Genetics in pulmonary arterial hypertension in a large homogeneous Japanese population.

Pulmonary arterial hypertension (PAH) is a rare but serious disease with a grave prognosis. Bone morphogenetic protein type 2 receptor (BMPR2) gene is a strong pathogenic factor for PAH. As a collaborative team from Kyorin University and Keio University in Japan, we have analyzed the BMPR2 gene in 356 probands and more than 50 family members, including secondary patients. Importantly, the study population is a racially, ethnically, and socially homogeneous population. In PAH patients, there is a high incidence of unique mutations in BMPR2, and several mutations are frequently observed in the Japanese population, suggesting that these common and recurring mutations may be highly pathogenic or have high penetrance, explaining why they are found frequently throughout the world. We have also mapped each breakpoint of exonic deletions/duplications and found that most break and rejoining points are in the Alu elements. Reviewing the distribution of the reported mutations on each exon of BMPR2 revealed that the number and frequency of mutations are imbalanced among exons. The penetrance of BMPR2 gene mutations was 3-fold higher in females than males. Full elucidation of BMPR2-mediated pathogenic mechanisms in PAH requires persistent efforts to achieve precision or individualized medicine as a therapeutic strategy for PAH.

Expression of a splice variant of choline acetyltransferase in magnocellular neurons of the tuberomammillary nucleus of rat.

A splice variant of choline acetyltransferase mRNA has recently been identified in the pterygopalatine ganglion of rat. An antibody against this variant protein (designated pChAT) was demonstrated to immunolabel peripheral cholinergic neurons. In the present study, we investigated the expression of pChAT in rat brain. Amongst the brain regions examined, magnocellular neurons in the tuberomammillary nucleus of the posterior hypothalamus were immunohistochemically labelled with anti-pChAT antibody, whilst no immunolabelling was detected in cholinergic neurons in the basal forebrain or striatum. RT-PCR analysis confirmed the expression of pChAT mRNA in the posterior hypothalamus. The distribution of pChAT-positive neurons in the tuberomammillary nucleus was compared with that of neurons positive for adenosine deaminase, which is contained in all neurons of this nucleus. After colchicine treatment to inhibit axonal transport of enzyme, virtually all pChAT-positive cells contained adenosine deaminase. Conversely, about 85% of adenosine deaminase-positive cells contained pChAT in the ventral area, whilst 19% of adenosine deaminase-positive cells were pChAT-positive in the dorsal area. Long axonal projections of pChAT-positive cells in the tuberomammillary nucleus were shown by retrograde labelling of these cells after injection of cholera-toxin B subunit into the cerebral cortex. This study demonstrates that a splice variant of choline acetyltransferase is expressed in the tuberomammillary nucleus of rat. The results raise the possibility that some of the known diverse projection areas of this nucleus may have a cholinergic component.

Development of growth-associated protein-43 and neurotrophin receptor immunoreactivities in the rat urinary bladder.

Little is known about how the prenatal innervation of the urinary bladder develops. The objective of this study was to define the timing and pattern of innervation of the developing rat bladder using growth-associated protein-43 (GAP-43) immunohistochemistry. In addition, we examined the development of two different classes of transmembrane receptor proteins, the Trk family of tyrosine kinases and neurotrophin receptor p75 immunoreactivities. GAP-43-positive nerves were first detected in the bladder at embryonic day (E) 16. They were growing rapidly to the bladder dome and covered the whole bladder by E18. The density of GAP-43-containing nerves increased in the muscle layer by postnatal day (P) 0. Both Trk receptor-positive and p75 neurotrophin receptor-positive fibers were also first seen at E16. The number of Trk immunoreactive nerves reached a peak at E18, and then decreased over the following days. In contrast, p75-labeled fibers were abundant at E18-P14. There were few GAP-43 or neurotrophin receptor-positive fibers in the adult. GAP-43 immunohistochemistry provided us with a picture of innervation in the developing rat bladder. Furthermore, the demonstration of neurotrophin receptors positive fibers in the prenatal and early postnatal bladders suggests that neurotrophins may contribute to the development of the peripheral nervous system in the urinary bladder.

Nitric oxide synthase expression in lungs of pulmonary hypertensive patients with heart disease.

