Carrie L. Walters

Human Vascular Endothelium Heterogeneity A Comparative Study of Cerebral and Peripheral Cultured Vascular Endothelial Cells

BACKGROUND AND PURPOSE Hormones, neurotransmitters, and autacoids play a key role in the regulation of vascular tone as a result of their interaction with the endothelium. The aim of this study was to compare selected properties of three human endothelial cell lines isolated from cerebral pial arteries (PEC) and two peripheral vessels, the superficial temporal (SEC) and omental (OEC) arteries. METHODS Intracellular free calcium concentration ([Ca2+]i) and receptor protein expression were measured in characterized primary cultures of human endothelial cells. RESULTS All cell lines labeled positively for factor VIII/von Willebrand factor. Growth rate and constitutive release of endothelin-1, expressed as a function of protein, were both significantly lower in cerebral cells (PEC) than in endothelial cells derived from peripheral vessels. Basal [Ca2+]i measured with the fluorescent calcium indicator fura 2-AM (2 mumol/L) did not differ in either of the three cell lines. Although PEC responded to endothelin-1 (0.1 mumol/L) and vasoactive intestinal peptide (1 mumol/L) by a twofold to threefold increase in [Ca2+]i, OEC were unresponsive to these peptides. Moreover, the calcium response to alpha-thrombin (10 nmol/L) was greater in cerebral (PEC) than in peripheral (SEC, OEC) endothelial cells, while bradykinin (100 nmol/L) increased [Ca2+]i to a similar level in all three cell types. CONCLUSIONS This study demonstrates that endothelial cells from different sites of the vasculature exhibit different growth rates and vary in their response to agonists.

Intrathecal vincristine. Report of a fatal case despite CNS washout

The clinical course of a two‐year‐old girl with acute lymphocytic leukemia (ALL) who inadvertently received intrathecal vincristine is presented. Despite aggressive treatment with parenteral folinic acid and extensive central nervous system washout, the outcome was fatal. The pharmacologic aspects of vincristine and folinic acid that may explain why treatment was unsuccessful, are reviewed.

Weakness of Sympathetic Neural Control of Human Pial Compared With Superficial Temporal Arteries Reflects Low Innervation Density and Poor Sympathetic Responsiveness

BACKGROUND AND PURPOSE The primary goal of these studies was to understand and investigate the capacity of perivascular nerves to influence the tone of human pial arteries and to compare them with other human cephalic arteries, the superficial temporal and middle meningeal. METHODS Responses to electrical activation of intramural nerves and related features of fresh segments of human cephalic arteries-the pial (PA; 478+/-34 microm ID), middle meningeal (MMA; 540+/-41 microm ID), and superficial temporal (STA; 639+/-49 microm ID)-obtained from patients aged 15 to 82 years during surgical procedures were studied on a resistance artery myograph. RESULTS The PA segment responses to electrical nerve activation and to norepinephrine (NE; 10[-5] mol/L) were 1% and 21% of tissue maximum, respectively, compared with 6% and 34% for the MMA and 14% and 90% for the STA. Tissue maximum was defined as the force increase to 127 mmol/L KCl plus arginine vasopressin (1 microm). All arteries dilated well to acetylcholine. Possible explanations for the PA marginal neurogenic responses were assessed. NE ED50 was 5.4+/-2.2 X 10(-7) mol/L and did not vary with age or diameter. NE responsiveness did not increase in vessels with spontaneous or raised potassium-induced tone. Relaxation to isoproterenol was variable and propranolol did not increase the neurogenic response. Neither N(G)-monomethyl-L-arginine, N(G)-nitro-L-arginine methyl ester, endothelium removal, nor indomethacin consistently influenced the contractions to NE or neurogenic reactivity. The weak PA neurogenic response is in keeping with its poor innervation. As determined by catecholamine histofluorescence, innervation in the PA is sparse, with density increasing in the order PA, MMA, and STA. The incidence of nerve structures in the PA adventitio-medial junction was only 3% of those in the STA, and these were situated more than 3 microm from the closest smooth muscle cell. CONCLUSIONS We conclude that the weak neurogenic response of adult human pial artery reflects its poor innervation and responsiveness to NE, implying that these features are not important in the regulation of its diameter.

