(Hypertension. 1996;28:583-592.)
© 1996 American Heart Association, Inc.
Articles |
Impairment of Endothelium-Dependent Relaxation by Increasing Percentages of Glycosylated Human Hemoglobin
Possible Mechanisms Involved
Departamento de Farmacologia y Terapeutica, Facultad de Medicina, Universidad Autonoma de Madrid, and Unidad de Investigacion y Servicio de Geriatria, Hospital Universitario de Getafe (Spain) (L.R.-M.).
Correspondence to Dr Carlos F. Sanchez-Ferrer, Departamento de Farmacologia y Terapeutica, Facultad de Medicina, Universidad Autonoma, c/Arzobispo Morcillo, 4. 28029, Madrid, Spain. E-mail carlosf@mvax.fmed.uam.es.
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High levels of glycosylated human hemoglobin impair nitric oxide–mediated responses. However, the percentage of glycosylation for which this effect is observed and the mechanisms involved are unknown. We tested endothelium-dependent relaxations caused by acetylcholine in rat aortic segments either in control conditions or after preincubation with increasing percentages of glycosylated human hemoglobin. Human hemoglobin (1 and 10 nmol/L) inhibited endothelium-dependent relaxations only when glycosylated at 9% or higher. We evaluated the effect of 14% glycosylated human hemoglobin on acetylcholine-evoked responses in vessels preincubated with scavengers of superoxide anions, hydroxyl radical, or hydrogen peroxide (superoxide dismutase, deferoxamine, and catalase, respectively); with inhibitors of xanthine oxidase, cyclooxygenase, or thromboxane synthase (allopurinol, indomethacin, and dazoxiben, respectively); with blockers of thromboxane A2/prostaglandin H2 or endothelin receptors (SQ 30741 and BQ-123); and with the precursor of nitric oxide synthesis L-arginine. Superoxide dismutase abolished the effect of glycosylated hemoglobin, and the other substances did not have any effect. Glycosylated hemoglobin at 14% did not modify either the vasoconstrictions induced by the blocker of nitric oxide synthase NG-nitro-L-arginine methyl ester or the relaxations evoked in deendothelialized vessels by sodium nitroprusside and 8-bromo-cGMP. However, it inhibited the vasodilations evoked by exogenous nitric oxide. Superoxide dismutase abolished this latter effect. We conclude that the threshold for glycosylated human hemoglobin (Hb A1) to inhibit endothelium-dependent relaxation is 9%. This effect is due to interference with endothelial nitric oxide by means of superoxide anion production.
Key Words: endothelium • diabetes • hemoglobin A, glycosylated • nitric oxide • vascular diseases
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Diabetes mellitus is a prevalent disease with devastating consequences when vascular complications are present.1 2 Vascular diseases are the cornerstone of long-term complications in diabetes; indeed, atherosclerosis, microangiopathy, and hypertension are more frequently observed in diabetic individuals than in the general population. However, the mechanisms involved in the development of vascular complications in diabetes are poorly understood. The seminal work of Fortes et al3 regarding the existence of an abnormal endothelial modulation of vascular responses has raised an important issue in the pathophysiology of vascular disease in diabetes. The impairment of endothelium-dependent relaxations has been reported in animal models and in diabetic patients.4 5 6 Different mechanisms have mediated these defective responses, namely, aldose reductase, protein kinase C, Na+,K+-ATPase, thromboxane A2/prostaglandin H2, oxygen-derived free radicals, and AGEs.7 8 9 10 11 12 13
Classic studies demonstrated that micromolar or higher (nonphysiological) concentrations of free oxyhemoglobin bind and inactivate NO.14 In previous work, we demonstrated that HHb at nanomolar (physiological) concentrations impairs endothelium-dependent responses only when glycosylated in a pathological range (Hb A1=14%).15 However, nanomolar concentrations of nonglycosylated or normally glycosylated HHb (Hb A1=7.3%) have no effects on endothelium-dependent relaxations.15 We therefore have postulated that GHHb may participate in the endothelial dysfunction described in diabetes, contributing to the development of vascular complications. Thus, GHHb could be useful, not only as a marker of glycemic control, but also as a contributor to the pathophysiological mechanisms producing the diabetic vascular complications.
