Computer-assisted Videomicroscopy Research Articles

Human ocular surface microcirculation quantified by in vivo computer assisted video microscopy and diffuse reflectance spectroscopy

Non-invasive imaging techniques are increasingly used to objectively quantify anterior segment structures of the eye. In this study, we apply the novel oxygen delivery index (ODIN) concept that, quantifies microvascular capacity for oxygen delivery, to the ocular surface in healthy humans.The purpose of the study was to test the applicability of the technologies used for data acquisition from the human ocular surface. We also validated whether the ODIN concept has sufficient sensitivity to detect and differentiate between microvascular structure and function in limbal and bulbar conjunctiva.Multiple ocular surface measurements using computer-assisted video microscopy (field of view: 1.6 mm × 0.9 mm) and diffuse reflectance spectroscopy (measuring volume: ∼0.1 mm3) were obtained from limbal and bulbar conjunctiva in 20 healthy volunteers. Three parameters were extracted during analyses: Functional capillary density, capillary flow velocity, and microvascular oxygen saturation.Functional capillary density was higher at limbus than in bulbar conjunctiva (11.2 ± 1.8 c/mm versus 5.2 ± 1.2 c/mm, p < 0.01), and microvascular oxygen saturation was lower at limbus (77 ± 8%) as compared to bulbar conjunctiva (89 ± 6%), p < 0.01. More than 80% of scored capillaries had continuous blood flow and no difference was seen between the recording sites (p = 0.68).In conclusion, the ODIN concept is applicable for the assessment of human ocular surface microvascular function and has sufficient sensitivity to detect increased capillary density and oxygen extraction at limbus as compared with bulbar conjunctiva.

Ocular surface microcirculation is better preserved with pulsatile versus continuous flow during cardiopulmonary bypass-An experimental pilot.

Non-pulsatile cardiopulmonary bypass (CPB) may induce microvascular dysregulation. In piglets, we compared ocular surface microcirculation during pulsatile versus continuous flow (CF) bypass. Ocular surface microcirculation in small tissue volumes (~0.1 mm3 ) at limbus (high metabolic rate) and bulbar conjunctiva (low metabolic rate) was examined in a porcine model using computer assisted video microscopy and diffuse reflectance spectroscopy, before and after 3 and 6h of pulsatile (n=5 piglets) or CF (n=3 piglets) CPB. Functional capillary density, capillary flow velocity and microvascular oxygen saturation were quantified. At limbus, velocities improved with pulsatility (p < 0.01) and deteriorated with CF (p < 0.01). In bulbar conjunctiva, velocities were severely reduced with CF (p < 0.01), accompanied by an increase in capillary density (p < 0.01). Microvascular oxygen saturation decreased in both groups. Ocular surface capillary densities and flow patterns are better preserved with pulsatile versus CF during 6h of CPB in sleeping piglets.

Hemodynamic evaluation of a new pulsatile blood pump during low flow cardiopulmonary bypass support.

The VentriFlo® True Pulse Pump (VentriFlo, Inc, Pelham, NH, USA) is a new pulsatile blood pump intended for use during short-term circulatory support. The purpose of this study was to evaluate the feasibility of the VentriFlo and compare it to a conventional centrifugal pump (ROTAFLOW, Getinge, Gothenberg, Sweden) in acute pig experiments. Pigs (40-45kg) were supported by cardiopulmonary bypass (CPB) with the VentriFlo (n=9) or ROTAFLOW (n=5) for 6h. Both VentriFlo and ROTAFLOW circuits utilized standard CPB components. We evaluated hemodynamics, blood chemistry, gas analysis, plasma hemoglobin, and microcirculation at the groin skin with computer-assisted video microscopy (Optilia, Sollentuna, Sweden). Pigs were successfully supported by CPB for 6h without any pump-related complications in either group. The VentriFlo delivered an average stroke volume of 29.2±4.8ml. VentriFlo delivered significantly higher pulse pressure (29.1±7.2mmHg vs. 4.4±7.0mmHg, p<0.01) as measured in the carotid artery, with mean aortic pressure and pump flow comparable with those in ROTAFLOW. In blood gas analysis, arterial pH was significantly lower after five hours support in the VentriFlo group (7.30±0.07 vs. 7.43±0.03, p=0.001). There was no significant difference in plasma hemoglobin level in both groups after six hours of CPB support. In microcirculatory assessment, VentriFlo tended to keep normal capillary flow, but it was not statistically significant. VentriFlo-supported pigs showed comparable hemodynamic parameters with significantly higher pulse pressure compared to ROTAFLOW without hemolysis.

