Regular ArticleInvolvement of Nitric Oxide and Cyclooxygenase Products in Photoactivation-Induced Microvascular Occlusion
Abstract
Photoactivation of intravascular dyes with high doses of light is a technique used clinically to treat tumors. This procedure results in arteriolar constriction, mast cell degranulation, platelet thrombus formation, and, ultimately, microvascular stasis. In vivo microscopy was utilized in the current study to examine if the endothelial release of prostaglandins and nitric oxide could participate in the microvascular effects of photoactivation. Diameter changes and thrombus formation of arterioles and venules of the cremaster muscle of male Sprague-Dawley rats were quantitated during continuous light activation of intravascular fluorescein isothiocyanate conjugated to bovine serum albumin. Vasoconstriction and thrombus development were assessed separately, using the relationships between the width of the red blood cell column, the inner wall diameter, and the thickness of the plasma layer. Venular photoactivation resulted in thrombus growth which reached 30% of the maximum size by 16.8 ± 3.71 min and a subsequent growth rate of 6.2 ± 1.64 μm/min. In arterioles, 30% thrombus growth occurred at 14.0 ± 2.02 min with a growth rate of 3.0 ± 0.57 μm/min. Continuous arteriolar photoactivation led to a vasoconstriction of 34.4 ± 6.87% of the initial vessel diameter. Thirty percent of the maximal constriction occurred after 10.6 ± 1.26 min of photoactivation. Constriction proceeded at a rate of 3.8 ± 1.32 μm/min. Topically applied mefenamic acid (a cyclooxygenase inhibitor) and NW -nitro-L-arginine methyl ester (L-NAME, a nitric oxide synthase inhibitor) each enhanced both the arteriolar and the venular thrombus growth due to photoactivation. Photoactivation-induced arteriolar constriction was augmented by L-NAME and inhibited by mefenamic acid. These data suggest that the photoactivation of intravascular dyes is accompanied both by the release of nitric oxide, which inhibits thrombus development and arteriolar constriction, and by the release of cyclooxygenase products, which inhibit thrombus growth and induce vasoconstriction. Rats treated with busulfan to induce thrombocytopenia exhibited a 90% decrease in circulating platelets. In these animals, photoactivation caused significantly delayed thrombus growth in arterioles and venules, while arteriolar constriction remained unaltered, suggesting that the vasoconstrictor prostanoid is not of platelet origin.
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Microvascular consequences of thrombosis in small venules. An in vivo microscopic study using a novel model in the ear of the hairless mouse
2000, Thrombosis ResearchLittle is known of the development of chronic microvascular alterations following small vessel thrombosis, which is probably due to the lack of appropriate experimental models. Herein we report the first results on thrombosis-associated long-term changes of microvascular permeability and vessel tortuosity and diameter and blood perfusion using the ear of the hairless mouse, and intravital fluorescence microscopy. Thrombosis was induced photochemically in small venules (diameter: 75 to 100 μm) using light/dye exposure (fluorescein isothiocyanate-dextran 150,000), and the microcirculation compromised by the blockade of blood drainage was analyzed before and 30 minutes after induction of thrombosis as well as repeatedly over a 28-day observation period. Thrombus formation resulted in a marked increase (p<0.05) of microvascular permeability (0.85±0.11) when compared with baseline values (0.46±0.04). Permeability remained elevated (p<0.05) at days 1 (0.67±0.07), 3 (0.58±0.02), and 7 (0.60±0.06), but returned to normal after 28 days (0.43±0.03). Tortuosity, diameter, and red blood cell velocity of venular segments, located upstream of thrombus formation, were found unchanged during the entire 28-day observation period. This was probably due to the fact that blood flow from the thrombosis-affected tissue was frequently drained into nonaffected tissue via preexisting “through-fare” channels, serving as venulo-venular collaterals. In accordance, in 10 to 20% of these venular segments the direction of blood perfusion was found changed, while those changes were only rarely observed in venular vessel segments of the nonthrombotic contralateral ears. We conclude that thrombosis in small cutaneous venules is primarily characterized by an increased vascular permeability, reflecting an inflammatory response, similar to what is known from thrombophlebitis in patients. The model presented herein may be a versatile tool to study pathogenesis of chronic microcirculatory derangements in microthrombosis and their prevention by novel therapeutic strategies.