Several studies have evaluated the effect of improved glycemic control on the outcome of diabetic patients.16 17 18 19 20 21 In those studies, patients with better glycemic control, measured by hemoglobin glycosylation, exhibited fewer vascular complications than those with poorer control. Furthermore, some of these studies raise the possibility that the glycosylation of hemoglobin has a threshold for the presence of vascular complications, although it is not perceived as an agent involved in the pathophysiology of such vascular complications.16 17 18 19 20 21
We hypothesize that the harmful vascular effects of hyperglycemia are related at least partially to hemoglobin glycosylation and subsequent endothelial dysfunction. To support this, we designed experiments (1) to determine the possible existence of a threshold percentage of GHHb from which impaired endothelium-dependent relaxation could be observed and (2) to characterize the possible mechanism or mechanisms involved in the endothelial alteration induced by GHHb.
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Experimental Animals
Male Sprague-Dawley rats (300 to 450 g) were used in this study. Rats were anesthetized with diethyl ether and blood samples obtained for determination of Hb A1c levels by immunoturbidimetric assay.22 Afterwards, rats were killed by bleeding, and the aorta was carefully excised, cleaned of excess fat and connective tissue, placed in a Petri dish containing Krebs-Henseleit solution (KHS) at 4°C, and divided into cylindrical segments 4 to 5 mm long.
Drug Effects on Vascular Tone
For isometric tension recording, each vascular segment was set up in an organ bath according to a method previously described.15 The organ chamber contained 5 mL KHS at 37°C continuously bubbled with a 95% O2/5% CO2 mixture, which gave a pH of 7.4. Two horizontally arranged stainless steel pins were passed through the lumen of the vascular segment. One pin was fixed to the organ bath wall, and the other was connected vertically to a strain gauge for isometric tension recording. The isometric contraction was recorded through a force-displacement transducer (FTO3C, Grass Instrument Co) connected to a polygraph (Grass model 7D). The segments were subjected to a tension of 1.5 g (optimal resting tension), which was readjusted every 15 minutes during a 90-minute equilibration period before drug administration.
At the beginning of the experiment, the vessels were exposed to 75 mmol/L K+ so that their functional integrity could be checked. After a washout period, each segment was contracted with 10 nmol/L norepinephrine. Once a stable plateau was reached, a concentration-response curve to acetylcholine (10 nmol/L to 10 µmol/L) was obtained. Segments with relaxant responses to 10 µmol/L acetylcholine greater than 50% of the previous contraction were considered to have an intact endothelium.
To test the effect of glycosylation of HHb on the acetylcholine responses, we preincubated the segments with 7.3%, 8%, 9%, 10%, 12%, or 14% GHHb (1 and 10 nmol/L) for 15 minutes. In another set of experiments, we studied the interference of 10 nmol/L of 14% GHHb on acetylcholine responses in vessels previously treated with 10 µmol/L indomethacin (20 minutes), 100 µmol/L dazoxiben (60 minutes), 1 µmol/L SQ 30741 (15 minutes), 1 µmol/L BQ-123 (15 minutes), 100 µmol/L L-arginine (20 minutes), 100 U/mL SOD (15 minutes), 600 U/mL catalase (15 minutes), 100 µmol/L deferoxamine (20 minutes), or 100 µmol/L allopurinol (20 minutes). The effective concentrations of these agents were tested in previous studies from our laboratory23 24 and others.25 26 27 28
To analyze the effects of 14% GHHb (10 nmol/L) and/or SOD (100 U/mL) on the concentration-dependent relaxations induced by NO (1 nmol/L to 30 µmol/L), we conducted experiments in endothelium-denuded vessels preincubated (15 minutes) with SNP (10 nmol/L to 100 µmol/L) or 8-bromo-cGMP (100 nmol/L to 300 µmol/L). Vessels were deendothelialized by treatment with saponin (0.3 mg/mL KHS oxygenated at 37°C) for 15 minutes.15 Endothelium removal was systematically checked by testing the loss of acetylcholine-induced relaxations. The effects of 14% GHHb (10 nmol/L) on L-NAME (1 µmol/L to 1 mmol/L) concentration-dependent contractions were evaluated in vessels with endothelium preincubated with GHHb for 15 minutes.
