Search

Vascular contractility and endothelium-dependent vasodilatation were studied in mesenteric, aorta and coronary vasculature from male and female LDL receptor deficient (LDLR(-/-)) and wild type C57BL/6 mice fed either a high-fat Western Diet (WD) or regular animal chow (RD). Endothelium-dependent vasodilatation was also studied in small mesenteric arteries and aorta from C57BL/6 mice following a 20 h exposure in vitro to 30 mM glucose. Compared with RD-fed animals, WD-fed LDLR-/- animals had increased body weights, elevated triglycerides and total cholesterol, but not glucose. Control C57BL6 animals had elevated body weight without increased cholesterol, triglyceride or glucose levels. The contractile sensitivity to cirazoline (pD(2)) of small mesenteric arteries was the same for RD-fed LDLR-/- and RD-fed C57BL6 mice, but was reduced in WD-fed male LDLR-/- and WD-fed female C57BL/6 mice. Maximum mesenteric contractile values for cirazoline (Emax) were unchanged; however, the Emax for phenylephrine in the aorta from WD-fed male C57BL/6 (but not LDLR-/- or female C57BL/6) mice was reduced. The Emax for acetylcholine-mediated endothelium-dependent vasodilatation in micro- and macro vessels (small mesenteric artery, coronary artery and aorta) from WD-fed LDLR-/- and C57BL/6 mice was unaltered, in contrast to the reduction in Emax for glucose-exposed tissues. Furthermore, the component of acetylcholine-mediated vasodilatation resistant to the combination of inhibitors of nitric oxide synthase, cyclooxygenase and guanylyl cyclase (nitro L-arginine methyl ester - 100 microM; indomethacin 10 microM and 1H-[1,2,4]-oxadiazolo[4,3,-a]quinoxalin-1-one, ODQ - 10 microM, respectively) was generally greater in WD-fed mice. Thus, vasculature from WD-fed mice with short-term dyslipidaemia do not exhibit reduced endothelium-dependent vasodilatation, but the WD is associated with changes in the overall endothelial-dependent relaxation and contractile responses thus suggesting an impact of diet rather than dyslipidaemia on cellular signalling pathways in vascular tissue. In contrast, acute hyperglycaemia resulted in endothelial dysfunction in both small mesenteric arteries and thoracic aorta.

Endothelial dysfunction is considered as a major risk factor of cardiovascular complications of type I and type II diabetes. Our previous studies have demonstrated that endothelial dysfunction in the small mesenteric arteries from 12-16 week old type II diabetic mice was associated with decreased bio-availability of nitric oxide whereas endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxation was preserved. The objective of the present study was to characterize EDHF-mediated relaxations of small mesenteric arteries from db/db mice. A depolarizing concentration of KCl or tetraethylammonium (TEA, 10 mM) significantly inhibited the EDHF-mediated relaxation to acetylcholine and bradykinin in small mesenteric arteries from both db/+ and db/db mice. Charybdotoxin or iberiotoxin alone and a combination of ouabain and barium significantly reduced the maximal relaxation to acetylcholine in small mesenteric arteries from db/db mice and charybdotoxin or iberiotoxin either alone or in combination with apamin reduced the sensitivity to the EDHF-mediated component of the relaxation response to bradykinin. 17-octadecynoic acid, but not catalase, significantly reduced the sensitivity to EDHF-mediated responses to bradykinin in db/db mice; 17-octadecynoic acid had no effect on acetylcholine-mediated relaxations. No differences were, however, detected for mRNA expression levels of calcium-activated potassium channels or connexins 37, 40, 43 and 45. Collectively, these data suggest that bradykinin-induced, EDHF-dependent relaxation in small mesenteric arteries from db/db mice is mediated via cytochrome P450 product that activates the large conductance calcium-activated potassium (BK(Ca) or Slo) channel, whereas the acetylcholine-induced, EDHF-mediated relaxation involves neither cytochrome P450 product nor hydrogen peroxide.

