Published online before print February 12, 2004, doi: 10.1161/01.ATV.0000122362.44628.09
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© 2004 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Diabetic Mouse Angiopathy Is Linked to Progressive Sympathetic Receptor Deletion Coupled to an Enhanced Caveolin-1 Expression
From the Department of Experimental Pharmacology (M.B., F.R., V.B., A.D.L., C.C., G.C.), Faculty of Pharmacy, University of Naples, Italy; Boyer Center for Molecular Medicine (M.I.L., W.C.S.), Yale University, New Haven, Conn; Department of Pharmaceutical Sciences (A.P.), Faculty of Pharmacy, University of Salerno, Italy; and Dipartimento di Medicina Sperimentale (S. Farneti, S. Fiorucci), Università di Perugia, Italy.
Correspondence to Dr Giuseppe Cirino, Professor of Pharmacology, Department of Experimental Pharmacology, University of Naples, Federico II, via Domenico Montesano 49, 80131 Naples, Italy. E-mail cirino{at}unina.it
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Abstract |
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Objective— Clinical studies have demonstrated that hyperglycaemia represents a major risk factor in the development of the endothelial impairment in diabetes, which is the first step in vascular dysfunction. Using non-obese diabetic mice, we have evaluated the role of the adrenergic system and eNOS on progression of the disease
Methods and Results— When glycosuria is high (20 to 500 mg/dL), there is a selective reduction in the response to 
1 and ß2 agonists but not to dopamine or serotonin. When glycosuria is severe (500 to 1000 mg/dL), there is a complete ablation of the contracture response to the 1 receptor agonist stimulation and a marked reduced response to ß2 agonist stimulation. This effect is coupled with a reduced expression of 1 and ß2 receptors, which is caused by an inhibition at transcriptional level as demonstrated by RT-PCR. In the severe glycosuria (500 to 1000 mg/dL), although eNOS expression is unchanged, caveolin-1 expression is significantly enhanced, indicating that high glucose plasma levels cause an upregulation of the eNOS endogenous inhibitory tone. These latter results correlate with functional data showing that in severe glycosuria, there is a significant reduction in acetylcholine-induced vasodilatation.
Conclusions— Our results show that in diabetes development, there is a progressive selective downregulation of the 1 and ß2 receptors. At the same time, there is an increased expression of caveolin-1, the endogenous eNOS inhibitory protein. Thus, caveolin-1 could represent a new possible therapeutic target in vascular impairment associated with diabetes.
Key Words: eNOS • caveolin-1 • adrenergic system • vascular impairment • diabetes
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Introduction |
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It is well established that the appearance of cardiovascular disorders is much more frequent and severe in diabetic than in healthy subjects.1,2 Clinical studies have shown that hyperglycaemia represents a major risk factor in the development of the endothelial impairment, which is the first step in vascular dysfunction.3–7 Up to 80% of deaths in patients with diabetes are closely associated with vascular diseases,8 including coronary atherosclerosis, macroangiopathy, autonomic neuropathy, and diabetic cardiomyopathy.9,10
Type 1 diabetes, or insulin-dependent diabetes mellitus (IDDM), is an autoimmune disease characterized by a islet inflammation or insulitis, followed by progressive destruction of pancreatic ß cells, followed by insulin secretion deficiency resulting in hyperglycaemia.11,12 The majority of current knowledge on the mechanisms involved in the hyperglycaemia-induced endothelial dysfunction has been obtained from vessels harvested from streptozocin-induced or alloxan-induced diabetic rabbits,13,14 rats,15–17 and mice.18,19 It has to be considered that these widely used animal models rely on a rapid and extensive Langerhans islet ß cell damage, characterized by the onset of high-level glycemia. This rapid shift from normal to high glycemia levels represents a critical limitation for these experimental models of IDDM because the appearance of hyperglycemia is not gradual as it is in humans. An alternative experimental approach is represented by non-obese diabetic mice (NOD/Ltj), a strain that spontaneously has autoimmune diabetes development with remarkable analogy to human IDDM.20,21 The disease is characterized by lymphocyte infiltration into the pancreatic islets, which progressively induces pancreatic ß cell necrosis leading to IDDM.22 Diabetes develops gradually in mice and its onset is 
week 15. Thus, this mouse strain represents an elective tool in investigating the vascular complications linked to diabetes development, because it is possible to follow-up the disease from its onset as well as during its progression. The relation between the disease progression and the onset of the vascular impairment has never been investigated in details. By using NOD mice, we have investigated on the role of sympathetic system by using aortas isolated from mice with different levels of glycosuria. In addition, we have also evaluated the involvement of eNOS and of its endogenous regulatory protein, caveolin-1 (CAV-1).