Skin trauma rapidly induces thermoregulatory plexus hyperemia, while an increased nutritive papillary capillary function can be detected after 24h.

Clinical assessments and laser Doppler perfusion measurements (LDPM) of skin microcirculation have limited value, as they fail to capture events regulated by local metabolic needs at a papillary capillary level. This study aimed to examine the ability of computer-assisted video microscopy (CAVM) and diffuse reflectance spectroscopy (DRS) to assess skin nutritive perfusion-compared to LDPM. Healthy volunteers (n=10) were examined after (≈1 and ≈24h) an incision (5×1mm) on the forearm, at 0.1mm (only with CAVM), 2-3mm, and 30mm from the trauma. No changes were detected by CAVM after ≈1h. After ≈24h, 0-1mm from the trauma, both CAVM parameters were increased: functional capillary density (capillary crossings/mm, 11.8±1.4 vs. 7.3±1.2, p<.01) and capillary flow velocities (CFV, %capillaries with brisk flow, 10±6.8 vs. 1±1, p<.01). At a distance of 2-3mm, only CFV was increased (6.2±6.1 vs. 1±1, p<.05). DRS and LDPM measurements increased 2-3mm from the trauma line in relation to baseline after both ≈1 and ≈24h, that is, with DRS (%microvascular oxygen saturation): 45.8±7.4% (baseline), 70.0±12.5% (≈1h), and 73.1±10.4% (≈24h), p<.01 and with LDPM (a.u.): 7.2±2.5 (baseline), 28.3±18.7 (≈1h), and 45.9±16.3 (≈24h), p<.01. ≈24h after skin trauma, an increased function of the nutritive papillary capillaries can be detected by CAVM.

Quantification of ocular surface microcirculation by computer assisted video microscopy and diffuse reflectance spectroscopy

In piglets we tested the applicability of digital video microscopy and diffuse reflectance spectroscopy for non-invasive assessments of limbal and bulbar conjunctival microcirculation. A priori we postulated that the metabolic rate is higher in limbal as compared to bulbar conjunctiva, and that this difference is reflected in microvascular structure or function between the two locations. Two study sites, Oslo University Hospital (OUH), Norway and Cleveland Clinic (CC), USA, used the same video microscopy and spectroscopy techniques to record limbal and bulbar microcirculation in sleeping piglets. Recordings were analyzed with custom-made software to quantify functional capillary density, capillary flow velocity and microvascular oxygen saturation in measuring volumes of approximately 0.1 mm3. The functional capillary density was higher in limbus than in bulbar conjunctiva at both study sites (OUH: 18.1 ± 2.9 versus 12.2 ± 2.9 crossings per mm line, p < 0.01; CC: 11.3 ± 3.0 versus 7.1 ± 2.8 crossings per mm line, p < 0.01). Median categorial capillary blood flow velocity was higher in bulbar as compared with limbal recordings (CC: 3 (1–3) versus 1 (0–3), p < 0.01). Conjunctival microvascular oxygen saturation was 88 ± 5.9% in OUH versus 94 ± 7.5% in CC piglets.Non-invasive digital video microscopy and diffuse reflectance spectroscopy can be used to obtain data from conjunctival microcirculation in piglets. Limbal conjunctival microcirculation has a larger capacity for oxygen delivery as compared with bulbar conjunctiva.