Determination of AGEs
AGE formation in nonglycosylated HHb or 14% GHHb solutions was analyzed according to a previously described protocol29 with polyclonal antiserum to AGE epitopes, which were formed in vitro after the incubation of bovine pancreatic ribonuclease in the presence of 0.5 mol/L glucose for 60 days. The antiserum was obtained from female New Zealand White rabbits receiving four primary and one booster immunization of ribonuclease or AGE-ribonuclease emulsified in Freund's complete adjuvant.29 The antiserum used in this work was kindly provided by Dr Richard Bucala (Rockefeller University, New York, NY). The antibody response was monitored by enzyme-linked immunosorbent assay (EL-340, Bio-Tek Instruments Inc).
AGE content was determined with AGEs from bovine serum albumin as a standard antigen. Increasing concentrations of bovine serum albumin AGEs (0.001 to 0.1 mg/mL) were used to establish a standard curve of antibody binding to the antigen. The semilogarithmic transformation of these data yielded a regression line with r2=.985.
Drugs Used
The composition of the KHS (mmol/L) was NaCl 115, CaCl2 25, KCl 4.6, KH2PO4 1.2, MgSO4·7H2O 1.2, NaHCO3 25, glucose 11.1, and Na2EDTA 0.03. Drugs used were norepinephrine hydrochloride, acetylcholine chloride, human hemoglobin, glycohemoglobin A1 control-E (14% glycosylation), glycohemoglobin A1 control-N (7.3% glycosylation), indomethacin, saponin, CuZn SOD (EC 1.15.1.1) from bovine erythrocytes, catalase from bovine liver, deferoxamine mesylate, allopurinol, L-arginine, L-NAME, SNP, 8-bromo-cGMP, bovine pancreatic ribonuclease, bovine serum albumin (all from Sigma Chemical Co), SQ 30741 (Bristol-Myers Squibb Pharmaceutical Research Institute), BQ-123 (Neosystem Laboratoires), NO (Sociedad Espanola del Oxigeno), and dazoxiben (Pfizer). Drug solutions were made in distilled water, except norepinephrine, which was prepared in saline (0.9% NaCl)/ascorbic acid (0.01% wt/vol), and indomethacin, which was dissolved in distilled water with Na2CO3 (1.5 mmol/L). All of the oxyhemoglobins studied were prepared by reduction of commercial compounds with sodium dithionite, which were subsequently dialyzed and stored in vials at -70°C. Oxyhemoglobin concentration was determined spectrophotometrically before every experiment.30 Intermediate percentages were obtained by addition of human hemoglobin to glycohemoglobin A1 control-E at the same concentration. NO was prepared from a saturated gas solution in deoxygenated (by bubbling with argon) distilled water at room temperature.
Statistical Evaluation
The experiments performed with SNP, 8-bromo-cGMP, and L-NAME were carried out in a nonpaired manner. Adjacent rings from the same vessels were used as controls and preincubated segments. The remaining experiments were systematically paired. In most cases, three concentration-response curves to acetylcholine or NO were obtained in each segment. The first was always adopted as a control, and different protocols were applied to the second and third curves (see legends to the figures and tables). The results are expressed as mean±SE. Deviations from the mean regarding the curves were statistically analyzed with factorial two-way ANOVA for nonpaired experiments and two-way ANOVA for repeated measures for paired experiments. Student's t tests were used in the statistical comparison of maximal relaxations and pD2 values, defined as the negative log of the effective dose (moles per liter) required to produce a half-maximal effect. A value of P<.05 was considered significant.
