Diabetic Arterioles Research Articles

Requisite roles of LOX-1, JNK, and arginase in diabetes-induced endothelial vasodilator dysfunction of porcine coronary arterioles

Diabetes is associated with cardiac inflammation and impaired endothelium-dependent coronary vasodilation, but molecular mechanisms involved in this dysfunction remain unclear. We examined contributions of inflammatory molecules lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), stress-activated kinases (c-Jun N-terminal kinase [JNK] and p38), arginase, and reactive oxygen species to coronary arteriolar dysfunction in a porcine model of type 1 diabetes. Coronary arterioles were isolated from streptozocin-induced diabetic pigs and control pigs for vasoreactivity and molecular/biochemical studies. Endothelium-dependent nitric oxide (NO)-mediated vasodilation to serotonin was diminished after 2 weeks of diabetes, without altering endothelium-independent vasodilation to sodium nitroprusside. Superoxide scavenger TEMPOL, NO precursor L-arginine, arginase inhibitor nor-NOHA, anti-LOX-1 antibody or JNK inhibitors SP600125 and BI-78D3 improved dilation of diabetic vessels to serotonin. However, hydrogen peroxide scavenger catalase, anti-IgG antibody or p38 kinase inhibitor SB203580 had no effect. Combined inhibition of arginase and superoxide levels did not further improve vasodilation. Arginase-I mRNA expression, LOX-1 and JNK protein expression, and superoxide levels were elevated in diabetic arterioles. In conclusion, sequential activation of LOX-1, JNK, and L-arginine consuming enzyme arginase-I in diabetes elicits superoxide-dependent oxidative stress and impairs endothelial NO-mediated dilation in coronary arterioles. Therapeutic targeting of these adverse vascular molecules may improve coronary arteriolar function during diabetes.

Tetrahydrobiopterin rescues impaired responses of cerebral resistance arterioles during type 1 diabetes.

Our goal was to test the hypothesis that administration of tetrahydrobiopterin (BH4) would improve impaired endothelial nitric oxide synthase-dependent dilation of cerebral arterioles during type 1 diabetes. In addition, we examined the influence of BH4 on levels of superoxide in brain tissue. In vivo diameter of cerebral arterioles in nondiabetic and diabetic rats was measured in response to endothelial nitric oxide synthase-dependent agonists (acetylcholine and adenosine 5'-diphosphate) and an endothelial nitric oxide synthase-independent agonist (nitroglycerine) before and during application of BH4 (1.0 µM). We also measured levels of superoxide from cortex tissue in nondiabetic and diabetic rats under basal states and during BH4 Acetylcholine and adenosine 5'-diphosphate dilated cerebral arterioles in nondiabetic rats, but this vasodilation was significantly impaired in diabetic rats. In contrast, nitroglycerine produced similar vasodilation in nondiabetic and diabetic rats. Application of BH4 did not enhance vasodilation in nondiabetic rats but improved impaired cerebral vasodilation in diabetic rats. Basal superoxide levels were increased in cortex tissue from diabetic rats, and BH4 reduced these levels to that found in nondiabetic rats. Thus, BH4 is an important mediator of endothelial nitric oxide synthase-dependent responses of cerebral arterioles in diabetes and may have therapeutic potential for the treatment of cerebral vascular disease.

Inactivation of Endothelial Small/Intermediate Conductance of Calcium-Activated Potassium Channels Contributes to Coronary Arteriolar Dysfunction in Diabetic Patients.

BackgroundDiabetes is associated with coronary arteriolar endothelial dysfunction. We investigated the role of the small/intermediate (SKCa/IKCa) conductance of calcium-activated potassium channels in diabetes-related endothelial dysfunction.Methods and ResultsCoronary arterioles (80 to 150 μm in diameter) were dissected from discarded right atrial tissues of diabetic (glycosylated hemoglobin = 9.6±0.25) and nondiabetic patients (glycosylated hemoglobin 5.4±0.12) during coronary artery bypass graft surgery (n=8/group). In-vitro relaxation response of precontracted arterioles was examined in the presence of the selective SKCa/IKCa activator NS309 and other vasodilatory agents. The channel density and membrane potential of diabetic and nondiabetic endothelial cells was measured by using the whole cell patch-clamp technique. The protein expression and distribution of the SKCa/IKCa in the human myocardium and coronary arterioles was examined by Western blotting and immunohistochemistry. Our results indicate that diabetes significantly reduced the coronary arteriolar response to the SKCa/IKCa activator NS309 compared to the respective responses of nondiabetic vessels (P<0.05 versus nondiabetes). The relaxation response of diabetic arterioles to NS309 was prevented by denudation of endothelium (P=0.001 versus endothelium-intact). Diabetes significantly decreased endothelial SKCa/IKCa currents and hyperpolarization induced by the SKCa/IKCa activator NS309 as compared with that of nondiabetics. There were no significant differences in the expression and distribution of SKCa/IKCa proteins in the coronary microvessels.ConclusionsDiabetes is associated with inactivation of endothelial SKCa/IKCa channels, which may contribute to endothelial dysfunction in diabetic patients.

