Modifications of adhesion properties and proteoglycan structure in rat embryo fibroblast cultures with increasing passages.

Adhesion properties of rat embryo fibroblast cultures and proteoglycans (PGs) produced both in the growth medium and in the cell layer were investigated with increasing passages. Both cell-cell and cell-substrate adhesion increased with increasing subculture number. Cell adhesion properties were improved by cell treatment with chondroitinase ABC. The increase in subculture number was coupled with a constant increase of PG molecular size, which was particularly evident in cell layer extracts. The ratio HS-PGs/DS-PGs increased with increasing passages. PG modifications are likely to represent evidence of changes in extracellular matrix organization and could play a role in the increase of cell adhesion properties.


Treatment of childhood encopresis--a review.

Children with encopresis may present to a number of different professionals. The literature on different treatment methods is reviewed. The roles of verbal psychotherapy, physical treatment, behaviour therapy and mixed treatment programmes are discussed.


Endotoxin stimulates interleukin-6 production by human Kupffer cells.

Interleukin-6 (IL-6) induces acute-phase protein synthesis in human hepatocytes. We evaluated whether the contiguous hepatic macrophages, human Kupffer cells (HKC), produce IL-6 in response to an inflammatory stimulus. HKC were harvested from collagenase-digested normal liver biopsies and purified (greater than 95% by phagocytosis) by adherence. Following overnight culture, 5 x 10(5) HKC were repleted with fresh media with or without 2.5 micrograms/ml of endotoxin (LPS). Parallel cultures contained polymyxin-B (10 micrograms/ml) or antihuman-IL-6 antibody (4 units/ml). Timed supernatants were collected and IL-6 levels (ng/ml) measured (B9.9 proliferative bioassay). Data analysis was by the paired Student's t test. Unstimulated HKC produced negligible IL-6 levels (less than 0.150 ng/ml). Endotoxin invoked early and sustained HKC production of IL-6, which was completely (P less than 0.001) abrogated by the addition of the anti-IL-6 antibody. Polymyxin B, an LPS-inhibitor, also blocked (P less than 0.001) IL-6 production, indicating the specificity of the response to the inflammatory stimulus. This is the first evidence that HKC can produce IL-6 in response to LPS. Local intrahepatic production of IL-6 may provide a necessary paracrine signal for HKC to amplify directly neighboring hepatocyte acute-phase responses during inflammation in man.


Effect of positive end-expiratory pressure on pulmonary vascular pressure-flow characteristics in canine endotoxin shock.

The vascular pulmonary pressure-flow (P-Q degree) relationships were studied in anesthetized dogs in order to characterize the distribution of total resistance in the pulmonary bed with respect to incremental resistance and critical closure prior to and after endotoxin insult. Incremental resistance was computed as the slope of the P-Q degree relation, whereas critical closure was referred to as the extrapolated pressure intercept at zero flow. P-Q degree coordinates were obtained by varying Q degree through graded inflation of right atrial balloon. The gradients across the arterial segment (Pa = Ppa - Pc) and across the venous segment (Pv = Pc - Pw) of the pulmonary vasculature were defined by the computation of effective capillary pressure (Pc) obtained from the analysis of the transient decay of pulmonary artery pressure (Ppa) toward wedge pressure (Pw) after arterial occlusion. Six group E dogs were infused with endotoxin at a rate of 0.25 microgram/kg min, while six additional animals served as control (group C). Endotoxin induced increases in flow resistance from 0.056 to 0.096 mm Hg/ml/min/kg due to arterial vasoconstriction and increases in critical closure from 2.3 to 8.4 mm Hg due to a venous waterfall. Before and after endotoxin insult, we assessed effects of each of three levels of static lung inflation (PEEP) on P-Q degree relationships.(ABSTRACT TRUNCATED AT 250 WORDS)


Hepatic insulin resistance during chronic hyperdynamic sepsis.

Disturbances in normal glucose metabolism and homeostasis which manifest as hyperglycemia and glucose intolerance are often observed during clinical sepsis. Skeletal and myocardial muscle as well as whole body insulin resistance have been demonstrated in this laboratory and others during experimental and clinical sepsis. The existence of hepatic insulin resistance in sepsis has yet to be fully elucidated. This study was undertaken to assess hepatic insulin resistance during chronic hyperdynamic sepsis. Animals were randomly assigned to a septic (n = 7), sham (n = 7), or control (n = 7) group. Sepsis was induced in anesthetized dogs via a midline laparotomy whereby a fecal-soaked gauze sponge was placed amid the intestines. Sham animals underwent a laparotomy with mechanical manipulation of the intestines but no fecal implant. Control animals had no previous surgery. Sham and control dogs were pair-fed with the septic dogs. On postoperative day 7, after an overnight fast, animals were anesthetized, intubated, and ventilated. Via a left subcostal laparotomy, electromagnetic flow probes were placed to measure hepatic arterial and portal venous blood flows. Cannulae were placed in femoral, portal, and hepatic veins and femoral artery and used to calculate hepatic outputs of glucose, lactate, and oxygen during a basal period and hyperinsulinemic-euglycemic clamps which used intravenous insulin infusions which ranged from 0.4 to 4,000 mU/min. Mean arterial blood pressure decreased with increasing insulin concentrations in septic animals while no change was seen in control or sham animals. In control and sham animals, net hepatic glucose output (NHGO) decreased in response to increasing insulin levels. Septic animals showed no such inverse relationship and, moreover, showed no change in glucose output response to any insulin infusion, i.e., hepatic insulin unresponsiveness during sepsis. Net hepatic lactate output during basal pre-insulin period during sepsis was negative. This was in contrast to the positive outputs in control and sham animals. Glucose infusion rates (GIR) increased during insulin infusion but were not different between groups at any insulin infusion rate. These data demonstrated a hepatic insulin resistance (unresponsiveness) during chronic hyperdynamic, hypermetabolic sepsis.


Role of extracellular Ca2+ in ischemia-reperfusion injury in the isolated perfused rat liver.

The influx of extracellular Ca2+ has been postulated to be one of the mediators of ischemia-reperfusion injury. A possible link between Ca2+ influx and oxygen radical generation has also been suggested. In the present study, using the isolated perfused rat liver, we evaluated the role of extracellular Ca2+ on oxygen radical generation, liver damage, and lipid peroxidation during 30 min ischemia and 60 min of reperfusion. Oxygen radical generation in the liver was continuously monitored by lucigenin-enhanced chemiluminescence. Liver damage and lipid peroxidation were evaluated by measuring lactate dehydrogenase (LDH) and thiobarbituric acid reactive substances (TBARS) release into the effusate, respectively. In the absence of extracellular Ca2+ (much less than 30 microM) oxygen radical generation from the liver increased gradually over 2 hr and there were concomitant increases in LDH and TBARS release. When livers were made ischemic and then reperfused, oxygen radical generation increased at the onset of reperfusion and then decreased over 30 min of reperfusion. After 30 min of reperfusion, livers reperfused with low Ca2+ buffer showed a linear increase in oxygen radical generation as well as progressive increases in LDH and TBARS release. On the other hand, livers reperfused with Ca2+ containing (1.25 mM) buffer showed no further increase in oxygen radical generation and no evidence of progressive liver damage and lipid peroxidation. These results suggest that Ca2+ overload is not a primary cause of liver ischemia-reperfusion injury and that the presence of extracellular Ca2+ during reperfusion is necessary to maintain normal liver function.


Acute hemodynamic effects of hypertonic (7.5%) saline infusion in patients with cardiogenic shock due to right ventricular infarction.

The hemodynamic effects, after infusion of 4 ml/kg of hypertonic (7.5%) saline solution (HS), were evaluated in six patients (mean age = 56.6 years) with cardiogenic shock (CS) due to right ventricular infarction (RVI). Basal condition data (mean +/- SEM) were as follows: cardiac index (CI) = 1.9 +/- 0.1 1/min/m2, arterial pressure (AP) = 66.5 +/- 0.9 mmHg, and systemic vascular resistance (SVR) = 31.3 +/- 1.0 mmHg/1/min/m2. Five- and 240-minute post-HS infusion data (respectively) revealed: CI = 3.3 +/- 0.1* and 2.9 +/- 0.1* 1/min/m2, AP = 87.7 +/- 1.6* and 80.7 +/- 2.2* mmHg, and SVR = 22.5 +/- 0.6* and 24.5 +/- 1.1* mmHg/1/min/m2 (*P less than 0.05 compared to baseline values). These data suggest that small-volume infusion of HS induced an important acute and sustained hemodynamic improvement in these patients with CS due to RVI.


Low dose calcium administration increases mortality during septic peritonitis in rats.

Calcium is essential for normal cellular function. However, increases in cell calcium are also involved in cellular dysfunction and death by activating destructive enzymatic processes such as proteases, nucleases, and lipases. Since ionized hypocalcemia is common during sepsis, calcium is often administered in an attempt to normalize circulating levels. Previous studies using endotoxin and large amounts of calcium demonstrated increased mortality in calcium-treated animals. This study extends these preliminary data to endogenous sepsis (cecal ligation) and low dose calcium therapy. Sprague Dawley rats (n = 9 per group) had jugular catheters placed and their cecums ligated. They were randomized to an infusion of D5W at 1 ml/hr, calcium chloride at 4 mg/ml/hr (Low Ca), or calcium chloride at 6 mg/ml/hr (High Ca) for 24 hr. An additional group of animals received calcium chloride at 6 mg/ml/hr but had no surgery. Animals were followed for 24 hr survival. A separate group of animals (n = 6 per group) was prepared in a similar manner. Blood was obtained 6 hr following cecal ligation for circulating endotoxin concentrations, and animals were sacrificed. Calcium infusion increased ionized calcium levels slightly (low Ca, 1.12 +/- .06 to 1.21 +/- .09; high Ca, 1.06 +/- .04 to 1.16 +/- .05; P less than 0.05). Survival (24 hr) was 100% in the nonseptic controls and D5W septic animals, 67% in the low Ca septic group, and 44% (P less than 0.05) in the high Ca septic group.(ABSTRACT TRUNCATED AT 250 WORDS)


Child development: what do parents expect?

Expectations of future development were assessed in parents of non-disabled children (n = 240) using the Predictive Adaptive Ability Questionnaire (PAAQ). The reliability estimate for the overall PAAQ was 0.90, with a test-retest reliability of 0.99 and an interrater reliability of 0.83. Parental expectations did not significantly differ from normal adaptive ability developmental standards. Longitudinal examination of parental expectations and their influence on child development and the impact of chronic illness on such expectations is recommended.


Continuous measurement of lactate concentration in skeletal muscle and liver interstitium using a microdialysis method after acute hemorrhage in anesthetized rats.

Interstitial lactate concentration in rat skeletal muscle was continuously measured using a microdialysis method following an acute hemorrhage. For comparison, similar measurements were also performed for liver. Normal lactate levels of skeletal muscle and liver interstitium were 0.58 +/- 0.10 mM and 1.23 +/- 0.04 mM, respectively. After withdrawal of 30% of the estimated total blood volume within 3 min, a constant high blood lactate level was achieved. The lactate concentrations in muscle and liver interstitium also increased to 270 and 210% of the basal levels, respectively, but they did not exceed the blood level. These findings led us to hypothesize that skeletal muscle as well as liver functions as a lactate consumer rather than a producer after acute hemorrhage.