Since little is known about the contribution of endothelial nitric oxide synthase (e-NOS) to the mechanism of pulmonary vasospasm and the development of pulmonary vascular occlusive disease, we elucidate how e-NOS is expressed in lung biopsy specimens obtained from operative patients with pulmonary hypertension. Lung biopsy specimens were obtained from 17 patients who underwent open-heart operations for various heart diseases. A piece of normal lung specimen was also obtained from the resected lungs of three lung cancer patients as a control. e-NOS expression was visualized with a monoclonal antibody against e-NOS, and the level of expression was partially quantified. Significantly high levels of e-NOS expression were seen in adult patients, whose preoperative mean pulmonary arterial pressures were greater than 20 mm Hg. In contrast, e-NOS expression in pediatric patients with the same levels of mean pulmonary arterial pressure was the same as that in the controls and in low pulmonary arterial pressure. There was a statistically significant positive correlation between the level of e-NOS expression and Heath--Edwards grading. These data suggest that the e-NOS expression in lung tissue is induced when pulmonary vascular obstructive diseases progress.

Immunohistochemical demonstration of choline acetyltransferase of a peripheral type (pChAT) in the enteric nervous system of rats.

Using a recently developed antiserum against a splice variant (pChAT) of choline acetyltransferase, the enzyme which synthesizes acetylcholine, we carried out an immunohistochemical examination in the digestive canal of rats. Positive staining was exclusively localized to neuronal cells and fibers. Positive somata were distributed widely in the intramural ganglia throughout the digestive tract from the esophagus to the rectum. Double staining indicated that, in the rat, virtually all pChAT immunoreactive somata exhibited histochemical activity for acetylcholinesterase but not for NADPH-diaphorase. In the guinea pig, however, there were a few neurons possessing both pChAT and NADPH-diaphorase. We also found a few neuronal somata which were positive for acetylcholinesterase but not for pChAT. The results suggest that pChAT immunohistochemistry is useful for studying the enteric cholinergic system.

Enhancement of progenitor cell division in the dentate gyrus triggered by initial limbic seizures in rat models of epilepsy.

PURPOSE: Mitogenic effects of seizures on granule cell progenitors in the dentate gyrus were studied in two rat models of epilepsy. We investigated which stage of epileptogenesis is critical for eliciting progenitor cell division and whether seizure-induced neuronal degeneration is responsible for the enhancement of progenitor cell division. METHODS: Seizures were induced by either kainic acid (KA) administration or electrical kindling. Neurogenesis of dentate granule cells was evaluated using the bromodeoxyuridine (BrdU) labeling method, and neuronal degeneration was assessed by in situ DNA fragmentation analysis. RESULTS: After injection of KA, the number of BrdU-positive granule cells began to increase at day 3 after the treatment, peaked at day 5, and returned to baseline at day 10. By day 13, the values were lower than control. After kindling, the number of BrdU-positive cells began to increase after five consecutive experiences of stage I seizures. The increase occurred from day 1 to day 3 after the last electrical stimulation, but returned to baseline by day 7. After generalized seizures were well established, repeated stimulation did not facilitate division of granule cell progenitors. DNA fragmentation was noted in pyramidal neurons in the CA1, CA3, and hilus regions at 18 h after KA injection, but not in the kindling model. CONCLUSIONS: These observations indicate that a mechanism in epileptogenesis boosts dentate progenitor cell division, but progenitor cells may become unreactive to prolonged generalized seizures. Pyramidal neuronal degeneration is not necessary for triggering the upregulation. It is suggested that newly born granule cells may play a role in the network reorganization that occurs during epileptogenesis.

Immunohistochemical localization of choline acetyltransferase of a peripheral type in the rat larynx.

As shown in the accompanying paper, choline acetyltransferase, so far the best histochemical marker for identifying cholinergic structures, has at least one alternative splice variant. The variant, termed pChAT because of its preferential expression in peripheral organs, encouraged us to study peripheral, probably cholinergic, cells and fibers by immunohistochemistry using an antiserum against a peptide specific for pChAT. We chose the larynx of the rat, since cholinergic innervation in this organ has been well established by physiological studies, but not sufficiently by chemical neuroanatomy. Neuronal somata positive for pChAT were found in the intralaryngeal ganglia. Our double staining study indicated that these somata always possessed acetylcholinesterase activity, while the reverse did not hold true. Nerve fibers positive for pChAT were distributed widely in the intrinsic laryngeal muscles, laryngeal glands, blood vessels and laryngeal mucosa. In the intrinsic laryngeal muscles, pChAT-positive terminals were apposed closely to motor end-plates which were stained positively for acetylcholinesterase activity. Denervation experiments revealed that there were three types of pChAT-positive fibers in the larynx: (1) special visceral efferent fibers to the intrinsic laryngeal muscles, which decreased dramatically in number after vagotomy; (2) parasympathetic postganglionic fibers near the laryngeal glands and blood vessels, which appeared unaffected after vagotomy or cervical sympathectomy: and (3) afferent fibers innervating the laryngeal mucosa, which reduced markedly in number after vagotomy performed distal, but not proximal, to the nodose ganglion. Such afferent fibers remained unchanged following the neonatal capsaicin treatment, suggesting their independence from those containing substance P.