1 Vascular reactivity to the alpha-adrenoceptor agonist phenylephrine (PE) was enhanced in small mesenteric arteries (SMA) from diabetic (db/db) mice under both high and low in vitro oxygen conditions. 2 Mechanical removal of the endothelium significantly attenuated the enhanced vascular reactivity of SMA from db/db mice. 3 Acute incubation of the SMA with sepiapterin, a precursor of tetrahydrobiopterin, and Nω-nitro L-arginine (L-NA), an inhibitor of nitric oxide (NO) synthase (NOS), resulted in no significant change in the enhanced vascular reactivity to PE in db/db mice. Endothelial nitric oxide synthase (eNOS) mRNA and protein levels in SMA were not different between db/+ and db/db mice. 4 Acute incubation of SMA with a combination of polyethylene glycol superoxide dismutase and catalase significantly reduced the enhanced contraction to PE in db/db mice. There were higher levels of malondialdehyde, a marker of lipid peroxidation and basal superoxide as measured by dihydroethidium staining, in SMA from db/db mice compared to db/+ mice. 5 Acute incubation with indomethacin, a nonselective inhibitor of cyclooxygenase, SQ 29548, a selective thromboxane receptor antagonist and furegrelate, a thromboxane synthesis inhibitor, significantly attenuated the enhanced contraction to PE in SMA from db/db mice. 6 This study demonstrates that the enhanced contractility of SMA from db/db mice to PE was endothelium dependent and involves elevated reactive oxygen species, cyclooxygenase activity and thromboxane synthesis, but not changes in the eNOS/NO pathway. British Journal of Pharmacology (2005) 144, 953–960. doi:10.1038/sj.bjp.0706121

Vascular disease remains a major cause of morbidity and mortality in diabetes mellitus, in spite of recent improvements in outcome, some of which may be modulated by improved endothelial function. Therapeutic strategies aimed directly at preventing, or minimising the extent of, these sequelae are required as an adjunct to treatments directed at normalising the metabolic milieu. The microvasculature, and the endothelium in particular, are early contributors to vascular dysfunction, thus raising the question as to how best to specifically target the endothelium. However, the expansive nature of the microvasculature, the varying demands that tissues have in terms of blood flow, and the heterogeneity that exists amongst cell types in different sites raises potential problems as to the practicality of such an approach. Further-more, temporal and genetic factors in the genesis of diabetic microvascular dysfunction may impact on therapeutic strategies. It is suggested that a systematic approach is required to understand the heterogeneity of the microvasculature, with particular emphasis on relating differences in gene and protein expression with functional properties. Such an approach may then provide the necessary information to allow exploitation of endothelial cell heterogeneity for unique targeted interventions, as well as providing the necessary rationale for pharmacological interventions (both prophylactic and corrective) aimed at the endothelium as a whole.

Endothelial dysfunction is considered as a major risk factor of cardiovascular complications of type I and types II diabetes. Impaired endothelium-dependent vasodilatation can be directly linked to a decreased synthesis of the endothelium-derived nitric oxide (NO) and/or an increase in the production of reactive oxygen species such as superoxide. Administration of tetrahydrobiopterin, an important co-factor for the enzyme nitric oxide synthase (NOS), has been demonstrated to enhance NO production in prehypertensive rats, restore endothelium-dependent vasodilatation in coronary arteries following reperfusion injury, aortae from streptozotocin-induced diabetic rats and in patients with hypercholesterolemia. Tetrahydrobiopterin supplementation has been shown to improve endothelium-dependent relaxation in normal individuals, patients with type II diabetes and in smokers. These findings from different animal models as well as in clinical trials lead to the hypothesis that tetrahydrobiopterin, or a precursor thereof, could be a new and an effective therapeutic approach for the improvement of endothelium function in pathophysiological conditions. In addition to NO, the endothelium also produces a variety of other vasoactive factors and a key question is: Is there also a link to changes in the synthesis/action of these other endothelium-derived factors to the cardiovascular complications associated with diabetes? Endothelium-derived hyperpolarizing factor, or EDHF, is thought to be an extremely important vasodilator substance notably in the resistance vasculature. Unfortunately, the nature and, indeed, the very existence of EDHF remains obscure. Potentially there are multiple EDHFs demonstrating vessel selectivity in their actions. However, until now, identity and properties of EDHF that determine the therapeutic potential of manipulating EDHF remains unknown. Here we briefly review the current status of EDHF and the link between EDHF and endothelial dysfunction associated with diabetes. (Mol Cell Biochem 263: 21-27, 2004).

