Mammalian Arteries Research Articles

Abnormal arterial-venous fusions and fate specification in mouse embryos lacking blood flow

The functions of blood flow in the morphogenesis of mammalian arteries and veins are not well understood. We examined the development of the dorsal aorta (DA) and the cardinal vein (CV) in Ncx1−/− mutants, which lack blood flow due to a deficiency in a sodium calcium ion exchanger expressed specifically in the heart. The mutant DA and CV were abnormally connected. The endothelium of the Ncx1−/− mutant DA lacked normal expression of the arterial markers ephrin-B2 and Connexin-40. Notch1 activation, known to promote arterial specification, was decreased in mutant DA endothelial cells (ECs), which ectopically expressed the venous marker Coup-TFII. These findings suggest that flow has essential functions in the DA by promoting arterial and suppressing venous marker expression. In contrast, flow plays a lesser role in the CV, because expression of arterial-venous markers in CV ECs was not as dramatically affected in Ncx1−/− mutants. We propose a molecular mechanism by which blood flow mediates DA and CV morphogenesis, by regulating arterial-venous specification of DA ECs to ensure proper separation of the developing DA and CV.

Development of an electrospun biomimetic polyurea scaffold suitable for vascular grafting.

The optimization of biomechanical and biochemical properties of a vascular graft to render properties relevant to physiological environments is a major challenge today. These critical properties of a vascular graft not only regulate its stability and integrity, but also control invasion of cells for scaffold remodeling permitting its integration with native tissue. In this work, we have synthesized a biomimetic scaffold by electrospinning a blend of a polyurea, poly(serinol hexamethylene urea) (PSHU), and, a polyester, poly-ε-caprolactone (PCL). Mechanical properties of the scaffold were varied by varying polymer blending ratio and electrospinning flow rate. Mechanical characterization revealed that scaffolds with lower PSHU content relative to PCL content resulted in elasticity close to native mammalian arteries. We also found that increasing electrospinning flow rates also increased the elasticity of the matrix. Optimization of elasticity generated scaffolds that enabled vascular smooth muscle cells (SMCs) to adhere, grow and maintain a SMC phenotype. The 30/70 scaffold also underwent slower degradation than scaffolds with higher PSHU content, thereby, providing the best option for in vivo remodeling. Further, Gly-Arg-Gly-Asp-Ser (RGD) covalently conjugated to the polyurea backbone in 30/70 scaffold resulted in significantly increased clotting times. Reducing surface thrombogenicity by the conjugation of RGD is critical to avoiding intimal hyperplasia. Hence, biomechanical and biochemical properties of a vascular graft can be balanced by optimizing synthesis parameters and constituent components. For these reasons, the optimized RGD-conjugated 30/70 scaffold electrospun at 2.5 or 5 mL/h has great potential as a suitable material for vascular grafting applications. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 278-290, 2018.

Zebrafish Model for Functional Screening of Flow-Responsive Genes.

Atherosclerosis is initiated at branches and bends of arteries exposed to disturbed blood flow that generates low shear stress. This mechanical environment promotes lesions by inducing endothelial cell (EC) apoptosis and dysfunction via mechanisms that are incompletely understood. Although transcriptome-based studies have identified multiple shear-responsive genes, most of them have an unknown function. To address this, we investigated whether zebrafish embryos can be used for functional screening of mechanosensitive genes that regulate EC apoptosis in mammalian arteries. First, we demonstrated that flow regulates EC apoptosis in developing zebrafish vasculature. Specifically, suppression of blood flow in zebrafish embryos (by targeting cardiac troponin) enhanced that rate of EC apoptosis (≈10%) compared with controls exposed to flow (≈1%). A panel of candidate regulators of apoptosis were identified by transcriptome profiling of ECs from high and low shear stress regions of the porcine aorta. Genes that displayed the greatest differential expression and possessed 1 to 2 zebrafish orthologues were screened for the regulation of apoptosis in zebrafish vasculature exposed to flow or no-flow conditions using a knockdown approach. A phenotypic change was observed in 4 genes; p53-related protein (PERP) and programmed cell death 2-like protein functioned as positive regulators of apoptosis, whereas angiopoietin-like 4 and cadherin 13 were negative regulators. The regulation of perp, cdh13, angptl4, and pdcd2l by shear stress and the effects of perp and cdh13 on EC apoptosis were confirmed by studies of cultured EC exposed to flow. We conclude that a zebrafish model of flow manipulation coupled to gene knockdown can be used for functional screening of mechanosensitive genes in vascular ECs, thus providing potential therapeutic targets to prevent or treat endothelial injury at atheroprone sites.