Ethical Issues
This work was performed according to European regulations. The study was approved by the Local Committee of Investigation.
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Acetylcholine-Induced Vasorelaxant Responses
The Hb A1c values in those rats from which aortas were excised ranged from 4.1% to 5.2% (mean±SE, 4.72±0.18%). We carried out control experiments to exclude the effect of repetition on the vasodilator concentration-response curves evoked by acetylcholine (10 nmol/L to 10 µmol/L). These effects were not modified after three successive curves (Table 1
). Acetylcholine-evoked relaxations were abolished after endothelium removal (data not shown). To exclude the potential confounding effect of different contractile responses to norepinephrine on the acetylcholine-induced vasodilation, we added several concentrations of norepinephrine (10 to 30 nmol/L) to the bath when needed, thereby obtaining similar contractions in every curve conducted in the same arterial segment. The contractile response evoked by 10 nmol/L norepinephrine before any experiment had a mean value of 1198±48.7 mg (n=308).
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Effect of GHHb Percentage
In the present experimental conditions, preincubation with two different concentrations (1 and 10 nmol/L) of GHHb at several percentages (7.3% to 14%) did not induce significant changes on the norepinephrine-evoked contractions (data not shown).
GHHb did not exert any effect on the vasodilator responses to acetylcholine at 7.3% or 8% (Fig 1, Table 1
). However, when vessels were preincubated with between 9% and 14% GHHb, a significant shift to the right was observed in the acetylcholine-induced vasodilator curve (Fig 2, Table 1).
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In the presence of 10 nmol/L GHHb, the maximal relaxation to acetylcholine was significantly reduced at 10% glycosylation or higher. Expressed as mean±SE of the percentage of the previous contraction elicited by norepinephrine, these data are as follows: control curve, 11.1±2.2%; curve with 10 nmol/L of 9% GHHb, 17.0±3.9% (n=10, P=.053); control curve, 18.3±3.6%; curve with 10 nmol/L of 10% GHHb, 23.6±4.7% (n=11, P<.05); control curve, 8.3±2.7%; curve with 10 nmol/L of 12% GHHb, 17.6±1.8% (n=11, P<.01); control curve, 12.8±1.0%; curve with 10 nmol/L of 14% GHHb, 21.4±1.3% (n=103, P<.01).
To eliminate accumulative effects of GHHb, we checked that the successive preincubation with identical concentrations of 14% GHHb yielded similar impairments of acetylcholine-induced relaxations (Table 1). Furthermore, the effects of GHHb were clearly reversible: When acetylcholine was added in a control third curve after a second curve in the presence of 10 nmol/L of 14% GHHb, the acetylcholine-evoked relaxations were similar to those of the first control curve (Table 1).
Mechanisms Involved in the Effect of 14% GHHb on Acetylcholine-Induced, Endothelium-Dependent Responses
To evaluate the role of cyclooxygenase-derived prostanoids and endothelin, we obtained concentration-response curves to acetylcholine in vessels treated with 10 nmol/L of 14% GHHb. After washout and stabilization periods, an identical protocol was repeated in the same vessels preincubated with 10 µmol/L indomethacin (inhibitor of cyclooxygenase), 100 µmol/L dazoxiben (inhibitor of thromboxane synthase), 1 µmol/L SQ 30741 (antagonist of thromboxane A2/prostaglandin H2 receptors), and 1 µmol/L BQ-123 (endothelin antagonist for the endothelin-A receptor subtype). Preincubation with these substances did not modify the basal tone, contractile response to norepinephrine, or vasodilator response to acetylcholine in control conditions (data not shown). In addition, indomethacin, dazoxiben, SQ 30741, and BQ-123 did not exert any effect on the inhibition produced by 14% GHHb on acetylcholine-evoked vasorelaxations (Fig 3, Table 2).
