IntroductionCardiac tissues are extensively innervated by autonomic nerves. The cardiac sympathetic nerve plays an important role in modulating heart rate, conduction velocity, myocardial contraction, and relaxation. Although several molecules that regulate the development of the heart have been well characterized, little is known about the mechanism that regulates sympathetic innervation of the heart. The cardiac sympathetic nerve extends from the sympathetic neuron in stellate ganglia (SG), which is derived from the neural crest (1). Nerve growth factor (NGF) is a prototypic member of the neurotrophin family, members of which are critical for the differentiation, survival, and synaptic activity of the peripheral sympathetic and sensory nervous systems (2, 3). Levels of NGF expression within innervated tissues roughly correspond to innervation density (4). The volume of sympathetic ganglion is reduced by at least 80% at postnatal day 3 in mice with a disruption of the NGF gene. In mice that lack the NGF receptor TrkA, no neurons remain at postnatal day 9 (2). Deletion of a single copy of the NGF gene results in a 50% reduction in sympathetic neurons (5), while overexpression of NGF in the heart results in cardiac hyperinnervation and hyperplasia in SG neurons (6). These results demonstrate the importance of NGF in the regulation of sympathetic neuron development and innervation.In pathological states, NGF production in the heart is variable. In ischemic hearts, an increase in cardiac NGF leads to regeneration of sympathetic nerves (7,8). In a previous experiment, we found that NGF was upregulated in streptozotocin-induced diabetic murine hearts (9). In contrast, it was reported that NGF and sympathetic innervation were reduced in congestive heart failure (10). Despite their importance, the molecular mechanisms that regulate NGF expression and sympathetic innervation in the heart remain poorly understood.Endothelin-1 (ET-1) is believed to play a critical role in the pathogenesis of cardiac hypertrophy, hypertension, and atherosclerosis. Gene targeting of ET-1 and its receptor endothelin-A (ET A ) resulted in unexpected craniofacial and cardiovascular abnormalities. These phenotypes are consistent with interference of neural crest differentiation. The influence of ET-1 on neural crest development remains undetermined (11-13).We hypothesized that ET-1 could affect the induction of neurotrophic factors, and that its disruption might contribute to the immature development of neural crest-derived cells. In this study, we found ET-1-specific induction of NGF in cardiomyocytes, idenEndothelin-1 regulates cardiac sympathetic innervation in the rodent heart by controlling nerve growth factor expression The cardiac sympathetic nerve plays an important role in regulating cardiac function, and nerve growth factor (NGF) contributes to its development and maintenance. However, little is known about the molecular mechanisms that regulate NGF expression and sympathetic innervation of the heart. In an effort to identify regulato...

Background and Purpose-Platelets play pivotal roles in the development of ischemic cerebrovascular disease (CVD).The platelet glycoprotein (GP) Ib/IX/V complex is a receptor for von Willebrand factor, which plays a major role in the initial phase of platelet activation under high shear stress conditions. This study was designed to investigate the association between a genetic variation of this receptor and the prevalence of CVD. Methods-Two hundred patients with ischemic CVD, as confirmed by brain CT and/or MRI, and 317 age-and sex-matched control subjects without clinical evidence of CVD or cardiovascular disease were analyzed for their genotype frequencies of the 145 Thr/Met dimorphism of the ␣-chain of GPIb (GPIb␣). Results-Genotypes with145 Met (T/M and M/M) were more frequently found in the CVD patients (26.5%) than in control subjects (14.2%, Pϭ0.0005). The genotype effect was more obvious in those Ͻ60 years of age or without acquired cardiovascular risk factors. The odds ratio for nonsmoking women Ͻ60 years of age was 10.6 (95% confidence intervals, 2.2 to 51.7). Although the number of patients studied was small (nϭ24), transient ischemic attack showed the highest odds ratio (4.3, Pϭ0.0004), followed by lacunar infarction (ORϭ2.2, Pϭ0.0024) and atherothrombotic infarction (ORϭ1.5, Pϭ0.3143). Logistic regression analysis revealed that the presence of Met-allele was independently associated with CVD. Conclusions-Our study suggests that the platelet GPIb␣ genotype is a genetic risk factor for ischemic CVD.