1. The current study examined the hypothesis that endothelial production of hydrogen peroxide (H2O2) mediates relaxations to acetylcholine (ACh) in aorta and small mesenteric arteries (SMA) from mice. 2. Relaxations to ACh (0.01-10 microM) and H2O2 (0.1-1000 microM) were produced in aorta and SMA isolated from wild-type C57BL/6 mice and type II diabetic mice (db/db). In SMA, relaxations to ACh were produced in the presence of N omega-nitro-L-arginine methyl ester (100 microM) and indomethacin (Indo, 10 microM). 3. 1-H[1,2,4]oxadiazolo[4,3-]quinoxalin-1-one (10 microM) significantly reduced ACh-induced relaxations in SMA, abolished responses in aorta, but had no effect on relaxations induced by H2O2. Catalase (2500 U ml-1) abolished responses to H2O2, but did not alter relaxations to ACh in the SMA and only caused a small rightward shift in responses to ACh in the aorta. 4. ACh-, but not H2O2-, mediated relaxations were significantly reduced by tetraethylammonium (10 mM), the combination of apamin (1 microM) and charybdotoxin (100 nM), and 25 mm potassium chloride (KCl). Higher KCl (60 mM) abolished relaxations to both ACh and H2O2. Polyethylene glycolated superoxide dismutase (100 U ml-1), the catalase inhibitor 3-amino-1,2,4-triazole (3-AT, 50 mM) and treatment with the copper chelator diethyldithiolcarbamate (3 mM) did not affect relaxations to ACh. 5. H2O2-induced relaxations were endothelium-independent and were not affected by ethylene diamine tetraacetic acid (EDTA 0.067 mM), 4-aminopyridine (1 mM), ouabain (100 microM) and barium (30 microM), 3-AT or Indo. 6. Although the data from this study show that H2O2 dilates vessels, they do not support the notion that H2O2 mediates endothelium-dependent relaxations to ACh in either aorta or SMA from mice.

We previously reported that acute incubation with tetrahydrobiopterin (BH4) or sepiapterin, a cofactor for endothelial nitric oxide synthase and a stable precursor of BH4, respectively, enhanced the acetylcholine (Ach)-induced relaxation of isolated small mesenteric arteries (SMA) from diabetic (db/db) mice. In this study, we investigated the effect of chronic oral supplementation of sepiapterin (10 mg kg−1 day−1) to db/db mice on endothelium function, biopterin levels and lipid peroxidation in SMA. Oral dietary supplementation with sepiapterin had no effect on glucose, triglyceride, cholesterol levels and body weight. SMA from db/db mice showed enhanced vascular reactivity to phenylephrine, which was corrected with sepiapterin supplementation. Furthermore, Ach, but not sodium nitroprusside-induced relaxation, was improved with sepiapterin supplementation in db/db mice. BH4 levels and guanosine triphosphate cyclohydrolase I activity in SMA were similar in db/+ and db/db mice. Sepiapterin treatment had no effects on BH4 or guanosine triphosphate cyclohydrolase I activity. However, the level of dihydrobiopterin+biopterin was higher in SMA from db/db mice, which was corrected following sepiapterin treatment. Thiobarbituric acid reactive substance, malondialdehyde, a marker of lipid peroxidation, was higher in SMA from db/db mice, and was normalized by sepiapterin treatment. These results indicate that sepiapterin improves endothelial dysfunction in SMA from db/db mice by reducing oxidative stress. Furthermore, these results suggest that decreased biosynthesis of BH4 may not be the basis for endothelial dysfunction in SMA from db/db mice. British Journal of Pharmacology (2003) 140, 701–706. doi:10.1038/sj.bjp.0705476