Training of head control in the sitting and semi-prone positions.

The purpose of this study was to compare the semi-prone (SP) and sitting (SIT) training positions with respect to head control in children with cerebral palsy, before and after 5 weeks biofeedback training using a head position trainer (HPT). Four children were randomly assigned to each of two training groups: (a) SP on a prone board inclined 45 degrees above the horizontal and (b) SIT in their personal wheelchair and orthotic device. The HPT, secured to the child's head, controlled a video-cassette player, turning it off when the head deviated beyond 25 degrees from the vertical (termed an error). The time in error and the number of errors during test periods of 3 minutes, without feedback and completed in both the SP and the SIT positions, were determined immediately before and after, and at 16 weeks after training. The SIT trained group performed significantly better immediately post-training in three of four comparisons (P < 0.01), but the groups performed similarly in the other eight comparisons--four immediately pre-training and four at 16 weeks post-training (P > 0.05). Post-training scores for the total group (n = 8) were significantly improved over pre-training scores, regardless of the test position or the criterion measurement (P < 0.05). Biofeedback training with a HPT can be effective in either the SIT or the SP positions, with improvement lasting at least 16 weeks after training is discontinued.


Passive immunization with antibodies against tumor necrosis factor (TNF-alpha) protects from the lethality of splanchnic artery occlusion shock.

Splanchnic artery occlusion shock was induced in anesthetized rats by clamping splanchnic arteries for 45 min. Survival rate, serum and macrophage tumor necrosis factor (TNF-alpha), peritoneal macrophage phagocytosis, and killing activities were evaluated. Shocked rats died within 2 hr, whilst all sham-shocked rats survived more than 6 hr. Serum and macrophage TNF-alpha was undetectable in sham-shocked rats while shocked rats exhibited increased serum (110 +/- 5 U/ml 90 min after release of occlusion) and macrophage levels (122 +/- 4.5 U/ml 90 min after release of occlusion) of TNF-alpha. Furthermore, splanchnic artery occlusion shock produced cardiovascular changes, reduced macrophage phagocytosis (23 +/- 4.6%) and killing (6 +/- 1.1%) activities, and induced a massive necrosis of the ileum. A passive immunization with a hyperimmune serum containing antibodies against murine TNF-alpha significantly protected rats from the lethal effects of splanchnic artery occlusion shock, lowered serum TNF-alpha (6 +/- 2.1 U/ml), and completely reverted the impairment in peritoneal macrophage phagocytosis (48 +/- 4.8%) and killing (13 +/- 1.5%) activities. In addition passive immunization had beneficial effects on the cardiovascular changes occurring during splanchnic artery occlusion shock and prevented necrosis of the ileum induced by this model of shock. By contrast, pretreatment with polymyxin B, an "antiendotoxin" antibiotic, did not modify the lethal effects and the TNF-alpha production induced by splanchnic artery occlusion shock. Furthermore, endotoxin was undetectable in the blood of splanchnic artery occlusion shocked rats. These findings are consistent with the involvement of TNF-alpha in splanchnic artery occlusion shock and suggest that the cytokine represents an important mediator of non-septic shock.


Portage guide to early intervention: cross-cultural aspects and intra-cultural variability.

The issues arising from implementing an early intervention service, developed in the rural United States in the late 1960s in a range of different cultural contexts over a period of a quarter of a century, are explained. Services from India, Bangladesh, Jamaica and the United Kingdom are compared. As well as considering cross-cultural aspects of Portage, variability within one country, the United Kingdom, is considered by comparing one service in an inner-city area and one in a rural area.


Kupffer cell exacerbation of hepatocyte hypoxia/reoxygenation injury.

In order to examine the possible contribution of the Kupffer cell to the generation of hypoxia/reoxygenation injury in the liver, primary cultures of hepatocytes, either alone or in coculture with Kupffer cells, were exposed to 90 min of sublethal hypoxia followed by 120 min of reoxygenation. Prolonged incubation of cocultured hepatocytes and Kupffer cells resulted in increased release of lactic dehydrogenase (LDH) indicating cell injury even under normoxic conditions. LDH release was further increased by the presence of Kupffer cells during hypoxia/reoxygenation. To determine whether or not this effect of Kupffer cells might be the result of oxygen-derived free radical production, we assessed the efficacy of the enzymatic scavengers superoxide dismutase (SOD) + catalase in ameliorating the Kupffer cell mediated injury. SOD + catalase was effective in preventing free radical injury generated by hypoxanthine + xanthine oxidase. However, SOD + catalase did not ameliorate hepatocyte injury caused by Kupffer cells. Thus, activation of Kupffer cells may be an important factor in the genesis of liver injury, but the mediator of Kupffer cell exacerbation of hepatocyte injury appears to be a mechanism other than free radicals released into the medium. These results indicate that chemical substances from the activated Kupffer cells may cause hepatocyte damage, which cannot be blocked by SOD + catalase, and suggest that these substances at reflow may be important for the genesis of reperfusion injury in vivo.


Sleep clinics with health visitors: a 2-year evaluation.

This study was undertaken to evaluate a service for sleep clinics over a time span of 2 years. The service was operated jointly by health visitors and clinical psychologists and based on a previous model run by the same team of psychologists and child development advisors. The outcome was not supportive of this type of service in terms of referral rates over a period of time, improvement rate, continued existence or cost-effectiveness of such a service. The reasons for the difficulties of such clinics are discussed.


Cardiovascular effects of thyrotropin-releasing hormone in normotensive and hypotensive rats: role of rostral ventrolateral medulla.

To ascertain the central mechanism of the effects of thyrotropin-releasing hormone (TRH) on the cardiovascular system, we evaluated the effects of this neuropeptide on unit fires in the rostral ventro-lateral medulla (RVL), mean arterial pressure (MAP), and heart rate (HR) in normotensive and hypotensive rats. Intracerebroventricular injection (i.c.v.) of 10 micrograms TRH significantly increased MAP and HR in both normotensive and hypotensive rats. No similar effects were observed after saline injection. If electrolytic lesions of bilateral RVL were made, the cardiovascular effects of TRH i.c.v. failed to occur. TRH i.c.v. markedly increased the firing frequency of most units in the RVL. In particular, TRH i.c.v. increased the firing frequency of most units excitatory to a fall in MAP and decreased the firing frequency of most units inhibitory to a fall in MAP in normotensive rats. Moreover, a drop of MAP as low as 40 mmHg for 10 min resulted in an increase of the firing frequency of most units. The effect of TRH i.c.v. on the RVL units in the hypotensive rats was similar to that in the normotensive rats. Our findings suggest that TRH is able to intensify the cardiovascular activities and the RVL plays a key role in the effects of TRH on the cardiovascular system in both normotensive and hypotensive rats.


Increase in renal magnesium overflow following aortotomy-induced hemorrhage in pigs.

Hemorrhage causes an increase in plasma Mg++ levels. This article identifies some of the tissues contributing to the hemorrhage induced increase in plasma Mg++. Anesthetized, splenectomized pigs were subjected to a 5 mm aortotomy. Blood samples from the abdominal aorta, inferior vena cava, pulmonary artery, hepatic portal vein, and renal vein were sampled for Mg++ changes caused by this uncontrolled hemorrhage. Aortotomy produced a rapid drop in arterial pressure and cardiac output, both of which remained depressed for the following 120 minutes (P less than 0.05). By 120 minutes, 6 of the 8 pigs survived, but three of the six survivors showed signs of cardiovascular decompensation. A significant increase in arterial plasma Mg++ was indicated by a paired t-test (P less than 0.05). Analysis of the venous samples also revealed a significant increase during the post-aortotomy period. Renal venous and hepatic portal venous plasmas both showed increases greater than that in the arterial plasma. The renal and splanchnic tissues, then, were partly responsible for the increase in the arterial Mg++ which follows hemorrhage.


The cardiovascular effects of gamma-hydroxybutyrate following hemorrhage.

We have previously shown that gamma-hydroxybutyrate (GHB) protects the small intestine against ischemia/reperfusion injury. This study examined the effects of GHB on cardiovascular function and intestinal microcirculation following hemorrhage. Hypotension was induced in control group of hamsters by controlled hemorrhage to a mean arterial pressure (MAP) of 40 mm Hg. Following 60 minutes of hypovolemia the shed blood was returned. This procedure resulted in complete intestinal mucosal microvascular stasis 2 hours following the return of shed blood. A second group of animals was treated with GHB (600 mg/kg body weight) and, despite the loss of 37% of total blood volume, GHB treatment completely prevented the microcirculatory stasis, following the reinfusion of shed blood. In male Wistar rats treated with GHB (200 mg/kg) after the induction of hemorrhage, blood pressure rapidly increased to pre-hemorrhage levels following treatment, even though the shed blood was not returned. Cardiac output (CO) also increased to pre-hemorrhage levels. Sodium chloride solution, in the same molar concentration as GHB (23% NaCl), produced much smaller, but statistically significant, increases in MAP and CO. In animals given an equal volume of normal saline, a gradual increase in MAP was observed, reaching statistical significance at 75 minutes following treatment. Three hours following hemorrhage, serum levels of creatine kinase were 3-fold higher, whereas aspartate aminotransaminase and alanine aminotransferase levels were 2-fold higher in both normal saline and hypertonic saline-treated animals than in GHB-treated animals. These experiments suggest that GHB can prevent ischemic complications following a hypovolemic episode and may improve survival following severe hemorrhage.


Favourable effect of defibrotide in lipid A-induced shock in pigs.

Defibrotide (DEF), a compound previously found to stimulate vascular prostacyclin (PGI2) formation, has been investigated in an experimental model of septic shock. Anesthetized pigs were subjected to i.v. infusion of lipid A (1.5 mg/kg per hr for 4 hr). DEF (50 mg/kg per hr) or vehicle were infused i.v. throughout the experiments, starting 1 hr prior to lipid A. Two out of 7 pigs receiving vehicle survived lipid A infusion for 4 hr, whereas 6 out of 7 DEF treated animals survived this period (P less than 0.05). DEF delayed the shock-induced depression of platelet count and preserved platelet secretory function (collagen-induced ATP-secretion). DEF increased plasma PGI2 by 45% (P less than 0.05) during lipid A infusion and tended to reduce thromboxane levels. DEF did not change eicosanoid formation in sham-shock pigs (n = 4 per group). In vivo treatment with DEF significantly increased the stimulatory effect of bradykinin (1 microM) and arachidonic acid (100 microM) on PGI2 formation ex vivo of mesenteric and iliac artery segments. The improvement of survival in lipid A-induced shock by DEF may be related to an enhancement of vascular PGI2 generation, potentially due to a reduction of shock-induced platelet activation and microcirculatory dysfunction.


Protective effect of platelet-activating factor antagonist on hepatic energy metabolism following transient hepatic inflow occlusion in rabbits.