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Using an identical protocol, we observed that the precursor of NO synthesis L-arginine (100 µmol/L) did not change the basal tone, contractile response to norepinephrine, or vasodilator response to acetylcholine in control conditions (data not shown). Also, the impairment of 10 nmol/L of 14% GHHb on acetylcholine-evoked vasorelaxations was unaltered (Fig 4, Table 2). In another set of experiments, the blocker of NO synthase L-NAME (1 µmol/L to 1 mmol/L) evoked concentration-dependent vasoconstrictions, which were unaffected by preincubation with 14% GHHb (Fig 4).
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Preincubation with 100 U/mL SOD (scavenger of superoxide anion) also had no effect on basal tone and norepinephrine-evoked contractions (data not shown) as well as on acetylcholine-induced vasodilation in control conditions (Fig 5), but when the vascular segments were preincubated with SOD, the inhibition of endothelium-mediated relaxations evoked by 14% GHHb were abolished (Fig 5, Table 2). The SOD-induced effects were not modified by previous administration of 600 U/mL catalase (Fig 6, Table 2).
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Pretreatment of the vascular segments with 600 U/mL catalase, 100 µmol/L deferoxamine, or 100 µmol/L allopurinol did not modify basal tone, norepinephrine-induced contractions, or acetylcholine-evoked relaxations (data not shown). Furthermore, these compounds did not affect the inhibition of the acetylcholine-induced vasodilations produced by 14% GHHb (Fig 6, Table 2).
Effect of 14% GHHb on Relaxations Induced by Exogenous NO, SNP, and 8-Bromo-cGMP
Concentration-dependent relaxations in deendothelialized aortic segments were evoked by SNP (10 nmol/L to 10 µmol/L), 8-bromo-cGMP (100 nmol/L to 300 µmol/L), and exogenous NO (1 nmol/L to 30 µmol/L) (Figs 6 and 7, Table 3). Three repetitive curves to exogenous NO yielded identical relaxations (Table 3).
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Preincubation with 14% GHHb did not affect the responses induced by SNP or 8-bromo-cGMP (Fig 7) but significantly reduced those evoked by exogenous NO (Fig 8, Table 3). To discard accumulative effects of GHHb, we checked that successive preincubation with identical concentrations of 14% GHHb yielded similar impairments of NO-induced relaxations (Table 3). Furthermore, the effects of GHHb were also reversible: When NO was added in a control third curve after a second curve in the presence of 10 nmol/L of 14% GHHb, the NO-evoked relaxations were similar to those of the first control curve (Table 3).
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In the presence of 10 nmol/L of 14% GHHb, the maximal relaxation to NO was significantly reduced. Expressed as mean±SE of the percentage of the previous contraction elicited by norepinephrine, these data are as follows: control curve, -33.5±9.8%; and curve with 10 nmol/L of 14% GHHb, 2.3±12.3% (n=9, P<.01).
Preincubation with SOD (100 U/mL) for 15 minutes did not affect the relaxations induced by NO in a control situation but abolished its inhibition by 14% GHHb (Fig 8, Table 3).
AGE Content in Nonglycosylated HHb and 14% GHHb Solutions
Measurements of AGEs in nonglycosylated HHb and 14% GHHb (100 nmol/L and 1 µmol/L) were made at least three times and yielded antibody binding values lower than the minor range observed in the standard curve (0.001 mg/mL bovine serum albumin AGEs) performed simultaneously.