Loss of endothelial caveolae leads to eNOS uncoupling and impaired flow‐mediated dilation in human coronary arterioles in diabetes

Diabetes mellitus (DM) leads to impaired endothelial synthesis of nitric oxide (NO). We aimed to investigate the role of caveolae ‐ cholesterol‐rich membrane lipid rafts ‐ in leading to eNOS uncoupling in human coronary arterioles in DM. Flow‐mediated dilation (FMD) of isolated, pressurized coronary arterioles was compared between DM (n=18) and non‐DM (n=16) patients undergoing heart surgery. Coronary arterioles of non‐DM patients exhibited NO‐dependent FMD (35±8%), which was abolished in DM patients (−7±4%). Incubation of DM arterioles with sepiapterin (a tetrahydrobiopterin (BH4) precursor) or peroxynitrite scavengers, uric acid or FeTMPyP restored FMD in an NO‐dependent manner. In DM patients, increased protein 3‐nitrotyrosine (3‐NT) formation was detected, with a greater degree of colocalization with caveolin‐1. Electron microscopy analysis revealed a prominent 3‐NT staining in endothelial cell membrane and demonstrated a reduced number of caveolae in coronary arteries of DM patients. Also, aorta from mice lacking caveolae showed a reduced NO‐dependent dilation to acetylcholine, which was restored by sepiapterin. Thus, we suggest that impaired NO‐mediated FMD of human coronary arterioles in diabetes is due to the peroxynitrite‐dependent destruction of caveolae, which leads to decreased bioavailability of BH4 and eNOS uncoupling.

糖尿病の細小血管障害マーカーとしての指動脈スティフネス指数

細小動脈硬化は動脈硬化の最も初期の徴候であるかもしれない。光電指尖容積脈波から導出され，血圧には依存しない指動脈スティフネス指標(FSI)が，指部動脈における圧－容積関係の指数関数モデルに基づいて開発されてきた。本研究では，細動脈の成分により一層関連する新しい指標として高経壁圧範囲におけるFSI（FSIH）を考案し，糖尿病患者において検査された。研究協力者は中年の眼科患者31名で，糖尿病を有する7 名の糖尿病群(DM), 糖尿病と高血圧を合併する12名の高血圧合併群(DH), 基礎疾患のない12 名の統制群(CT)の3群に割り振られた。FSIとFSIHは，健常青年集団では50 ± 10となるようにすでに標準化されていた。FSIはCT 群(54.7 ± 11.8)に比べてDH群(73.8 ± 11.3)で有意に高く，DM 群(65.0 ± 14.6)はその中間であった。FSIHはCT 群(61.0 ± 12.1)に比べ，DM群(91.2 ± 22.9)とDH群(83.8 ± 31.1)の患者で有意に高かった。FSI とFSIH はそれぞれ指の小動脈と細動脈のスティフネスと関連することが示唆される。

Decreased contractile response to endothelin-1 of peripheral microvasculature from diabetic patients