Role of Ca2+-Activated K+ Channels in the Regulation of Membrane Potential and Tone of Smooth Muscle in Human Pial Arteries

Smooth muscle cells (SMCs) in 58% of human pial arteries obtained during surgery showed no spontaneous contractions and displayed a stable resting membrane potential (MP) of -54.7 +/- 1.5 mV. Those that exhibited periodic spontaneous contractions associated with periodic depolarization and generation of spontaneous action potentials (APs) had a less negative MP of -43.1 +/- 0.5 mV (42%). Inhibition of calcium-activated potassium (KCa) channels in the silent arteries by charybdotoxin (CTX) and tetraethylammonium ions (TEA) induced dose-dependent depolarization, AP generation, and contraction. TEA and CTX enhanced the spontaneous depolarization and force in arteries that exhibited spontaneous activity. They also prolonged the spontaneous APs up to several times and increased their upstroke amplitude. Both TEA and CTX failed to produce significant depolarization in arteries treated with nifedipine. It is concluded that KCa channels are important regulators of human pial artery SMC resting MP and tone. They are also involved in the control of AP amplitude and duration and the associated contractions. These data suggest that alterations in the activity of SMC KCa channels could be responsible for the appearance of spontaneous activity in human pial arteries in vitro and that impaired function of these channels might be related to vasospastic phenomena in human cerebral circulation.

Aminergic histofluorescence and contractile responses to transmural electrical field stimulation and norepinephrine of human middle cerebral arteries obtained promptly after death.

The responses of cerebral arteries to catecholamines and sympathetic nerve stimulation show wide variation between animal species. We examined the catecholaminergic histofluorescence and the contractile responses elicited by transmural electrical field stimulation and norepinephrine (NE) in proximal segments of human middle cerebral artery (MCA) obtained during autopsy. Twenty-four percent of the specimens were obtained within 2 hours and 76% within 4 hours of death. A moderately dense catecholaminergic histofluorescence was seen in all segments of human MCA using the glyoxylic acid technique, counterstained with pontamine sky blue. However, only seven of 35 (20%) MCA segments tested showed tetrodotoxin-blocked transmural electrical field stimulation contractions, and all of these were harvested within 4 hours of death. The responses were mostly seen in the most proximal MCA segments and, at 32 Hz, only achieved 6 +/- 1% of the maximal tissue contraction. NE caused two distinct responses in human MCA segments. At low concentrations, it acts via an alpha-like adrenoreceptor to cause contractions 20 +/- 3% of the maximal tissue response. The NE ED50s for the three successive segments were not different from each other; the value for the most-proximal segment was 7.9 +/- 0.2 x 10(-7) M. At concentrations above 10(-5) M, this catecholamine acts on low-affinity sites resistant to alpha-adrenergic antagonists causing contractions that at 10(-3) M reach 52 +/- 5% of the maximal tissue response.(ABSTRACT TRUNCATED AT 250 WORDS)

Diameter Dependence of Myogenic Tone of Human Pial Arteries Possible Relation to Distensibility

BACKGROUND AND PURPOSE Responses to changes in intraluminal pressure of isolated human pial arteries (200 to 1200 microns i.d.) obtained from patients undergoing neurosurgery were measured. METHODS The vessels were cannulated and pressurized (60 mm Hg); vascular diameter and intraluminal pressure were recorded simultaneously. After spontaneous development of steady state tone, intraluminal pressure was changed to both higher and lower levels in random sequence. RESULTS Human pial arteries exhibited myogenic responses and maintained their diameter over the pressure range of 20 to 100 mm Hg. The level of myogenic tone observed at 30 mm Hg did not vary significantly with artery diameter. In contrast, at 60 and 90 mm Hg, the extent of myogenic tone increased as the diameter decreased (up to 70% to 80% of maximum in 200-microns i.d. arteries). The arteries contracted to KCl 30 mmol/L, norepinephrine 1 mumol/L, and vasopressin 0.1 mumol/L and relaxed to acetylcholine 3 mumol/L. The extent of these responses did not vary with the diameter of the artery. Arterial distensibility, represented by the slope of the tangent of the passive pressure-diameter curve at lower pressures (5 to 50 mm Hg), increased as arteries became smaller. This is consistent with the possibility that the level of myogenic tone is related to vessel distensibility. Human omental arteries of comparable size did not develop myogenic tone but contracted to KCl and norepinephrine and relaxed to acetylcholine to an extent similar to pial arteries. CONCLUSIONS There is a specific gradient of myogenic responsiveness in human pial arteries that varies inversely with their diameter. This tone does not develop in all vascular beds. These levels of tone in the pial circulation would be expected to be of profound functional significance by allowing blood flow to vary widely.