Cooling Effect on Skin Microcirculation in Asphyxiated Newborn Infants with Increased C-Reactive Protein

Background: Therapeutic hypothermia is presumed to suppress inflammatory processes after perinatal asphyxia. In a previous study of neonatal hypoxic-ischemic encephalopathy (HIE) we found altered skin microcirculation in about a third of the infants after rewarming. We speculated whether this could be linked to increased inflammatory responses, such as high C-reactive protein (CRP). The present study further explored this question. Objective: The aim of this study was to explore the differences in skin microcirculation and its oxygen delivery ability during cooling and after rewarming in HIE infants with or without high CRP. Methods: A previously studied population of 28 HIE infants was divided into two subgroups depending on low or high CRP (repeated values above 30 mg/l for more than 24 h). The differences between the two groups regarding laser Doppler perfusion measurements (LDPMs), computer-assisted video microscopy and diffuse reflectance spectroscopies during cooling on days 1 and 3 and after rewarming on day 4 were assessed. Results: After rewarming, infants with high CRP showed significantly higher skin LDPM perfusion, lower functional vessel density and larger heterogeneity of capillary flow velocities as compared to infants with low CRP, while no such differences were found during cooling. Conclusion: Skin microcirculatory responses differed significantly after rewarming, but not during cooling, between asphyxiated neonates with or without high CRP. We speculate whether cooling influences the inflammatory skin microcirculatory response and the ability of oxygen delivery to the cells. Further studies are needed to investigate this as well as its applicability to other vascular beds in the body.

Resveratrol Prevents Reactive Oxygen Species-Induced Effects of Light-Emitting Diode-Generated Blue Light in Human Skin Fibroblasts.

Light-emitting diode-generated blue light (LED-BL) is part of the visible light spectrum that does not cause DNA damage and may represent a safer alternative to ultraviolet phototherapy. Previous research demonstrated that LED-BL can inhibit adult human skin fibroblast proliferation and migration speed and is associated with increased reactive oxygen species (ROS) generation in a dose-dependent manner. In addition, resveratrol possesses potent intracellular antioxidative effects on ROS-free radicals in human skin fibroblasts. The authors studied the effects on migration speed as a surrogate to measure LED-BL effects on fibroblast function. The authors hypothesized that resveratrol, a potent scavenger of ROS, could prevent the effects of LED-BL on fibroblast migration speed. This would implicate ROS as the mechanistic driver of LED-BL effects on human skin fibroblasts. To demonstrate that resveratrol could prevent the effects of LED-BL (415-nm), fibroblasts were incubated with resveratrol (Sigma-Aldrich, St. Louis, MO) at concentrations of 0.001% and 0.0001% for 24 hours and then irradiated with LED-BL at fluences of 30, 45, and 80 J/cm. Postirradiation fibroblast migratory speed was assayed in an environment-controlled computer-assisted video microscopy system. Reactive oxygen species levels were measured by flow cytometric analysis of dihydrorhodamine. Statistical analyses with analysis of variance and Student t-test were performed to compare individual treatment arms and matched controls. The experimental results demonstrate that pretreatment of skin fibroblasts with resveratrol at concentrations of 0.001% and 0.0001% prevents the effects of 30, 45, and 80 J/cm of LED-BL on fibroblast migration speed. The authors found that LED-BL at a fluences of 30, 45, and 80 J/cm significantly increased ROS, whereas pretreatment with 0.001% resveratrol significantly reduced ROS generation. The findings demonstrate that LED-BL-induced decreases in fibroblast migration speed can be prevented by pretreating cells with resveratrol. This finding supports the hypothesis that ROS generation is the most likely driver of LED-BL-induced alterations in migration speed and suggests that ROS generation may be responsible for a number of other alterations seen after LED-BL phototherapy, such as decreases in cellular migration, cytokine levels, and myofibroblast differentiation. The authors hypothesize that their findings may result in greater understanding of the fundamental mechanisms underlying visible light interaction with skin and they hope dermatologists and other researchers may use these pathways for patient benefit.

Noninvasive assessments of oxygen delivery from the microcirculation to skin in hypothermia-treated asphyxiated newborn infants.