We compared the contractile responses to endothelin-1 (ET-1) with and without the inhibition of ET-A receptors and protein kinase C-alpha (PKC-α) in the human peripheral microvasculature of diabetic and case-matched, nondiabetic patients. Chest wall skeletal muscle was harvested from patients with and without diabetics undergoing cardiac surgery. Peripheral arterioles (90-180 μm in diameter) were dissected from the harvested tissue. Microvascular constriction was assessed by videomicroscopy in response to ET-1 with and without an endothelin-A (ET-A) receptor antagonist, an endothelin B (ET-B) antagonist, or a PKC-α inhibitor. ET-1 induced a dose-dependent contractile response of skeletal muscle arterioles from diabetic and nondiabetic patients. The contractile response of diabetic arterioles from both prebypass and postbypass to ET-1 (10(-9) mol/L) was decreased compared with those of nondiabetic patients (P < .05). The contractile responses of microvessels of both diabetics and nondiabetics to ET-1 were inhibited in the presence of either ET-A receptor antagonist BQ123 (10(-7) mol/L) or the PKC-α inhibitor safingol (2×10(-5) mol/L, P < .05, respectively). In contrast, the ET-1-induced vasoconstriction was not affected by the administration of the ET-B receptor antagonist BQ788 (10(-7) mol/L). There were no differences in skeletal muscle levels of the ET-A and ET-B receptors between diabetic and nondiabetic groups. Diabetic patients demonstrated a decreased contractile response to ET-1 in human peripheral microvasculature. The contractile response of diabetic vessels to ET-1 occurs via activation of ET-A receptors and PKC-α. These results provide novel mechanisms of ET-1-induced contraction in vasomotor dysfunction in patients with diabetes.

Retinal arterioles have impaired reactivity to hyperoxia in type 1 diabetes

Diabetes has adverse effects on the retinal microvasculature. The purpose of this study was to compare the effects of inhalation of hypoxic, hyperoxic and normoxic-hypercapnic gas mixtures on retinal vessel diameter in people with and without diabetes. Sixty-one participants (aged 24-50 years) 29 with (male : female ratio 2.6 : 1) and 32 without (male : female ratio 0.7 : 1) diabetes, inhaled hypoxic, hyperoxic and normoxic-hypercapnic gas mixtures for 3-5 mins. The diameters of arterioles and venules were measured using digital retinal images taken before and after gas inhalation. There was no significant difference in the diameters of arterioles and venules prior to gas inhalation in people with and without diabetes. Inhalation of the hyperoxic gas mixture caused a statistically significant decrease in arteriolar and venular diameters without altering mean arterial pressure significantly. Arteriolar vasoconstriction in response to the hyperoxic gas mixture was significantly reduced in people with diabetes (3.95% versus 7.75%; p = 0.04), but venular vasoconstriction did not differ significantly. A hypoxic gas mixture caused increased arteriolar and venular diameter and a normoxic-hypercapnic gas mixture had no significant effect on vessel diameter. Responses to hypoxic and normoxic-hypercapnic gas did not differ significantly between diabetes and non-diabetes subjects. Type 1 diabetes impairs retinal arteriolar responses to hyperoxia. Abnormalities in retinal arteriolar reactivity in response to oxygen may play a role in the development of diabetic retinopathy and this technique may represent a simple means of identifying early abnormalities in the reactivity of retinal arterioles in diabetes.

Retinal arteriolar endothelial dysfunction in early stage of diabetes in porcine model

Hyperglycemia is implicated in retinal vascular dysfunction associated with the development of diabetic retinopathy. Although release of vasodilators from the endothelium of retinal arterioles is known to play an important role in the regulation of retinal blood flow, the impact of hyperglycemia on vasodilator reactivity of these microvessels remains unclear. Herein, using a streptozotocin‐induced type‐1 diabetes model in the pig, we examined nitric oxide (NO) synthase (NOS)‐ and cyclooxygenase (COX)‐mediated dilation of isolated retinal arterioles at 2 and 6 wk of hyperglycemia (485±64 mg/dL vs. 81±23 mg/dl in age‐matched control). In diabetic retinal arterioles, dilation to endothelial NOS‐mediated agent bradykinin but not to endothelium‐independent NO donor sodium nitroprusside or COX‐mediated agent forskolin was significantly reduced in a similar manner at 2‐ and 6‐wk hyperglycemia. Dihydroethidium staining showed markedly increased levels of superoxide in diabetic arterioles, while protein expression of NAD(P)H oxidase subunit p47phox was elevated. These findings show that acute hyperglycemia selectively impairs endothelial NOS‐mediated dilation of retinal arterioles in a diabetic pig model. Enhanced production of NAD(P)H oxidase‐derived superoxide likely contributes to the reduction of NO bioavailability. Support: EY018420 (TWH); Scott &amp; White Research Foundation (LK).