To investigate whether or not platelet-activating factor (PAF) is a mediator of the liver injury resulting from transient hepatic inflow occlusion (Pringle's maneuver), the effect of pretreatment with a potent PAF antagonist (CV6209) on hepatic energy metabolism was evaluated following 30 min of inflow occlusion in rabbits. At 60 min after declamping, the arterial ketone body ratio (AKBR; acetoacetate/3-hydroxybutyrate), reflecting hepatic mitochondrial redox state (NAD+/NADH), increased to 1.10 +/- 0.05 (mean +/- SEM) in the CV6209 (5 mg/kg)-pretreated group (group 1, n = 5) compared with 0.72 +/- 0.06 (P less than 0.01) in the untreated group (group 2, n = 5). Hepatic energy charge at 60 min after declamping was significantly higher in group 1 than in group 2 (0.871 +/- 0.010 vs. 0.800 +/- 0.023; P less than 0.05). Pretreatment with CV6209 had no significant influence on these parameters in sham-operated animals. The present study demonstrates that pretreatment with CV6209 has a protective effect against the impairment of hepatic energy metabolism following transient hepatic inflow occlusion.


Production of tumor necrosis factor-alpha and interleukin-6 in mice infected with group B streptococci.

Group B streptococci (GBS) are a leading cause of sepsis and meningitis in neonates. Since cytokines are thought to play an important role in septic shock, we have studied serum levels of tumor necrosis factor-alpha (TNF alpha) and interleukin-6 (IL-6) in BALB/c mice infected with type III GBS. TNF alpha and IL-6 were detected by the L929 cytotoxicity and the B9 proliferation assays, respectively, in serial serum samples obtained after infection. After i.p. challenge with an LD50, serum TNF alpha rose above baseline values as early as 3 hr, peaked at 7 hr, and returned to baseline values at 20 hr. IL-6 serum levels rose concomitantly with TNF alpha, peaking 8 hr after challenge. No serum TNF alpha activity was detected in the course of sublethal infections. However, a transient rise in TNF alpha levels was observed after i.v. inoculation of high numbers (greater than or equal to 1 x 10(8) of heat-killed GBS. When groups of mice were injected i.v. with a single dose of anti-TNF alpha rabbit serum 2 hr before challenge with an LD90 or LD30, no effect was noted in terms of survival, although the serum TNF alpha peak was completely abrogated. Serum TNF alpha does not seem to play an obligatory role in GBS-induced lethality of adult mice. However, further studies are needed to assess better the role of this cytokine in the pathogenesis of GBS sepsis.


Circulating cytokines: the tip of the iceberg?

Circulating cytokines, particularly TNF alpha, IL-1, IL-6 and IL-8, can be detected during severe infections consecutively to an exacerbate release by activated cells. The presence or absence of cytokines within biological fluids reflects a rather complex balance between enhancing and inhibitory signals acting on producer cells, between production and catabolism, and between their binding to the target cells and the modulation of their receptors on the cell surface. Furthermore, their presence does not necessarily parallel their activity and a possible interplay between the cytokines and their corresponding inhibitors should be considered. On the contrary, the absence of detectable circulating cytokines does not indicate an absence of production by activated cells. We discuss the concept that circulating cytokines represent the tip of the iceberg.


Efficacy of monoclonal antibody against tumor necrosis factor alpha in an endotoxemic baboon model.

Tumor necrosis factor alpha (TNF) has been described as a primary mediator of the pathophysiology associated with bacterial sepsis/endotoxemia. We tested the efficacy and possible mechanisms of protection of a monoclonal antibody against TNF (TNF Mab) in a low mortality (29%), endotoxemic baboon model. A number of parameters were monitored at days 0, 1, 2 and 5-7 after challenge with 2 mg E. coli endotoxin/kg. TNF Mab pretreatment (15 mg/kg) prevented the increase in detectable serum TNF and the early perturbations in cardiovascular function which occurred in the control group. Early metabolic dysfunction was delayed in the TNF MAb group and was attenuated thereafter. Dysfunction of the kidney, liver, and coagulation systems and the increased IL-6 levels were significantly attenuated in the TNF MAb group; neutrophil activation was not affected by TNF MAb. No deaths occurred in the TNF MAb group. These results support the hypothesis that TNF plays a central role in the pathophysiology of endotoxic shock.


Polymyxin-B suppresses endotoxin-induced insulin hypersecretion in pancreatic islets.

Endotoxin induces insulin hypersecretion in vivo, which results in hyperinsulinemia and glucose dyshomeostasis. Polymyxin-B (PMX-B), an inhibitor of protein kinase C (PKC), has been shown to ameliorate the consequences of endotoxin-induced hyperinsulinemia in vivo. To explore the mechanism for this effect in vitro, this study determined whether PMX-B could alter endotoxin-induced insulin hypersecretion in isolated pancreatic islets of Langerhans. Pancreases were obtained from fasted, male, Sprague-Dawley rats treated with either saline (control) or endotoxin (S. enteritidis B, 16.7 mg/kg, i.v.). Three hours after the experimental treatment, islets were isolated by collagenase digestion and then incubated for 1 hr in Krebs Ringer bicarbonate buffer containing 0.5% bovine albumin, 10 mM HEPES, 300 mg/dl D-glucose, phorbol 12-myristate 13-acetate (PMA, 1 microM when present), and PMX-B (1 or 10 mM when present). In the absence of PMA and PMX-B, "endotoxic" islets hypersecreted immunoreactive insulin (IRI) relative to control islets. PMA, the prototypical PKC activator, significantly increased IRI secretion from both control and "endotoxic" islets. The additional inclusion of PMX-B (1 mM or 10 mM) in the incubation media significantly reduced insulin secretion from both control and "endotoxic" islets and suppressed the insulin hypersecretion observed in "endotoxic" islets. Since insulin secretion occurs at least partially through mechanisms dependent on PKC activation, the ability of PMX-B to suppress insulin hypersecretion in "endotoxic" islets suggests that activation of PKC within pancreatic beta-cells may play a role in the excess insulin secretion and hyperinsulinemia associated with endotoxicosis.


Regulation of glucose metabolism by free fatty acid availability in septic and nonseptic rats.

An inhibitory effect of fatty acid oxidation on glucose uptake and oxidation has been demonstrated in heart and skeletal muscle under certain experimental conditions. This reciprocal relationship has been termed the glucose-fatty acid cycle. The purpose of the present study was to determine under in vivo conditions whether this interaction was operational in various nonmuscle tissues, and whether infection altered the activity of this cycle. Oral administration of alpha-methylpalmoxirate (MPA; 75 mg/kg), a known inhibitor of long-chain fatty acid oxidation, suppressed hepatic glucose production by 54% and increased whole body glucose disappearance by 15% in nonseptic rats. In contrast, MPA produced a larger reduction of glucose output in septic rats, but did not enhance glucose disposal. In vivo glucose uptake (Rg) by individual tissues was determined using the tracer 2-deoxyglucose technique. In nonseptic animals, MPA increased Rg in "working" muscles (heart and diaphragm; 12-fold and two-fold respectively), but not in "resting" skeletal muscles. MPA increased the Rg of heart and diaphragm to the same level in septic animals. Inhibition of fatty acid oxidation in nonseptic rats also enhanced Rg in liver (174%), spleen (158%), lung (153%), ileum (52%), skin (28%), kidney (115%), and epididymal fat (135%). In septic rats, MPA only increased Rg in the ileum (23%) and kidney (50%). This increased glucose uptake was independent of increases in plasma glucose and insulin concentrations. The infusion of heparin and intralipid, which increased circulating levels of fatty acids, failed to produce consistent changes in either the whole body glucose turnover or glucose uptake by individual tissues. We conclude that under basal in vivo conditions the inhibition of fatty acid oxidation suppresses glucose production and increases peripheral glucose disposal in nonseptic animals. These data support the presence of the glucose-fatty acid cycle in nonmuscle tissues and emphasizes its importance in whole body glucose homeostasis in situations where fatty acid oxidation is impaired. Infection increases glucose uptake by nonmuscle tissues which, for the most part, cannot be further enhanced by the inhibition of lipid oxidation.


Regulation of platelet-derived growth factor ligand and receptor gene expression by alpha-thrombin in vascular smooth muscle cells.

Since the expression of genes for platelet-derived growth factor (PDGF)-A and PDGF beta-receptor are reciprocally regulated in vascular wall cells after balloon injury, we have investigated the ability of specific vasoactive molecules or growth factors to reproduce the injury pattern of gene expression in cultured rat smooth muscle cells (SMCs) and assessed the effect of inactivating alpha-thrombin on injury-induced expression of PDGF-A mRNA by vascular wall cells in vivo. The molecules investigated, to which vascular SMCs may be locally exposed after mechanical injury, included vasoactive factors (alpha- and beta-adrenergic agonists, serotonin, histamine, angiotensin II, and endothelin) and growth factors (PDGF-AA, PDGF-BB, basic fibroblast growth factor, insulin-like growth factor, epidermal growth factor, and alpha-thrombin). In cultured rat SMCs, only alpha-thrombin (0.1-100 nM), among these compounds, produced the pattern of transiently increased PDGF-A and decreased PDGF beta-receptor mRNA. PDGF-B chain mRNA levels remained undetectable in these cultured SMCs. The dependence of these changes in gene expression on the proteolytic activity of alpha-thrombin was shown by the interruption of altered gene expression or DNA synthesis after incubating the cultured SMCs with covalently inactivated alpha-thrombin using D-Phe-Pro-Arg chloromethyl ketone, a synthetic direct active-site irreversible inhibitor of alpha-thrombin. Continuous intravenous infusion of this synthetic antithrombin into baboons for 6 hours (100 nmol/kg per minute maintaining constant plasma levels of 3.0 +/- 0.5 microns/ml) after inducing balloon-catheter arterial injury also prevented the threefold increase in expression of PDGF-A mRNA characteristically exhibited by untreated mechanically injured vessels.(ABSTRACT TRUNCATED AT 250 WORDS)


Effects of serotonin on intracellular pH and contraction in vascular smooth muscle.

Serotonin (5-HT) and other contractile agonists stimulate Na(+)-H+ exchange in vascular smooth muscle. Since intracellular alkalinization, per se, stimulates contraction, we tested whether 5-HT-induced contraction was associated with an increased pHi. In HCO3(-)-free buffer (pHo 7.4), 5-HT (10(-5) M) increased pHi, as measured by 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein fluorescence, from 7.10 +/- 0.03 to 7.34 +/- 0.03 (p < 0.01) in primary cultures of canine femoral artery vascular smooth muscle cells grown to confluence in the presence of 10% fetal calf serum. In HCO3- buffer (24 mM, pHo 7.4), resting pHi was 7.26 +/- 0.04 (p < 0.01 versus HCO3(-)-free buffer) but was not altered by 5-HT. In both types of buffer, 5-HT stimulated 5-(N-ethyl-N-isopropyl)amiloride-sensitive 22Na+ uptake (Na(+)-H+ exchange). In HCO3- buffer and in Na(+)- and HCO3(-)-free buffer, 5-HT increased 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid-sensitive 36Cl- uptake, suggesting that 5-HT stimulated Na(+)-independent Cl(-)-HCO3- and Cl(-)-Cl- exchange activities, respectively. Individual vascular smooth muscle cells were then cultured on rat tail tendon collagen gels in the presence of 0.5% fetal calf serum, and cell length and pHi were measured by video and epifluorescence microscopy. 5-HT contracted cells in a dose-dependent, reversible, and ketanserin-inhibitable manner. These cells, like cells grown in 10% fetal calf serum, exhibited Na(+)-H+ and Na(+)-independent Cl(-)-HCO3- exchange. In HCO3- buffer, 5-HT contracted cells without an associated change in pHi. We concluded the following: 1) 5-HT stimulated both Na(+)-H+ and Na(+)-independent Cl(-)-HCO3- exchange activities in cultured vascular smooth muscle cells in parallel. 2) As a result of enhanced H+ and HCO3- efflux, pHi was not altered. 3) In the presence of HCO3-, 5-HT-induced contraction was not associated with a change in pHi.


