Functional consequences of inhibiting exocytosis of Weibel-Palade bodies in acute renal ischemia.

Exocytosis of Weibel-Palade bodies (WPB) represents a distinct response of endothelial cells to stressors, and local release of WPB contents leads to systemic escalation of this response. We synthesized a glycine-(Nα-Et)lysine-proline-arginine (ITF 1697) peptide that has a potential to inhibit exocytosis of WPB and protect microcirculation. Here, we confirmed an inhibitory effect of ITF 1697 using intravital videoimaging and point-tracking of individual organelles. In an in vivo study, mice were implanted with Alzet osmotic pumps (10 µg ITF 1697·kg(-1)·min(-1) at volume of 1 µl/h) and subjected to renal ischemia (IRI). IRI resulted in marked renal injury and elevation of serum creatinine in mice treated with a vehicle. In contrast, renal injury and elevation of creatinine were significantly ameliorated in mice subjected to IRI and receiving ITF 1697. ITF 1697 prevented a systemic response to IRI: a significant surge in the levels of eotaxin and IL-8 (KC; both components of WPB), IL-1α, IL-1ß, and RANTES was all prevented or blunted by the administration of ITF 1697, whereas the levels of an anti-inflammatory, IL-10, and macrophage inflammatory protein-1α were upregulated in ITF 1697-treated animals. En face staining of aortic endothelial cells showed that WPB were depleted after 40-180 min post-IRI, and this was significantly blunted in aortic preparations obtained from mice treated with ITF 1697. WPB exocytosis contributed to IRI-associated mobilization of endothelial progenitor cells and hematopoietic stem cells, and ITF 1697 blunted their mobilization. Unexpectedly, 1 mo after IRI, mice treated with ITF 1697 showed a significantly more pronounced degree of scarring than nontreated animals. In conclusion, 1) application of ITF 1697 inhibits exocytosis of WPB and IRI; 2) the systemic inflammatory response of IRI is in part due to the exocytosis of WPB and its blockade blunts it; and 3) ITF 1697 improves short-term renal function after IRI, but not the long-term fibrotic complications.

pO(2) and ROS/RNS measurements in the microcirculation in hypoxia.

We expose methods for in vivo assessment of oxygen, nitric oxide (NO), and reactive oxygen species (ROS)/reactive nitrogen species (RNS), in the microcirculation during normoxia and hypoxia. We provide an example of the related mechanisms of ROS/RNS and oxygen level in the process of regulating capillary perfusion. Namely, we discuss the real time pO(2) measurements in vivo in the microvessels and tissues of the hamster cheek pouch and window chamber preparations during normoxia and hypoxia, as well as the corresponding changes in ROS/RNS in systemic blood during normoxia and hypoxia under conditions where NO availability is maximally reduced.

Melatonin reduces microvascular damage and insulin resistance in hamsters due to chronic intermittent hypoxia.

Obstructive sleep apnea (OSA) causes intermittent hypoxia (IH) associated with hypertension, insulin resistance and a systemic inflammatory response. We evaluated the effects of melatonin on vasodilation, capillary perfusion in hamster cheek pouch and insulin resistance, hypertension, and reactive oxygen species (ROS) and nitrate/nitrite levels after IH for 4 wk. Syrian hamsters were divided into four groups: control group (CON), IH group, and melatonin (10 mg/kg) intraperitoneally administered daily for 4 wk/30 min before intermittent air (MEL) or IH (IH + MEL) exposure. IH alone caused elevated blood pressure, increased hematocrit, fasting hyperglycemia, elevated ROS and nitrite/nitrate levels, and vasoconstriction and reduced microvascular perfusion. Melatonin treatment of IH-exposed animals decreased blood pressure, blood glucose, and ROS and nitrite/nitrate levels, and increased vasodilation and capillary perfusion. An oral glucose tolerance test was performed after 4 wk of IH. During the last 30 min of the hyperinsulinemic euglycemic clamp, blood glucose, and insulin levels were identically matched between groups, but the glucose infusion rate was significantly reduced in IH (29.9 +/- 1.9 mg/kg/min) versus IH + MEL group (45.4 +/- 1.5 mg/kg/min, P < 0.05) demonstrating a decrease in insulin sensitivity. These results suggest that ROS and nitrite/nitrate levels play important roles in the microvascular dysfunction in IH and that this process is attenuated by melatonin. In conclusion, protection induced by melatonin against functional and metabolic impairment in IH is related to the regulation of ROS and nitrite/nitrate levels in the microcirculation. These observations may have importance to OSA pathological changes.