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Methods |
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Phenilephrine-Induced Vasoconstriction Is Reduced in NOD Aortic Rings and Is Closely Related to Glycosuria
To investigate whether diabetes causes changes in vascular reactivity, phenilephrine (PE)-induced cumulative concentration–response curve was performed in control and NOD mice that were divided in 3 groups according to glycosuria/glycemia levels (Figure 1). Because NOD group 1 mice demonstrated a similar pattern of response as did CD-1 mice, indicating that at this stage of the disease no pathological changes occurred, CD-1 and NOD group 1 mice have been considered as normal control response. In NOD groups 2 and 3, the PE-induced cumulative concentration–response curve was significantly reduced when compared with NOD group 1 and CD-1 mouse aortic rings (Figure 2A). In particular, PE-induced contractions in NOD group 2 were significantly reduced compared with NOD group 1 and CD-1 mouse aortic rings. In addition, NOD group 3 PE-induced contractions were abolished. Conversely, 5-HT-induced (Figure I, available online at http://atvb.ahajournals.org; 5-HT [A] and DA [B] on aortic rings harvested from NOD mice. 5-HT–induced and DA-induced cumulative concentration–response curves [10 nM to 30 µmol/L] were not significantly different in NODs and CD-1 mice. SNP-induced [C] cumulative concentration–response curves showed no significant difference between NOD groups 1, 3, and CD-1 mice. Data are presented as mean±SEM of percent of vasorelaxation, n=8 for each group) and DA-induced cumulative concentration–response curves were not significantly different between aortic rings from all NOD mouse groups and were similar to CD-1 mouse aortic rings. These observations showed that with diabetes progression, as assessed by glycosuria, NOD mice have a selective deficit to
1 adrenergic stimulation.
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Isop-Induced Vasorelaxation Is Reduced in NOD Aortic Rings and Is Closely Related to Glycosuria
Isop-induced cumulative–concentration response curve was performed to investigate whether the reduced response of 1 adrenergic receptors was shared with ß2 adrenergic receptors. As shown in Figure 2C, Isop-induced cumulative concentration–response curve was significantly reduced compared with NOD group 1 and CD-1 mice aortic rings. In particular, Isop-induced vasorelaxation in NOD group 2 results were significantly curtailed when compared with NOD group 1 and CD-1 mice aortic rings. Again, in NOD group 3, there was a major effect and Isop-induced vasorelaxation was markedly reduced. Thus, with diabetes progression, NOD mice appear to be less responsive to ß2 adrenergic stimulation.
RT-PCR and Western Blot Studies
The effects observed in the functional experiments suggested hypo-functionality of 1 and ß2 adrenergic receptors or a reduction in number of the adrenergic receptors. To further verify this hypothesis, we performed a Western blot analysis on NOD groups 1, 2, 3, and CD-1 aortas for 1 adrenergic receptor. As shown in Figure 3, 1 (Figure 3A) and ß2 (Figure 3B) receptor expression was reduced. This reduction was related to diabetes progression, with a remarkable reduction of receptor expression in NOD group 3 compared with NOD group 1 and CD-1 aortas (Figure 3A and 3B). These results reinforce the finding that with diabetes progression, an impairment of adrenergic function occurs in 1-mediated vasoconstriction and ß2-mediated vasorelaxation caused by a reduction of receptor expression. This view was further confirmed by the RT-PCR experiment (Figure 3C). PCR analysis demonstrated a significantly decreased 1 and ß2 mRNA expression in NOD 3 aortas in comparison with NOD 1 mice, indicating that the reduction of receptor expression was caused by an inhibition of their transcription.