Therapeutic hypothermia (TH) has become standard treatment for severe and moderate hypoxic-ischemic neonatal encephalopathy (HIE). Our group has developed an optically based, noninvasive concept of assessing the capacity for oxygen delivery from the microcirculation to the cells of a tissue under investigation. The hypothesis was that mechanisms of reduced oxygen delivery due to reduced metabolism in cooled asphyxiated neonates could be characterized with this concept. The skin of 28 asphyxiated newborn infants was studied on days 1 and 3 during TH and on day 4 following rewarming with laser Doppler perfusion measurements (LDPM), computer-assisted video microscopy (CAVM), and diffuse reflectance spectroscopy (DRS). Twenty-five healthy neonates served as a control group. The LDPM decreased during cooling (P < 0.01). Functional capillary density was higher both during and following TH compared with control infants (P < 0.01). Capillary flow velocities were reduced during TH (P < 0.05). The heterogeneity of the flow velocities was larger in the HIE infants than in the control infants. Tissue oxygen extraction was higher during TH (P < 0.01). This study indicates that assessments of skin microvascular density, capillary flow velocity, and oxygen extraction can be used to characterize reduced oxygen delivery to cells during TH.

324 Skin Microcirculation in Healthy Newborns - Assessments of Morphology, Perfusion and Oxygenation

Background and aims: Measurements of central hemodynamics, blood gases and urinary output are commonly used to monitor patients, but values within reference range do not guarantee adequate cellular nutrition and oxygenation. We have examined skin microvascular perfusion, morphology and oxygenation in healthy newborns. Material and Methods: Twenty-five healthy newborns (gestational age >37 weeks) were enrolled within 24 hours after delivery. Skin microvascular assessments were performed day one, two and three, on the chest and the hand, using three different techniques: Computer-assisted video microscopy, CAVM (morphology); Laser Doppler Perfusion Monitoring, LDPM (perfusion); Diffuse Reflectance Spectroscopy, DRS (oxygen saturation of capillary erythrocytes). Results: (Mean with SD) CAVM: Microvascular density (number of microvessels crossing a grid of lines/mm line): Chest: 11.3 (1.5), 11.0 (1.7), 10.7 (1.6). Hand: 13.2 (2.0), 13.2 (1.9), 12.4 (1.6).LDPM: Perfusion (arbitrary units): Chest: 110.4 (32.1), 102.7 (27.8), 100.5 (28.1). Hand: 58.5 (26.2), 55.1 (17.5), 46.6 (14.9).DRS: Microvascular oxygen saturation (%): Chest: 71.9 (7.3), 73.5 (12.2), 74.5 (9.2). Hand: 68.3 (11.5), 63.1 (11.6), 65.5 (11.7). Conclusions: We were able to obtain noninvasive reproducible data for skin microvascular morphology, perfusion and oxygen saturation. The number of skin vessels is fewer in chest than in hand, with a slight reduction from day 1 to day 3. There is higher perfusion in the chest than in the hand, probably due to higher skin temperature (+2.8°). Since all the infants has arterial oxygenation >97%, oxygen extraction in the skin microvessels were approximately 20% in the chest and 30% in the hand.

Characterization of adrenergic and purinergic contractile responses in rat mesenteric arteries and veins

Norepinephrine (NE) and adenosine 5’-triphosphate (ATP) are neurotransmitters released from sympathetic neurons that act to alter net vascular tone in the mesentery via activation of adrenergic (α1 and α2) and purinergic (P2X and P2Y) receptors. This study was designed to identify adrenergic and purinergic receptors in third order mesenteric arteries and veins in male Sprague-Dawley rats. Agonists and antagonists of adrenergic and purinergic receptors were exogenously applied to vessels and contractile responses were measured using computer assisted video microscopy. NE and ATP both caused contractions of mesenteric arteries and veins. The selective α1 antagonist prazosin attenuated NE-derived constriction of the vessels. The selective α1 agonist phenylephrine was a more efficacious constrictor of both mesenteric arteries and veins than the selective α2 agonist clonidine. The P2X/P2Y1 receptor antagonist pyridoxal-phosphate-6-azophenyl-2’,4-disulfonic acid (PPADS) caused a rightward shift in the ATP dose response curve in mesenteric arteries but not veins. These data indicate that the α1 adrenergic receptors are the primary adrenoreceptors mediating contraction to NE in mesenteric vessels. Additionally, these data suggest that the P2X/P2Y1 receptors mediate substantial contractile responses to ATP in mesenteric arteries but not veins.