HCO3(-)-dependent intracellular pH regulation in the premature myocardium.

This study investigated developmental changes in Na(+)-H+ exchange and HCO3(-)-Cl- exchange activities in newborn and adult rabbit hearts. pHi was measured using the fluorescent dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein in isolated myocytes. Myocardial mechanical function was measured in the isolated ventricular preparation. Intracellular acidosis with normal pHo was induced by an NH4Cl (10 mM) prepulse technique. Upon removal of NH4Cl, pHi fell transiently and then recovered toward the control level. In the HCO3-/CO2-buffered solution, the rate of recovery of pHi in the newborn was greater than in the adult. In the HCO3-/CO2-buffered solution, 5-(N-ethyl-N-isopropyl)amiloride (EIPA), an inhibitor of Na(+)-H+ exchange, inhibited the recovery of pHi completely in the adult. In the newborn, however, significant recovery of pHi was observed in the presence of EIPA. In the presence of both EIPA and 4-acetamido-4'-isothiocyanatostilbene-2',2'-disulfonic acid (SITS), an inhibitor of HCO3(-)-Cl- exchange, the recovery of pHi was not observed in the two age groups. In the HEPES-buffered solution that did not contain HCO3-/CO2, the rate of recovery of pHi after NH4Cl removal was similar in the two age groups. In the HEPES-buffered solution, the recovery of pHi was completely inhibited by EIPA in the two age groups. In the presence of EIPA in the HCO3-/CO2-buffered solution, contractile function decreased during acidosis after NH4Cl removal and did not recover in the adult. In the newborn, significant recovery of contractile function was observed after NH4Cl removal in the presence of EIPA. The recovery of mechanical function observed in the presence of EIPA in the newborn was inhibited by SITS. These data suggest that, although there is no developmental change in the Na(+)-H+ exchange activity, HCO3(-)-Cl- exchange is more active in the premature myocardium. The presence of the HCO3(-)-Cl- exchanger is important in maintaining myocardial contractile function during acidosis, especially when Na(+)-H+ exchange is inhibited and may partly explain the greater resistance of the premature myocardium to acidosis.


Activation of ATP-sensitive K+ channels by cromakalim. Effects on cellular K+ loss and cardiac function in ischemic and reperfused mammalian ventricle.

Pharmacological modulation of [K+]o accumulation and action potential changes during acute myocardial ischemia is under evaluation as a promising new antiarrhythmic and cardioprotective strategy during myocardial ischemia and reperfusion. We studied the effects of cromakalim, a K+ channel opener that activates ATP-sensitive K+ channels, in isolated arterially perfused rabbit interventricular septa subjected to ischemia and reperfusion and, through use of the patch clamp technique, in inside-out membrane patches excised from guinea pig ventricular myocytes. During aerobic perfusion, 5 microM cromakalim shortened action potential duration (APD) from 217 +/- 7 to 201 +/- 10 msec, had no effect on [K+]o, and reduced tension by 17 +/- 3% (n = 11). During ischemia, pretreatment with 5 microM cromakalim resulted in 1) more rapid APD shortening (71 +/- 9 versus 166 +/- 7 msec at 10 minutes and 63 +/- 12 versus 122 +/- 8 msec at 30 minutes), 2) similar [K+]o accumulation after 10 minutes (8.9 +/- 0.3 versus 9.6 +/- 0.5 mM) but a trend toward increased [K+]o accumulation after 30 minutes (11.0 +/- 1.7 versus 9.6 +/- 1.0 mM), and 3) similar times for tension to decline to 50% of control (2.14 +/- 0.16 versus 2.14 +/- 0.19 minutes) but shorter time to fall to 20% of control (4.34 +/- 0.33 versus 4.90 +/- 0.22 minutes; p = 0.003). After 60 minutes of reperfusion following 30 minutes of ischemia, recovery of function was similar, with a trend toward better recovery of developed tension (to 58 +/- 9% versus 39 +/- 10% of control; p = 0.18) and tissue ATP levels in cromakalim-treated hearts but no differences in APD or rest tension. Thus, 5 microM cromakalim had mild effects in normal heart but greatly accelerated APD shortening during ischemia without markedly increasing [K+]o accumulation, possibly because the more rapid APD shortening reduced the time-averaged driving force for K+ efflux through ATP-sensitive K+ channels. A significant cardioprotective effect during 30 minutes of ischemia plus 60 minutes of reperfusion could not be demonstrated in this model. In excised membrane patches studied at room temperature, the ability of cromakalim to activate ATP-sensitive K+ channels was significantly potentiated by 100 microM but not 15 microM cytosolic ADP, suggesting that in addition to the modest fall in cytosolic ATP during early ischemia, the rapid increases in cytosolic ADP may further sensitize cardiac ATP-sensitive K+ channels to activation by cromakalim.(ABSTRACT TRUNCATED AT 400 WORDS)


Decreased myofilament responsiveness in myocardial stunning follows transient calcium overload during ischemia and reperfusion.

The purpose of this study was to test the hypothesis that abnormal intracellular calcium handling characterizes myocardial stunning. Isolated, isovolumic, buffer-perfused ferret hearts were loaded with the bioluminescent calcium indicator aequorin for simultaneous measurement of individual calcium transients and left ventricular pressure. After 15 minutes of global ischemia and 20 minutes of reperfusion, left ventricular developed pressure was significantly reduced (75 +/- 7 versus 93 +/- 6 mm Hg, p < 0.05). During ischemia, [Ca2+]i levels were significantly elevated compared with preischemic levels, both during systole (1.38 +/- 0.31 versus 0.88 +/- 0.2 microM, p < 0.05) and end diastole (0.85 +/- 0.16 versus 0.38 +/- 0.13 microM, p < 0.05). Early during reperfusion, [Ca2+]i was also significantly elevated during systole (1.63 +/- 0.44 versus 0.88 +/- 0.20 microM, p < 0.05) and end diastole (0.75 +/- 0.15 versus 0.38 +/- 0.13 microM, p < 0.05). After 20 minutes of reperfusion, myocardial stunning occurred, but [Ca2+]i was not significantly different from preischemic levels. Thus, myocardial stunning does not result from decreased levels of activator calcium. The force-pCa relation generated by the stunned hearts was shifted downward compared with that generated by the control hearts, consistent with a decrease in maximum calcium-activated force (Fmax). At steady state during tetanus, the decrease in Fmax was confirmed, but there was no significant difference in the slope of the force-pCa relation of the stunned hearts versus controls. Thus, we conclude that stunned myocardium is characterized by decreased Fmax without desensitization of the myofilaments to [Ca2+]i.(ABSTRACT TRUNCATED AT 250 WORDS)


Age-related changes in fibronectin expression in spontaneously hypertensive, Wistar-Kyoto, and Wistar rat hearts.

The effects of age and blood pressure on fibronectin expression in the rat heart were studied in the normotensive Wistar and Wistar-Kyoto (WKY) strains and in the spontaneously hypertensive rat (SHR). Fibronectin mRNA expression decreased threefold between 10 and 40 weeks of age in Wistar hearts, with changes of similar magnitude occurring between 6 and 24 weeks in WKY rats. In contrast, no decrease in fibronectin mRNA was observed in SHR hearts during this time span. These results are in contrast to changes observed previously in the aorta, where an increase in fibronectin mRNA occurred with age in all three rat strains. Ribonuclease protection analysis showed a small age-specific increase in the relative content of EIIIA+ fibronectin mRNA isoforms in hearts from Wistar rats, whereas no change was found in the relative amount of either EIIIA or EIIIB isoforms in SHR hearts. Changes similar to those observed for fibronectin mRNA, although of different magnitudes, were observed in mRNA levels for collagen alpha 1(III) and beta 1 integrin. In Wistar hearts, collagen alpha 1(III) mRNA levels decreased fivefold to sixfold between 10 and 40 weeks of age, whereas a twofold to threefold decrease in beta 1 integrin was observed in WKY hearts between 6 and 24 weeks of age. Western blot analysis revealed a positive correlation between fibronectin mRNA and protein for age-dependent changes in ventricular tissue but not in the atria, suggesting that the regulation of fibronectin expression during the changes common to both aging and hypertrophy could involve both transcriptional and posttranscriptional mechanisms.


Body surface mapping of ectopic left ventricular activation. QRS spectrum in patients with prior myocardial infarction.

To improve electrocardiographic localization of the site of origin of ectopic left ventricular (LV) impulse formation in the heart with prior myocardial infarction, 62-lead body surface QRS integral maps were studied during LV pacing at a total of 221 endocardial sites in 14 patients with previous anterior (AMI), inferior (IMI), lateral (LMI), or anterior and inferior (AMI/IMI) myocardial infarction. The anatomic location of each pacing site was computed using digitized biplane fluoroscopic images and plotted on standardized LV endocardial polar projections. A data base of characteristic AMI and IMI mean QRS integral maps was developed after visually selecting subgroups with nearly identical QRS integral morphology from the ectopic activation sequences produced at 110 sites in eight patients with AMI and at 66 sites in four patients with IMI. Intrasubgroup pattern uniformity and intersubgroup pattern variability were statistically verified. The endocardial pacing site locations belonging to each AMI and IMI subgroup were depicted as segments on the respective LV polar projections. In patients with AMI, a total of 18 typical mean QRS integral patterns were obtained, whereas 22 different mean total QRS integral patterns showing more substantial intersubgroup variation were acquired in patients with IMI. Posterolateral regions exhibited a relatively low electrocardiographic sensitivity (six AMI and five IMI patterns) as compared with anteroseptal regions (12 AMI and 17 IMI patterns). Total QRS integral patterns obtained at 24 sites in one patient with LMI were largely compatible with the IMI mean total QRS integral patterns, whereas the majority of total QRS integral patterns acquired at 21 sites in one patient with AMI/IMI corresponded with the AMI mean total QRS integral patterns. The results show that total body surface QRS integral maps generated during LV pacing in patients with prior myocardial infarction cluster by pattern and that each QRS integral pattern is related to a circumscribed endocardial segment of ectopic impulse formation. The relation between a given QRS integral pattern and the position and size of the corresponding paced segment is dependent on infarct location. The present infarct-specific data base of characteristic total body surface QRS integral patterns provides a clinical tool to obtain detailed electrocardiographic localization of ventricular arrhythmias in patients with previous myocardial infarction.


Ventricular loading is coupled with DNA synthesis in adult cardiac myocytes after acute and chronic myocardial infarction in rats.

