for this receptor subtype. Attenuation of changes in ET receptor profile with bosentan treatment suggests that ET-1 has a positive feedback on the expression of its receptors in the cerebrovasculature. These results emphasize that ET receptor antagonism may yield different results in healthy and diseased states.

Comparison of selective versus dual endothelin receptor antagonism on cerebrovascular dysfunction in diabetes

Abstract Objectives: Cerebrovascular tone plays a key role in controlling cerebral blood flow. Our studies have demonstrated that the endothelin system is upregulated in type 2 diabetes leading to increased sensitivity to endothelin-1 and decreased relaxation in basilar artery. While chronic endothelin A receptor blockade restored relaxation, selective endothelin B receptor blockade caused paradoxical constriction in diabetes. Whether this effect was due to activation of endothelin A receptors in the presence of endothelin B receptor blockade or due to the loss of vasculoprotective effects of endothelin B receptors remained unknown. The current study hypothesizes that due to the antagonism of the vasculoprotective endothelin receptor B, dual blockade will not be as effective as selective endothelin receptor A antagonism in improving cerebrovascular dysfunction in type 2 diabetes. Methods: These studies were done in non-obese, type 2 diabetic Goto-Kakizaki rats administered either vehicle, selective endothelin receptor A antagonist Atrasentan (5 mg/kg) or dual endothelin antagonist Bosentan (100 mg/kg) for 4 weeks. At termination, basilar arteries were collected and mounted on a wire myograph and cumulative dose-response curves to endothelin-1 (1-500 nM) and acetylcholine (1 nM-5 μm) were studied. Results: Basilar artery was highly sensitive to endothelin-1-mediated constriction in diabetic animals. While neither Atrasentan nor Bosentan affected endothelium-dependent vascular relaxation in control animals, both treatments improved the maximum dilatation in diabetes and Atrasentan also improved sensitivity to acetylcholine. Conclusion: In light of our previous data which showed that endothelin B receptors are vasculoprotective and blockade of this receptor worsens relaxation, current findings suggest that when blocked simultaneously with the endothelin receptor A, the endothelin receptor B antagonism is protective by reducing the hyperreactivity and improving cerebrovascular function in diabetes.

Dual Endothelin Receptor Antagonism Prevents Remodeling of Resistance Arteries in Diabetes

Vascular remodeling, characterized by extracellular matrix deposition and increased media-to-lumen (M/L) ratio, contributes to the development of microvascular complications in diabetes. We have previously shown in type 2 diabetic Goto-Kakizaki (GK) rats that selective ETA receptor blockade prevents medial thickening of mesenteric arteries via regulation of matrix metalloproteases (MMP), whereas selective ETB receptor blockade augments this thickening. The goal of this study was to determine the effect of combined ETA and ETB receptor blockade on resistance vessel remodeling. Vessel structure, MMP activity, and extracellular matrix proteins were assessed in control Wistar and diabetic GK rats treated with vehicle or bosentan (100 mg/kg per day) for 4 weeks (n = 7-9 per group). Bosentan completely prevented the increase in M/L ratio and MMP-2 activity in diabetes but paradoxically increased M/L ratio and MMP activation in control animals. Collagenase (MMP-13) activity and protein levels were significantly decreased in diabetes. Accordingly, collagen deposition was augmented in GK rats. Dual ET receptor antagonism improved enzyme activity and normalized MMP-13 levels in diabetic animals but blunted MMP-13 activity in control animals. In summary, current findings suggest that diabetes-mediated remodeling of resistance arteries is prevented by dual blockade of ETA and ETB receptors and that the relative role of ET receptors in the regulation of vascular structure differs in the control and disease states.