Propofol Restores Brain Microvascular Function Impaired by High Glucose via the Decrease in Oxidative Stress

Vascular dysfunction induced by hyperglycemia has not been studied in cerebral parenchymal circulation. The current study was designed to examine whether high glucose impairs dilation of cerebral parenchymal arterioles via nitric oxide synthase, and whether propofol recovers this vasodilation by reducing superoxide levels in the brain. Cerebral parenchymal arterioles in the rat brain slices were monitored using computer-assisted videomicroscopy. Vasodilation induced by acetylcholine (10 to 10 m) was obtained after the incubation of brain slices for 60 min with any addition of l-glucose (20 mm), d-glucose (20 mm), or propofol (3 x 10 or 10 m) in combination with d-glucose (20 mm). Superoxide production in the brain slice was determined by dihydroethidium (2 x 10 m) fluorescence. Addition of d-glucose, but not l-glucose, reduced arteriolar dilation by acetylcholine, whereas the dilation was abolished by the neuronal nitric oxide synthase inhibitor S-methyl-l-thiocitrulline (10 m). Both propofol and the superoxide dismutase mimetic Tempol (10 m) restored the arteriolar dilation in response to acetylcholine in the brain slice treated with d-glucose. Addition of d-glucose increased superoxide production in the brain slice, whereas propofol, Tempol, and the nicotinamide adenine dinucleotide phosphate (NAD[P]H) oxidase inhibitor apocynin (1 mm) similarly inhibited it. Clinically relevant concentrations of propofol ameliorate neuronal nitric oxide synthase-dependent dilation impaired by high glucose in the cerebral parenchymal arterioles via the effect on superoxide levels. Propofol may be protective against cerebral microvascular malfunction resulting from oxidative stress by acute hyperglycemia.

Prevention and reversal of vasospasm and ultrastructural changes in basilar artery by continuous infusion of CGS 35066 following subarachnoid hemorrhage.

Endothelin-1, a potent vasoconstrictive peptide, has been implicated in the pathogenesis of cerebral vasospasm following subarachnoid hemorrhage (SAH). The goal of this study was to evaluate the effect of continuous intravenous infusion of a highly selective endothelin-converting enzyme-1 inhibitor, CGS 35066, on the prevention and reversal of cerebral vasospasm following SAH. New Zealand white rabbits were subjected to SAH by injecting autologous arterial blood into the cisterna magna. Infusion of CGS 35066 at dosages of 1, 3, or 10 mg/kg/ day was initiated either 1 hr and 24 hrs later in the prevention and reversal protocols, respectively. Animals were sacrificed by perfusion-fixation 48 hrs after SAH induction. The cross-sectional areas of basilar arteries were measured using computer-assisted videomicroscopy. Ultrastructural changes in basilar arteries were determined using electron microscopy. CGS 35066 significantly prevented and reversed the arterial narrowing after SAH in all three groups. The mean cross-sectional areas of arteries from animals in both the prevention and reversal protocol groups that received 10 mg/kg/day of CGS 35066 did not differ significantly from those of the healthy controls. Histological studies of the basilar artery in the 10 mg/kg/day treatment group did not show pathomorphological changes, such as corrugation of the endothelium seen at 2 days after SAH induction or vacuole formation in the endothelial cells noted in the vehicle-treated SAH group. These findings suggest that CGS 35066 is a promising therapeutic agent for the prevention and reversal of cerebral vasospasm after SAH. It also prevents the pathological changes in vascular walls due to SAH.

Increased substance P content in nerve fibers associated with mesenteric veins from deoxycorticosterone acetate (DOCA)-salt hypertensive rats