Abstract 5256: Increased Endothelin-A Receptor-Protein Expression/Localization and Contractile Response to Endothelin-1 of Coronary Microvasculature From Diabetic Patients

Objectives: Hyperglycemia is a potent stimulus for endothelin-1 (ET-1) production. Increased ET-1 exposure of coronary vasculature and myocytes in diabetics can hamper blood flow delivery to the vulnerable myocardium and exacerbate contractile function. In the present study, we investigated the protein expression/localization of ET-A receptors, the contractile response to ET-1 and the inhibition of ET-A receptors in the human coronary microvasculature in diabetic and case-matched non-diabetic patients. Methods: Right discarded atrial tissues were harvested pre-CPB from 30 patients with and without diabetics undergoing cardiac surgery. Coronary arterioles (90 – 180 micrometer in diameter) were dissected from the harvested tissue and placed in an organ bath containing Krebs solution. Microvascular constriction was assessed by videomicroscopy in response to ET-1, with and without an endothelin-A (ET-A) receptor antagonist. The expression and localization of ET-A receptors were also examined using immunoblot and confocal immunofluorescence microscopy. Results: ET-1 caused a dose-dependent contractile response of coronary arterioles from diabetics and non-diabetics. The contractile response of diabetic arterioles to ET-1 (10 −9 M) was significantly increased compared with those of non-diabetics (15±3% vs. 8±2%, P&lt;0.05). The contractile response of microvessels from diabetics and non-diabetics to ET-1 was significantly inhibited in the presence of the ET-A receptor antagonist BQ123 (10 −7 M, P&lt;0.05, respectively). The protein expression of ET-A receptors was significantly elevated from 1 fold of non-diabetics to 2.5 folds of diabetics (P&lt;0.01). Immunofluorescent staining displayed stronger ET-A receptors signals of smooth muscle from diabetics than those of non-diabetics. Conclusion: Hyperglycemia up-regulates ET-A-receptor protein expression/localization and increases the contractile response to ET-1 in the diabetic coronary microvasculature. The contractile response to ET-1 is via activation of ET-A receptors in diabetics since blockade of ET-A receptors prevented ET-1-induced vasoconstriction. These findings support the notion that diabetic patients face an increased ET over-stimulation.

Quantitative retinal vascular calibre changes in diabetes and retinopathy: the Singapore Malay eye study

To describe the relationship of retinal vascular calibre with diabetes and diabetic retinopathy in an Asian population. A total of 3280 (78.7% response) subjects, aged 40-80 years, of Malay ethnicity residing in Singapore participated in this population-based, cross-sectional study. Retinal vascular calibre was measured and summarized using a validated computer programme from digital retinal photographs. Diabetic retinopathy signs were graded from photographs using the modified Airlie House classification. Of the 3004 subjects with data for this analysis, 682 (22.7%) had diabetes, of whom 194 (28.4%) had retinopathy. After multivariable adjustment, retinal arteriolar calibre was significantly wider in persons with diabetes (141 vs139 microm, P<0.001); venular calibre was not associated with diabetes (P=0.93). Among participants with diabetes, venular calibre increased from 218.7 microm in those without retinopathy to 231.1 microm in those with moderate and 231.4 microm in those with severe retinopathy (Pfor trend=<0.001); arteriolar calibre was not associated with diabetic retinopathy. This study shows wider retinal arterioles in diabetes and wider venules in those with diabetic retinopathy in an Asian population. These findings confirm earlier data on white population, supporting the concept that a quantitative assessment of retinal vasculature may provide further insights into early diabetic microvascular damage.

Diabetes Downregulates Large-Conductance Ca 2+ -Activated Potassium β1 Channel Subunit in Retinal Arteriolar Smooth Muscle