Intermittent hypoxia modulates nitric oxide-dependent vasodilation and capillary perfusion during ischemia-reperfusion-induced damage.

The microvascular function of nitric oxide (NO) during ischemia-reperfusion (I/R) in intermittent hypoxia (IH)-pretreated hamsters was analyzed using 20 mg/kg of the nonselective NO inhibitor N(omega)-nitro-l-arginine methyl ester (l-NAME) and 5 mg/kg of the preferential inducible NO inhibitor S-methylisothiourea sulphate (SMT) injected before I/R. Studies were made in the hamster cheek pouch microcirculation (intravital fluorescence microscopy). IH consisted of 6 min of 8% O(2) breathing followed by 6 min of 21% O(2) for every 8 h for 21 days. Normoxia controls (NCs) were exposed to room air for the same period. The effects were characterized in terms of systemic hemodynamics, diameter, flow, wall shear stress in arterioles, capillary perfusion, and the concentrations of thiobarbituric acid-reactive substances (TBARS) and plasma NO, assessed as nitrite/nitrate (NOx) levels. IH did not change arterial blood pressure and increased hematocrit and shear stress. IH increased NOx and TBARS levels and reduced arterial diameter, blood flow, and capillary perfusion versus the NC. Conversely, TBARS and NOx were lower during I/R in IH-pretreated hamsters, resulting in vasodilation and the increase of capillary perfusion and shear stress. After IH, capillary perfusion was reduced by 24% (2.3%) and enhanced by 115% (1.7%) after I/R (P < 0.05). Both modalities of NO blockade decreased NOx generation and increased TBARS versus IH. l-NAME and SMT induced a significant decrease in arteriolar diameter, blood flow, and capillary perfusion (P < 0.05). l-NAME enhanced TBARS more than SMT and aggravated I/R damage. In conclusion, we demonstrated that preconditioning with IH greatly reduces oxidative stress and stimulates NO-induced vasodilation during I/R injury, thus maintaining capillary perfusion.

ITF1697, a stable Lys-Pro-containing peptide, inhibits weibel-palade body exocytosis induced by ischemia/reperfusion and pressure elevation.

A number of Lys-Pro-containing short peptides have been described as possessing a variety of biological activities in vitro. Because of limited metabolic stability, however, their efficacy in vivo is uncertain. To exploit the pharmacological potential of Lys-Pro-containing short peptides, we synthesized a series of chemically modified forms of these peptides. One of them, ITF1697 (Gly-(Nalpha-Et)Lys-Pro-Arg) was stable in vivo and particularly efficacious in experimental models of disseminated endotoxemia and of cardiovascular disorders. Using intravital fluorescence microscopy, we studied the peptide cellular and molecular basis of protection in the Syrian hamster cheek pouch microcirculation subjected to ischemia/reperfusion (I/R) and in pressure elevation-induced proinflammatory responses in isolated Sprague-Dawley rat lungs. Continuous intravenous infusion of ITF1697 at 0.1 to 100 mug/kg/min nearly completely protected the cheek pouch microcirculation from I/R injury as measured by decreased vascular permeability and increased capillary perfusion. Adhesion of leukocytes and platelets to blood vessels was strongly inhibited by the peptide. ITF1697 exerted its activity at the early stages of endothelial activation and inhibited P-selectin and von Willebrand factor secretion. Further mechanistic studies in the rat lung preparation revealed that the peptide inhibited the intracellular Ca(2+)-dependent fusion of Weibel-Palade bodies with the plasma membrane. The ability of ITF1697 to inhibit the early functions of activated endothelial cells, such as the exocytosis of Weibel-Palade bodies, represents a novel and promising pharmacological tool in model of pathologies of a variety of microvascular disorders.

Mechanisms by which low-intensity ultrasound improve tolerance to ischemia-reperfusion injury.

Recent studies show that low-intensity ultrasound (US) increases endothelial nitric oxide (NO) levels in different models both in vitro and in vivo. Ischemia-reperfusion (I/R) injury is characterized by endothelial cell dysfunction, mainly as a result of altered shear stress responses associated with vasoconstriction, reduced capillary perfusion and excessive oxidative stress. This review provides an overview of the microvascular effects of low-intensity US and suggests that US exposure can be a method to provide tolerance to I/R damage. The hamster cheek pouch, extensively used in studies of I/R-induced injury, has been characterized in terms of changes of arteriolar diameter, flow and shear stress. The low-intensity US exposure reduces vasoconstriction and leukocyte adhesion and increases capillary perfusion during postischemic reperfusion. These effects may be the result of enhanced fluctuations in shear stress exerted by the flowing blood on the vessel wall. The fluctuations in turn are due to mechanical perturbations arising from the difference in acoustical impedance between the endothelial cells and the vessel content. We believe that periodic pulses of US may also cause a sustained reduction of oxidative stress and an enhanced endothelial NO level by increasing oscillatory shear stress during postischemic reperfusion. Low-intensity US exposure may represent a safe and novel important therapeutic target for patients with acute coronary syndromes and for treatment of chronic myocardial ischemia.