This study examined sensory nerves associated with mesenteric arteries and veins in sham and deoxycorticosterone acetate (DOCA)-salt hypertensive rats. Reactivity of arteries and veins to substances released from sensory nerves was also studied in vitro using computer-assisted video microscopy. Co-localization of substance P (SP) and calcitonin gene-related peptide (CGRP) immunoreactivity (ir) was used to evaluate perivascular sensory nerves. Radioimmunoassay was used to quantify SP-and CGRP-ir content. Immunohistochemical studies revealed a plexus of SP/CGRP-ir nerves associated with arteries and veins. The intensity of SP-ir, but not CGRP-ir labeling was greater in arteries and veins from DOCA-salt compared to sham rats. RIA measurements revealed that the CGRP-ir content of arteries and veins was higher than the SP-ir content but there was a significant increase in SP-ir, but not CGRP-ir, content in arteries and veins from DOCA-salt rats. SP (0.03–1 μM) contracted veins and the NK-3 receptor agonist, senktide, mimicked this effect. There were no differences in SP or senktide reactivity of veins from sham or DOCA-salt rats. SP, but not senktide, relaxed KCl (40 mM) preconstricted arteries. CGRP (0.3 μM), acetylcholine (10 μM) and capsaicin (1 μM) relaxed KCl-preconstricted arteries and veins. The NK-1 receptor agonist, substance P methyl ester relaxed arteries but not veins. These data indicate that DOCA-salt hypertension is associated with upregulation of SP content in perivascular nerves. NK-3 receptors mediate venoconstriction which is unchanged in DOCA-salt hypertension. Increased release of SP from perivenous nerves might contribute to the increased venomotor tone in DOCA-salt hypertension.

3D culture model and computer-assisted videomicroscopy to analyze migratory behavior of noninvasive and invasive bronchial epithelial cells

To date, most of the studies in the field of cell migration have been applied to two-dimensional (2D) models. To mimic the three-dimensional (3D) conditions similar to those observed in vivo during tumor invasion, we developed a 3D model of cell migration in which cells were embedded in a collagen I matrix placed in a double-compartment chamber. Using time-lapse videomicroscopy and interactive cell tracking in a four-dimensional data set, we determined the cell trajectories and their migration kinetics. We compared the 2D and 3D migratory behavior of a noninvasive cell line (16HBE) with the migratory behavior of an invasive cell line (BZR). Our results show that the 3D migration kinetics of the noninvasive cell line were lower than the migration kinetics of the invasive cell line. In contrast, in 2D models, no significant difference was observed between the two cell lines. To validate our 3D model, we further investigated the effect of epidermal growth factor (EGF), a promoter of tumor cell motility and invasion on the noninvasive cell line (16HBE). EGF increased significantly the migration kinetics of the noninvasive cell line. Our results show that the 3D model of cell migration allowed us to differentiate the migratory behavior of invasive and noninvasive cells and that such a model can help in the development of molecular targeted therapy as it approaches the in vivo conditions.

Vascular reactivity of mesenteric arteries and veins to endothelin-1 in a murine model of high blood pressure

We characterized vascular reactivity to endothelin-1 (ET-1) in mesenteric vessels from DOCA-salt hypertensive and SHAM control mice and assessed the effect that endothelial-derived vasodilators have on ET-1-induced vasoconstriction. Changes in the diameter of unpressurized small mesenteric arteries and veins (100- to 300-μm outside diameter) were measured in vitro using computer-assisted video microscopy. Veins were more sensitive than arteries to the contractile effects of ET-1. There was a decrease in arterial maximal responses ( E max) compared to veins, this effect was larger in DOCA-salt arteries. The selective ET B receptor agonist, sarafotoxin 6c (S6c), contracted DOCA-salt and SHAM veins but did not contract arteries. The ET B receptor antagonist, BQ-788 (100 nM), but not the ET A receptor antagonist, BQ-610 (100 nM), blocked S6c responses. BQ-610 partially inhibited responses to ET-1 in mesenteric veins from DOCA-salt and SHAM mice while BQ-788 did not affect responses to ET-1. Co-administration of both antagonists inhibited responses to ET-1 to a greater extent than BQ-610 alone suggesting a possible functional interaction between ET A and ET B receptors. Responses to ET-1 in mesenteric arteries were completely inhibited by BQ-610 while BQ-788 did not affect arterial responses. Nitric oxide synthase inhibition potentiated ET-1 responses in veins from SHAM but not DOCA-salt mice. There was a prominent role for ET-mediated nitric oxide release in DOCA-salt but not SHAM arteries. In summary, these studies showed a differential regulation of ET-1 contractile mechanisms between murine mesenteric arteries and veins.