Retinal vasoconstriction and reduced retinal blood flow precede the onset of diabetic retinopathy. The pathophysiological mechanisms that underlie increased retinal arteriolar tone during diabetes remain unclear. Normally, local Ca(2+) release events (Ca(2+)-sparks), trigger the activation of large-conductance Ca(2+)-activated K(+)(BK)-channels which hyperpolarize and relax vascular smooth muscle cells, thereby causing vasodilatation. In the present study, we examined BK channel function in retinal vascular smooth muscle cells from streptozotocin-induced diabetic rats. The BK channel inhibitor, Penitrem A, constricted nondiabetic retinal arterioles (pressurized to 70mmHg) by 28%. The BK current evoked by caffeine was dramatically reduced in retinal arterioles from diabetic animals even though caffeine-evoked [Ca(2+)](i) release was unaffected. Spontaneous BK currents were smaller in diabetic cells, but the amplitude of Ca(2+)-sparks was larger. The amplitudes of BK currents elicited by depolarizing voltage steps were similar in control and diabetic arterioles and mRNA expression of the pore-forming BKalpha subunit was unchanged. The Ca(2+)-sensitivity of single BK channels from diabetic retinal vascular smooth muscle cells was markedly reduced. The BKbeta1 subunit confers Ca(2+)-sensitivity to BK channel complexes and both transcript and protein levels for BKbeta1 were appreciably lower in diabetic retinal arterioles. The mean open times and the sensitivity of BK channels to tamoxifen were decreased in diabetic cells, consistent with a downregulation of BKbeta1 subunits. The potency of blockade by Pen A was lower for BK channels from diabetic animals. Thus, changes in the molecular composition of BK channels could account for retinal hypoperfusion in early diabetes, an idea having wider implications for the pathogenesis of diabetic hypertension.

Pharmacological vascular reactivity in isolated diabetic rabbit ciliary artery

Impairment of the ocular circulation induced by diabetes mellitus has not been fully defined, but is thought to be related to hemodynamic changes in the ocular circulation. The purpose of the present study is to investigate the functional and morphological changes occurring in the ciliary artery wall of rabbits with alloxan-induced diabetes mellitus. A single intravenous bolus injection of alloxan (100 mg/kg) was given to each of 26 10-week-old rabbits and 16 sham-injected control rabbits. Twenty weeks later, control rabbits and diabetic rabbits were sacrificed, and their ciliary arteries were mounted in a myograph system. The responses of these arteries to high K + solution (K-Krebs solution), phenylephrine and carbachol were investigated using isometric tension recording. L-NAME ( NG-nitro- l-arginine methyl ester; 100 μM) and indomethacin (1 μM) were also used to test the mechanism causing the carbachol induced relaxation. The arteries were also examined morphologically. The maximum tensions induced by K-Krebs solution in this tissue were not significantly different: 17.2 ± 0.8 mN ( n = 16) in the control rabbits and 17.6 ± 0.8 mN ( n = 23) in the diabetic rabbits ( P = 0.36). Phenylephrine caused dose-dependent contraction with EC 50 values of 1.3 ± 0.4 μM ( n = 6) in the control and 5.1 ± 2.3 μM ( n = 6) in the diabetic rabbits, but there was no significant difference between the two ( P = 0.36). Carbachol induced dose-dependent relaxations in segments precontracted with K-Krebs solution. These relaxations were significantly reduced in the diabetic rabbits. The maximum relaxation induced by carbachol was 77.0 ± 2.4% (10 μM) and 66.4 ± 2.5% (100 μM) in the control and diabetic rabbits, respectively. These values were significantly different ( P = 0.0076). The IC 50 value for carbachol was 396.3 ± 58.4 nM ( n = 16) in the control, and 443.6 ± 141.1 nM ( n = 23) in the diabetic rabbit ( P = 0.87). Application of a 100 μM nitric oxide synthase inhibitor, L-NAME, significantly inhibited the amplitude of relaxations evoked by carbachol ( P = 0.0066). However, these relaxations were not inhibited by pretreatment with 1 μM indomethacin ( P = 0.60). Histologically, the frequency of invaginations was less in the diabetic arterioles with a flattening of the lamina in the diabetic rabbits than in the controls. The cytoplasm of endothelial cells contained large vacuoles, indicating weak adhesion to the lamina. Some endothelial cells even showed vacuolar degeneration due to breakdown of the cell membranes. However, the smooth muscle cells were well preserved in the diabetic rabbit. These results suggest that the mechanism of impairment of ocular circulation induced by diabetes mellitus is mainly the reduction of NO synthase due to endothelial cell dysfunction. Furthermore, the characteristics of rabbits with alloxan-induced diabetes mellitus probably make them a useful model for investigating ocular complications induced by diabetic mellitus.