Melatonin reduces ventricular arrhythmias and preserves capillary perfusion during ischemia-reperfusion events in cardiomyopathic hamsters.

Earlier studies showed that melatonin has powerful antioxidative effects on ischemia-reperfusion (I/R) injury in healthy hamsters. In the present study, the possible protective effects of melatonin in 10-month-old cardiomyopathic (CM) hamsters were evaluated in a model of I/R in the cheek pouches observed by intravital microscopy. In CM (BIO 14.6) hamsters diameter, red blood cell (RBC) velocity and flow in arterioles as well as lipid peroxide and nitrite/nitrate concentrations in the systemic blood, perfused capillary length, vascular permeability, and leukocyte adhesion were measured after melatonin injection (6 mg/kg intraperitoneally daily for 3 weeks), and after I/R. The influence of melatonin on the incidence of postischemic-reperfusion-induced ventricular tachycardia (VT) and ventricular fibrillation (VF) were also measured. Changes in the arteriolar response to NG-monomethyl-L-arginine (L-NMMA), a nitric oxide inhibitor, norepinephrine (NE), and angiotensin II (ANG II) were studied before and after melatonin injection (10 mg/kg intravenously). In CM hamsters, melatonin restored normal arteriolar responses to L-NMMA, NE, and ANG II. I/R elevated lipid peroxide and nitrate/nitrite levels, and vascular permeability while arteriolar diameter, RBC velocity, flow and capillary perfusion were reduced. These effects were more marked in CM versus healthy hamsters. During I/R melatonin reduced oxidative and nitrosative stress, vasoconstriction, leukocyte adhesion, and vascular permeability and increased capillary perfusion. Melatonin reduced the incidence of VT while VF during reperfusion disappeared totally. In conclusion, melatonin prevents both microvascular injury and ventricular arrhythmias during postischemic reperfusion by modulating the lipid peroxide overproduction and nitrative stress which are involved in the development of cardiomyopathy.

Increase in capillary perfusion following low-intensity ultrasound and microbubbles during postischemic reperfusion.

OBJECTIVES: We postulated that the increase in shear stress caused by microbubbles in the presence of low-intensity ultrasound increases vasodilation in ischemia/reperfusion. DESIGN: Prospective, randomized, and blinded experimental study. SETTING: Research laboratory. SUBJECTS: Forty hamsters were subjected to ischemia/reperfusion and observed by intravital microscopy. INTERVENTIONS: Ultrasound (2.5 MHz, 1.3 mechanical index, 2.0 peak pressure) was applied to the hamster cheek pouch in ischemia/reperfusion with and without microbubbles (Levovist or Sono Vue) at baseline (15 mins) and at the beginning (15 mins) of reperfusion after ischemia (30 mins). MEASUREMENTS AND MAIN RESULTS: Arterial diameter (A2-A3, 38.5 +/- 5.3 microm; A4,15.0 +/- 7.0 microm), red blood cell velocity, wall shear stress, permeability, perfused capillary length, and adherent leukocytes in venules were evaluated. Lipid peroxides were also determined in the systemic blood. Ultrasound and microbubbles in reperfusion significantly increased the diameter (A2-A3 Sono Vue, 33%; Levovist, 53% vs. ischemia/reperfusion, p < .05; A4, Sono Vue, 93%; Levovist, 104% vs. ischemia/reperfusion, p < .05), red blood cell velocity, flow, and shear stress in both A4 and A2-A3 arterioles. Shear stress was significantly higher with Levovist (A2-A3, 105%; A4, 185%) and Sono Vue (A2, 108%; A4, 140% vs. ischemia/reperfusion, p < .05) than ultrasound alone in arterioles. With ischemia/reperfusion, perfused capillary length was reduced significantly, whereas it increased with Levovist and Sono Vue (43%, 41% vs. ischemia/reperfusion p < .05). Lipid peroxides increased early during reperfusion and remained at increased levels throughout reperfusion. Lipid peroxides were unchanged after ultrasound alone or ultrasound with Sono Vue or Levovist during ischemia/reperfusion. With ultrasound there was a significant increase in vascular permeability vs. ischemia/reperfusion. Treatment with Sono Vue (-36%) and Levovist (-57%) decreased permeability vs. ischemia/reperfusion in reperfusion (p < .001). Ischemia/reperfusion had significantly increased leukocyte adhesion. Ultrasound alone (-39%) or with Sono Vue (-64%) and Levovist (-57%) caused smaller increases in leukocyte adhesion than ischemia/reperfusion (p < .05). CONCLUSIONS: Ultrasound and microbubbles equilibrate microvascular shear stress, thus avoiding the failure of capillary perfusion in postischemic reperfusion.