Micro- and macrovascular complications are the leading causes of mortality in Types I and II diabetes. Hyperglycemia results in increased advanced glycosylation end (AGE) products, oxidative stress, increased sorbitol levels, and increased activation of protein kinase C. These effects of hyperglycemia eventually lead to impaired endothelium-dependent relaxation to vasoactive substances such as acetylcholine and bradykinin. Increased oxidative stress, which will reduce levels of nitric oxide (NO), and/or decreased bioavailability of tetrahydrobiopterin (BH 4 ), a cofactor for endothelial NO synthase (eNOS), may lead to a phenomenon called uncoupling of eNOS and this leads to endothelial dysfunction. Uncoupled NOS produces superoxide anions which lead to a further reduction in NO bioavailability. Thus, restoring BH4 levels and antioxidant activity could prove to be novel approaches for the treatment of endothelial dysfunction in Type I and II diabetes.

Endothelium-dependent and -independent regulation of vascular tone in small mesenteric arteries (SMA) from control (db/db +/?) and diabetic (db/db −/−) mice was compared. Phenylephrine-induced maximum contraction, but not sensitivity, of SMA in db/db −/− compared to db/db +/? was enhanced. Acetylcholine (ACh), but not sodium nitroprusside (SNP), -induced relaxation was reduced in SMA from db/db −/− compared to db/db +/?. ACh-induced relaxation of SMA was inhibited by a combination of Nω-nitro-L-arginine and indomethacin in db/db +/?, but not in db/db −/−. Acute incubation of SMA with tetrahydrobiopterin (BH4, 10 μM) and sepiapterin (100 μM) enhanced ACh-induced relaxation in SMA from db/db −/−, but not from db/db +/? 2,4-diamino-6-hydroxypyrimidine, an inhibitor of GTP cyclohydrolase I, (10 mM), impaired the sensitivity of SMA from db/db +/? to ACh, which was restored by co-incubation with BH4 (10 μM). BH4 and superoxide dismutase (SOD, 150 u ml−1), either alone or in combination, had no effect on either ACh or SNP-induced relaxation in SMA from eNOS −/− mice. Incubation of SMA with SOD (150 iu ml−1), catalase (200 iu ml−1) and L-arginine (1 mM) had no effect on ACh-induced relaxation of SMA. However, the combination of polyethylene glycol-SOD (200 iu ml−1) and catalase (80 u ml−1) improved the sensitivity of ACh-induced relaxation in db/db −/−, but not in db/db +/?. These data suggest that increased production of superoxide anions and decreased availability of BH4 result in an ‘uncoupling’ of nitric oxide synthase and endothelial dysfunction in SMA from db/db −/− mice. British Journal of Pharmacology (2002) 136, 255–263; doi:10.1038/sj.bjp.0704683

The Type 2 diabetic db/ db mouse experiences vascular dysfunction typified by changes in the contraction and relaxation profiles of small mesenteric arteries (SMAs). Contractions of SMAs from the db/ db mouse to the α1-adrenoceptor agonist phenylephrine (PE) were significantly enhanced, and acetylcholine (ACh)-induced relaxations were significantly depressed. Drug treatment of db/ db mice with a nonthiazolidinedione peroxisome prolifetor-activated receptor-γ agonist and insulin sensitizing agent 2-[2-(4-phenoxy-2-propylphenoxy)ethyl]indole-5-acetic acid (COOH) completely prevented the changes in endothelium-dependent relaxation, but, with the discontinuation of therapy, endothelial dysfunction returned. Dysfunctional SMAs were found to specifically upregulate the expression of a 35-kDa isoform of sarcolemmal membrane-associated protein (SLMAP), which is a component of the excitation-contraction coupling apparatus and implicated in the regulation of membrane function in muscle cells. Real-time PCR revealed high SLMAP mRNA levels in the db/ db microvasculature, which were markedly downregulated during COOH treatment but elevated again when drug therapy was discontinued. These data reveal that the microvasculature in db/ db mice undergoes significant changes in vascular function with the endothelial component of vascular dysfunction specifically correlating with the overexpression of SLMAP. Thus changes in SLMAP expression may be an important indicator for microvascular disease associated with Type 2 diabetes.