Functional Changes in Human Pial Arteries (300 to 900 μm ID) Within 48 Hours of Aneurysmal Subarachnoid Hemorrhage

Background and Purpose —Animal studies of cerebral arteries 2 to 3 days after experimental subarachnoid hemorrhage (SAH) provide evidence of arterial change such as hyperresponsiveness to contractile agonists. There is evidence that small arteries, as well as those large enough to be seen on angiography, may be involved. To directly test these possibilities, the contractile and dilator responses of pial artery segments taken from patients up to 48 hours after SAH were compared with those from patients having elective surgery for an aneurysm (Clip) and with those from normal brain vessels overlying tumors (controls). Methods —Segments were mounted on a resistance artery myograph for measurements of wall force changes. Results —There were no differences in maximum contractility (Emax) of the 3 groups of segments. The responses of the SAH segments to K+ (30 mmol/L) were 60.7±4.6% of Emax (n [number of vessels]=18), which was significantly greater than those of controls (29.9±5% Emax) (n=20). Clip responses were the same as control. Contractions of SAH segments to norepinephrine (1 μmol/L) were 54.3±7.9% Emax (n=12), and these were significantly greater than those of controls (15.1±6.2% Emax) (n=25). All SAH segments showed spontaneous contractile activity of varying patterns. Spontaneous activity did not occur in the Clip group and occurred in only 50% of control segments. Dilation to acetylcholine was numerically less in SAH and Clip segments than in controls, but differences were not statistically significant. The change in agonist responsiveness could result from exposure to agents that damage the blood vessel wall, resulting in partial depolarization of endothelial and smooth muscle cells. Conclusions —Small human pial arteries are hyperresponsive to contractile agents and show spontaneous contractile activity within 48 hours of SAH. Such effects could result in narrowed resistance arteries and reduction in cerebral blood flow. These effects emphasize the wisdom of early therapeutic intervention.

As human pial arteries (internal diameter 200–1000 μm) get smaller, their wall thickness and capacity to develop tension relative to their diameter increase

Pial arteries play a key role in the regulation of human cerebral blood flow. However, many of the features and mechanisms that regulate the tone and diameters of these vessels cannot be studied in situ. One approach is to study in vitro segments of arteries obtained during neurosurgical procedures. The ratios of arterial media thickness to lumen diameter and of the capacity to develop wall force to lumen diameter have important functional consequences and are known to change in disease. Experiments were carried out on pial arteries from normotensive humans to determine the way in which these parameters vary with vessel size. Vessel dimensions--media thickness and lumen diameter were derived from fixed sections using quantitative morphometry. Wall force was measured using a resistance artery myograph. The ratio of media thickness to lumen diameter and of maximum tension developed to lumen diameter both increased as vessel diameter decreased. These ratios do not change over the age range of 15-75 years. These findings show that although in vivo intralumenal pressure falls as human pial arteries become smaller, their media thickness and capacity to develop tone increase.

Is There a Neurogenic Influence on the Diameter of Human Small Pial Arteries

In vitro studies of neural influences on cerebral arteries have for the most part been carried out on segments of the basilar artery, the Circle of Willis, or the origins of the major cerebral arteries (for example, refs. 1-4). In both animals and humans, constriction on electrical activation of intramural nerves has been observed, whereas dilation has been demonstrated only in nonhuman species. The relative size of neurogenic dilator and constrictor effects, and the variety of putative neurotransmitters and mechanisms, are remarkably different between species.4–6