Characterization of Gastrin-Induced Proangiogenic Effects In vivo in Orthotopic U373 Experimental Human Glioblastomas and In vitro in Human Umbilical Vein Endothelial Cells

This study aims to investigate the role of gastrin-17 (G17) on angiogenesis features in gliomas both in vitro and in vivo. The influences of G17 and G17 receptor antagonists were characterized in vitro in terms of angiogenesis on human umbilical vein endothelial cell (HUVEC) tubulogenesis processes on Matrigel and in vivo with respect to U373 orthotopic glioma xenografts. The influence of phosphatidylinositol 3'-kinase, protein kinase C, and nuclear factor-kappaB inhibitors was characterized in vitro on G17-mediated HUVEC tubulogenesis. G17-mediated release of interleukin (IL)-8 from HUVECs and G17-induced modifications in nuclear factor-kappaB DNA binding activity were characterized by means of specific enzyme-linked immunosorbent assays. The influence of G17 on E- and P-selectin expression was determined by means of computer-assisted microscopy, whereas the influence of E- and P-selectin on HUVEC migration was approached by means of antisense oligonucleotides. The chemotactic influence of G17 and IL-8 on HUVEC migration was characterized by means of computer-assisted videomicroscopy with Dunn chambers. Messenger RNAs for cholecystokinin (CCK)A, CCKB, and CCKC receptors were present in HUVECs and microvessels dissected from a human glioblastoma. Whereas G17 significantly increased the levels of angiogenesis in vivo in the U373 experimental glioma model and in vitro in the HUVECs, the CCKB receptor antagonist L365,260 significantly counteracted the G17-mediated proangiogenic effects. G17 chemoattracted HUVECs, whereas IL-8 failed to do so. IL-8 receptor alpha (CXCR1) and IL-8 receptor beta (CXCR2) mRNAs were not detected in these endothelial cells. Gastrin significantly (but only transiently) decreased the level of expression of E-selectin, but not P-selectin, whereas IL-8 increased the expression of E-selectin. Specific antisense oligonucleotides against E- and P-selectin significantly decreased HUVEC tubulogenesis processes in vitro on Matrigel. The present study shows that gastrin has marked proangiogenic effects in vivo on experimental gliomas and in vitro on HUVECs. This effect depends in part on the level of E-selectin activation, but not on IL-8 expression/release by HUVECs.

Lidocaine Impairs Vasodilation Mediated by Adenosine Triphosphate-Sensitive K+ Channels but Not by Inward Rectifier K+ Channels in Rat Cerebral Microvessels

Vasodilator effects of adenosine triphosphate (ATP)-sensitive, as well as inward rectifier, K+ channel openers have not been well demonstrated in cerebral microvessels. Although lidocaine impairs vasorelaxation via ATP-sensitive K+ channels in the rat aorta, the effects of this compound on K+ channels in the cerebral circulation have not been shown. We designed the present study to examine whether ATP-sensitive and inward rectifier K+ channels contribute to vasodilator responses in cerebral microvessels and whether the vasodilation mediated by these channels is inhibited by lidocaine. Rat brain slices were monitored using a computer-assisted videomicroscopy. Cerebral parenchymal arterioles (diameter, 5-10 microm) were contracted with prostaglandin F(2alpha), and thereafter potassium chloride (KCl), levcromakalim, or sodium nitroprusside was added to the perfusion chamber. Levcromakalim and KCl produced vasodilation of the cerebral parenchymal arterioles, which was abolished by an ATP-sensitive K+ channel antagonist, glibenclamide, or an inward rectifier K+ channel antagonist, barium chloride, respectively. Lidocaine (10(-5) to 3 x 10(-5) M) inhibited the dilation produced by levcromakalim but not by KCl or sodium nitroprusside. In parenchymal arterioles of the cerebral cortex, lidocaine seems to reduce vasodilation mediated by ATP-sensitive K+ channels but not by inward rectifier K+ channels.

Increased reactivity of murine mesenteric veins to adrenergic agonists: functional evidence supporting increased alpha1-adrenoceptor reserve in veins compared with arteries.