In this review we discuss the contribution of NO, prostacyclin and endothelium-derived relaxing factor--endothelium-derived hyperpolarizing factor, or EDHF, to vascular function. We also explore the hypotheses (1): that tissues can store NO as nitrosothiols (RSNOs) and (2) that such RSNO stores can be modulated by physiological and pathophysiological processes. Notably in the microcirculation, EDHF appears to play an important role in the regulation of vascular tone. Leading candidates for EDHF include extracellular potassium (K+), an epoxygenase product, hydrogen peroxide and/or a contribution from myoendothelial gap junctions. Data from our laboratory indicate that in mouse vessels, different endothelium-dependent vasodilators, such as acetylcholine and protease-activated receptor (PAR) agonists, release different endothelium-derived relaxing factors. The combination of two K-channel toxins, apamin and charybdotoxin, inhibits EDHF activity in most protocols. Endothelial dysfunction is considered as the major risk factor and a very early indicator of cardiovascular disease including the cardiovascular complications of type I & types II diabetes. Impaired endothelium-dependent vasodilatation results primarily from a decreased synthesis of endothelium-derived nitric oxide (NO) and/or an increase in the production of reactive oxygen species such as superoxide. We have shown that the administration of tetrahydrobiopterin, an important co-factor for nitric oxide synthase (NOS) partially restores endothelial function (1) in leptin-deficient mice (db/db) with spontaneous type II diabetes, as well as (2) in human vascular tissue harvested for coronary artery bypass grafting (CABG). These data suggest that a deficiency in the availability of tetrahydrobiopterin plays an important role in vascular dysfunction associated with Type II diabetes. In addition, changes in the contribution of EDHF occur in vascular tissue from the db/db mice suggesting a compensatory increase in EDHF production; whether this alteration in EDHF production is physiological or pathophysiological remains controversial.

The endothelium is a source of many substances that play important roles in the short- and long-term regulation of the cardiovascular system. In this review we focus on endothelium-derived hyperpolarizing factor, or EDHF. EDHF is often referred to as the “third pathway” as, in addition to nitric oxide (NO) and prostacyclin (PGI2) EDHF seems to play an important role as the “third” endothelium-derived relaxing factor. Considerable debate is on going concerning the nature of EDHF and, indeed, whether a unique molecule even exists. EDHF, by definition, mediates its action by directly, or indirectly, opening K-channels. In most instances the action of EDHF is abolished by the combination of two K-channel toxins, apamin and charybdotoxin; however, the channels that these inhibitors interact with would seem to be located on endothelial rather than vascular smooth muscle cells. The cellular mechanisms whereby EDHF mediates vascular smooth muscle hyperpolarization may involve myo-endothelial gap junctions thus negating the role for a true chemical mediator. Endothelial dysfunction is a common feature of cardiovascular disease, including the cardiovascular dysfunction associated with diabetes, and the role that changes in the nature/function of EDHF play in this process is currently an area of considerable interest. Therapeutic and dietary interventions that restore endothelial function may prove to be of tremendous benefit in the treatment of cardiovascular disease.

1. The current study examined the hypothesis that endothelial production of hydrogen peroxide (H2O2) mediates relaxations to acetylcholine (ACh) in aorta and small mesenteric arteries (SMA) from mice. 2. Relaxations to ACh (0.01-10 microM) and H2O2 (0.1-1000 microM) were produced in aorta and SMA isolated from wild-type C57BL/6 mice and type II diabetic mice (db/db). In SMA, relaxations to ACh were produced in the presence of N omega-nitro-L-arginine methyl ester (100 microM) and indomethacin (Indo, 10 microM). 3. 1-H[1,2,4]oxadiazolo[4,3-]quinoxalin-1-one (10 microM) significantly reduced ACh-induced relaxations in SMA, abolished responses in aorta, but had no effect on relaxations induced by H2O2. Catalase (2500 U ml-1) abolished responses to H2O2, but did not alter relaxations to ACh in the SMA and only caused a small rightward shift in responses to ACh in the aorta. 4. ACh-, but not H2O2-, mediated relaxations were significantly reduced by tetraethylammonium (10 mM), the combination of apamin (1 microM) and charybdotoxin (100 nM), and 25 mm potassium chloride (KCl). Higher KCl (60 mM) abolished relaxations to both ACh and H2O2. Polyethylene glycolated superoxide dismutase (100 U ml-1), the catalase inhibitor 3-amino-1,2,4-triazole (3-AT, 50 mM) and treatment with the copper chelator diethyldithiolcarbamate (3 mM) did not affect relaxations to ACh. 5. H2O2-induced relaxations were endothelium-independent and were not affected by ethylene diamine tetraacetic acid (EDTA 0.067 mM), 4-aminopyridine (1 mM), ouabain (100 microM) and barium (30 microM), 3-AT or Indo. 6. Although the data from this study show that H2O2 dilates vessels, they do not support the notion that H2O2 mediates endothelium-dependent relaxations to ACh in either aorta or SMA from mice.