To determine whether the overload associated with myocardial infarction and ventricular failure in rats is coupled with activation of DNA synthesis in the remaining left and right ventricular myocytes, flow cytometric analysis was performed on myocyte nuclei prepared from both ventricles 7 and 30 days after coronary occlusion. In addition, oral captopril was administered in separate groups of control and experimental rats to establish whether a relation existed between attenuation of ventricular loading and magnitude of DNA synthesis in myocytes. Results demonstrated that left ventricular failure and right ventricular dysfunction at 7 days after infarction were biventricularly associated with marked increases in the number of myocyte nuclei in the G2M phase of the cell cycle. In contrast, the fraction of nuclei in the G0+G1 phase decreased. In comparison with the earlier time point, the 30-day interval was characterized by a significant magnitude of cardiac hypertrophy, a moderate amelioration of ventricular pump function, and a decrease in the percentage of myocyte nuclei in the G2M phase in both ventricles. However, 30 days after infarction, the number of right ventricular myocyte nuclei in the S and G2M phases remained elevated with respect to control animals. Captopril therapy reduced the extent of ventricular loading and the population of myocyte nuclei in the cell cycle at 7 days. In conclusion, DNA synthesis in myocyte nuclei may represent an important adaptive component of the myocardial response to infarction.


Age-related appearance of outward currents may contribute to developmental differences in ventricular repolarization.

Ventricular repolarization significantly influences contractility, refractoriness, and ion channel state. Factors affecting repolarization will thus affect these secondary phenomena. To understand the influence of age on ventricular repolarization, we studied neonatal, young, and adult dogs using electrocardiogram, action potential, and whole-cell voltage-clamp recordings from single epicardial myocytes. Hearts of neonatal and 57-58-day-old dogs require a significantly longer time for repolarization than those of adult dogs, as determined by analysis of rate-corrected QT and JT (QT minus QRS) intervals. Epicardial action potentials of neonates are significantly longer than those of adults, as determined by measurements of duration at 50% and 90% repolarization. The adult action potential is characterized by a large phase 1 notch that is absent from neonatal recordings. This notch develops between 58 and 64 days of age, and by 64-68 days of age, it is equal to that in adults. In addition, action potentials recorded from adult and young epicardial muscle are more greatly affected by rapid pacing and superfusion of 2 mM 4-aminopyridine than are potentials recorded from neonatal tissue. Whole-cell voltage-clamp recordings reveal a 4-aminopyridine-sensitive transient outward current in adult myocytes that is absent from neonatal myocytes. The correlation between developmental changes in the 4-aminopyridine-sensitive current, the action potential, and the QT interval suggests that the transient outward current may be an important determinant in the relation between age and repolarization.


Accumulation and assembly of myosin in hypertrophic cardiomyopathy with the 403 Arg to Gln beta-myosin heavy chain mutation.

The sarcomeric proteins and organization of cardiac myofibrils appeared intact in multiple unrelated patients with hypertrophic cardiomyopathy. In two subjects demonstrating the missense mutation at position 403 (Arg to Gln) in the beta-myosin heavy chain gene, total myosin and immunoreactive beta-myosin heavy chain levels were similar to those found in other patients with hypertrophic cardiomyopathy and various disease control subjects. No alteration in expression of the cardiac alpha-myosin heavy chain gene was observed. These results are consistent with the examined myosin heavy chain mutation, permitting proper accumulation and assembly of myosin while primarily impairing contractile function. The characteristic myocyte disarray would appear likely to be a secondary consequence of the mutations.


Lysophosphatidylcholine inhibits surface receptor-mediated intracellular signals in endothelial cells by a pathway involving protein kinase C activation.

Lysophosphatidylcholine (lysoPC) transferred from oxidatively modified low density lipoprotein (Ox-LDL) to the endothelial surface membrane has been shown to produce a selective unresponsiveness to cell surface receptor-regulated endothelium-dependent relaxation (EDR) in the rabbit aorta. To determine its mechanism we examined the effects of lysoPC on endothelial surface receptor-mediated transmembrane signals. Incubation for 1 minute with palmitoyl lysoPC (5-10 microM) decreased thrombin (Th, 2 units/ml)- or histamine (His, 0.1 mM)-stimulated inositol 1,4,5-trisphosphate (IP3) production in primary cultures of human umbilical vein endothelial cells (HUVECs). LysoPC also decreased Th- or His-induced intracellular calcium ([Ca2+]i, fura 2) elevation. Pretreatment with protein kinase C (PKC) inhibitors staurosporine (100 nM) or H-7 (50 microM) prevented the inhibitory actions of lysoPC, but HA-1004 had no effect. Incubation for 5 minutes with phorbol 12-myristate 13-acetate (PMA, 100 nM) produced the inhibitory actions on the Th- or His-induced intracellular signals, which closely mimic those exhibited by lysoPC. However, the inhibitory effect of lysoPC was lost in cells that were depleted of PKC by pretreatment for 24 hours with 100 nM PMA. Furthermore, incubation of the cells for 1 minute with lysoPC stimulated PKC activity in the membrane fraction. In organ chamber experiments with porcine coronary artery rings, pretreatment with staurosporine (20 nM) attenuated lysoPC-induced impairment of EDR in response to Th. These results indicate that lysoPC, which accumulates in Ox-LDL and atherosclerotic arterial walls, inhibits the early transmembrane signaling pathway in endothelial cells, and PKC activation could at least partially be involved in the negative regulation by lysoPC.(ABSTRACT TRUNCATED AT 250 WORDS)


Endothelium-derived relaxing factor modulates platelet aggregation in an in vivo model of recurrent platelet activation.

It has been shown that endothelium-derived relaxing factor (EDRF) may inhibit platelet aggregation in vitro through activation of platelet-soluble guanylate cyclase. To assess whether EDRF may also affect platelet function in vivo, intravascular platelet aggregation was initiated by placing an external constrictor around endothelially injured rabbit carotid arteries. Carotid blood flow velocity was measured continuously by a Doppler flow probe placed proximal to the constrictor. After placement of the constrictor, cyclic flow reductions (CFRs), due to recurrent platelet aggregation, developed at the site of the stenosis. After CFRs were observed for 30 minutes, a solution of authentic nitric oxide (NO, n = 10) was infused into the carotid artery via a small catheter placed proximally to the stenosis. Before infusion of NO, CFR frequency averaged 18.3 +/- 2.9 cycles per hour, and CFR severity (lowest carotid blood flow as percentage of baseline values) was 6 +/- 1%. NO completely inhibited CFRs in all animals, as shown by the normal and constant pattern of carotid blood flow (CFR frequency, 0 cycles per hour, p < 0.001; carotid blood flow, 92 +/- 5%, p = NS versus baseline). These effects were transient; CFRs were restored spontaneously within 10 minutes after cessation of NO infusion. After CFRs returned, S-nitroso-cysteine (S-NO-cys), a proposed form of EDRF, was infused into the carotid artery. S-NO-cys also abolished CFRs in all animals but at a significantly lower dose than NO (0.3 +/- 0.1 versus 12 +/- 4 nmol/min). The role of endogenously released EDRF in modulating in vivo platelet function was then tested in additional experiments. In 10 animals, endogenous release of EDRF was stimulated by infusing acetylcholine into the aortic root during CFRs. Infusion of acetylcholine was also associated with a complete inhibition of CFRs, similar to that observed during exogenous infusion of NO or S-NO-cys. These antithrombotic effects of acetylcholine were completely lost when EDRF synthesis was prevented by administration of the L-arginine analogue NG-monomethyl L-arginine (L-NMMA). Furthermore, in six additional rabbits the basal release of EDRF was blocked by L-NMMA after CFRs had been previously abolished with aspirin or the combination of aspirin and ketanserin, a serotonin S2 receptor antagonist. L-NMMA caused restoration of CFRs in all animals, indicating that even the basal release of EDRF is important in modulating platelet reactivity in vivo. Taken together, the data of the present study demonstrate that endogenous EDRF might importantly contribute to the modulation of platelet function in vivo.


Coronary blood flow after the regression of pressure-overload left ventricular hypertrophy.

Abnormal coronary blood flow (CBF) in long-standing left ventricular (LV) pressure-overload hypertrophy has been associated with ischemia and LV dysfunction. Thus, goals of therapy in pressure overload are not only the relief of the overload itself but also regression in hypertrophy and subsequent improvement in CBF. However, little is known about CBF in humans or in large mammals after the relief of pressure overload, when the hypertrophy has regressed. This study was performed to test the hypothesis that, even 6 months after the relief of pressure overload in the dog, CBF would still be abnormal. Three groups of dogs were studied: 1) normal control dogs (NL group), 2) dogs with LV pressure-overload hypertrophy (LVH group), and 3) dogs that had developed LV pressure-overload hypertrophy but in whom the pressure overload was relieved 6 months before the final study (LVH Reg group). CBF was studied in conscious dogs by use of the radiolabeled microsphere technique at rest, during rapid atrial pacing, and during maximum coronary vasodilation produced by adenosine infusion. The ratio of LV weight (g) to body weight (kg) (LVBW) was 4.2 +/- 0.3 in the NL group, 7.1 +/- 0.6 in the LVH group, and 7.7 +/- 0.5 in the LVH Reg group before pressure-overload relief (p = NS, LVH versus LVH Reg). Six months after removal of the pressure overload, the LVBW in the LVH Reg group had fallen to 5.5 +/- 0.3 (p < 0.05), but this LVBW was still greater than that in the NL group (p < 0.05). During rapid atrial pacing, endocardial and epicardial CBF rose significantly in NL dogs. However, during rapid atrial pacing, endocardial CBF fell from 1.18 +/- 0.22 to 0.7 +/- 0.20 ml/min per gram in the LVH group (p < 0.05) and did not rise in the LVH Reg group. During adenosine infusion, endocardial blood flow increased in NL dogs from 1.63 +/- 0.13 to 4.0 +/- 0.3 ml/min per gram and increased to a similar level in the LVH Reg group. Although CBF increased during adenosine infusion in the LVH group, the increase was less than that in the NL or LVH Reg group (p < 0.05). Minimum coronary vascular resistance was similar in NL dogs (14 +/- 2 units) and LVH Reg dogs (18 +/- 3 units, p = NS) but was significantly elevated (32 +/- 10 units) in LVH dogs (p < 0.05).(ABSTRACT TRUNCATED AT 400 WORDS)


Characterization of angiotensin II receptor subtypes in rat heart.

Angiotensin II exerts positive inotropic and chronotropic effects on the mammalian heart by binding to specific membrane receptors. Recently, two subtypes of angiotensin II receptors (AT1 and AT2) have been distinguished by using the nonpeptide antagonists losartan (previously known as DuP 753) and PD123177. To evaluate the tissue distribution and subtypes of angiotensin II receptors in rat heart, we performed a 125I-[Sar1,Ile8]angiotensin II in situ binding assay on tissue sections obtained from adult Sprague-Dawley rats (10 and 14 weeks old). Binding specificity was verified by competition with unlabeled [Sar1]angiotensin II. Distribution of AT1 and AT2 receptors was determined by competition with losartan and PD123177, respectively, and the density of the receptors was quantified by emulsion autoradiography. Angiotensin II receptors were widely distributed throughout the heart, with each receptor subtype accounting for approximately 50% of the specific binding. Binding density was comparable in the atria, right and left ventricles, intraventricular septum, and sinoatrial node, whereas it was significantly greater in the atrioventricular node. The AT1 receptor appears to interact with guanidine nucleotide regulatory proteins, because GTP-gamma-S causes dissociation of the radioligand from this receptor. In contrast, the AT2 receptor does not appear to directly interact with guanine nucleotide regulatory proteins, inasmuch as radioligand dissociation from this receptor subtype is not affected by GTP-gamma-S. Because angiotensin II has been reported to have growth-potentiating effects in several tissues, we examined angiotensin II receptors in fetal (embryonic days 16 and 19) and neonatal (1-, 2-, 3-, and 10-day-old) rats.(ABSTRACT TRUNCATED AT 250 WORDS)


Coronary collateral development in swine after coronary artery occlusion.

