Histamine decreases myogenic tone in rat cerebral arteries by H-2-receptor-mediated K-v channel activation, independent of endothelium and cyclic AMP

The effect of histamine on the pressure-induced constriction was characterized in rat cerebral arteries and mechanisms were investigated. Rat cerebral arteries were pressurized to 70 mm Hg in an arteriograph and the effect of histamine on myogenic tone was studied. Histamine and amthamine, a selective histamine H-2-receptor agonist, concentration-dependently decreased myogenic tone, which was unchanged in the absence of endothelium. 2-(2-aminoethyl) pyridine, a selective histamine H-1-receptor agonist, produced concentration-dependent constriction of arteries that was significantly increased in the absence of endothelium. Imetit, a selective histamine H-3-receptor agonist, has no effect on myogenic tone. The dilation to histamine was antagonized by tiotidine, a selective antagonist of histamine H-2-receptor subtype, giving a pK(B) of 7.86 that was not altered in the absence of endothelium. The histamine-mediated dilation was significantly antagonized by NF449, a reversible inhibitor of Gs-protein activation but was not affected by ODQ and SQ 22536. Dilations to histamine and amthamine were accompanied by a decrease in arterial wall calcium measured by fura-2 ratios. The dilation to histamine was significantly reduced by partial depolatization of smooth muscle by 25 mM KCl (control 91 +/- 5%, 25 mM KCI 53 +/- 5%, P <0.002) and was not observed in the presence of strongly depolarizing 60 mM KCI. The histamine dilation was not affected by iberiotoxin, barium chloride and glibenclamide but was strongly antagonized by 4-aminopyridine (0.3 mM) and tetraethylammonium chloride (10 mM) (pEC(50): control: 5.6 +/- 0.14-aminopytidine: 4.1 +/- 0.1 (P <0.001); tetraethylammonium, chloride: 3.2 +/- 0.2 (P <0.0001)). These results suggest that histamine-mediated reversal of myogenic tone in rat cerebral arteries is endothelium-independent, mediated by histamine H-2-receptor subtype with no involvement of guanylyl cyclase or adenylyl cyclase activation and most likely involves activation of K-V potassium channels. (c) 2006 Elsevier B.V. All rights reserved

Impaired mitochondria-dependent vasodilation in cerebral arteries of Zucker obese rats with insulin resistance

Mitochondria affect cerebrovascular tone by activation of mitochondrial ATP-sensitive K+ (KATP) channels and generation of reactive oxygen species (ROS). Insulin resistance accompanying obesity causes mitochondrial dysfunction, but the consequences on the cerebral circulation have not been fully identified. We evaluated the mitochondrial effects of diazoxide, a putative mitochondrial KATP channel activator, on cerebral arteries of Zucker obese (ZO) rats with insulin resistance and lean (ZL) controls. Diameter measurements showed diminished diazoxide-induced vasodilation in ZO compared with ZL rats. Maximal relaxation was 38 ± 3% in ZL vs. 21 ± 4% in ZO rats (P < 0.05). Iberiotoxin, a Ca2+-activated K+ channel inhibitor, or manganese(III) tetrakis(4-benzoic acid)porphyrin chloride, an SOD mimetic, or endothelial denudation diminished vasodilation to diazoxide, implicating Ca2+-activated K+ channels, ROS, and endothelial factors in vasodilation. Inhibition of nitric oxide synthase (NOS) in ZL rats diminished diazoxide-induced vasodilation in intact arteries, but vasodilation was unaffected in endothelium-denuded arteries. In contrast, NOS inhibition in ZO rats enhanced vasodilation in endothelium-denuded arteries, but intact arteries were unaffected, suggesting that activity of endothelial NOS was abolished, whereas factors derived from nonendothelial NOS promoted vasoconstriction. Fluorescence microscopy showed decreased mitochondrial depolarization, ROS production, and nitric oxide generation in response to diazoxide in ZO arteries. Protein and mRNA measurements revealed increased expression of endothelial NOS and SODs in ZO arteries. Thus, cerebrovascular dilation to mitochondria-derived factors involves integration of endothelial and smooth muscle mechanisms. Furthermore, mitochondria-mediated vasodilation was diminished in ZO rats due to impaired mitochondrial KATP channel activation, diminished mitochondrial ROS generation, increased ROS scavenging, and abnormal NOS activity