Cardiovascular design in fin whales: high-stiffness arteries protect against adverse pressure gradients at depth

Fin whales have an incompliant aorta, which, we hypothesize, represents an adaptation to large, depth-induced variations in arterial transmural pressures. We hypothesize these variations arise from a limited ability of tissues to respond to rapid changes in ambient ocean pressures during a dive. We tested this hypothesis by measuring arterial mechanics experimentally and modelling arterial transmural pressures mathematically. The mechanical properties of mammalian arteries reflect the physiological loads they experience, so we examined a wide range of fin whale arteries. All arteries had abundant adventitial collagen that was usually recruited at very low stretches and inflation pressures (2-3 kPa), making arterial diameter largely independent of transmural pressure. Arteries withstood significant negative transmural pressures (-7 to -50 kPa) before collapsing. Collapse was resisted by recruitment of adventitial collagen at very low stretches. These findings are compatible with the hypothesis of depth-induced variation of arterial transmural pressure. Because transmural pressures depend on thoracic pressures, we modelled the thorax of a diving fin whale to assess the likelihood of significant variation in transmural pressures. The model predicted that deformation of the thorax body wall and diaphragm could not always equalize thoracic and ambient pressures because of asymmetrical conditions on dive descent and ascent. Redistribution of blood could partially compensate for asymmetrical conditions, but inertial and viscoelastic lag necessarily limits tissue response rates. Without pressure equilibrium, particularly when ambient pressures change rapidly, internal pressure gradients will develop and expose arteries to transient pressure fluctuations, but with minimal hemodynamic consequence due to their low compliance.

New analytic solutions for wave propagation in flexible, tapered vessels with reference to mammalian arteries

AbstractNovel, closed-form, analytic solutions for the pressure and velocity fields are derived for the linear problem of wave propagation inside a tapered flexible vessel of conical shape. It is shown that pressure and velocity can be written in terms of Bessel functions of orders $1/ 3$ and $4/ 3$ respectively. An expression is also derived that quantifies the effect of the cone angle on the wave propagation velocity. The analytic solutions are general and valid for tube variations at any length scale in relation to the wavelength of the wave. In other words, the requirement that the changes in vessel properties with distance should take place over a length scale large compared to the wavelength of the wave, is not employed or needed. This is the basic condition for the application of WKB theory to tapered vessels. However, this condition is not satisfied in pressure pulses propagating in mammalian arteries. The general expressions derived in this paper are directly applicable to the cardiovascular system of mammals. It is further shown that the presented solution naturally tends to the asymptotic WKB solution when the assumptions of the theory are applied to the general expressions. An explicit formula is provided for the time-averaged energy flux of the wave that shows clearly the effect of the continuous reflection of the wave from the vessel wall. Viscous effects are incorporated by coupling the derived analytic solution with the radial velocity profile of Womersley. The results are compared with full nonlinear fluid–structure interaction simulations and very good agreement is found (maximum differences are ${\ensuremath{\sim} }1\hspace{0.167em} \% $ and 1.6 % for area-averaged pressure and velocity respectively, and 4–6 % for local velocity values).

The immunomorphological analysis of innervation of paraganglian chromaffin cells of mammalian arteries and heart

Innervation of chromaffin cells of paraganglia of the wall of mammalian large arterial vessels and heart (in rat, cat, and human) was studied by neuromorphological and immunohistochemical methods. There is established similarity in structure of specialized, “basket“-like nerve endings of the chromaffin cells (ChC) with pericellular nerve apparatuses of sympathetic and parasympathetic autonomic neurons. It is proposed to use immunohistochemical reaction for synaptophysin as method of selective detection of ChC of paraganglia and adrenal medulla. The conclusion is made that synaptophysin-positive terminals (SPPT) found on bodies of ChC and postganglionic neurons represent efferent, rather than afferent, synapses formed by myelinated axons of preganglionic fibers. It is suggested that ChC of paraganglia alongside with their characteristic endocrine function participate in complex mechanisms of chemoreceptor regulation of tissue homeostasis of mammalian blood vessels and heart.

