George Osol

Mechanical behavior of pressurized in vitro prearteriolar vessels determined with a video system

The muscular resistance arteries of the mesentery and brain serve two different control functions in the cardiovascular system. The former are representative vessels of vascular beds that influence total peripheral resistance and blood pressure; the latter are a good model of vessels in beds that demonstrate blood flow autoregulation. Our purpose was to develop a versatile myographic system appropriate for the in vitro study of 75–250 μm diameter vessels and to explore different physiological properties of cerebral and mesenteric arteries. In this paper the system is described in detail, examples of its use in determining the dynamic responses of the vessels to electrical stimulation are provided, and certain measures indicative of the extent of myogenic behavior are characterized.Cylindrical artery segments about 3-mm long were dissected from Wistar-Kyoto rats and mounted in a chamber filled with physiological saline solution maintained at 37°C. The same solution was perfused via a syringe into one end of the vessel through a microcannula. The other end was then occluded so that experiments could be made over a wide range of transmural pressures without flow. The vessel was viewed through a microscope coupled with a TV camera, and the video output signal of a selected scan line was processed by an electronic dimension analyzing system. This permitted simultaneous digital presentation and analog voltage outputs of the vessel wall thicknesses and lumen diameter. We further incorporated servo control of the syringe using a motor drive. In this way, vessel tests could be carried out at constant pressure or constant diameter, and vessel responses could be obtained following either pressure or diameter command signals.Using the methods presented in this study, small vessels can be maintained under conditions that approximate their in vivo state more closely than other in vitro techniques using ring segments on wires. We also find that the opto-electronic instrumentation is ideally suited for studying the dynamic vessel properties that underlie the control of vascular smooth muscle.

Pressure-induced actin polymerization in vascular smooth muscle as a mechanism underlying myogenic behavior

We hypothesize that actin polymerization within vascular smooth muscle (VSM) in response to increased intravascular pressure is a novel and previously unrecognized mechanism underlying arterial myogenic behavior. This hypothesis is based on the following observations. 1) Unlike skeletal or cardiac muscle, VSM contains a substantial pool of unpolymer‐ized globular (G) actin whose function is not known. 2) The cytosolic concentration of G‐actin is significantly reduced by an elevation in intravascular pressure, demonstrating the dynamic nature of actin within VSM and implying a shift in the F:G equilibrium in favor of F‐actin. 3) Agents that inhibit actin polymerization and stabilize the cytoskeleton (cytochalasins and latruncu‐lin) inhibit the development of myogenic tone and decrease the effectiveness of myogenic reactivity. 4) Depolymerization of F‐actin with cytochalasin D causes VSM relaxation and increased G‐actin content, whereas polymerization of F‐actin with jasplakinolide causes VSM contraction and decreased G‐actin content. These results are consistent with observations in other cell types in which actin dynamics have been implicated in contractility and/or motility. Actin filament formation in VSM may therefore underlie mechanotransduction and, by providing additional sites for interaction with myosin, enhance force production in response to pressure. Although the mechanism by which actin polymerization is stimulated by pressure is not known, it likely occurs via integrin‐mediated activation of signal trans‐duction pathways previously associated with VSM contraction (e.g., PKC activation, Rho A, and tyrosine phosphorylation).—Cipolla, M. J., Gokina, N. I., Osol, G. Pressure‐induced actin polymerization in vascular smooth muscle as a mechanism underlying myogenic behavior. FASEB J. 16, 72–76 (2002)

Mechanotransduction by Vascular Smooth Muscle

Mechanotransduction by vascular smooth muscle (VSM) is defined as a cellular response (contraction, secretion, growth, division) to transmural pressure or stretch. This review includes an overview of the physical forces VSM cells experience in vivo, consideration of experimental techniques used to study VSM mechanotransduction, and a discussion of the scientific literature pertinent to the individual cellular components that have been implicated in the transduction of physical forces. These include: the extracellular matrix, integrins, ion channels, the sarcoplasmic reticulum, second messenger systems, contractile proteins, and the cytoskeleton.