Blockade of NADPH Oxidase Restores Vasoreparative Function in Diabetic CD34+ Cells

Administration of autologous CD34+ cells with restored reparative function by ex vivo inhibition of NADPH oxidase may represent a safer therapeutic approach to correct the multitude of vascular abnormalities in diabetic patients

Diabetic eNOS-Knockout Mice Develop Accelerated Retinopathy

The results of this study demonstrate that diabetic eNOS−/− mice develop an accelerated diabetic retinopathy, including increased retinal vessel leakage, gliosis, an increased number of acellular retinal capillaries, and basement membrane thickening. Moreover, these pathologic changes occur at an accelerated rate compared with those in wild-type STZ-treated diabetic mice, supporting a role for the deficiency in eNOS-derived NO production in the pathogenesis of diabetic retinopathy

Functional characterization of alpha(1)-adrenoceptor subtypes in human skeletal muscle resistance arteries

1. α(1)-adrenoceptor subtypes in human skeletal muscle resistance arteries were characterized using agonists noradrenaline (non-selective) and A61603 (α(1A)-selective), the antagonists prazosin (non-selective), 5-methyl-urapidil (α(1A)-selective) and BMY7378 (α(1D)-selective) and the alkylating agent chloroethylclonidine (preferential for α(1B)). 2. Small arteries were obtained from the non-ischaemic skeletal muscle of limbs amputated for critical limb ischaemia and isometric tension recorded using wire myography. 3. Prazosin antagonized responses to noradrenaline with a pA(2) value of 9.18, consistent with the presence of α(1)-adrenoceptors, although the Schild slope (1.32) was significantly different from unity. 4. 5-Methyl-urapidil competitively antagonized responses to noradrenaline with a pK(B) value of 8.48 and a Schild slope of 0.99, consistent with the presence of α(1A)-adrenoceptors. In the presence of 300 nM 5-methyl-urapidil, noradrenaline exhibited biphasic concentration response curves, indicating the presence of a minor population of a 5-methyl-urapidil-resistant subtype. 5. Contractile responses to noradrenaline were not affected by 1 μM chloroethylclonidine suggesting the absence of α(1B)-adrenoceptors. Maximum responses to noradrenaline and A61603 were reduced to a similar extent by 10 μM chloroethylclonidine, suggesting an effect of chloroethylclonidine at α(1A)-adrenoceptors at the higher concentration. 6. BMY7378 (10 and 100 nM) had no effect on responses to noradrenaline. BMY7378 (1 μM) poorly shifted the potency of noradrenaline giving a pA(2) of 6.52. These results rule out the presence of the α(1D)-subtype. 7. These results show that contractile responses to noradrenaline in human skeletal muscle resistance arteries are predominantly mediated by the α(1A)-adrenoceptor subtype with a minor population of an unknown α(1)-adrenoceptor subtype

Vasoreparative Dysfunction of CD34⁺ Cells in Diabetic Individuals Involves Hypoxic Desensitization and Impaired Autocrine/Paracrine Mechanisms

<div><p>We hypothesized that endothelial progenitor cells derived from individuals with diabetes would exhibit functional defects including inability to respond to hypoxia and altered paracrine/autocrine function that would impair the angiogenic potential of these cells. Circulating mononuclear cells isolated from diabetic (n = 69) and nondiabetic (n = 46) individuals were used to grow endothelial colony forming cells (ECFC), early endothelial progenitor cells (eEPCs) and isolate CD34⁺ cells. ECFCs and eEPCs were established from only 15% of the diabetic individuals tested thus directing our main effort toward examination of CD34⁺ cells. CD34⁺ cells were plated in basal medium to obtain cell-free conditioned medium (CM). In CM derived from CD34⁺ cells of diabetic individuals (diabetic-CM), the levels of stem cell factor, hepatocyte growth factor, and thrombopoietin were lower, and IL-1β and tumor necrosis factor (TNFα) levels were higher than CM derived from nondiabetic individuals (nondiabetic-CM). Hypoxia did not upregulate HIF1α in CD34⁺ cells of diabetic origin. Migration and proliferation of nondiabetic CD34⁺ cells toward diabetic-CM were lower compared to nondiabetic-CM. Attenuation of pressure-induced constriction, potentiation of bradykinin relaxation, and generation of cGMP and cAMP in arterioles were observed with nondiabetic-CM, but not with diabetic-CM. Diabetic-CM failed to induce endothelial tube formation from vascular tissue. These results suggest that diabetic subjects with microvascular complications exhibit severely limited capacity to generate ex-vivo expanded endothelial progenitor populations and that the vasoreparative dysfunction observed in diabetic CD34⁺ cells is due to impaired autocrine/paracrine function and reduced sensitivity to hypoxia.</p></div