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Factors that contribute to vascular complications in diabetes have different mechanisms, varying from genetic determinants to several hyperglycemia-linked events. During the last several years, impairment of endothelial function has emerged as an added mechanism, among others, in producing diabetic vascular dysfunction. An explanation for such an effect is the interference of glycosylated proteins with endothelium-derived substances, mainly NO. It is well known that hemoglobin is a protein susceptible to nonenzymatic glycosylation. Moreover, hemoglobin exhibits some properties that would allow us to postulate its role in the impairment of endothelial function in diabetes when glycosylated: It is sensitive to changes in glycemic level31 ; it circulates freely in plasma at nanomolar concentrations32 ; and it penetrates into the vascular wall.33
In this study, we demonstrate that impairment by GHHb of endothelium-dependent vasodilator responses is observed at around 9% glycosylation, measured as Hb A1. The inhibitory effect of GHHb on endothelium-dependent relaxations was observed after a brief period of preincubation with this compound, with concentrations of GHHb in the lower physiological limit of circulating free hemoglobin (
0.16 µmol/L)32 and with a conductance vessel, such as the aorta. These factors may explain why the effect, although significant, is quantitatively moderate. We also analyzed the difference in the response to acetylcholine in control and GHHb-preincubated vessels by determining pD2 values. We observed differences similar to those reported by other authors in diabetic versus control vessels.34 In addition, our results can be entirely ascribed to the percentage of hemoglobin glycosylation, because nonglycosylated HHb at these nanomolar concentrations does not exert any effect on endothelium-dependent vasodilation.15
The significance of a glycosylation percentage threshold for the effect of GHHb derives from the assumption that the glycosylation of hemoglobin plays a role in diabetic endothelial dysfunction. If GHHb is not only a marker of glycemic control but also a pathogenic factor, it is reasonable to assume that a threshold for its deleterious effect exists. Furthermore, this threshold should be near to that observed in diabetic patients with fewer vascular complications.
However, controversy exists over whether a threshold of hemoglobin glycosylation is related to the development of diabetic microangiopathy. The results of the Diabetes Control and Complications Trial (DCCT) research group do not support the existence of a target value for glycosylation of hemoglobin,16 although the absence of a threshold for hemoglobin glycosylation in the DCCT study has been questioned.35 More recently, other studies have described the existence of such a threshold for the development of microalbuminuria and retinopathy in diabetic patients.36 37 In these studies, the complications associated with diabetes were related to either the duration of diabetes or degree of hyperglycemia, which is measured by the level of Hb A1 (10.1%). A threshold close to that observed in our experimental conditions has also been found in other studies examining the effect of strict glycemic control on the manifestations of long-term complications in diabetes.17 18 19 21
It is important to note that although vascular complications are less frequent in patients with a low level of hemoglobin glycosylation, these patients are not free of such complications. This fact supports the existence of many other factors contributing to the development of these events.38 Moreover, better glycemic control not only prevents hemoglobin glycosylation but also other metabolic consequences of the disease that may play a role in vascular and endothelial function. To avoid these factors, we conducted our experiments in aorta from nondiabetic rats (Hb A1c ranging from 4.1% to 5.2%). This allowed us to analyze the effect of a single factor (the percentage of GHHb) on the endothelium-dependent relaxations, excluding the participation of other pathophysiologically significant factors that are present when vessels from diabetic animals are used. Furthermore, the HHb concentrations used in the experiments (1 and 10 nmol/L) are similar to those found in plasma from humans in physiological conditions.32
Few studies have evaluated the endothelium-dependent responses in diabetic patients in vivo.4 5 6 39 Most have found an impairment of endothelium-dependent relaxations, although no link has been established between hemoglobin glycosylation and endothelial dysfunction. However, it is remarkable that a defect in the stimulated endothelium-dependent vasodilation was found in the studies in which the patients had higher levels of GHHb5 6 but not in those in which patients had a near-normal level of GHHb.4 Given these studies, an association between hemoglobin glycosylation and defective endothelium-dependent responses in diabetic patients cannot be excluded.