Protein kinase C modulates basal myogenic tone in resistance arteries from the cerebral circulation.

The objective of this study was to determine whether myogenic tone in the cerebral circulation can be modified by agents that interact with protein kinase C (PKC), a modulator of intracellular calcium sensitivity. Pial arteries (194 +/- 8 microns at 125 mm Hg) were isolated from Wistar-Kyoto rats and mounted on glass microcannulas in a specialized arteriograph. Simultaneous recordings of transmural pressure and lumen diameter were made with a video-electronic system. Myogenic tone, which developed at transmural pressures above 50 mm Hg, reduced lumen diameter by 29 +/- 3%, to 136 +/- 5 microns. Staurosporine (a PKC inhibitor) or indolactam (a PKC activator) was added cumulatively to segments of arteries obtained from each animal. Staurosporine induced progressive and eventually complete dilation, with half-maximal inhibition of myogenic tone occurring at a concentration of 1.32 +/- 0.10 nM. Conversely, indolactam augmented basal tone, reducing diameter by a maximum of 62 +/- 3%, with half-maximal effects at 0.4 +/- 1.0 microM. The effects of indolactam on arterial responses to acute increases in transmural pressure were also determined to test whether this dynamic and possibly separate mechanism could be potentiated by PKC stimulation. Although basal tone was augmented, diameter responses to increased pressure were not altered. In summary, these results implicate PKC in the regulation of basal myogenic tone and resistance artery caliber, which is a major determinant of blood flow. PKC modulation did not affect diameter responses to sudden changes in transmural pressure, however, suggesting the existence of a separate sensing/transduction mechanism that has yet to be identified.

Pregnancy-induced changes in the three-dimensional mechanical properties of pressurized rat uteroplacental (radial) arteries

OBJECTIVE The purpose of this study was to describe the effects of pregnancy on the size and three-dimensional mechanics of uterine radial arteries. STUDY DESIGN Measurements of lumen diameter, wall thickness, and axial and radial distensibility were made in situ and in pressurized segments of excised vessels from nonpregnant (n = 29) and late-pregnant (days 19 to 21, n = 19) Sprague-Dawley rats. RESULTS In the unpressurized state, the overall length of the radial artery segment of the arcade increased approximately 4.8 times during gestation. Lumen diameter increased by 60%, as did distensibility in both the radial and axial directions. However, there was no measurable change in the thickness of the vascular wall, which was muscular in appearance and comprised approximately two layers of circumferentially oriented smooth muscle, a relatively thick internal elastic lamina, and a well-defined endothelial layer. Cross-sectional area increased significantly during pregnancy (1.37 times at 50 mm Hg), as did the overall volume of the vascular wall (6.86 times at 50 mm Hg), primarily as a result of arterial growth in the longitudinal (axial) direction. CONCLUSIONS Remodeling of the radial artery segment of the uterine vasculature clearly occurs during gestation, resulting in vessels that are of a larger caliber and are longer and more distensible in both the axial and radial directions.

Loss of SM-B myosin affects muscle shortening velocity and maximal force development

We used an exon-specific gene-targeting strategy to generate a mouse model deficient only in the SM-B myosin isoform. Here we show that deletion of exon-5B (specific for SM-B) in the gene for the heavy chain of smooth muscle myosin results in a complete loss of SM-B myosin and switching of splicing to the SM-A isoform, without affecting SM1 and SM2 myosin content. Loss of SM-B myosin does not affect survival or cause any overt smooth muscle pathology. Physiological analysis reveals that absence of SM-B myosin results in a significant decrease in maximal force generation and velocity of shortening in smooth muscle tissues. This is the first in vivo study to demonstrate a functional role for the SM-B myosin isoform. We conclude that the extra seven-residue insert in the surface loop 1 of SM-B myosin is a critical determinant of crossbridge cycling and velocity of shortening.