Hydrogen Peroxide Increases K V Currents by Altering Intracellular Glutathione Redox Status in Rat Mesenteric Arterial Smooth Muscle Cell

Hydrogen peroxide (H2O2) was reported as an endothelium-derived hyperpolarizing factor in mammalian arteries. However, the effect of H2O2 on voltage-gated K+ (KV) currents, which are reportedly the primary regulator of resting membrane potential in many arterial smooth muscles including rat mesenteric artery, is unclear. In this study, using whole-cell patch-clamp technique, we examined the effect of H2O2 on the Kv currents of rat mesenteric arterial smooth muscle cells (MASMCs). H2O2 increased the Kv currents in major portion of MASMCs, whereas a slight decreasing effect of H2O2 on the Kv currents was evident in minor portion of MASMCs. As a reason for this heterogeneity, we hypothesized that heterogeneity exists in the basal intracellular redox status of MASMCs and that the effects of H2O2 on the Kv currents are intracellular redox-dependent. Pipette application of either H2O2 or oxidized glutathione (GSSG) resulted in the increased Kv currents compared with control. Under that condition, subsequent bath application of H2O2 decreased the Kv currents. Pipette application of glutathione reductase and NADPH for blocking conversion of GSH to GSSG prevented the increasing effect of H2O2 on the Kv currents. In organ chamber mechanics experiment, bath application of H2O2 relaxed arterial rings precontracted with norepinephrine. Pretreatment of dithiothreitol, a thiol-specific reducing agent prevented the relaxation by H2O2. Present results indicate that H2O2 activates KV channels under reduced, physiologic conditions whereas it inhibits the channels under oxidized conditions in MASMCs. The glutathione redox system seems to primarily mediate the H2O2-regulation of the Kv channels. Key words: Hydrogen peroxide; Mesenteric arterial smooth muscle cell; Kv channels; Redox; Glutathione.

Increased oxidative stress in atherosclerosis-predisposed regions of the mouse aorta

Aims The localization of atherosclerotic lesions to predictable regions in mammalian arteries has been recognized for over a century. We sought to investigate the association between oxidative stress and regional susceptibility of the mouse aorta to atherosclerosis. Main methods En face confocal microscopy was employed to assess oxidative stress in the aortic intima of atherosclerosis-susceptible and protected regions of wild-type C57BL/6 mouse. Expression of reactive oxygen species and antioxidant producing genes were compared in endothelial cells from the susceptible and protected regions. Key findings In vivo administration of redox-sensitive fluorescent dyes revealed an increase in the production of reactive oxygen species (ROS) in the atherosclerosis-susceptible regions relative to the protected regions. In contrast, Hoechst a redox-insensitive dye distributed evenly in the susceptible and protected regions. Accumulation of superoxide in the susceptible regions of the aorta was significantly blocked by the administration of Diphenyleneiodonium, a flavoprotein inhibitor. mRNA levels of superoxide-producing and scavenging enzymes were significantly increased in the regions predisposed to atherosclerosis. The regional difference in oxidative stress was at a lesser magnitude in BALB/c than the atherosclerosis-susceptible mouse (C57BL/6). Significance Our study for the first time demonstrated an augmented oxidative stress in atherosclerosis-susceptible regions of the normal mouse aorta.

Relation of Nitrite to Structural and Mechanical Adaptation of Arteries During Postnatal Development

Mammalian arteries undergo rapid remodeling during postnatal growth and development. The high wall shear stress at birth is an important mediator of postnatal endothelial nitric oxide (NO) and consequently of growth and remodeling. The objective of this study was to quantify the NO production in relation to geometric and mechanical remodeling of aorta and pulmonary artery during postnatal development. Fifty-one C57BL/6 mice aged from 1 to 33 days were divided into 8 age groups for measurements of nitrite (NO(x)). Systematic measurements of NO(x) in each rings were made in the main pulmonary artery and primary branch as well as along the length of aorta using the combination of a diazo coupling method and high-performance liquid chromatography. The NO(x) data on the aorta were correlated with data on the geometry (diameter, wall thickness) and mechanical properties (stress, strain, elastic modulus) in the same strain of mice under the same conditions. Our findings show postnatal age and vessel size affects the NO production; i.e., the NO(x) decreased with age and diameter. Furthermore, there is a significant positive correlation between strain and NO(x) but negative correlation between both wall thickness and elastic modulus and NO(x) levels. These findings suggest an important interplay between NO(x) and geometric and mechanical remodeling during postnatal growth and development.