We have quantified the development of the coronary collateral circulation in the pig. The collateral circulation was induced to grow by placing an ameroid occluder on the left circumflex coronary artery. Two to 16 weeks after ameroid placement, the coronary collateral circulation was identified after the injection of several colors of a silicone polymer into the coronary arteries and the aorta. We identified intercoronary and extracardiac collaterals and quantified their number, location, size, and wall thickness. Intercoronary collaterals grew to a level that represents a 14-fold increase in normal collateral blood flow under resting conditions compared with the values in an animal not subjected to coronary artery occlusion. Extracardiac collaterals could potentially supply approximately 30% of resting flow. The sources of the extracardiac collaterals were the bronchial and internal mammary arteries. Coronary collateral morphometry and DNA synthesis in the pig heart also were examined. Coronary collaterals had significantly less smooth muscle than did normal arterioles. This may account, in part, for the reduced response of the coronary collaterals to vasodilators. We observed intense DNA synthesis in endothelial and smooth muscle cells in the first 2 or 3 weeks of ischemia. However, DNA synthesis rapidly ceased after this time, coincident with coronary collateral reserve values (ischemic/nonischemic regional blood flow ratios during maximal vasodilation) reaching their maximum level. This suggests that failure of the vessels to continue proliferating accounts for the occurrence of the plateau in blood flow levels.


Effect of inhibition of endopeptidase 24.11 on responses to angiotensin II in human volunteers.

The effects of endopeptidase 24.11 inhibition on angiotensin-induced changes in plasma angiotensin II, aldosterone, and atrial natriuretic factor concentrations and blood pressure were assessed in normal volunteers. Two groups, each consisting of eight normal volunteers, received stepwise infusions of angiotensin II (2, 4, and 8 ng/kg per minute) on day 5 of dose administration with 25 mg every 12 hours (group 1) or 100 mg every 12 hours (group 2) of an oral inhibitor of endopeptidase 24.11 (UK 79300, candoxatril) or placebo in balanced randomized, double-blind, placebo-controlled crossover studies. Both doses of candoxatril significantly enhanced achieved plasma angiotensin II concentrations during infusions (group 1, p < 0.001; group 2, p < 0.01; overall treatment effect for combined data, p < 0.001). This effect was most pronounced at the highest dose of angiotensin II (treatment-time interaction, p < 0.0001 for combined data) and tended to be more marked with the higher dose of candoxatril (treatment-group interaction, p = 0.08). The pressor response to angiotensin II was clearly enhanced by the lower dose of candoxatril; peak systolic and diastolic pressures exceeded placebo values by approximately 10 mm Hg (p < 0.001 and p < 0.05 for systolic and diastolic pressures, respectively). This effect of candoxatril was absent in group 2, which (unlike group 1) had exhibited a modest natriuretic response (sustained cumulative negative sodium balance, -70 +/- 21 mmol; p < 0.01) to the higher dose of inhibitor. Baseline plasma aldosterone concentrations and the incremental aldosterone response to angiotensin II infusions were not significantly altered by low-dose (group 1) candoxatril.(ABSTRACT TRUNCATED AT 250 WORDS)


Optimization of retroviral vector-mediated gene transfer into endothelial cells in vitro.

Retroviral vector-mediated gene transfer into endothelial cells is relatively inefficient with transduction rates as low as 1-2% in vitro and even lower in vivo. To increase the efficiency of gene transfer into endothelial cells, we used retroviral vectors expressing beta-galactosidase and urokinase and measured endothelial cell transduction efficiencies with quantitative assays for beta-galactosidase and urokinase protein. We evaluated several techniques reported to improve the efficiency of retroviral transduction in vitro, including 1) extended periods of exposure to vector, 2) repeated exposures to vector, 3) maximization of the ratio of vector particles to endothelial cells by increasing the volume and concentration of vector particles or by decreasing the number of endothelial cells exposed, 4) cocultivation of endothelial cells with vector-producing cells, and 5) variation of the type and concentration of polycation used with the retroviral vector. Only the use of more concentrated (higher titer) vector-containing supernatant and the use of the polycation DEAE-dextran improved the efficiency of gene transfer into endothelial cells in vitro. In an optimized transduction protocol, a 60-second exposure to 1 mg/ml DEAE-dextran followed by a single 6-hour exposure to supernatant of a titer of 10(5)-10(6) colony-forming units/ml resulted in transduction efficiencies of 50-90% with both vectors. Decreasing the time of the supernatant exposure to 15 minutes permitted transduction efficiencies of 15-20% while significantly minimizing the duration of the transduction. Therefore, the optimized protocol allows high efficiency in vitro gene transfer into endothelial cells within several hours. The briefer protocol may prove useful for in vivo gene transfer in which the time of exposure to the supernatant is limited.


Kinetics of C5a release in cardiac lymph of dogs experiencing coronary artery ischemia-reperfusion injury.

Previous studies of myocardial ischemia suggest that complement activation may play a central role in the inflammatory response during reperfusion. Our previous work has demonstrated neutrophil chemotactic activity to be present in reperfusion canine cardiac lymph after myocardial ischemia and infarction. To evaluate the contribution of the complement-dependent anaphylatoxin C5a to this neutrophil chemotactic activity, rabbit antiserum to canine C5a was prepared. At dilutions > 1:500 but < 1:2,000, the antiserum abolished the ability of zymosan-activated dog serum to induce a ruffled, bipolar morphology in isolated neutrophils used as a bioassay of chemotactic stimulation. This antiserum did not affect similar morphological changes in neutrophils exposed to platelet activating factor (10(-7)-10(-6) M) or recombinant human interleukin-8 (10(-9)-10(-8) M); thus, we deemed it functionally specific for canine C5a. In a pattern similar to what we previously reported, cardiac lymph collected before a 1-hour ligation of the left circumflex coronary artery had little ability to alter the morphology of canine neutrophils (shape change index, 11.3 +/- 4.6, mean +/- SEM; n = 7), but by 1 hour of reperfusion, lymph activated neutrophils significantly in five of seven dogs (mean shape change index, 72.6 +/- 17.7; p < 0.01). At 2 hours of reperfusion, neutrophil activation by lymph occurred in six of seven dogs (mean shape change index, 103.1 +/- 22.2). At 3 hours of reperfusion, cardiac lymph of only three of six dogs caused neutrophil activation, and at 4 hours of reperfusion, this activity was evident in lymph from only two of five dogs.(ABSTRACT TRUNCATED AT 250 WORDS)


Platelet-derived growth factor-BB-induced suppression of smooth muscle cell differentiation.

Previously, we demonstrated that treatment of postconfluent quiescent rat aortic smooth muscle cells (SMCs) with platelet-derived growth factor (PDGF)-BB dramatically reduced smooth muscle (SM) alpha-actin synthesis. In the present studies, we focused on the expression of two other SM-specific proteins, SM myosin heavy chain (SM-MHC) and SM alpha-tropomyosin (SM-alpha TM), to determine whether the actions of PDGF-BB were specific to SM alpha-actin or represented a global ability of PDGF-BB to inhibit expression of cell-specific proteins characteristic of differentiated SMCs. SM-MHC and SM-alpha TM expression were assessed by one- or two-dimensional gel electrophoretic analysis of proteins from cells labeled with [35S]methionine, as well as by Northern analysis of mRNA levels. Synthesis of both SM-specific proteins was decreased by 50-70% in PDGF-BB--treated cells as compared with cells treated with PDGF vehicle. Treatment of cells with 10% fetal bovine serum, which produced a mitogenic effect equivalent to that of PDGF-BB, decreased SM-MHC synthesis by 40% but increased SM-alpha TM synthesis. SM-MHC and SM-alpha TM mRNA expression was decreased by 80% at 24 hours in PDGF-BB--treated postconfluent SMCs, whereas treatment with 10% fetal bovine serum did not decrease the expression of SM-alpha TM mRNA but did inhibit SM-MHC mRNA expression by 36%. Consistent with the absence of detectable PDGF alpha-receptors on these cells, PDGF-AA had no effect on either mitogenesis or expression of SM-MHC or SM-alpha TM.(ABSTRACT TRUNCATED AT 250 WORDS)


Active skeletal muscle mass and cardiopulmonary reserve. Failure to attain peak aerobic capacity during maximal bicycle exercise in patients with severe congestive heart failure.

In addition to depressed cardiac reserve, peripheral factors may contribute to limit maximal exercise capacity in patients with congestive heart failure (CHF). To investigate the role of reduced active skeletal muscle mass, peak oxygen uptake (VO2, milligrams per kilogram per minute) was determined during maximal symptom-limited exercise involving the lower limbs (LL) alone and the lower limbs and upper limbs (LL+UL) combined in patients with CHF and in normal subjects of similar age and sex. LL bicycle exercise was performed upright with a ramp protocol and continuous expired gas analysis. When respiratory exchange ratio (RER) reached 1.0, UL exercise was initiated at constant load with the use of a cranking device positioned at shoulder level. LL exercise alone and combined LL+UL exercise were performed on separate days in randomized order by 24 patients with CHF and seven normal subjects. In patients with CHF, peak VO2 was greater during combined LL+UL exercise than during LL exercise alone, i.e., 15.8 +/- 0.8 versus 14.2 +/- 0.9 ml.kg-1.min-1 (p < 0.001), whereas in normal subjects, maximal VO2 was similar during the two tests, i.e., 26.7 versus 26.2 ml.kg-1.min-1 (NS). The increase in peak VO2 during combined LL+UL exercise relative to LL exercise alone was almost exclusively observed in patients with peak VO2 < 15 ml.kg-1.min-1 (mean increase, 21.7 +/- 4.1%). Peak VO2 during combined LL and UL exercise did not increase relative to LL exercise alone in patients with peak VO2 > 15 ml.kg-1.min-1 and in normal subjects of similar age and sex, i.e., 0.1 +/- 4.0% and 2.0 +/- 2.3% respectively. In contrast to normal subjects and patients with moderate CHF, patients with severe CHF do not exhaust their cardiopulmonary reserve during symptom-limited maximal LL exercise on a bicycle.


Positron emission tomography detects metabolic viability in myocardium with persistent 24-hour single-photon emission computed tomography 201Tl defects.