Decreased Endothelium-Dependent Vasodilation in Diabetic Female Rats: Role of Prostanoids

Overproduction of vasoconstrictor prostanoids and reduced prostacyclin levels have been related to the male diabetic-linked vascular dysfunction. However, it is not clear yet if these changes also occur in diabetic females. The aim of this study was to verify the role of prostanoids in the vascular dysfunction of diabetic female rats. The parameters studied were the mesenteric arteriolar reactivity (intravital microscopy and isolated perfused arteriolar bed), prostanoid measurement (enzyme immunoassay), superoxide generation (intravital fluorescence microscopy), and the presence of peroxynitrite (Western blot for nitrotyrosine-containing proteins). The response to acetylcholine was decreased in arterioles of diabetic female rats and diclofenac, but not ridogrel, corrected the altered response. The unstimulated (basal) release of thromboxane B₂ (TXB₂), but not prostaglandin F₂_α (PGF₂_α) or 6-keto-PGF₁_α, was increased in the mesenteric perfusate from diabetic female rats. Increased production of PGF₂_α and 6-keto-PGF₁_α, but not TXB₂, was induced by acetylcholine in diabetic arterioles. The superoxide generation was increased in diabetic female rats and diclofenac corrected it. Diabetes increased nitrotyrosine-containing proteins in mesenteric microvessels. In conclusion, our data show that the increase of constrictor prostanoid release, most likely PGF₂_α, could be involved in the reduced endothelium-dependent vasodilation of diabetic female rats. In addition, the enhanced activation of cyclooxygenase may be a source of superoxide anion generation in this model.

Superoxide-NO interaction decreases flow- and agonist-induced dilations of coronary arterioles in Type 2 diabetes mellitus.

Type 2 diabetes mellitus (T2-DM) markedly increases the incidence of ischemic heart disease (IHD) and, consequently, mortality. However, the underlying mechanisms leading to IHD in T2-DM are not completely understood. We hypothesized that in T2-DM the regulation of coronary microvascular resistance by local mechanisms is altered. Thus, in coronary arterioles (diameter: approximately 80 microm) isolated from male mice with T2-DM (C57BL/KsJ-db/db) and control littermates, responses to changes in intraluminal pressure, flow, and agonists with known mechanisms of action were studied. Increases in pressure (from 20 to 120 mmHg) resulted in similar myogenic responses of coronary arterioles of control and db/db mice, whereas dilations in response to cumulative concentrations of ACh and the nitric oxide (NO) donor NONOate were significantly decreased compared with those of control vessels. On the other hand, responses to adenosine were not different between vessels of control and db/db mice. Increases in flow (0-20 microl/min) resulted in dilations of control vessels (maximum: 38 +/- 4%) that were inhibited by the NO synthase inhibitor N omega-nitro-L-arginine methyl ester (L-NAME). In contrast, arterioles of db/db mice exhibited greatly reduced dilations to flow (maximum: 4 +/- 6%) that were unaffected by L-NAME. In carotid arteries of db/db mice, superoxide dismutase (SOD)-sensitive, enhanced superoxide production was detected by dihydroethydine staining and lucigenin enhanced chemiluminescence. Correspondingly, intraluminal administration of SOD significantly augmented flow-, ACh-, and NONOate-induced dilations of diabetic arterioles, and then flow- and ACh-induced responses could be inhibited by L-NAME. Collectively, these findings suggest that in T2-DM, due to an enhanced superoxide production, NO mediation of agonist- and flow-induced dilations of coronary arterioles is reduced. This alteration in the regulation of coronary microvascular resistance may contribute to the development of IHD in T2-DM.

Lack of Nitric Oxide Mediation of Flow-Dependent Arteriolar Dilation in Type I Diabetes Is Restored by Sepiapterin