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Results |
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Ach-Induced, but not SNP-Induced Vasorelaxation Is Reduced in NOD Aortic Rings
Ach causes an endothelium nitric oxide (NO)-dependent relaxation. In NOD group 3, the Ach-induced cumulative concentration response curve was significantly reduced compared with NOD groups 1, 2, and CD-1 mouse aortic rings (Figure 2D). Because PE-induced contraction in NOD groups 2 and 3, mouse aortic rings were weaker (see previous) when compared with CD-1 mice. Ach cumulative concentration–response curve was performed on 5-HT precontracted rings (Figure 2D). To verify the integrity of smooth muscle, SNP-induced vasorelaxation (NO donor) was also investigated. SNP-induced cumulative concentration–response curves showed no significant difference among aortic rings from all NOD groups and CD-1 mice (Figure I), indicating that diabetes does not directly influence smooth muscle relaxation mediated by NO. To define the involvement of basal NO release in Ach-induced vasorelaxation, the tonic tone was removed by using a NO inhibitor. Increase in tension development generated by the NO synthase inhibitor L-NAME (100 µmol/L) on 5-HT precontracted rings obtained from NOD groups 2 and 3 mice was significantly reduced compared with NOD group 1 and CD-1 mice (Figure 2B). This effect is more consistent in NOD group 3 aortic rings, suggesting that severe diabetic conditions result in an impairment of basal NO release.
Basal Release of NO, eNOS, and Caveolin-1 Involvement in NOD Aortic Rings
To investigate if the reduction of Ach-induced vasorelaxation was related to a reduction in eNOS expression, we performed Western blot analysis of eNOS expression on NOD groups 1, 2, 3, and CD-1 aortas. As shown in Figure 4A, there was no difference of eNOS expression in the NOD group 3 compared with the NOD groups 1, 2, and CD-1 aortas, whereas caveolin-1 expression was significantly enhanced in NOD group 3 mice (Figure 4B and 4C). This result suggests that the impairment of Ach-induced vasorelaxation in NODs is not caused by a reduced eNOS expression but by an enhanced negative modulation of eNOS by caveolin-1.
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Cell Experiments with Normal and High Glucose Environment
To test the effect of high glucose on eNOS activity (eg, NO production), we used 2 cell lines, BAEC and HEK293, stable transfected with eNOS.24 BAEC in normal glucose environment produces a basal release of NO of 2 nM of nitrite, whereas on stimulation with calcium ionophore A-23187 nitrite levels shift to 13 nM with a 6-fold increase in the response. When cells are cultured in high glucose levels, the basal level of NO release was not significantly affected whereas the stimulated release by A-21387 was significantly reduced by 50% (Figure 5A). This reduced NO release correlates with the increase in caveolin-1 expression in BAEC (Figure 5B). In the same experimental setting, NOS activity was determined by monitoring conversion of labeled arginine in citrulline. Treatment of BAEC with calcium ionophore A-23187 (10-5 M) in high-glucose environment caused a reduction of NOS activity of 70%, shifting the conversion of L-(3H)-arginine to L-(3H)-citrulline from 247±47 pmol/min per milligram of protein to 65±57 pmol/min per milligram of protein. These results further confirm that the effect observed in high-glucose environment in stimulated conditions is related to a reduction in eNOS activity rather than other mechanisms such as NO quenching. Next, to further demonstrate that the reduced NO release was caused by glucose effect on eNOS, we used HEK293 stably transfected with eNOS. As expected in these cells, the basal release of NO was higher and it was significantly increased by treatment with A-23187. When HEK293 were cultured in high-glucose environment, there was a marked reduction in NO basal production as well as in the stimulated condition (eg, A-23187-stimulated), thereby establishing that high-glucose environment negatively modulates eNOS activity (Figure 5C).