We previously reported that acute incubation with tetrahydrobiopterin (BH4) or sepiapterin, a cofactor for endothelial nitric oxide synthase and a stable precursor of BH4, respectively, enhanced the acetylcholine (Ach)-induced relaxation of isolated small mesenteric arteries (SMA) from diabetic (db/db) mice. In this study, we investigated the effect of chronic oral supplementation of sepiapterin (10 mg x kg-1 x day-1) to db/db mice on endothelium function, biopterin levels and lipid peroxidation in SMA. Oral dietary supplementation with sepiapterin had no effect on glucose, triglyceride, cholesterol levels and body weight. SMA from db/db mice showed enhanced vascular reactivity to phenylephrine, which was corrected with sepiapterin supplementation. Furthermore, Ach, but not sodium nitroprusside-induced relaxation, was improved with sepiapterin supplementation in db/db mice. BH4 levels and guanosine triphosphate cyclohydrolase I activity in SMA were similar in db/+ and db/db mice. Sepiapterin treatment had no effects on BH4 or guanosine triphosphate cyclohydrolase I activity. However, the level of dihydrobiopterin+biopterin was higher in SMA from db/db mice, which was corrected following sepiapterin treatment. Thiobarbituric acid reactive substance, malondialdehyde, a marker of lipid peroxidation, was higher in SMA from db/db mice, and was normalized by sepiapterin treatment. These results indicate that sepiapterin improves endothelial dysfunction in SMA from db/db mice by reducing oxidative stress. Furthermore, these results suggest that decreased biosynthesis of BH4 may not be the basis for endothelial dysfunction in SMA from db/db mice.

1. Endothelium-dependent and -independent regulation of vascular tone in small mesenteric arteries (SMA) from control (db/db +/?) and diabetic (db/db -/-) mice was compared. 2. Phenylephrine-induced maximum contraction, but not sensitivity, of SMA in db/db -/- compared to db/db +/? was enhanced. 3. Acetylcholine (ACh), but not sodium nitroprusside (SNP), -induced relaxation was reduced in SMA from db/db -/- compared to db/db +/?. 4. ACh-induced relaxation of SMA was inhibited by a combination of N(omega)-nitro-L-arginine and indomethacin in db/db +/?, but not in db/db -/-. 5. Acute incubation of SMA with tetrahydrobiopterin (BH(4), 10 microM) and sepiapterin (100 microM) enhanced ACh-induced relaxation in SMA from db/db -/-, but not from db/db +/? 2,4-diamino-6-hydroxypyrimidine, an inhibitor of GTP cyclohydrolase I, (10 mM), impaired the sensitivity of SMA from db/db +/? to ACh, which was restored by co-incubation with BH(4) (10 microM). 6. BH(4) and superoxide dismutase (SOD, 150 u ml(-1)), either alone or in combination, had no effect on either ACh or SNP-induced relaxation in SMA from eNOS -/- mice. 7. Incubation of SMA with SOD (150 iu ml(-1)), catalase (200 iu ml(-1)) and L-arginine (1 mM) had no effect on ACh-induced relaxation of SMA. However, the combination of polyethylene glycol-SOD (200 iu ml(-1)) and catalase (80 u ml(-1)) improved the sensitivity of ACh-induced relaxation in db/db -/-, but not in db/db +/?. 8. These data suggest that increased production of superoxide anions and decreased availability of BH(4) result in an 'uncoupling' of nitric oxide synthase and endothelial dysfunction in SMA from db/db -/- mice.