The mechanisms leading to microangiopathy in diabetes mellitus have still not been clearly elucidated. We hypothesized that type I diabetes mellitus affects the endothelium and alters flow-dependent dilation of arterioles, an important mechanism involved in local regulation of blood flow. Isolated, pressurized gracilis muscle arterioles (inside diameter approximately 150 µm at 80 mm Hg) from rats with streptozotocin (STZ)-induced diabetes mellitus exhibited reduced dilations induced by increases in perfusate flow compared to those of normal rats (plasma glucose: 25.7 ± 0.7 vs. 6.4 ± 0.5 mmol/l; maximum increase in diameter: 15 ± 4 vs. 31± 3 µm, p < 0.05). In control arterioles, both nitric oxide (NO) and prostaglandins mediated the flow-dependent dilation, whereas flow-induced dilations of diabetic arterioles were unaffected by N^ω-nitro-L-arginine methyl ester (L-NAME) and were abolished by indomethacin. Sepiapterin – precursor of the endothelial NO synthase (eNOS) cofactor tetrahydrobiopterin (BH₄) – restored the L-NAME-sensitive portion of flow-dependent dilations of diabetic arterioles. Furthermore, depletion of BH₄ by 2,4-diamino-6-hydroxypyrimidine (DAHP) in control arterioles also resulted in reduced flow-dependent dilations, which were restored by intraluminal sepiapterin [but not with superoxide dismutase (SOD) plus catalase (CAT) (SOD+CAT)] and then could be inhibited by L-NAME. Dilations induced by the NO donor sodium nitroprusside (SNP) were unaffected by L-NAME in diabetes mellitus arterioles or when eNOS was activated by intraluminal flow in DAHP-treated arterioles (with or without SOD+CAT). In contrast, pyrogallol (known to produce reactive oxygen species) substantially reduced acetylcholine- and SNP-induced dilation in a SOD+CAT-reversible manner. Collectively, these findings suggest that in diabetic arterioles, due to the reduced bioavailability of BH₄, the synthesis of NO by eNOS is limited, resulting in a reduced flow-induced dilation, a mechanism that may also be responsible for the development of diabetic microangiopathy and exacerbation of other vascular diseases.

Mediation of vascular relaxation in epineurial arterioles of the sciatic nerve: effect of diabetes in type 1 and type 2 diabetic rat models.

Previously, the authors have reported that acetylcholine-induced vascular relaxation in epineurial arterioles of the sciatic nerve is mediated by nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF). Furthermore, they have demonstrated that acetylcholine-induced vasodilation in these vessels is impaired in streptozotocin-induced (type 1) and ZDF obese (type 2) diabetic rats. In the present study, the authors sought to determine the effect of diabetes on NO- and EDHF-mediated vasodilation in epineurial arterioles. In epineurial arterioles from nondiabetic Sprague-Dawley rats, NO and EDHF are equivalent in regard to their contribution to acetylcholine-induced vascular relaxation. In contrast, NO accounts for a greater portion of acetylcholine-induced vascular relaxation in normal glycemic ZDF lean rats. Following 4 weeks of hyperglycemia, the EDHF component of acetylcholine-induced vascular relaxation was totally inhibited in both streptozotocin-induced and ZDF obese diabetic rats. Vasodilation mediated by NO was still active in epineurial arterioles from both type 1 and type 2 diabetic rat models. These data suggest that diabetes causes an impairment in EDHF-mediated vascular relaxation and that interventions directed at improving EDHF production or bioactivity may improve vascular function in epineurial arterioles in diabetes.

The SOD mimetic tempol restores vasodilation in afferent arterioles of experimental diabetes

Endothelium-dependent vasodilation is impaired in large conduit vessels in diabetes mellitus. Oxygen radicals contribute to the impaired endothelium-dependent vasodilation. We tested the hypothesis that stimulated endothelium-dependent vasodilation is reduced in renal afferent arterioles in diabetes and is caused by an increase in vascular superoxide (O2(-)). Renal afferent arterioles from normal and insulin-treated alloxan-diabetic rabbits were microdissected and microperfused in vitro for the study of luminal diameter responses to acetylcholine (Ach; 10(-11) to 10(-6) mol/L). The blood glucose concentration of insulin-treated alloxan-diabetic rabbits was elevated fourfold compared with normal rabbits (319 +/- 23 vs. 79 +/- 6 mg/dL, P < 0.001). In norepinephrine (NE)-preconstricted afferent arterioles of normal rabbits, Ach significantly (P < 0.001) increased luminal diameter by 165 +/- 44%. The nitric oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester (10(-4) mol/L) blocked this Ach-induced vasodilation. In marked contrast, in NE-preconstricted arterioles of diabetic rabbits, Ach significantly (P < 0.01) decreased luminal diameter by 41 +/- 11%. Pretreatment of diabetic afferent arterioles with the superoxide dismutase (SOD) mimetic tempol (10(-3) mol/L) restored a vasodilator response to Ach. In NE-preconstricted diabetic afferent arterioles treated with tempol, Ach significantly (P < 0.001) increased luminal diameter by 25 +/- 6%. Ach-induced afferent arteriolar vasodilation is dependent on nitric oxide and is impaired in diabetes. O2(-) contributes to the impaired Ach-induced vasodilation in renal afferent arterioles in diabetes.