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Discussion |
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Szentivanyi and Pek first showed an altered vasodilatory response in IDDM patients in the arterial bed of the conjunctiva in 1973.23 Since then, in several studies it has been demonstrated that a substantial link between diabetes and vascular disorders culminate in atherosclerosis, with the most common complication being diabetes.24–28 In the past 10 years, much attention has been focused on a particular strain of mice whose diabetes strongly resembles the human disease (the NOD mice).29 However, these mice have been used to study diabetes on elevation of blood glucose over the physiological limit at different pathological values of glycemia or glycosuria and were selected for specific different experimental protocols. Thus, most of the results are not comparable, and studies on the linkage between diabetes progression and vascular functionality on this particular strain of mice are still lacking.
Vascular tone is driven by a dynamic process in which several signals that are either cell-mediated or receptor- mediated are involved. A major role is played by vascular adrenoreceptors, and this effect is coupled to the regulatory role played by the endothelium through mediator release. This complex and finely tuned interplay is disrupted by diabetes resulting in endothelial dysfunction. Our study shows that NOD group 1 mice, with null or low glycosuria, behave as normal outbred CD-1 mice. This result implies that it is important to use NOD mice with consistent glycosuria/glycemia to study angiopathy associated with diabetes. Indeed, in NOD mice with high glycosuria (NOD group 2 and group 3), an impaired vascular reactivity of the adrenergic system was consistently observed. In particular, high glycosuria (NOD group 2 mice) is associated with a reduction in 1 receptor expression, whereas a severe glycosuria (NOD group 3 mice) causes a complete ablation of the contractile response associated with a further reduction of 1 receptor expression. Similarly, the relaxing response to isoproterenol (ß2) is reduced in high and severe glycosuria associated with a reduction in ß2 receptor expression. PCR analysis performed on NOD group 3 mice aortas demonstrated that high glucose levels downregulated both receptors at transcriptional level. This experimental evidence finds a match with the human disease, in which it has been shown that in non-complicated IDDM there is a chronic decrease in sympathetic drive.30 In addition, it has been shown that insulin infusion increases sympathetic activity,31 suggesting that the lack of insulin in diabetes type 1 in humans is directly related to a reduction of the adrenergic function. Thus, it appears that diabetes progression is linked to a selective disruption of the sympathetic drive. To evaluate if in this animal model there is such selectivity, we have assessed if vascular reactivity impairment in NOD mice is specific for adrenergic receptors by using another 2 contracture agents, eg, 5-HT and DA. Aortic rings from NOD group 3, in which there is a complete lack of response to 1 stimulation, still contracted to 5-HT and DA, further stressing that diabetic conditions caused a selective impairment of the adrenergic function. It is also important to note that NOD group 2, in which glycosuria ranges between 20 mg/dL and 500 mg/dL, displays the same impaired response to adrenergic stimulation and reduced expression of 1 and ß2 receptors.