These studies examined adrenergic reactivity of mesenteric arteries and veins from deoxycorticosterone acetate-salt (DOCA-salt) hypertensive and sham control mice. We measured constrictions in unpressurized arteries and veins by monitoring vessel diameter using computer-assisted video micros-copy in vitro. Veins were more sensitive than arteries to the constricting effects of norepinephrine (NE) and phenylephrine (PE), but the alpha2-agonists clonidine and UK 14,304 [5-bromo-6-(2-imidazolin-2-yl-amino)-quinoxaline] did not constrict arteries or veins. Reactivity was not altered in arteries or veins from DOCA-salt mice. We next investigated the mechanism of increased venous reactivity to NE and PE by studying desensitization to maximum concentrations of NE and PE. Sham arteries desensitized to NE and PE more than DOCA-salt arteries, whereas DOCA-salt and sham veins maintained 80% of the initial NE and PE constriction. To determine whether the increased reactivity and resistance to desensitization in veins was due to a greater alpha-adrenoceptor reserve, vessels were incubated with the alkylating agent phenoxybenzamine (PBZ; 0.3, 3, 10, and 30 nM). The NE-elicited initial constriction was reduced by PBZ (3, 10, and 30 nM) in sham but only by PBZ (30 nM) in DOCA-salt veins. All doses of PBZ blocked NE responses in sham and DOCA-salt arteries. These data suggest that mesenteric veins express more alpha1-adrenoceptors than arteries, accounting for greater reactivity and resistance to desensitization compared with arteries.

Upregulation of galectins-1 and -3 in human colon cancer and their role in regulating cell migration.

To probe the potential contribution of beta-galactoside-contributing epitopes and receptor proteins (gal-1 and gal-3) to colon malignancy, we first examined the expression of galectins and binding sites in clinical specimens by lectin and immunohistochemistry. Sixty-seven colonic surgical resections were studied, including 10 normal, 10 mild dysplasias, 10 severe dysplasias and 37 cancers. gal-1 and gal-3 were expressed in variable amounts in the epithelial cells and the connective tissue of normal colon. Their expression significantly increased with the degree of dysplasia, suggesting that gal-1 and gal-3 and their binding sites are related to malignant progression, while gal-8 has been associated with suppressor activity. To study the functional aspects, the influence of these galectins on the migration of 4 human colorectal cancer cell lines (HCT-15, LoVo, DLD-1, CoLo201) was studied. In agreement with histopathologic monitoring, these tumor cells were found to produce gal-3, while only CoLo201 was positive for gal-1. Except for DLD-1 and gal-1, the lines exhibited gal-1 binding sites on the surface, prompting study by computer-assisted videomicroscopy of the effect on cell migration of the presence of galectin on the culture substrate. The level of cell migration for HCT-15, LoVo and CoLo201 cells was significantly reduced by 0.15 microg/cm(2) gal-1, and the presence of a blocking antibody at least reduced this effect. gal-3 significantly reduced cell migration in all 4 of the in vitro cell lines.

Extracellular S100A4 stimulates the migration rate of astrocytic tumor cells by modifying the organization of their actin cytoskeleton

In previous studies, we have shown that numbers of S100 calcium-binding proteins (including S100A4) are expressed differentially in astrocytic tumors according to their levels of malignancy. S100A4 is involved in tumor progression, cell migration and metastasis. This protein is able to play extracellular roles such as neuritogenic and angiogenic activities. The present study aims to investigate the possible role played by extracellular S100A4 in the in vitro migration of astrocytic tumor cells. The speed and rate of migration of living cells were measured using computer-assisted videomicroscopy. In parallel, we also analyzed the effects of extracellular S100A4 on the organization of the actin cytoskeleton and the expression of a number of its molecular regulators. These included small Rho-GTPases (RhoA, Rac1 and Cdc42) and some of their direct effectors (mDia and N-WASP), and also actin-binding proteins such as profilin and α-actinin. Our data demonstrate the influence of S100A4 on astrocytic tumor cells with respect to these different aspects. Indeed, we show that extracellular S100A4 treatments decrease both the amount of polymerized F-actin and the levels of expression of RhoA, mDia and profilin. While a decrease in the Cdc42 and N-WASP expression was also observed, the Rac1 expression remained unchanged. All these activities, which result in the stimulation of cell motility, contribute to the understanding of the extracellular role of S100A4.