A recent report is consistent with our proposal for a pathogenic role of glycosylated hemoglobin in vascular alterations associated with experimental diabetes. In streptozotocin-induced diabetic rats, circulating glycosylated hemoglobin increases to around 14% after 8 or 12 weeks, together with an impairment of acetylcholine-induced relaxations of aortic segments similar to that obtained in our experimental conditions.40 After pancreatic islet transplantation, a normal level of glycosylated hemoglobin and recovery of endothelial dysfunction were observed.40
Regarding the mechanism by which GHHb impairs endothelium-dependent vasodilation, we have previously demonstrated that GHHb (Hb A1=14%) decreases the responses elicited by exogenous NO in vessels without endothelium, suggesting that the inhibition by GHHb on endothelial modulation is mediated by interaction with NO.15 However, we did not exclude the potential participation of other mechanisms, such as endothelium-derived vasoconstrictor substances. Thus, several authors postulate that the impairment of endothelium-dependent relaxations observed in diabetes is due to an increased production of endothelium-derived prostanoids. Main candidates include thromboxane A2 and/or prostaglandin H2.11 41 42 43 The increased production of vasoconstrictor prostanoids has been shown mostly in vessels preincubated in media containing a very high concentration of glucose (
22 mmol/L11 42 or 300 mg/dL43 ). We evaluated the effects of preincubating the vessels with inhibitors of cyclooxygenase (indomethacin), thromboxane synthase (dazoxiben), and a specific thromboxane A2/prostaglandin H2 receptor blocker (SQ 30741). None of these substances modified the inhibitory effect of GHHb, excluding the participation of vasoconstrictor prostanoids. In agreement with our findings, the thromboxane A2/prostaglandin H2 receptor blocker SQ 29548 does not avoid endothelial dysfunction in diabetic mesenteric resistance arteries.44
Endothelin, a vasoconstrictor peptide also produced by the endothelium, exerts its effects by binding vascular smooth muscle cell endothelin-A receptors, although a minor effect on endothelin-B receptors cannot be ignored, especially at low endothelin concentrations.45 46 Endothelin production is increased in diabetic animals47 and patients.48 Nevertheless, a role for endothelin in the effect of GHHb can also be dismissed in the present experiments because the endothelin-A receptor antagonist BQ-123 did not modify the inhibition induced by GHHb.
Therefore, the participation of vasoconstrictor substances released by the endothelium as a result of the effects induced by GHHb can be excluded, leaving the interference of GHHb with endothelial NO as the only effect likely to be involved. Some different mechanisms for such interference can be proposed: (1) impairment of NO synthesis inside the endothelial cells; (2) direct inactivation of the released NO; and (3) interference with the NO action on the underlying vascular smooth muscle cells.
The effects of GHHb and the synthesis of NO are not likely to be related. L-Arginine, the precursor of NO synthesis,24 did not prevent the GHHb interference with acetylcholine-induced relaxations, whereas the endothelium-dependent contractions evoked by L-NAME, the blocker of NO synthase,24 were unaffected by GHHb. Although these experiments are not conclusive, they strongly suggest a lack of relation, taking into account the difficulty for a molecule such as hemoglobin to enter the endothelial cells in order to impair NO synthase activity.
On the other hand, GHHb did not modify the vasodilations induced by SNP, the direct activator of guanylate cyclase,24 or the permeable cGMP analog 8-bromo-GMP.24 This indicates that this compound does not alter the intracellular vasodilator mechanisms in the smooth muscle of the vascular wall. However, the vasorelaxant effects of exogenous NO were inhibited by GHHb in a way similar to those of acetylcholine. These facts are consistent with a direct inactivation by GHHb of the NO, either added exogenously or released by endothelial cells in response to acetylcholine stimulation.
