Mary D. S. Frame

l-Arginine–Induced Conducted Signals Alter Upstream Arteriolar Responsivity to l-Arginine

Our purpose was to determine whether L-arginine was involved in vascular communication between downstream and upstream locations within a defined microvascular region. Arteriolar diameter was measured for the branches along a transverse arteriole in the superfused cremaster of anesthetized (pentobarbital sodium, 70 mg/kg i.p.) hamsters (N = 53). The upstream branch arterioles dilated significantly to locally applied L-arginine (100 mumol/L pipette concentration) only if the downstream branches (approximately 1400 microns away) were preexposed. With exposure order downstream to upstream, diameter change was last branch, -3.8 +/- 1.5% (of baseline); third, +58.1 +/- 27%; first, +92 +/- 26% (n = 5); with exposure order upstream to downstream: first branch, -0.4 +/- 3%; third, +5 +/- 11%; last, -5.6 +/- 7.5% (n = 4). Thus, downstream preexposure to L-arginine altered the responsivity upstream to locally applied L-arginine. Downstream-applied L-arginine also induced a conducted vasodilation (+17.8 +/- 2.8%; n = 14) 1327 +/- 166 microns upstream. This response was completely blocked by simultaneous sucrose (600 mOsm), halothane (0.0345%), or N omega-nitro-L-arginine (L-NNA, 100 mumol/L) exposure to the feed vessel (second micropipette) midway between the downstream site of L-arginine exposure and the upstream observation site. An acetylcholine-induced conducted vasodilation (+18.1 +/- 2.6%, n = 8) was also completely blocked by sucrose, halothane, or L-NNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Flow‐Induced Cytoskeletal Changes in Endothelial Cells Growing on Curved Surfaces

Objective: Our purpose was to investigate the effect of the shape of the growth surface (curved versus flat) on flow‐induced F‐actin organization in endothelial cells.

Network Vascular Communication Initiated by Increases in Tissue Adenosine

Vascular communication of vasomotor signals appears to coordinate the distribution of tissue blood flow. This study was performed to determine whether elevated tissue concentrations of adenosine or nitric oxide could induce vascular communicating signals. To test this, remote arteriolar responses were tested when drugs were applied either directly to an arteriole (∼20 μm diameter), or into the tissue in a region (with no vessels over 10 μm in diameter) that was 500 μm away from the arteriole and that bore no defined relationship to the flow path of the remote arteriole. In anesthetized hamster cheek pouch (n = 25), or cremaster muscle (n = 10), remote arteriolar responses were measured in response to nitric oxide (NO) donors (10–5 to 10–3 M), adenosine (10–5 to 10–3 M), or papaverine (10–5 to 10–2 M) applied for 40–120 s. Papaverine caused no remote response when applied directly while adenosine and NO donors caused similar, late-onset (10–20 s), dose-dependent, remote responses in both preparations. Remarkably however, only adenosine initiated a consistent remote arteriolar dilation when applied to the tissue site. Thus, increases in tissue adenosine may be critical for vascular communication of metabolic demands without regard to the specific blood flow path.

Regulation of capillary perfusion by small arterioles is spatially organized.

To explore a mechanism for spatial recruitment of capillaries, this study determined whether the arterioles controlling capillary perfusion, which typically arise as sequential branches along a transverse arteriole, could respond differently from each other in situ in a spatially ordered way. Diameter changes were measured for these arterioles at a known location in the intact microvasculature in the cremaster muscle of anesthetized Golden hamsters (N = 67); each arteriole controls separate capillary groups. These arterioles all had the same concentration dependence to locally (by micropipette) applied norepinephrine (NE, 10(-9) to 10(-3) mol/L), and 10(-9) mol/L NE did not induce diameter changes when applied locally to individual vessels. However, 10(-9) mol/L NE added to the tissue superfusate, or 5% added superfusate oxygen (also locally subthreshold), each induced significant diameter changes (both constrictions and dilations), in different branches, that were presumably due to summation of individually subthreshold events that changed the prevailing conditions at the point of observation. These significant diameter changes were related to the maximal diameter or to initial tone of the branches, but these changes occurred in different ways for NE versus oxygen. With NE, the branch arterioles that constricted (versus dilated) were significantly larger (maximal diameter, 22.3 +/- 2.6 versus 15.9 +/- 2.1 microns) and had higher tone (fractional constriction, 0.53 +/- 0.05 versus 0.63 +/- 0.05); with oxygen, those that constricted were the same size as those that dilated (maximal diameter, 28.6 +/- 1.1 versus 30.5 +/- 2.7 microns), but constrictors had lower tone (fractional constriction, 0.49 +/- 0.04 versus 0.39 +/- 0.06).(ABSTRACT TRUNCATED AT 250 WORDS)