How to assess post-occlusive reactive hyperaemia by means of laser Doppler perfusion monitoring: application of a standardised protocol to patients with peripheral arterial obstructive disease.

The standardisation of manoeuvres to perform clinically discriminative microvascular flow reserve tests is still poorly developed, as well as the response analysis. The aim of this study was to establish a reproducible analysis method for the post-occlusive reactive hyperaemia (PORH) test measured using laser Doppler perfusion monitoring (LDPM). LDPM data were measured from the PORH response of 24 Fontaine class II-III peripheral atherosclerotic/arterial obstructive disease (PAOD) patients and 30 healthy subjects. The PORH response was recorded from the dorsum of the foot after 3 min of arterial occlusion at the thigh. The resulting tracings were analysed by describing their morphology through five defined parameters: resting flux (RF), time to RF level (tRF), maximum flux (MF) during reactive hyperaemia, time to maximum flux (tMF), and time to half recovery (tHR). While the time parameters were discriminative between patients and controls, flux parameters were not. The time to resting flux (tRF) led to the most discriminative model that correctly predicted 88.5% of the cases. Hence, we concluded that obtaining t(RF) with the presented procedures provides an optimal model to quantify the patient's microvascular condition from the PORH response.

Role of nitric oxide in capillary perfusion and oxygen delivery regulation during systemic hypoxia.

The role of nitric oxide (NO) and reactive oxygen species (ROS) in regulating capillary perfusion was studied in the hamster cheek pouch model during normoxia and after 20 min of exposure to 10% O2-90% N2. We measured PO2 by using phosphorescence quenching microscopy and ROS production in systemic blood. Identical experiments were performed after treatment with the NO synthase inhibitor NG-monomethyl-L-arginine (L-NMMA) and after the reinfusion of the NO donor 2,2'-(hydroxynitrosohydrazono)bis-etanamine (DETA/NO) after treatment with L-NMMA. Hypoxia caused a significant decrease in the systemic PO2. During normoxia, arteriolar intravascular PO2 decreased progressively from 47.0 +/- 3.5 mmHg in the larger arterioles to 28.0 +/- 2.5 mmHg in the terminal arterioles; conversely, intravascular PO2 was 7-14 mmHg and approximately uniform in all arterioles. Tissue PO2 was 85% of baseline. Hypoxia significantly dilated arterioles, reduced blood flow, and increased capillary perfusion (15%) and ROS (72%) relative to baseline. Administration of L-NMMA during hypoxia further reduced capillary perfusion to 47% of baseline and increased ROS to 34% of baseline, both changes being significant. Tissue PO2 was reduced by 33% versus the hypoxic group. Administration of DETA/NO after L-NMMA caused vasodilation, normalized ROS, and increased capillary perfusion and tissue PO2. These results indicate that during normoxia, oxygen is supplied to the tissue mostly by the arterioles, whereas in hypoxia, oxygen is supplied to tissue by capillaries by a NO concentration-dependent mechanism that controls capillary perfusion and tissue PO2, involving capillary endothelial cell responses to the decrease in lipid peroxide formation controlled by NO availability during low PO2 conditions.

Microvascular oxygenation and oxidative stress during postischemic reperfusion. PO2, ROS, and NO during reperfusion.