Characteristics of the myogenic behaviour of arteries of the common European frog (Rana temporaria)

Mammalian small arteries exhibit pressure-dependent myogenic behaviour characterised by an active constriction in response to an increased transmural pressure or an active dilatation in response to a decreased transmural pressure. This study aimed to determine whether pressure-dependent myogenic responses are a functional feature of amphibian arteries. Mesenteric and skeletal muscle arteries from the common European frog (Rana temporaria) were cannulated at either end with two fine glass micropipettes in the chamber of an arteriograph. Arterial pressure-diameter relationships (5-40 mmHg) were determined in the presence and absence of Ca2+. All arteries dilated passively with increasing pressure in the absence of Ca2+. In the presence of Ca2+ proximal mesenteric branches and tibial artery branches dilated with increasing transmural pressure but tone (p < 0.05) was evident in both arteries. A clear myogenic response to a step increase or decrease in pressure was observed in small distal arteries (6 of 13 mesenteric and 7 of 10 sciatic branches) resulting in significantly (p < 0.05) narrower diameters in Ca2+ in the range 10-40 mmHg in mesenteric and 20-40 mmHg in sciatic arteries, respectively. The results demonstrate that arteries of an amphibian can generate spontaneous pressure-dependent tone. This is the first study to demonstrate myogenic contractile behaviour in arteries of nonmammalian origin.

The peptidic urotensin‐II receptor ligand GSK248451 possesses less intrinsic activity than the low‐efficacy partial agonists SB‐710411 and urantide in native mammalian tissues and recombinant cell systems

Several peptidic urotensin-II (UT) receptor antagonists exert 'paradoxical' agonist activity in recombinant cell- and tissue-based bioassay systems, likely the result of differential urotensin-II receptor (UT receptor) signal transduction/coupling efficiency between assays. The present study has examined this phenomenon in mammalian arteries and recombinant UT-HEK (human embryonic kidney) cells.BacMam-mediated recombinant UT receptor upregulation in HEK cells augmented agonist activity for all four peptidic UT ligands studied. The nominal rank order of relative intrinsic efficacy was U-II>urantide ([Pen(5)-DTrp(7)-Orn(8)]hU-II(4-11))>SB-710411 (Cpa-c[DCys-Pal-DTrp-Lys-Val-Cys]-Cpa-amide)>>GSK248451 (Cin-c[DCys-Pal-DTrp-Orn-Val-Cys]-His-amide) (the relative coupling efficiency of recombinant HEK cells was cat>human>>rat UT receptor). The present study further demonstrated that the use of high signal transduction/coupling efficiency isolated blood vessel assays (primate>cat arteries) is required in order to characterize UT receptor antagonism thoroughly. This cannot be attained simply by using the rat isolated aorta, an artery with low signal transduction/coupling efficiency in which low-efficacy agonists appear to function as antagonists. In contrast to the 'low-efficacy agonists' urantide and SB-710411, GSK248451 functioned as a potent UT receptor antagonist in all native isolated tissues studied (UT receptor selectivity was confirmed in the rat aorta). Further, GSK248451 exhibited an extremely low level of relative intrinsic activity in recombinant HEK cells (4-5-fold less than seen with urantide). Since GSK248451 (1 mg kg(-1), i.v.) blocked the systemic pressor actions of exogenous U-II in the anaesthetized cat, it represents a suitable peptidic tool antagonist for delineating the role of U-II in the aetiology of mammalian cardiometabolic diseases.

Exponential taper in arteries: an exact solution of its effect on blood flow velocity waveforms and impedance

The dimensions and wall elasticity commonly taper along the length of mammalian arteries. The effects of taper on flow velocity waveforms can be included by either of two methods; to theoretically divide the artery into short sections wherein the properties are assumed constant (the approximate solution); or to find an exact solution incorporating the effects of taper. In this paper, an exact solution to the resulting, and previously unsolved nonlinear Ricatti equation for the impedance, is obtained by a process of substitutions. This solution is utilised to develop an exact expression for the flow velocity in the artery. The transmission line equations are then combined into a single integral expression for the entire artery and an exact solution to this is evaluated. This is the first solution to simultaneously account for both geometric and elastic taper, and it has been validated by comparing simulations of flow in the aorta of a dog to those using an infinitesimal approximate solution. The Pulsatility Index of the approximate solution requires at least 10 segments to converge to within 5% of that using the exact solution. The exact solution thus accurately accounts for the effects of exponential taper, and may be used to improve existing arterial models, which use the less accurate and more computationally cumbersome approximate solution.