Shear stress gradient over endothelial cells in a curved microchannel system

Our purpose was to test a scale model of the microcirculation by measuring the shear forces to which endothelial cells were exposed, and comparing this to computer simulations. In vitro experiments were performed to measure the 2-dimensional projected velocity profile along endothelial cell lined microchannels (D-shaped, 10-30 microns radius, n = 15), or in microchannels without endothelial cells (n = 18). Microchannels were perfused with fluorescently labeled microspheres (0.5 micron dia., < 1%) suspended in cell culture media. The velocity of individual microspheres was obtained off-line (videorecording), using an interactive software program; velocity was determined as the distance traveled in one video field (1/60 s). Mass balance was verified in the microchannels by comparing the microsphere velocities to the perfusion pump rate. In confluent endothelial cell lined microchannels, a velocity profile was obtained as microspheres passed an endothelial cell nucleus (identified by fluorescent dye), and again, for a paired region 100 microns away without nuclei (cytoplasm region). The velocity profile was significantly shifted and sharpened by the endothelial cell nucleus, as anticipated. Over the nucleus, data are consistent with a normal sized nucleus extending into the lumen, further confirming that this scale model can be used to determine the wall shear stress to which endothelial cells are exposed. Using the experimental bulk phase fluid parameters as boundary conditions, we used computational fluid dynamics (CFD) to predict the expected wall shear stress gradient along an endothelial cell lined D-shaped tube. The wall shear stress gradient over the nucleus was 2-fold greater in the radial versus axial directions, and was sensitive to lateral versus midline positioned nuclei.

Conducted signals within arteriolar networks initiated by bioactive amino acids

Our purpose was to determine the specificity of l-arginine (l-Arg)-induced conducted signals for intra- vs. extracellular actions ofl-Arg. Diameter and red blood cell velocities were measured for arterioles [18 ± 1.6 (SE) μm] in the cremaster muscle of pentobarbital sodium-anesthetized (Nembutal, 70 mg/kg) hamsters ( n = 53). Remote (conducted) responses were viewed ∼1,000 μm upstream from the local (micropipette) application. Six amino acids were tested:l-arginine,l-cystine,l-leucine,l-lysine,l-histidine, andl-aspartate (100 μM each). Only l-Arg induced a remote dilation; l-lysine andl-aspartate had no effect, and the others each induced a significant remote constriction. There is a second conducted signal initiated byl-arginine that preconditions the arteriolar network and upregulates a direct response ofl-arginine to dilate the remote site. This was blocked by inhibition ofl-arginine uptake at the local (preconditioning) site (100 μMl-histidine or 1 mM phenformin). Arginine-glycine-aspartate (100 μM)-induced remote dilations (+3.2 ± 0.3 μm) were not mimicked by a peptide control and were prevented by anti- integrin αv monoclonal antibody. Remote dilations were greater in animals with a higher wall shear stress for arginine-glycine-aspartate ( r 2 = 0.92) but not for l-arginine ( r 2 = 0.12). Thusl-arginine initiates separate conducted signals related to system y+ transport, integrins, and baseline flow.

Technique for using video microscopy and indicator dilution for repeated measurements of cardiac output in small animals.