YC-1, a nitric oxide-independent activator of soluble guanylate cyclase, inhibits platelet-rich thrombosis in mice
1997, European Journal of PharmacologyYC-1 (3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole), a nitric oxide (NO)-independent activator of soluble guanylate cyclase, has been shown to inhibit platelet activation and aggregation in vitro through the generation of cGMP. In the present study, we assessed the antithrombotic effect of YC-1 in models of experimental thrombosis in mice. YC-1 (10, 30 μg/g, i.p.)-treated mice showed a prolonged tail bleeding time 30 min after injection (from control 91.0±6.4 s to 208.6±22.7 s and 291.8±42.4 s, respectively). In contrast, aspirin at a dose of 30 μg/g (i.p.) prolonged the bleeding time to more than 600 s. Platelet-rich thrombus formation was induced by irradiation of the mesenteric venule with filtered light in mice pretreated intravenously with fluorescein sodium. YC-1 (30 μg/g, i.p.) markedly prolonged the occlusion time of irradiated venules (from control 146.1±19.0 s to 275.6±24.5 s) in heparinized (1 U/g) mice. In the same condition, aspirin (100 μg/g) only slightly prolonged the time required for occlusion (193.2±13.2 s). In a model of fatal pulmonary thromboembolism induced by intravenous injection of ADP (300 μg/g), YC-1 was effective in reducing mortality when administered intraperitoneally at doses of 10–30 μg/g. The antithrombotic effect of YC-1 was correlated with the inhibition of ADP-induced platelet aggregation ex vivo. In contrast, aspirin (30, 100 μg/g) did not inhibit ADP-induced pulmonary thromboembolism in vivo or platelet aggregation ex vivo. YC-1 (3, 10 μg/g) also exhibited profibrinolytic activity ex vivo, as revealed by shortening of the euglobulin clot lysis time. Therefore, YC-1 is an effective antithrombotic agent in preventing thrombosis in animal models, and its antiaggregating and additional profibrinolytic effects may be of potential clinical benefit in the treatment of thromboembolic diseases.
Interleukin-2 induces increased platelet-endothelium interactions: A potential mechanism of toxicity
1996, Journal of Laboratory and Clinical MedicineCancer immunotherapy with interleukin-2 (IL-2) is limited by side effects that may cause organ dysfunction. The role of platelets in the generation of IL-2-induced organ dysfunction has not been studied, although various studies have shown that IL-2 therapy activates both platelets and the vascular endothelium. We hypothesized that IL-2 therapy may enhance the thrombogenic response to inflammatory stimuli through increased platelet-endothelial interactions and that these effects could lead to the development of organ dysfunction. C57B1/6 mice were treated with IL-2 intraperitoneally for 2 hours (short term) or 2 to 5 days (long term) and prepared for in vivo microscopy of the ear microcirculation. Mice were injected intra-arterially with fluorescein isothiocyanate conjugated to bovine serum albumin (FITC-BSA). Blue light activation of the FITC-BSA in ear arterioles induced thrombus formation. The time to initial thrombus formation was measured as an index of thrombogenicity. Platelet function was analyzed by aggregometry and platelet expression of IL-2 receptors, and the adhesion molecule lymphocyte function-associated antigen-1 (LFA-1) was analyzed by flow cytometry. Organ dysfunction was evaluated by serum markers. The administration of both short-term and longterm IL-2 reduced the time to initial thrombus formation as compared with controls. In vitro platelet aggregometry revealed no acute alterations in platelet function; however, long-term IL-2 treatment resulted in decreased disaggregation rates. There were no platelet IL-2 receptors present, and the expression of the adhesion molecule LFA-1 was not altered by IL-2. Increased thrombogenicity occurred before the generation of organ dysfunction. These data suggest that increased platelet adherence induced by IL-2 is caused by effects on the endothelium that could result in microvascular thrombus formation and contribute to organ dysfunction.