Short‐Term Incubation with Physiological Level of Estrogen Impairs ß 1 ‐Adrenoceptor‐Mediated but Enhances ß 2 ‐Adrenoceptor Mediated Coronary Relaxation

Considerable evidence suggests that ß-adrenoceptors are subject to regulation by sex steroid hormones. It was reported that estrogen replacement potentiated the vascular responses mediated by ß-adrenoceptor activation by an endothelium-independent mechanism. ß-Adrenoceptor is present on both vascular smooth-muscle and endothelial cells. However, no experiment has examined the effect of acute exposure to physiological concentrations of estrogen on ß-adrenoceptor–mediated vasorelaxation in mammalian arteries. The major observation in this study is that the relaxant response to ß2-adrenoceptor activation with fenoterol was significantly enhanced by short-term incubation (1 hr) with 0.3 nM 17ß-estradiol in the isolated porcine coronary circumflex arteries. This effect was abolished by pretreatment with 10-μM tamoxifen. Preincubation with 17ß-estradiol (0.3 nM) reduced the relaxant response to dobutamine, a ß1-adrenoceptor agonist. The concentration of 0.3 nM falls into the reported circulating level of estrogen ranging between 0.1 and 1 nM in the body. Low concentrations of estrogen appeared to slightly enhance the relaxation induced by isoproterenol, a non-selective ß- (ß1 and ß2) adrenoceptor agonist, following 1-hr incubation. Physiological level of estrogen did not influence the relaxation induced by IBMX, an inhibitor of phosphodiesterase. In contrast, 20-min exposure to 17ß-estradiol (0.1–1 nM) was without effect on ß1- or ß2-adrenoceptor-mediated vascular responses. Our data indicate that acute exposure to physiological concentration of estrogen has differential effect on ß-adrenoceptor-mediated relaxation in porcine coronary arteries, increasing ß2-adrenergic response but decreasing ß1-adrenergic response. It would therefore be not surprising to observe a marginal effect of estrogen on relaxation induced by isoproterenol, an agonist that activates both ß1 and ß2-adrenoceptors in blood vessels. (supported by UPGC Direct Grant)

Inhibitory effect of tetrabutylammonium ions on endothelium/nitric oxide-mediated vasorelaxation

Apart from the well-described K + channel blocking effects in vascular smooth muscle cells, monovalent quaternary ammonium ions may also interact with endothelial cells in the endothelium-intact mammalian arteries. The present study was aimed to examine the effect of tetrabutylammonium ions on endothelium-dependent and –independent relaxation in the rat isolated aortic rings. Pretreatment with tetrabutylammonium concentration dependently reduced the endothelium-dependent relaxation induced by acetylcholine, cyclopiazonic acid and ionomycin. Tetrabutylammonium also inhibited endothelium-independent relaxation induced by hydroxylamine or nitroprusside. Pretreatment of endothelium-denuded rings with tetrabutylammonium did not affect relaxation induced by NS1619 or by diltiazem. In contrast, tetrabutylammonium significantly reduced the pinacidil- or cromakalim-induced relaxation. Tetrabutylammonium also inhibited the acetylcholine- but not nitroprusside-induced increase of tissue content of cyclic GMP in the aortic rings. The present study indicates that tetrabutylammonium ions could inhibit endothelial and exogenous nitric oxide-mediated aortic relaxation while it had no effect on relaxation induced by activation of Ca 2+-activated K + channels (by NS1619) or by inhibition of voltage-gated Ca 2+ channels (by diltiazem). The inhibitory effect on pinacidil- and cromakalim-induced relaxation suggests that tetrabutylammonium ions also inhibit ATP-sensitive K + channels in aortic smooth muscle cells.