The most likely hypothesis is that the inactivation of NO may be produced by free radicals released by GHHb. To confirm this possibility, we tested several scavengers of different free radicals, such as deferoxamine for hydroxyl radicals,10 49 catalase for hydrogen peroxide,10 25 and SOD for superoxide anions.10 25 Thus, hydroxyl radicals and hydrogen peroxide were dismissed by the lack of effect of deferoxamine or catalase in antagonizing the endothelial dysfunction caused by GHHb. However, superoxide anions seem to be responsible for those effects, because SOD reversed the inhibitory effect of GHHb on acetylcholine- and NO-evoked vasodilations. A nonspecific effect of SOD is very unlikely in this case because SOD did not modify the effect of acetylcholine or NO in control vessels that had not been preincubated with GHHb. An enhanced production of hydrogen peroxide by SOD50 was also dismissed because catalase did not alter the SOD blockade of GHHb effects. Furthermore, to exclude the possibility that GHHb may generate oxygen-derived free radicals through the activation of intracellular xanthine oxidase,51 we performed experiments in the presence of allopurinol, which did not modify GHHb effects. Therefore, we conclude that the endothelial dysfunction caused by GHHb is mediated by the direct release of superoxide anions, which inactivates the NO released by the endothelium.
Several reports have recently stressed the importance of free radicals in the impairment of the endothelium-dependent relaxations observed in diabetes.10 11 25 52 53 Among them, superoxide anion is most likely involved,10 11 although the participation of other free radicals also has been proposed.25 52 53 The mechanisms by which free radicals interfere with endothelium-dependent responses in diabetes are controversial. Direct inactivation of NO activity has been proposed in mesenteric branches as well as in aortas from diabetic rats,10 25 53 but a decrease of NO production related to cyclooxygenase or aldose reductase pathways also has been suggested.11 52 In our experimental approach, as discussed above, the main mechanism involved in the defective endothelium-dependent vasodilation produced by superoxide anions appears to be the direct inactivation of NO. The source of free radicals in diabetes is also controversial. In the present study, we suggest that GHHb is a source of superoxide anions. In agreement with this, Mullarkey et al50 demonstrated that superoxide generation occurs in glycosylated proteins almost 50-fold more than in nonglycosylated proteins but not in free glucose (10 mmol/L). This effect was observed after short-term incubation periods (1 day), and it was abolished by SOD, as measured by electron paramagnetic resonance spectra.
Recently, the role of nonenzymatic protein glycosylation has become an important issue in explaining the vascular alterations in diabetes. The so-called AGEs quench NO and are involved in the impairment of endothelium-dependent responses.12 54 Although GHHb, a typical Amadori product, has been described in vivo as an important precursor of AGEs,55 we could not find detectable levels of AGEs in either nonglycosylated HHb or 14% GHHb, indicating that these compounds are not likely to be involved in the vasoactive effects of GHHb, at least at the concentrations used in our experimental conditions. Compounds other than AGEs generated during the Maillard reaction may contribute to NO inactivation.12 These other compounds are probably free radicals generated by early products, including Schiff base and Amadori glycosylation products.49 56
In conclusion, GHHb produces an inhibitory effect on endothelium-dependent vasodilator responses at 9% or higher (Hb A1). GHHb exerts its effects on inactivating NO by the generation of superoxide anions. These findings may improve our knowledge of vascular complications in diabetes, creating new targets for therapeutic approaches.
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Acknowledgments |
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This work was supported by grants from Fondo de Investigaciones Sanitarias de la Seguridad Social (94/0162, 93/0916E, and 95/1954), Comunidad Autonoma de Madrid (I+D 0017/94), and Bayer Espana. We thank Alberto Sanchez-Ferrer and Jose L. Llergo for their excellent technical assistance; Dr Jose Luis Garcia Lopez for AGE determination; and Dr Gonzalo Costa for his help in some aspects of the work. We would like to acknowledge Michelle Sheehan for correcting the English style and grammar.
Received March 14, 1996; first decision March 26, 1996; accepted May 20, 1996.
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