Four-hour 201Tl redistribution images underestimate myocardial viability in patients with coronary artery disease (CAD). Because 4-hour defects often redistribute late, delayed imaging may enhance assessment of tissue viability. Myocardial metabolic activity was therefore assessed with positron emission tomography (PET) in 26 CAD patients with impaired ventricular function (ejection fraction, 32.1 +/- 13.9%) and 24-hour single-photon emission computed tomography (SPECT) 201Tl defects. On circumferential profile analysis, PET ischemia was defined by preserved glucose metabolism in hypoperfused myocardium, and PET infarction was defined by concordant reductions in perfusion and metabolism. On 19 stress-redistribution and seven rest-redistribution SPECT studies, four observers visually scored 201Tl activity in eight segments on a scale from 0 (normal) to 3 (complete defect). Using an improvement in visual score > or = 0.75 to define redistribution, there were 100 fixed, 17 partially reversible, and 12 completely reversible defects. PET identified tissue metabolic activity in 51 (51%) segments with fixed defects (21 PET ischemia, 30 PET normal) and nine (53%) segments with partially reversible defects (five PET ischemia, four PET normal). When grouped by 24-hour score, the proportion of fixed defects with metabolic activity varied from 84% (scores < or = 1.4) to 15% (scores > 2.6). For partially reversible defects, only 53% with scores < 2.0 and one of two with scores > or = 2.0 were considered metabolically viable on PET. Of 12 completely reversible defects, six (50%) were normal, five (42%) had PET ischemia, and one (8%) had PET infarction. The proportion of fixed defects with metabolic activity did not depend on whether a rest or stress study was performed or on the change in visual score used to define 201Tl redistribution (0.25, 0.50, 0.75, and 1.00). In CAD patients, PET identifies glucose metabolic activity in the majority of fixed 24-hour 201Tl defects. However, very severe (near-complete) 24-hour 201Tl defects are less likely to exhibit metabolic activity on PET imaging than are defects with less-pronounced reductions in segmental 201Tl activity.


Highly variable anticoagulant response after subcutaneous administration of high-dose (12,500 IU) heparin in patients with myocardial infarction and healthy volunteers.

In this study, the anticoagulant response of 12,500 IU heparin s.c. was investigated in patients with myocardial infarction and healthy volunteers to determine variabilities in response and modifying factors. On the fourth day after thrombolytic therapy, blood samples were taken before and at frequent intervals until 10 hours after the injection of 12,500 IU heparin s.c. Plasma anti-Xa activity, anti-IIa activity, and the activated partial thromboplastin time (APTT) were measured in addition to body weight and thickness of the abdominal subcutaneous fat layer. Contrary to expectations, the increase of anti-Xa activity, anti-IIa activity, and APTT compared with baseline (predrug) levels was very small, with an average maximal APTT of 42.6 seconds (SD, 12.4 seconds; range, 30.4-70.7 seconds). Subsequently, the influence of the length of the injection needle on the anticoagulant effect of 12,500 IU heparin s.c. was studied in 10 healthy volunteers to find a factor that could be responsible for the poor response in the patients. The length of the injection needle did not influence the anticoagulant effect of heparin. Large interindividual and intraindividual variabilities were seen in the volunteers. The majority of volunteers had minimal prolongation of the APTT, but very strong prolongation was also seen (maximal APTT, 163 seconds). There was no correlation between the abdominal skinfold thickness and anti-Xa activity, anti-IIa activity, or APTT (p > 0.05), but in the patient study, there was a correlation between weight and anti-Xa activity and anti-IIa activity (p < 0.05), and in the volunteer study, there was a correlation between weight and anti-Xa activity and APTT (p < 0.05). Subcutaneous administration of heparin in a fixed dose for prophylactic and therapeutic purposes may be inadequate because of the large interindividual and intraindividual variations in anticoagulant effect.


Precordial QT interval dispersion as a marker of torsade de pointes. Disparate effects of class Ia antiarrhythmic drugs and amiodarone.

Patients with a history of class Ia drug-induced torsade de pointes have been treated with chronic amiodarone without recurrence of torsade de pointes despite comparable prolongation of the QT interval. We hypothesized that in such patients, class Ia drugs cause nonhomogeneous prolongation of cardiac repolarization times, whereas amiodarone causes homogeneous prolongation of cardiac repolarization times. Thirty-eight consecutive patients who received both class Ia drug therapy and chronic amiodarone therapy were evaluated. Standard 12-lead ECGs at baseline and during each therapy were used to calculate precordial QT interval dispersion (maximum QT in leads V1 through V6 minus minimum QT leads V1 through V6) as a measure of regional variabilities in ventricular repolarization times. Nine of these patients had torsade de pointes during class Ia drug therapy. In these nine patients, class Ia drug therapy and amiodarone significantly prolonged the maximum QT interval to comparable extents. However, class Ia drug therapy but not amiodarone therapy significantly increased precordial QT interval dispersion (101 +/- 37 versus 49 +/- 26 msec; baseline, 44 +/- 12 msec; p = 0.002). In the 29 patients without class Ia drug-induced torsade de pointes, neither class Ia drug therapy nor amiodarone therapy significantly increased QT interval dispersion (50 +/- 6 versus 69 +/- 7 msec; baseline, 54 +/- 5 msec). None of the patients with class Ia drug-induced torsade de pointes had recurrent torsade de pointes during chronic amiodarone therapy. An increase in regional QT interval dispersion during class Ia antiarrhythmic drug therapy is associated with torsade de pointes. Chronic amiodarone therapy in patients with a history of class Ia drug-induced torsade de pointes produces comparable maximum QT interval prolongation but does not increase QT interval dispersion. This characteristic may explain its apparent safe use in patients with a history of class Ia drug-induced torsade de pointes.


Intravascular ultrasound evaluation of the effect of rotational atherectomy in obstructive atherosclerotic coronary artery disease.

High-speed rotational atherectomy uses a diamond-coated, elliptical burr to abrade occlusive atherosclerosis, especially noncompliant calcified plaque. Intravascular ultrasound (IVUS) was used to analyze 28 patients after atherectomy. Arteries treated and imaged were left main (three), left anterior descending (12), left circumflex (five), right coronary (seven), and saphenous vein graft (one). Twenty patients had adjunct balloon angioplasty. Twenty-two (79%) target lesions were calcified; the intimal arc of calcium was 160 +/- 126 degrees (range, 0-360 degrees). After atherectomy, the intima-lumen interface was unusually distinct and circular. The lumen was larger than the largest burr used for both stand-alone (1.19 +/- 0.19-fold the largest burr size) and adjunct balloon procedures (1.30 +/- 0.15-fold the largest burr). Three-dimensional reconstruction of the ultrasound images showed a smooth lumen, especially in calcified plaque. Deviations from cylindrical geometry occurred only in areas of soft plaque or superficial tissue disruption of calcified plaque. Five patients were studied before and after rotational atherectomy. IVUS showed an increase in lumen size, a decrease in plaque-plus-media area and in arc of target lesion calcification, and no change in target lesion external elastic membrane cross-sectional area. Rotational atherectomy causes atheroablation with only moderate evidence of barotrauma in heavily calcified arteries, even after adjunct balloon angioplasty. The lumen is cylindrical, especially in areas of calcified plaque, and somewhat larger than the largest burr tip used.


Restenosis after directional coronary atherectomy. Effects of luminal diameter and deep wall excision.

Deep wall excision during directional atherectomy has been reported in one study to increase the risk of subsequent restenosis. On the other hand, we have observed that the probability of late (6-month) restenosis is reduced by maximizing postprocedure luminal diameter. Although such maximal luminal enlargement by directional atherectomy has not increased procedural complications in our experience, it might well increase the incidence of subintimal (deep wall component) recovery. We performed this study to evaluate the relative influences of luminal enlargement and deep wall component excision on postatherectomy restenosis. Atherectomy resulted in a 7 +/- 15% residual stenosis with < 0.5% incidence of angiographic vessel perforation. The minimal luminal diameter of each lesion was measured before and after intervention in 413 lesions, 389 (94%) of which had histological analysis of the excised specimens. Specimens were categorized by the deepest layer retrieved: type I (recovery of intima alone, n = 141), type II (recovery of media, n = 79), and type III (recovery of adventitia, n = 65). Repeat angiographic measurement of minimal luminal diameter was available for 329 (80%) segments 6 months after atherectomy. Compared with the 32% restenosis rate for type I excision, there was no increase in restenosis (stenosis > 50%) for type II, type III, or types II+III (p = 0.86). Stratification by vessel characteristics also failed to show any association between restenosis and deep wall component recovery in any subgroup, including native coronary (p = 0.85), left anterior descending coronary artery (p = 0.70), right coronary artery (p = 0.51), saphenous graft (p = 0.78), or prior restenosis lesions (p = 0.98). Paradoxically, the recovery of adventitia (type III excision) was associated with a lower late percent stenosis (p = 0.03) and a trend toward less restenosis (p = 0.11) compared with type I excisions. A multiple logistic regression model was constructed that demonstrated immediate postprocedure luminal diameter (p = 0.02) to be an independent determinant of restenosis. In this model, the presence of deep wall components (type II+III) did not adversely affect (p = 0.86) restenosis, but the recovery of adventitia was associated with an independent trend toward reduced restenosis (p = 0.06). The immediate goal of directional atherectomy should be to safely provide the largest lumen possible in order to reduce restenosis. The recovery of deep wall components does not appear to jeopardize the beneficial effect that obtaining a large immediate postprocedure lumen diameter has on reducing the incidence of late restenosis.


Randomized trial of late elective angioplasty versus conservative management for patients with residual stenoses after thrombolytic treatment of myocardial infarction. Treatment of Post-Thrombolytic Stenoses (TOPS) Study Group.

After thrombolytic therapy for patients with acute myocardial infarction (MI), percutaneous transluminal coronary angioplasty (PTCA) is frequently performed because of the presence of a "significant" infarct vessel stenosis demonstrated at predischarge coronary angiography. Several studies have shown PTCA performed early after thrombolysis to be unnecessary or even harmful. However, PTCA in these trials was generally performed 1-3 days after MI, when the milieu in the infarct artery may be unsuited for PTCA, and the incidence of major ischemic complications was high. To date, no trial has assessed whether delayed PTCA (4-14 days) should be performed in patients without evidence of ischemia on stress testing. To test the hypothesis that delayed PTCA might provide clinical benefit compared with medical therapy alone, 87 patients treated within 6 hours of chest pain onset with thrombolytic therapy and with negative functional test were randomized between PTCA to be performed 4-14 days after MI versus no PTCA. Both groups received medical therapy. Patients with postinfarct angina or prior Q wave infarction in the infarct distribution were excluded. The primary study end point was increase in left ventricular ejection fraction with exercise measured by radionuclide studies 6 weeks after MI, a parameter known from other studies to correlate inversely with future ischemic events. Clinical outcome was also monitored for 12 months. There were no differences between the study groups for any prerandomization variable recorded. Mean age was 57 +/- 10 years, 84% of patients were male, 21% had prior MI, 36% had anterior MI, 7% had multivessel disease, and the infarct stenosis measured 70 +/- 17% before randomization. PTCA was successful in 38 of 42 patients (88%) but resulted in non-Q wave MI due to acute closure of the treated site in three of 42 (9.5%). There was no difference in 6-week resting ejection fraction or increase in ejection fraction with exercise between the two groups (47 +/- 12% and 6 +/- 8%, respectively, in the PTCA group; 49 +/- 10% and 5 +/- 9% in the no-PTCA group; p = NS for both.) There were no deaths in either group. Actuarial 12-month infarct-free survival was 97.8% in the no-PTCA group and 90.5% in the PTCA group (p = 0.07). There was no functional or clinical benefit from routine late PTCA after MI treated with thrombolytic therapy in this relatively low-risk cohort of patients. These data strongly suggest that patients with an uncomplicated MI after thrombolytic therapy, even if they have a "significant" residual stenosis of the infarct vessel, should be treated medically if they are without evidence of ischemia on stress testing before hospital discharge.