Increased formation of ROS on reperfusion after ischemia underlies ischemia reperfusion (I/R) damage. We measured, in real time, both oxygen tension in microvessels and tissue and oxidant stress during postischemic reperfusion in hamster cheek pouch microcirculation. We measured PO2 by using phosphorescence quenching microscopy and oxygen radical species (ROS) production in the systemic blood. We evaluated the effects of a NOS inhibitor (L-NMMA) and superoxide dismutase (SOD) on the oxidative stress during reperfusion. Microvascular injury was assessed by measuring diameter change, the perfused capillary length (PCL), and leukocyte adhesion. Our findings demonstrate that early reperfusion is characterized by low concentration of oxygen linked to increased production of ROS. After this initial transience in arterioles, the oxygen tension and production of ROS return to normal after reperfusion, while the blood flow and capillary perfusion decrease. The early increased ROS production, in turn, may impair oxygen consumption by endothelial cells, thus further promoting activation of oxygen to ROS. This event is substantiated by the finding that treatment with SOD maintains ROS at normal levels, which, in turn, should be effective to increase the production of endothelial NO. Conversely, a decrease in NO levels led to decreased ROS production during early reperfusion, which increased later during reperfusion, ultimately causing vasoconstriction and greatly increasing venular leukocyte adhesion on postcapillary venules during hypoxic conditions. Therefore, low-flow hypoxia is primarily responsible for vascular endothelial damage during reperfusion through changes in ROS and NO production.

Antioxidant activity of nitro derivative of aspirin against ischemia-reperfusion in hamster cheek pouch microcirculation.

Aspirin that has been chemically combined with a nitric oxide (NO) donor (NCX-4016) has been shown to inhibit cyclooxygenase and prostaglandin generation while maintaining the inhibitory effects of aspirin. The possible role of reactive oxygen species (ROS) in the action of NCX-4016 in ischemia-reperfusion (I/R) has not been studied. Furthermore, we were interested in comparing the effects of a conventional NO donor [2,2'-hydroxynitrosohydrazino-bis-etanamine (DETA/NO)] and NCX-4016 at the microvascular level in the hamster cheek pouch visualized by using an intravital fluorescent microscopy technique. Microvascular injury was assessed by measuring diameter change, the perfused capillary length (PCL), and leukocyte adhesion. Animals were treated with NCX-4016 (100 mg/kg or 30 mg.kg(-1).day(-1) for 5 days po) or DETA-NO (0.5 mg/kg). Mean arterial blood pressure increased slightly but significantly after NCX-4016 treatment. During 5- and 15-min reperfusion, lipid peroxides in the systemic blood increased by 72 and 89% vs. baseline, respectively, and were still higher than in basal conditions after 30-min reperfusion in the I/R group. Pretreatment with NCX-4016 maintained ROS at normal levels; increased arteriolar diameter, blood flow, and PCL; and decreased leukocyte adhesion (P < 0.05). DETA-NO decreased ROS during 30-min reperfusion; however, later there was a significant increase during reperfusion. DETA-NO decreased leukocyte adhesion (P < 0.05) but microvascular permeability increased after 30 min of reperfusion. In conclusion, NCX-4016 attenuates oxidative stress and prevents arteriolar constriction during I/R, whereas DETA-NO increases lipid peroxides in the systemic blood and permeability after reperfusion.

Blockade of P-selectin does not affect reperfusion injury in hamsters subjected to glutathione inhibition.

P-selectin antibody has been shown to prevent microvascular damage after ischemia reperfusion (I/R). We investigated whether the treatment with anti-P-selectin would attenuate the decrease in capillary perfusion after glutathione (GSH) inhibition in hamster cheek pouch microcirculation subjected to I/R. Animals were treated for 3 days with l-buthionine-[S,R]-sulfoximine (BSO) to inhibit GSH synthesis. P-selectin expression was determined by using an in situ immunofluorescence method in the microvessels. Ischemia was induced by clamping the cheek pouch for 30 min followed by 30 min of reperfusion. Changes in capillary perfusion, RBC velocity, and leukocyte and platelet adhesion on microvessels were measured after I/R. Hamsters subjected to I/R showed increased leukocyte and platelet adhesion as well as decreased capillary perfusion. The anti-P-selectin group showed a significant P-selectin expression, that occurs at the venular bifurcations within 15-30 min of reperfusion, as well as no increase in leukocyte and platelet adhesion on microvessels. BSO partially prevented P-selectin expression but the decrease in capillary perfusion and the increase in both platelet and leukocyte adhesion in microvessels were greater. GSH significantly prevented P-selectin expression as well as capillary perfusion decrease after I/R. In conclusion, GSH inhibition blunted the protective effects of anti-P-selectin treatment with marked leukocyte adhesion on postcapillary venules and platelet-endothelial cell interactions in arterioles and venules and decreased capillary perfusion at reperfusion, thus suggesting that the mechanism of I/R injury is not critically dependent on P-selectin.