The cardiac sympathetic nerve plays an important role in regulating cardiac function, and nerve growth factor (NGF) contributes to its development and maintenance. However, little is known about the molecular mechanisms that regulate NGF expression and sympathetic innervation of the heart. In an effort to identify regulators of NGF in cardiomyocytes, we found that endothelin-1 specifically upregulated NGF expression in primary cultured cardiomyocytes. Endothelin-1-induced NGF augmentation was mediated by the endothelin-A receptor, Gibetagamma, PKC, the Src family, EGFR, extracellular signal-regulated kinase, p38MAPK, activator protein-1, and the CCAAT/enhancer-binding protein delta element. Either conditioned medium or coculture with endothelin-1-stimulated cardiomyocytes caused NGF-mediated PC12 cell differentiation. NGF expression, cardiac sympathetic innervation, and norepinephrine concentration were specifically reduced in endothelin-1-deficient mouse hearts, but not in angiotensinogen-deficient mice. In endothelin-1-deficient mice the sympathetic stellate ganglia exhibited excess apoptosis and displayed loss of neurons at the late embryonic stage. Furthermore, cardiac-specific overexpression of NGF in endothelin-1-deficient mice overcame the reduced sympathetic innervation and loss of stellate ganglia neurons. These findings indicate that endothelin-1 regulates NGF expression in cardiomyocytes and plays a critical role in sympathetic innervation of the heart.

Abstract. Urinary steroid profile analysis using gas chromatography/mass spectrometry (GC/MS) has been reported for the diagnosis of abnormal steroidogenesis in newborn infants with some success. We tried to establish the reference values of 63 urinary steroids in Japanese newborn infant, using GC/MS in selected ion monitoring (SIM) that utilizes two characteristic mass ions for each steroid for definitive identification. We studied 36 healthy full-term newborn infants (1-56 days of age) on spot urine samples to define the reference values (mg/g creatinine, median and 10-90 percentile range) and to investigate the possible difference between daytime and nighttime levels. We also studied 23 healthy adult females (20-24 years of age) on 24-hour-urine for the comparison of the reference values of newborn infants. Fifty metabolites of DHEA, pregnenolone, 17-hydroxypregnenolone, androstenedione, progesterone, 17-hydroxyprogesterone, 21-deoxycortisone, corticosterone, 18-hydroxycorticosterone, aldosterone, 18-hydroxycortisol, 11-deoxycortisol, cortisone, cortisol, and estrogen in each infant were measurable without interference, but 13 metabolites of 11-hydroxyandrostenedione, pregnenolone, 11-deoxycorticosterone, corticosterone, 11-dehydrocorticosterone, 21-deoxycortisol, 11-deoxycortisol and cortisol were unmeasurable in each infant due to the interference of fetal cortex steroids as confirmed by abnormal peak area ratios of two mass ions. All 63 metabolites in each control adult were measurable without interference. 16a-, 16b-, and 15b-hydroxy metabolites of 3b-hydroxy-5-en-steroids, and 6b-, 18-hydroxy and 11-oxo-metabolites of corticosteroids were significantly higher in full-term newborn infants than those in adults as previously reported. Urinary steroids showed little circadian variation in the newborn infants, indicating that spot urine can substitute for 24-hour urine.

Elevated blood 17alpha-hydroxyprogesterone (17OHP) level, although widely used for the screening of classical 21-hydroxylase deficiency (21OHD) in neonates, has frequently been found in some neonates without classical 21OHD, particularly preterm neonates. We studied the diagnostic value of the metabolite of 21-deoxycortisol (pregnanetriolone, Ptl) and the metabolite of 17OHP (pregnanetriol, PT) in identifying 21OHD in term and preterm neonates with elevated blood 17OHP on the newborn screening. Spot urine samples from 59 classical 21OHD neonates (50 term, 9 preterm), 83 neonates without 21OHD having transiently elevated blood 17OHP (non-21OHD) (49 term, 34 preterm), and 62 control term neonates were studied using gas chromatography/mass spectrometry in selected ion monitoring analysis for Ptl, PT, 5beta-tetrahydrocortisone (betaTHE), and 5alpha-tetrahydrocortisone (alphaTHE). Ptl and Ptl/(betaTHE+alphaTHE) showed no overlap between 21OHD and non-21OHD, and 21OHD and controls, respectively (Ptl was 0.46-124 mg/g creatinine in 21OHD term, 0.80-26.9 mg/g creatinine in 21OHD preterm, < or = 0.08 mg/g creatinine in non-21OHD term, < or =0.06 mg/g creatinine in non-21OHD preterm, and < or = 0.07 mg/g creatinine in controls). PT and PT/(betaTHE+alphaTHE) showed significant overlap between 21OHD and non-21OHD. The above data indicate that spot urine Ptl is a highly specific marker of 21OHD with a cutoff value of 0.1 mg/g creatinine, yielding an unambiguous separation between 21OHD and non-21OHD in term and preterm neonates.