Randomized trial of thrombolysis versus heparin in unstable angina.

The clinical usefulness of intravenous thrombolytic therapy in unstable angina is currently unknown, despite the pathogenetic similarity of this entity to acute myocardial infarction, for which thrombolysis has enjoyed great success. To compare the clinical benefit of intravenous urokinase with that of conventional antithrombotic therapy in preventing the progression of unstable angina to new myocardial infarction, intractable angina, or death within the first 96 hours after hospitalization, 149 patients with unstable angina were randomized to one of two intravenous thrombolytic strategies. Forty-nine patients received 3 million units urokinase i.v. over 90 minutes plus intravenous heparin (group A); 47 patients received unblinded 3 million units urokinase i.v. plus 325 mg aspirin p.o. daily (group B); and 53 patients received placebo thrombolytic infusion plus full-dose heparin (group C). The primary end point of this trial was 96-hour clinical status. There were no significant differences in the baseline characteristics (age, sex, previous myocardial infarction, hypertension prevalence, diabetes, tobacco use, or previous revascularization) among the three groups. Despite an excess of minor untoward reactions for the urokinase groups (chills, 26.5% and 23.4% for groups A and B versus 0% for group C; p < 0.01), there was no significant difference with respect to major bleeds (two, none, and two for groups A, B, and C, respectively; p = NS). At 96 hours after presentation, no significant difference emerged in the incidence of new cardiac events: new myocardial infarctions developed in 10.2% of group A, 6.4% of group B, and 3.8% of group C (p = NS); intractable angina occurred in 6.1% of group A, 10.6% of group B, and 9.4% of group C (p = NS). There were no deaths. All three groups encountered a similar incidence of overall cardiac events: 16.3%, 17.0%, and 13.2% for groups A, B, and C, respectively (p = NS). Although trial enrollment was to extend to 600 patients, interim analysis led to early cessation of enrollment due to a negative trend in respect to outcome after thrombolysis. High-dose intravenous urokinase followed by either heparin or aspirin can be safely administered to a broad, unselected group of patients with unstable angina. However, this study suggests that no clinical advantage is conferred by urokinase, with either adjunctive antithrombotic therapy over standard heparin therapy alone, when given relatively late (mean, 8.7 hours) after admission for unstable angina. A possible detrimental effect cannot be excluded.


A randomized comparison of external and internal cardioversion of chronic atrial fibrillation.

Delivery of shocks within the right atrium has been reported to be more effective than conventional external shocks in converting atrial fibrillation (AF), but these two cardioversion techniques have never been compared prospectively. The purpose of this study was to compare the efficacies of external and internal cardioversion in patients with chronic AF unresponsive to prior attempts at electrical and/or pharmacological cardioversion. Low-dose amiodarone was used in all patients after cardioversion to suppress recurrences of AF. One hundred twelve patients with AF of at least 1 month in duration were randomly assigned to undergo external cardioversion with 300-360-J shocks or internal cardioversion with 200-300-J shocks delivered through a standard electrode catheter within the right atrium. The patients were treated with amiodarone (200 mg/day 5-7 days/week) for 1 month before electrical cardioversion and afterward if the cardioversion was successful. The patients were evaluated at regular intervals during 1 year of follow-up. The efficacy of internal cardioversion was significantly greater than that of external cardioversion (91% versus 67%, p = 0.002). The only variable that was associated with the outcome of cardioversion was body weight. Among patients in whom sinus rhythm was restored, AF recurred as often after internal and external cardioversion; at 1 year of follow-up, 37% of patients in whom external or internal cardioversion had been effective were still in sinus rhythm. Patients who had undergone an attempt at electrical cardioversion before entry into this study were less likely to remain in sinus rhythm after cardioversion. The only complications of cardioversion were one instance of cerebral thromboembolism after external cardioversion and one instance of transient pulmonary edema after internal cardioversion. Therapy with amiodarone was discontinued because of an adverse drug effect in only three patients. Internal cardioversion is more effective than external cardioversion in restoring sinus rhythm and is as safe as external cardioversion in patients with chronic AF. The recurrence rate of AF is the same after both types of cardioversion. If conventional electrical cardioversion is ineffective, internal cardioversion should be attempted. The combination of low-dose amiodarone and external or internal cardioversion may result in maintaining sinus rhythm long-term in patients with refractory AF.


Pharmacokinetics and pharmacodynamics of intravenous diltiazem in patients with atrial fibrillation or atrial flutter.

Diltiazem, a calcium channel blocker, has been shown to be safe and effective in the treatment of patients in atrial fibrillation and/or atrial flutter. However, there have been no pharmacokinetic/pharmacodynamic studies of diltiazem in these patients. The pharmacokinetics and pharmacodynamics of intravenous diltiazem were determined in 32 patients with atrial fibrillation or atrial flutter (mean +/- SD age, 66 +/- 7 years; mean baseline heart rate, 131 +/- 10 beats per minute) after 20 mg or 20 mg followed by 25-mg bolus doses and a 10 and 15 mg/hr infusion for 24 hours. After the 10 and 15 mg/hr infusions of diltiazem, mean +/- SD elimination half-life was 6.8 +/- 1.8 and 6.9 +/- 1.5 hours, volume of distribution was 411 +/- 151.8 and 299 +/- 70.8 I, and systemic clearance was 42 +/- 12.4 and 31 +/- 8.3 l/hr, respectively. Percentages of the plasma concentrations of the principal metabolites desacetyldiltiazem and N-desmethyldiltiazem to diltiazem were < 15% and < 10%, respectively. Thirty of 32 patients maintained response throughout the 24-hour infusion of diltiazem. Using a sigmoidal Emax pharmacodynamic model, a strong relation (mean +/- SD r2, 0.78 +/- 0.2) was observed between plasma diltiazem concentration and percent heart rate reduction. Mean +/- SD Emax (maximum percent reduction in heart rate from baseline) and EC50 (plasma diltiazem concentration that achieves half Emax) were 52 +/- 17% and 110 +/- 84 ng/ml, respectively. The model predicts that mean plasma diltiazem concentration of 79, 172, and 294 ng/ml are required to produce a 20%, 30%, and 40% reduction in heart rate, respectively. A relation between plasma diltiazem concentration and percent change in systolic blood pressure (SBP) or diastolic blood pressure (DBP) from baseline was not observed (mean +/- SD r2, SBP/DBP: 0.35 +/- 0.24/0.36 +/- 0.2). There were no untoward side effects observed. First, the pharmacokinetics of diltiazem in patients with atrial fibrillation or atrial flutter is nonlinear with an apparent dose-dependent decrease in systemic clearance with increasing infusion rate. Second, using a sigmoidal Emax model, there is a strong relation between plasma diltiazem concentration and percent heart rate reduction. Third, the plasma concentrations of the principal metabolites desacetyldiltiazem and N-desmethyldiltiazem are low and are not expected to contribute significantly to the pharmacodynamics of intravenous diltiazem in these patients.


Dynamic intraventricular obstruction during dobutamine stress echocardiography. A new observation.

The implications of hypotension occurring during dobutamine stress echocardiography have not been elucidated. We observed in some patients that hyperdynamic left ventricular function developed during dobutamine stress echocardiography and hypothesized that intracavitary obstruction was occurring and might account for hypotension in some patients. Fifty-seven consecutive patients undergoing dobutamine stress echocardiography underwent pulsed-wave and continuous-wave Doppler examination of the left ventricular cavity at rest and at peak dobutamine infusion. The development of an intraventricular gradient with dobutamine stress was defined as a late-peaking left ventricular Doppler velocity profile that exceeded basal velocity by at least 1 m/sec. During dobutamine stress testing, left ventricular outflow velocity or intracavitary velocity increased in all patients. Obstruction occurred in 12 patients (21%, group 1). Group 2 was the remaining 45 patients. Peak velocities in group 1 ranged from 2.0 to 5.0 m/sec (mean, 3.5 m/sec), and the mean increase from velocity at rest was 2.3 m/sec. The mean change in systolic blood pressure was significantly lower in patients in group 1 (-15 versus 4 mm Hg, p = 0.02). When the 18 patients with an ischemic response to stress testing (evidenced by new or worsening wall motion abnormalities) were excluded from analysis, systolic blood pressure response was still significantly different for the two groups (-19 versus 2 mm Hg, p = 0.03). Dynamic left ventricular obstruction is a new observation; it may develop frequently in patients undergoing dobutamine stress echocardiography. Obstruction rather than ischemia may explain a decrease in blood pressure during dobutamine stress echocardiography.


Real-time NMR beam-directed velocity mapping. V-mode NMR.

Currently available noninvasive techniques for measuring blood flow velocities are constrained by limited view orientations (Doppler ultrasound) or limited time resolution (magnetic resonance imaging, MRI). We describe an MRI technique for measuring flow velocities in real time at arbitrary orientations within a cylindrical volume or "beam": V-mode nuclear magnetic resonance (NMR). The technique was implemented on a standard 1.5-T clinical NMR imager with no special hardware and was tested on phantoms and human volunteers. The beam can be fired at rates up to 60 times per second, allowing measurements on a time scale that is appropriate for ungated cardiac studies. In phantoms, steady flow velocities were measured with the beam aligned along the direction of flow, and the measured velocities correlated well with the actual velocities (r > 0.99). The radial distribution of velocities in phantoms under constant flow conditions was also determined. In humans, flow of blood in the descending aortas of normal and aortic insufficiency subjects was measured. Distinctive backflow of blood because of aortic insufficiency was readily apparent. The V-mode NMR technique is capable of acquiring clinically relevant real-time blood flow information from any desired angle of view with no attenuation at bone or air-tissue interfaces.


Impaired circadian modulation of sympathovagal activity in diabetes. A possible explanation for altered temporal onset of cardiovascular disease.

Diabetic subjects have a high incidence of cardiovascular accidents, with an altered circadian distribution. Abnormalities in the circadian rhythm of autonomic tone may be responsible for this altered temporal onset of cardiovascular disease. To assess circadian changes of sympathovagal balance in diabetes, we performed 24-hour power spectral analysis of RR interval fluctuations in 54 diabetic subjects (age, 44 +/- 2 years) with either normal autonomic function or mild to severe autonomic neuropathy and in 54 age-matched control subjects. The power in the low-frequency (LF, 0.03-0.15 Hz) and high-frequency (HF, 0.18-0.40 Hz) bands was considered an index of relative sympathetic and vagal activity, respectively. Diabetic subjects with autonomic abnormalities showed a reduction in LF compared with control subjects (5.95 +/- 0.12 In-msec2 versus 6.73 +/- 0.11, p < 0.001) and an even greater reduction in LF, particularly during the night and the first hours after awakening (5.11 +/- 0.18 In-msec2 versus 6.52 +/- 0.14, p < 0.001). Day-night rhythm in sympathovagal balance was reduced or absent in diabetic subjects compared with control subjects. Diabetic subjects with or without signs of autonomic neuropathy have a decreased vagal activity (and hence a relatively higher sympathetic activity) during night hours and at the same time of the day, during which a higher frequency of cardiovascular accidents has been reported. These observations may provide insight into the increased cardiac risk of diabetic patients, particularly if autonomic neuropathy is present.