Spontaneously hypertensive rats are resistant to the development of hypercholesterolemia
1995, American Journal of HypertensionThe arterioles of young spontaneously hypertensive rats (SHR) are purported to have an enhanced sensitivity to nitric oxide (NO)-dependent vasodilators, relative to normotensive animals, while NO-related arteriolar responses are diminished in both mature SHR as well as hypercholesterolemic normotensive rats. Because endothelial production of NO relaxes vascular smooth muscle and inhibits platelet adhesion and aggregation, hypercholesterolemia may synergistically affect the development of genetic hypertension. The NO-mediated baseline vascular tone, acetylcholine-induced dilation, and inhibition of platelet thrombus formation were studied over time (10 weeks) in SHR and hypercholesterolemic SHR (HC-SHR). The in vivo microcirculation of the cremaster muscle was used to quantitate all observations. The HC-SHR became significantly hypercholesterolemic after 1 week on the cholesterol-supplemented diet, with serum cholesterol concentrations remaining elevated for the 10 weeks studied. However, the serum cholesterol concentrations of HC-SHR were significantly less than those of Sprague-Dawley and Wistar-Kyoto rats fed the same diet. Dietary hypercholesterolemia did not exacerbate the development of ge netic hypertension. Second- and third-order arterioles of SHR and age-matched HC-SHR constricted to the same extent when the NO synthase inhibitor Nω-nitro-L-arginine methyl ester (L-NAME) was applied. The third-order arterioles of both groups also dilated the same amount to acetylcholine and sodium nitroprusside. Platelet thrombus formation induced by light/dye photochemistry was not different between the SHR and HC-SHR groups either at 1 or 10 weeks of diet, and L-NAME decreased the time to thrombus occlusion of blood flow equally in both groups. This is in marked contrast to the previously reported hypercholesterolemia-induced decreases in vascular reactivity in Sprague-Dawley rats. These current findings demonstrate that SHR are resistant to the development of hypercholesterolemia and that NO-mediated vascular responses in SHR are not attenuated by hypercholesterolemia.
Capillary reperfusion after L-arginine, L-NMMA, and L-NNA treatment in cheek pouch microvasculature
1995, Microvascular ResearchThe effects of arginine (L-arg), promoter of nitric oxide (NO) production and NO synthesis inhibitors, Nψ-monomethyl-L-arginine (L-NMMA) and Nψ-nitro-L-arginine (L-NNA), on arteriolar responses and capillary perfusion after 30 min ischemia were studied in the cheek pouch preparation under pentobarbital anesthesia and intravenous drug infusion. Capillary density, venular leukocyte sticking, and vessel diameters were investigated by fluorescence microscopy. Damage due to photoactivation of intravascular dyes was investigated by injecting fluorescent dextran 150,000 MW prior to and after ischemia reperfusion. No difference was found indicating that effects were independent from exposure time to photoactivated dyes. Capillary perfusion reduction was always present after reperfusion in untreated, L-NMMA-treated, and L-NNA-treated animals, with increased venular leukocytes adhesion. Arteriolar vasomotion was induced by L-NMMA treatment. Capillary perfusion recovered in L-arg-treated hamsters, where capillary blood flow velocity was lower than in L-NMMA group and the number of adhering leukocytes was lower than in untreated controls, L-NMMA, and L-NNA groups. It is concluded that L-arg determines perfusion with increased blood flow heterogeneity while inhibition of NO preserves capillary perfusion causing appearance of vasomotion in the arterial network.
An overview of three promising mechanical, optical, and biochemical engineering approaches to improve selective photothermolysis of refractory port wine stains
2012, Annals of Biomedical Engineering