Another important role in controlling vascular homeostasis is played by eNOS-derived NO. Thus, we have investigated the role of endothelium by measuring NO-dependent response in the aortic rings. A significant reduction in Ach-induced vasorelaxation was observed in mice with severe glycosuria, whereas at all the other levels of glycosuria the response to Ach was unchanged. Aortas from NOD 1, 2, and 3 relaxed similarly with exogenous NO. These results imply that L-arginine/NO pathway is the last to be impaired by the progression of the disease; and vessels, even at very high levels of glycosuria, still maintain at least in part their ability to relax with endogenous agonist, eg, Ach. It is known that vessels generate a tonic amount of NO that is dynamically and constantly produced by eNOS and that removal of this tonic activity by addition of an NO inhibitor causes an increase in vascular tone in vitro and blood pressure in vivo. When rings from NOD group 2 and 3 mice were challenged with L-NAME, there was a marked and significant reduction in the increase in tension indicating that in the presence of high or severe glycosuria, an impairment of eNOS activation/activity occurs. Thus, in this mouse strain reduction in vascular adrenoreceptor expression is also coupled with a reduction in formation of eNOS-derived NO. Also, this finding fits with the demonstration that IDDM patients have an impairment in NO release.32–34 Western blot analysis of aortas of NOD groups and CD-1 demonstrated that there are no differences in eNOS expression, indicating that the reduction of NO release in diabetes is not linked to a reduced expression of the enzyme. This finding is further corroborated by the cell studies performed in high-glucose and normal glucose environments. Whereas in BAEC grown in high glucose levels basal NO release is not affected, a significant reduced production of NO in stimulated condition (eg, calcium ionophore A23917) is present. The finding that the high-glucose environment specifically modulates eNOS activity is further confirmed by the reduced conversion of labeled arginine in citrulline. In addition, in HEK 293 stably transfected with eNOS, there is a significant reduced production of NO in basal and stimulated conditions in the high-glucose environment. eNOS activity is regulated by dynamic subcellular targeting, regulatory protein–protein interactions,35,36 and protein phosphorylation,37,38 many of which can be modulated by stimuli in a calcium-dependent or calcium-independent manner.35,36 In general, in the basal state, eNOS is negatively regulated by several proteins, thus producing low levels of NO. eNOS is located within the plasma membrane microdomain caveolae, where it is complexed with the coat protein for this organelle, termed CAV-1. CAV-1 is a 22-kDa protein that decorates the cytoplasmic surface of caveolae and serves as the primary structural component of caveolae (50 to 100 nm invaginations of the plasma membrane), where it acts as a physiological inhibitor of eNOS.39,40 On activation of endothelial cells by a cadre of mediators, the caveolin-1 inhibitory clamp is diminished by the recruitment of several proteins that promote an activation complex.41 For this reason, we have evaluated whether diabetic conditions could modulate caveolin-1 expression downregulating through this mechanism of eNOS activity. Western blot analysis for caveolin-1 shows a significant increase of caveolin-1 expression in NOD group 3 aortas only, and this is in agreement with the functional data in which a significant reduction to Ach response has been found in NOD group 3 only. Furthermore, a similar increase in caveolin-1 coupled with an increase in NO production is present in BAEC cells in high-glucose environments. Thus, the reduction of NO release in IDDM can be ascribed to a reduction in eNOS activity through an enhanced expression of caveolin-1. This hypothesis is corroborated by a study in which it has been shown that in adipocyte plasma membrane, insulin receptor is localized in caveolae,41 the same microdomain where eNOS resides.42 In addition, it has been shown that caveolin-1, through its scaffolding domain, potently stimulated insulin receptor kinase activity, stabilizing the activated conformation of the regulatory region of the receptor in an activated state.43 Thus, it is feasible that IDDM, in which a progressive reduction of insulin production occurs, there is an increase of caveolin-1 expression that leads eNOS activity inhibition that in turn reduces NO production and promotes and stabilizes insulin receptor in its active conformation. This latter finding could be interpreted as a body-adaptive response to a reduction in insulin production, in which insulin receptor is more receptive to a minor concentration of insulin because of IDDM.
In conclusion, on progression of diabetes in NOD mice, there is a marked selective reduction in the adrenergic response associated with a reduction in the expression of 1 and ß2 receptors on vessels. When glycosuria is severe (eg, NOD group 3), a reduction in NO-dependent vasorelaxation occurs and this is coupled with an enhanced caveolin-1 expression. Our results indicate that studies on cardiovascular complications in diabetes performed using NOD mice should be performed while accounting for disease progression in mice. In addition, our results suggest that caveolin-1 may represent a new possible therapeutic target in diabetes.
Received July 16, 2003; accepted December 18, 2003.
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