High Glucose Concentrations Dilate Cerebral Arteries and Diminish Myogenic Tone Through an Endothelial Mechanism

BACKGROUND AND PURPOSE Diabetes is associated with cerebrovascular disease and impaired autoregulation of cerebral blood flow. The purpose of this study was to determine the effect of acute glucose exposure on basal tone and myogenic reactivity of isolated rat cerebral arteries. METHODS Posterior cerebral arteries (PCAs, n = 38) were dissected from male Wistar rats and mounted on glass cannulas in a system that allowed control of transmural pressure (TMP) and measurement of lumen diameter. Arteries were exposed to various concentrations of glucose, and the amount of basal tone and reactivity to TMP was measured. The effect of elevated glucose on cerebral endothelial modulation of basal tone was determined by mechanical denudation and the use of inhibitors of both nitric oxide and prostaglandin synthesis. RESULTS Arteries exposed to 44 versus 5.5 mmol/L glucose developed significantly less intrinsic tone (percent tone, 2 +/- 1% versus 28 +/- 2%; P < .01) and responded passively to increases in TMP. Preexisting tone present in 5.5 mmol/L glucose was eliminated on exposure to 44 mmol/L glucose, which decreased tone from 30 +/- 5% to 5 +/- 4% (P < .01). Glucose-induced dilations were concentration dependent such that half-maximal responses were obtained at 25 +/- 2 mmol/L. Endothelial removal abolished this effect, and the amount of tone was similar in 5.5 versus 44 mmol/L glucose (percent tone, 46 +/- 6% versus 49 +/- 5%; P > .05), as did inhibition of nitric oxide production with 0.3 mmol/L nitro-L-arginine (percent tone, 52 +/- 4% versus 46 +/- 3%; P > .05); however, blockade of the cyclooxygenase pathway with indomethacin (10(-5) mmol/L) only partially inhibited the dilation to glucose (percent tone, 32 +/- 3% in 5.5 mmol/L versus 12.4 +/- 3% in 44 mmol/L; P < .01). CONCLUSIONS Acute glucose exposure dilates arteries with intrinsic tone and impairs cerebrovascular reactivity to TMP via an endothelium-mediated mechanism that involves nitric oxide and prostaglandins.

Hypertrophic and hyperplastic effects of pregnancy on the rat uterine arterial wall

OBJECTIVE The purpose of this study was to characterize the cellular mechanisms (hypertrophy or hyperplasia) contributing to uterine arterial growth during pregnancy. STUDY DESIGN Vascular smooth muscle cells were enzymatically dispersed from radial uterine arteries from nonpregnant (n = 4) and late pregnant (day 20 to 21, n = 6) Sprague-Dawley rats, and the axial length was measured. Midpregnant (days 9 to 11, n = 5), late pregnant (n = 5), and nonpregnant (n = 5) Sprague-Dawley rats were injected with bromodeoxyuridine, and immunohistochemistry was used to visualize dividing and nondividing vascular smooth muscle cells and endothelial cells in both radial and main uterine arteries. RESULTS Vascular smooth muscle cells isolated from late-pregnant vessels were 21% longer than those from nonpregnant animals, increasing from 118 +/- 4 microns in nonpregnant rats to 150 +/- 7 microns in late-pregnant rats. Cell proliferation rates of both vascular smooth muscle cells and endothelial cells were quite low in nonpregnant animals but increased significantly during pregnancy in both radial and main uterine arteries. The pattern of changes in cell division rates over time, however, varied between the different arterial segments: in midpregnancy rates of both vascular smooth muscle cell and endothelial cell division were highest in the smaller radial arteries, whereas at term the mitotic index values were increased in main uterine arteries. CONCLUSIONS Gestational growth of the uterine vasculature is accomplished by a combination of both cellular hypertrophy and hyperplasia in the arterial wall. Furthermore, local differences in cell division rates at different times during gestation suggest the existence of spatially discrete growth mechanisms within the vascular network.