Background The authors developed an indicator dilution technique for small animals to repeatedly determine cardiac output and blood volume without cardiac instrumentation or blood sampling. Methods Observations were made in the hamster (N = 32, 70 mg/kg pentobarbital) cremaster using in vivo fluorescence videomicroscopy. Fluorescein isothiocyanate–conjugated bovine serum albumin (10 mg/ml) was injected as a bolus dose (right jugular) while video recording the light intensity in a 20-&mgr;m arteriole (intensified charge-coupled device [CCD] camera at fixed gain). The intensity signal was analyzed over time (background subtracted) and calibrated to the dye concentration. The ex vivo calibration was performed using a constant optical path length (20 &mgr;m) and a range of dye and hematocrit concentrations. In vivo tube hematocrit was determined using standard methods with fluorescently labeled erythrocytes. Thus, quenching of the fluorescence signal by hemoglobin was corrected for the calibration, and the plasma space in the arteriole was determined. The steady state dye concentration measured by the light intensity at 2 min was not different from the dye concentration found by direct spectrophotometric analysis of the plasma. Results Cardiac index was calculated as milliliters of blood per minute per kilogram body weight. The calculated cardiac index was 359 ± 18 ml · min−1 · kg−1, which is not different from the reported values for hamsters. Cardiac output was increased twofold when enough intravenous nitroprusside or nitroglycerine was injected to decrease mean arterial pressure from 90 to 70 mmHg. Cardiac output was elevated during dobutamine infusion (16 &mgr;g · kg−1 · min−1) and decreased during esmolol infusion (50, 75 · kg−1 · min−1). Blood volume determined from the steady state dye concentrations was 6.2 ± 0.5 ml/100 g body weight, within the normal range for hamsters. Conclusions Fluorescent dye dilution and video microscopy can be used to repeatedly determine cardiac output or blood volume in small animals.

Localized Adenovirus‐Mediated Gene Transfer Into Vascular Smooth Muscle in the Hamster Cheek Pouch

Objective: Our purpose was to develop a method for adenovirus delivery to the hamster cheek pouch to experimentally target gene transfer in tissue used for microvascular studies.

Shear rate gradient in arteriolar bifurcations: theoretical and experimental

Our purpose was to determine whether the shear rate gradient through arteriolar bifurcations in vivo could be predicted from a model of low Reynolds (Re) divergent flow. The computational model (CFD-ACE) numerically solved 3D Navier-Stokes equations for a range of bifurcation angles (30-150/spl deg/) at low Re (0.01). The branch to feed diameter ratio was 4/5; the segment intersection shape was not held constant. Velocity profiles were determined in the feed, and at the start and end of the intersection. Calculated shear rate (dv/dr) predicted a significant increasing gradient along the branch wall, but not along the corresponding lateral wall, especially for the 90/spl deg/ angles. In vivo data were obtained from cremaster preparations of the anesthetized hamster. Fluorescently labeled red blood cells were flow tracers; velocity profiles were obtained at corresponding positions through a sequential series of arteriolar bifurcations, sampling the same range of angle and vessel dimensions. Calculated dv/dr showed an increasing gradient along the branch wall when angle was 80-120/spl deg/, but not for smaller angles, which generally occur further downstream in the network. The data suggest that both bifurcation shape and location within a flow network, together determine the shear gradient in vivo.

Vitronectin receptor stimulation of remote vascular responses: role of connexin 43

We hypothesize that gap junctional communication along the vascular wall modulates remote responses. To test this, we employed both pharmacologic and genomic approaches to block connexin (C/spl times/)43 transcription, using the remote response to vitronectin receptor (VTR) stimulation as a model response. Arteriolar diameter and red blood cell velocity were determined in the cheek pouch preparation of anesthetized (pentobarbital, 70 mg/kg) hamsters (N=25). LM609 (VTR agonist) was micropipette applied to the termination of the arteriolar network; observations were made 1000 /spl mu/m upstream at the entrance to the network. Control responses consist of an immediate increase in velocity (shear rate) followed by a dilation with a decrease in shear rate. Gap junctions were pharmacologically inhibited with 18-alpha-glycyrrhetinic acid micropipette application to the middle of the network; in a dose dependent fashion, remote dilation was attenuated, but shear rate did not change. Prevention of C/spl times/43 transcription (double strand RNA inhibition) likewise blocked only the dilation in a dose dependent fashion, and not shear. Together this suggests that gap junctions are involved in this response, and that C/spl times/43 is required for flow dependent dilation following stimulation of the vitronectin receptor.