Role of tapering in aortic wave reflection: hydraulic and mathematical model study

Pressure and flow have been measured simultaneously at six locations along the aorta of an anatomically correct 1 : 1 scale hydraulic elastic tube model of the arterial tree. Our results suggest a discrete reflection point at the level of the renal arteries based on (i) the quarter-wavelength formula and (ii) the comparison of foot-to-foot ( c ff) and apparent phase velocity ( c app). However, separation of the pressure wave into an incident and reflected wave at all six locations indicates continuous reflection: a reflected wave is generated at each location as the forward wave passes by. We did a further analysis using a mathematical transmission line model with a simple tapering geometry (length 50 cm, 31 and 11 mm proximal and distal diameter, respectively) for a low (0.32 ml/mmHg), normal (1.6 ml/mmHg) and high (8 ml/mmHg) value of total arterial compliance. Using the quarter-wavelength formula, a discrete reflection point is found at x=33 cm, the level of the renal arteries, independent of the value of total compliance. However, local analysis comparing c ff and c app does not reveal a marked reflection site, and the analysis of incident and reflected waves merely suggests a continuous reflection. We therefore conclude that the measured in vivo aortic wave reflection indices are the result of at least two interacting phenomena: a continuous wave reflection due to tapering, and local reflections arising from branches at the level of the diaphragm. The continuous reflection is hidden in the input impedance pattern. Using the quarter-wavelength formula or the classical wave separation theory, it appears as a reflection coming from a single discrete site, confusingly also located at the level of the diaphragm. Therefore, the quarter-wavelength formula and the linear wave separation theory should be used with caution to identify wave reflection zones in the presence of tapering, i.e., in most mammalian arteries.

Phenotypic modulation of smooth muscle cells during formation of neointimal thickenings following vascular injury.

Smooth muscle cells build up the media of mammalian arteries and constitute one of the principal cell types in atherosclerotic and restenotic lesions. Accordingly, they show a high degree of plasticity and are able to shift from a differentiated, contractile phenotype to a less differentiated, synthetic phenotype, and then back again. This modulation occurs as a response to vascular injury and includes a prominent structural reorganization with loss of myofilaments and formation of an extensive endoplasmic reticulum and a large Golgi complex. At the same time, the expression of cytoskeletal proteins and other gene products is altered. As a result, the cells lose their contractility and become able to migrate from the media to the intima, proliferate, and secrete extracellular matrix components, thereby contributing to the formation of intimal thickenings. The mechanisms behind this change in morphology and function of the smooth muscle cells are still incompletely understood. A crucial role has been ascribed to basement membrane proteins such as laminin and collagen type IV and adhesive proteins such as fibronectin. A significant role is also played by mitogenic proteins such as platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF). An improved knowledge of the regulation of smooth muscle differentiated properties represents an important part in the search for new methods of prevention and treatment of vascular disease.

Differentiated Properties and Proliferation of Arterial Smooth Muscle Cells in Culture

The smooth muscle cell is the sole cell type normally found in the media of mammalian arteries. In the adult, it is a terminally differentiated cell that expresses cytoskeletal marker proteins like smooth muscle alpha-actin and smooth muscle myosin heavy chains, and contracts in response to chemical and mechanical stimuli. However, it is able to revert to a proliferative and secretory active state equivalent to that seen during vasculogenesis in the fetus, and this is a prerequisite for the involvement of the smooth muscle cell in the formation of atherosclerotic and restenotic lesions. A similar transition from a contractile to a synthetic phenotype occurs when smooth muscle cells are established in culture. Accordingly, an in vitro system has been used extensively to study the regulation of differentiated properties and proliferation of these cells. During the first few days after seeding, the cells are reorganized structurally with a loss of myofilaments and formation of a widespread endoplasmic reticulum and a prominent Golgi complex. In parallel, they lose their contractility and instead become competent to divide in response to a large variety of mitogens, including platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF). After entering the cell cycle, they start to produce these and other mitogens on their own, and continue to replicate in the absence of exogenous stimuli for a restricted number of generations. Furthermore, they start to secrete extracellular matrix components such as collagen, elastin, and proteoglycans. The mechanisms that control this change in morphology and function of the smooth muscle cells are still poorly understood. Adhesive proteins such as fibronectin and laminin apparently have an important role in determining the basic phenotypic state of the cells and exert their effects via integrin receptors. The proliferative and secretory activities of the cells are influenced by a multitude of growth factors, cytokines, and other molecules. Although much work remains before an integrated view of this regulatory machinery can be achieved, there is no doubt that the cell culture technique has contributed substantially to our knowledge of smooth muscle differentiation and growth. At the same time, it has been crucial in exploring the role of these cells in vascular disease and developing new therapeutic strategies to cope with major causes of human death and disability.

Ionic currents in single cells from human cystic artery.