Free radicals mediate endothelial dysfunction of coronary arterioles in diabetes.

Previous studies have demonstrated that vascular responses to acetylcholine (ACh) are impaired in diabetes mellitus (DM). Since reactive oxygen species (ROS) generation is increased in various disease states including DM, and a direct reaction between nitric oxide (NO) and superoxide anion has been demonstrated, we tested the hypothesis that inhibition of ROS will restore coronary microvascular responses to ACh in a dog model of DM (alloxan 60 mg/kg, i.v., 1 week prior to study). Changes in coronary microvascular diameters in diabetic (blood glucose >200 mg%) and normal animals to ACh (1-100 microM, topically) in the presence and absence of superoxide dismutase and catalase were measured using intravital microscopy coupled to stroboscopic epi-illumination and jet ventilation. In diabetic animals in the absence of ROS scavengers, ACh induced coronary microvascular dilation was impaired when compared to normal animals (ACh 100 microM: DM=25+/-5%; normal=64+/-13%, P<0.05). Topical application of SOD (250 U/ml) and catalase (250 U/ml) restored to normal ACh induced coronary microvascular responses in DM while having no affect in normal animals. Responses to adenosine and nitroprusside were not different between normal and diabetic groups. These data provide direct evidence that oxygen-derived free radicals contribute to impaired endothelium-dependent coronary arteriolar dilation in diabetic dogs in vivo.

Impaired Vascular Dynamics in Normotensive Diabetic Rats Induced by Streptozotocin: Tapered T-tube Model Analysis

This study is to explore the changes of arterial mechanical properties in streptozotocin (STZ)-diabetic rats, based on the exponentially tapered T-tube model. Rats given STZ 65 mgkg−1i.v. are compared with untreated weight- and age-matched controls. A high-fidelity pressure sensor and electromagnetic flow probe measured pulsatile pressure and flow waves in the ascending aorta, respectively. Diabetic rats exhibit isobaric vasodilatation that is characterized by an increase in cardiac output and no significant changes in aortic pressure. Total peripheral resistance of diabetic rats is lower than that of weight- and age-matched controls. Diabetic rats have higher total peripheral compliance (2.86±0.70 μlmmHg−1) than do weight- (1.77±0.34 μlmmHg−1) and age-matched (1.87±0.69 μlmmHg−1) controls. Aortic characteristic impedance is reduced from 0.017±0.003 mmHgminkgml−1in weight- and 0.020±0.004 mmHgminkgml−1in age-matched controls to 0.010±0.004 mmHgminkgml−1in diabetic rats. Moreover, diabetic rats show shorter wave transit time in lower body circulation (17.86±1.91 ms) than do weight- (20.45±1.91) and age-matched (23.05±2.04 ms) controls. Under isobaric vasodilatation, the decreased resistance and increased compliance in peripheral circulation suggest that the contractile dysfunction of the smooth muscle cells may occur in resistance arterioles in diabetes. With unaltered aortic pressure, an impairment in aortic distensibility of STZ-diabetic rats is manifest on the reduced wave transit time rather than on the diminished aortic characteristic impedance.

Microcirculatory changes following reperfusion insult in diabetic rat skeletal muscles.

We investigated the microcirculatory changes of ischemia/reperfusion injury in the diabetic rat cremaster muscle as well as the therapeutic effect of insulin. Streptozotocin-induced diabetic rats were maintained hyperglycemic for up to 8 weeks or were treated with insulin in the diabetic period. The rat cremaster muscle was prepared as an island flap and subjected to 2-h clamp ischemia followed by 1-h reperfusion. In nonischemic conditions, effective concentrations for 50% response (EC50) of serial orders of arterioles to norepinephrine were higher in diabetic muscles. Ischemia/reperfusion insult significantly decreased the EC50 of arterioles in the normal group, but not in the diabetic group. Light microscopy showed that the diabetic cremasters had more collapsed capillaries and smooth muscle-disarranged arterioles. Insulin therapy showed significant improvement in the diabetes-caused reduction of perfused capillary density, but not in the contractility of the diabetic arterioles. These results indicate that diabetes mellitus may damage the skeletal muscle microvasculature irreversibly and make it less responsive to autonomic regulation. Insulin therapy can improve capillary perfusion, but not the microvascular reactivity of diabetic muscles.