Protection of blood retinal barrier and systemic vasculature by insulin-like growth factor binding protein-3

Previously, we showed that insulin growth factor (IGF)-1 binding protein-3 (IGFBP-3), independent of IGF-1, reduces pathological angiogenesis in a mouse model of the oxygen-induced retinopathy (OIR). The current study evaluates novel endothelium-dependent functions of IGFBP-3 including blood retinal barrier (BRB) integrity and vasorelaxation. To evaluate vascular barrier function, either plasmid expressing IGFBP-3 under the regulation of an endothelial-specific promoter or a control plasmid was injected into the vitreous humor of mouse pups (P1) and compared to the non-injected eyes of the same pups undergoing standard OIR protocol. Prior to sacrifice, the mice were given an injection of horseradish peroxidase (HRP). IGFBP-3 plasmid-injected eyes displayed near-normal vessel morphology and enhanced vascular barrier function. Further, in vitro IGFBP-3 protects retinal endothelial cells from VEGF-induced loss of junctional integrity by antagonizing the dissociation of the junctional complexes. To assess the vasodilatory effects of IGFBP-3, rat posterior cerebral arteries were examined in vitro. Intraluminal IGFBP-3 decreased both pressure- and serotonin-induced constrictions by stimulating nitric oxide (NO) release that were blocked by L-NAME or scavenger receptor-B1 neutralizing antibody (SRB1-Ab). Both wild-type and IGF-1-nonbinding mutant IGFBP-3 (IGFBP-3NB) stimulated eNOS activity/NO release to a similar extent in human microvascular endothelial cells (HMVECs). NO release was neither associated with an increase in intracellular calcium nor decreased by Ca(2+)/calmodulin-dependent protein kinase II (CamKII) blockade; however, dephosphorylation of eNOS-Thr(495) was observed. Phosphatidylinositol 3-kinase (PI3K) activity and Akt-Ser(473) phosphorylation were both increased by IGFBP-3 and selectively blocked by the SRB1-Ab or PI3K blocker LY294002. In conclusion, IGFBP-3 mediates protective effects on BRB integrity and mediates robust NO release to stimulate vasorelaxation via activation of SRB1. This response is IGF-1- and calcium-independent, but requires PI3K/Akt activation, suggesting that IGFBP-3 has novel protective effects on retinal and systemic vasculature and may be a therapeutic candidate for ocular complications such as diabetic retinopathy

Paracrine angiogenic functions of CM obtained from nondiabetic and diabetic CD34⁺ cells.

<p>Mouse corporeal strips were suspended in matrigel and treated with different media for 5 days. Bright-field images were obtained at 4× or 10× magnification. Day 0 shows strips in liquid matrigel before polymerization at 37°C. <b>A</b>, Representative images of corporeal strips treated with basal medium show migration of endothelial cells into matrigel that tend to form poorly defined tubular structures at different time intervals. <b>B</b>, Treatment of corporeal strips with 100 nM VEGF stimulated migration of endothelial cells into the matrigel, with endothelial sprouting, and formation of tubular structures. <b>C</b>, Treatment of corporeal strips with nondiabetic-CM stimulated migration of endothelial cells into the matrigel that tended to form sprouts and tubular structures. <b>D</b>, Treatment of corporeal strips with diabetic-CM stimulated minimal migration of endothelial cells and cells tended to form clumps, which failed to form defined tubular structures. Scale bar measures 100 microns. <b>E</b>, Quantification of angiogenic response determined by tube length (microns): Response to the non-diabetic CM is higher than the response observed in the untreated strips (<i>P<0.001</i>, n = 5). Response to diabetic-CM was lower compared to the untreated (<i>P<0.0001</i>, n = 5) or nondiabetic-CM-treated strips (<i>P<0.0001</i>, n = 5).</p