The patch-clamp technique was used to study the electrophysiological properties of single smooth muscle cells obtained from the human cystic artery. These cells contracted on exposure to high K+ and had a mean resting potential of -36 +/- 7 mV. Under current clamp, regenerative responses could not be elicited when depolarizing pulses were applied. Voltage-clamp measurements demonstrated that a large fraction of the outward current was inhibited by tetraethylammonium (5-10 mM) or Ca2+ channel blockers and that it was enhanced by increasing [Ca2+]o, suggesting that it is a Ca(2+)-activated K+ current. In addition, spontaneous transient outward currents that were sensitive to extracellular Ca2+ were observed in some cells. In cell-attached patch-clamp recordings, Ca(2+)-activated K+ channels that had a conductance of 117 pS were consistently identified. At negative potentials (approximately -60 mV), these single-channel events deactivated completely and very quickly, suggesting that they do not control the resting membrane potential in healthy cystic artery cells. Ca2+ currents that were recorded using Ba2+ (10 mM) as the charge carrier were enhanced by the dihydropyridine agonist, Bay K 8644, and blocked by nifedipine (0.1 microM). Only one type of Ca2+ current, the L-type, could be identified in these cells. These results demonstrate that the major ionic currents in the human cystic artery are similar to other mammalian arteries and indicate that this tissue will be a useful model for studying the metabolic and pharmacological modulation of ionic currents in human vascular smooth muscle.

Dissociation between endothelium-dependent relaxations and increases in cGMP in systemic veins

In canine systemic veins, in contrast to what is observed in mammalian systemic arteries, endothelium-dependent relaxations to the calcium ionophore A23187 are not diminished by the inhibitor of soluble guanylate cyclase, methylene blue. Therefore, experiments were designed to determine whether these relaxations in the veins are associated with the accumulation of guanosine 3',5'-cyclic monophosphate (cGMP). Rings of canine femoral arteries and veins with and without endothelium were suspended for the measurement of isometric force in organ chambers; cGMP was measured by radioimmunoassay. In arteries and veins contracted with norepinephrine, the tissue content of cGMP was greater in rings with than without endothelium. This difference was decreased by methylene blue (10(-5) M). A23187 (3 X 10(-7) M, for 1 min) increased the accumulation of cGMP, which was temporally related with the onset of relaxation in tissues with endothelium. Methylene blue inhibited the accumulation of cGMP in both blood vessels but inhibited the relaxations only in the arteries. In rings without endothelium, sodium nitroprusside (3 X 10(-7) and 10(-5) M) initiated increases in cGMP, which followed the onset of relaxation. Neither response to sodium nitroprusside was reduced by methylene blue. These results suggest that in canine femoral arteries and veins, relaxation of the smooth muscle to sodium nitroprusside are mediated by a mechanism distinct from changes in cGMP. Likewise, in canine systemic veins, endothelium-derived factor(s) released in response to A23187 also can initiate relaxation of the smooth muscle by a mechanism distinct from changes in cGMP.

Endothelium‐derived relaxing and contracting factors

Endothelium-dependent relaxation of blood vessels is produced by a large number of agents (e.g., acetylcholine, ATP and ADP, substance P, bradykinin, histamine, thrombin, serotonin). With some agents, relaxation may be limited to certain species and/or blood vessels. Relaxation results from release of a very labile non-prostanoid endothelium-derived relaxing factor (EDRF) or factors. EDRF stimulates guanylate cyclase of the vascular smooth muscle, with the resulting increase in cyclic GMP activating relaxation. EDRF is rapidly inactivated by hemoglobin and superoxide. There is strong evidence that EDRF from many blood vessels and from cultured endothelial cells is nitric oxide (NO) and that its precursor is L-arginine. There is evidence for other relaxing factors, including an endothelium-derived hyperpolarizing factor in some vessels. Flow-induced shear stress also stimulates EDRF release. Endothelium-dependent relaxation occurs in resistance vessels as well as in larger arteries, and is generally more pronounced in arteries than veins. EDRF also inhibits platelet aggregation and adhesion to the blood vessel wall. Endothelium-derived contracting factors appear to be responsible for endothelium-dependent contractions produced by arachidonic acid and hypoxia in isolated systemic vessels and by certain agents and by rapid stretch in isolated cerebral vessels. In all such experiments, the endothelium-derived contracting factor appears to be some product or by-product of cyclooxygenase activity. Recently, endothelial cells in culture have been found to synthesize a peptide, endothelin, which is an extremely potent vasoconstrictor. The possible physiological roles and pathophysiological significance of endothelium-derived relaxing and contracting factors are briefly discussed.