Vascular Smooth Muscle Actin Cytoskeleton in Cerebral Artery Forced Dilatation

BACKGROUND AND PURPOSE We investigated the role of actin polymerization in regulating arterial diameter in response to increasing pressure and modulating forced dilatation of cerebral arteries at pressures above the upper limit of autoregulation. METHODS Posterior cerebral arteries (n = 12) were isolated and pressurized in a special arteriograph that allowed control of intravascular pressure and measurement of lumen diameter. Intact arteries in the absence (control) or presence of 3.0 mumol/L cytochalasin B (CB), an inhibitor of actin polymerization, were subjected to stepwise increases in pressure from 75 to 200 mm Hg. Lumen diameter was continuously recorded, as was the pressure at which forced dilatation (loss of tone) occurred. After a period of time at 200 mm Hg, pressure was returned to 75 mm Hg and the extent of tone recovery was evaluated. RESULTS Arteries with and without CB developed a similar amount of tone during equilibration at 75 mm Hg: percent tone = 27 +/- 3% for control versus 29 +/- 4% for CB arteries (P > 0.05). However, arteries in the presence of CB could not withstand pressure as well and underwent FD at significantly lower pressures: 168 +/- 5 mm Hg for control versus 142 +/- 5 mm Hg for CB arteries (P < 0.01). The amount of tone that arteries regained after FD when pressure was returned to 75 mm Hg was also less in CB arteries: percent tone = 34 +/- 3% for control versus 11 +/- 2% for CB arteries (P < 0.01). CONCLUSIONS Cytoskeletal integrity appears important for maintaining cerebral arterial diameter during changing intravascular pressure. In addition, the process of actin polymerization may be a significant contributor to development of myogenic tone after forced dilatation.

Gestation increases nitric oxide–mediated vasodilation in rat uterine arteries☆☆☆★★★

OBJECTIVE The purpose of this study was to determine the influence of endothelium-released nitric oxide on uterine arterial tone and reactivity during pregnancy. STUDY DESIGN The effects of pregnancy on endothelial function were evaluated in isolated pressurized rat uterine arteries from late-pregnant rats (day 19 to 20) versus age-matched nonpregnant controls. The effects of nitric oxide synthase inhibition (N(omega)-nitro-L-arginine) on arterial tone and reactivity under basal and activated (phenylephrine) conditions were determined, as was arterial reactivity to endothelium-dependent (acetylcholine) and endothelium-independent (sodium nitroprusside) vasodilators, by evaluating changes in lumen diameter. RESULTS (1) Maximal constriction to N(omega)-nitro-L-arginine was significantly enhanced under basal (nonstimulated) conditions in arteries from late-pregnant versus nonpregnant rats (changes in lumen diameter 37% +/- 8% vs 9.3 +/- 6.2%, respectively, p < 0.05). (2) Nitric oxide synthase blockade with 1 nmol/L N(omega)-nitro-L-arginine significantly increased phenylephrine sensitivity in arteries from late-pregnant animals (median effective concentration 115 +/- 23 nmol/L vs 33 +/- 8 nmol/L, control vs treated vessels, p < 0.05) but was without statistically significant effect on arteries from nonpregnant animals (control 255 +/- 164 nmol/L, treated 250 +/- 102 nmol/L, p > 0.05). (3) The threshold concentration of acetylcholine required to elicit endothelium-dependent dilation was significantly lower in late-pregnant versus nonpregnant arteries (1.4 +/- 0.2 nmol/L vs 12.2 +/- 3.8 nmol/L, p < 0.05). (4) Vascular smooth muscle sensitivity to an exogenous nitrodilator (sodium nitroprusside) was identical in late-pregnant versus nonpregnant vessels. CONCLUSION Endothelial vasodilator influences are augmented during pregnancy under basal, activated (phenylephrine), and chemically provoked (acetylcholine) conditions in uterine arteries by enhanced release of nitric oxide.