Two modes of corticospinal reinnervation occur close to spinal targets following unilateral lesion of the motor cortex in neonatal hamsters.

Although it has been shown that unilateral neonatal cortical ablation induces bilateral corticospinal projections, the explanation for the pathways responsible for this bilateral innervation remains controversial. We hypothesized that such reinnervation may be supplied from newly formed fibers sprouting at the level rostral to, or at, or caudal to the pyramidal decussation. In order to test our hypothesis, we examined the brain and spinal cord of young hamsters which had a unilateral ablation of the right motor cortex at six days postnatally, and then received an injection of an anterograde neuronal lectin tracer, Phaseolus vulgaris-leucoagglutinin, into the hindlimb area of the left motor cortex at 21 days postnatally. For the identification of motoneurons in the lumbar spinal cord, some of these animals also received an injection of cholera toxin subunit B, a retrograde tracer, into the gastrocnemius muscle. A quantitative analysis in the left gray matter of the lumbar spinal cord indicated that the lectin labeling was two to eight times higher in cortically ablated animals than in intact animals. Immunohistochemical detection of the lectin revealed that innervation of the left spinal cord occurred close to targets at lower levels in the spinal cord. Two modes of reinnervation (types I and II) by the intact corticospinal tract were recognized. The type I fibers consisted of recrossing axon collaterals sprouted from the intact dorsal funiculus near their targets, while the type II fibers were recrossing parent axons which entered the intact, right gray matter several levels rostral to their targets, and then changed direction toward the targets. The recrossing at lower spinal levels yielded a large number of ipsilaterally labeled axons and their terminals in the gray matter of the denervated lumbar cord, with a distribution pattern similar to that seen on the intact side. The present results indicate that such ipsilateral innervation may play an important role in the sparing and recovery of function following neonatal hemicortical injury.

Increase of low-affinity neurotrophin receptor p75 and growth-associated protein-43 immunoreactivities in the rat urinary bladder during experimentally induced nerve regeneration.

PURPOSE: Nerve regeneration in the urinary bladder after pelvic nerve plexus injury remains uncertain. The objectives were to establish a rat model of nerve regeneration in the bladder and to examine possible changes of low-affinity neurotrophin receptor p75 and growth-associated protein-43 (GAP-43) immunoreactivities during denervation and nerve regeneration. MATERIALS AND METHODS: Adult male rats were divided into 3 groups: controls, crush of the nerve bundles from the right major pelvic ganglion (MPG) to the bladder (nerve crush group) and removal of the right MPG (MPG removal group). Bladders were collected at 3, 7, 14, 30 and 60 days, and immunohistochemically stained for protein gene product 9.5 (PGP9.5: an axonal marker), p75 and GAP-43. RESULTS: In the nerve crush group, PGP9.5 positive nerves were decreased in number at 3 and 7 days, and then increased after 14 days in the muscle layer of the operated side. By 60 days, the density returned to control levels. However, MPG removal resulted in a decrease of the density of PGP9.5 positive nerves throughout the experimental periods. These findings indicate that nerve regeneration occurred in the nerve crush group. The density of p75 labeled fibers was significantly increased at 3 to 30 days postoperatively in the nerve crush group. p75 immunoreactivity showed smooth surface and cytoplasmic staining, indicating that Schwann cells were p75 positive. GAP-43 labeled fibers showed significantly greater density at 3 to 14 days postoperatively. Schwann cells were GAP-43 immunoreactive and, in particular, regenerating nerve fibers appeared to be GAP-43 positive at 14 days. CONCLUSION: The present study suggests that p75 and GAP-43 are involved in the mechanism(s) of nerve regeneration in the urinary bladder.

[Effects of ulinastatin on PMNL and vascular endothelial injury in patients undergoing open heart surgery with CPB].

Activated leukocytes are thought to contribute to respiratory dysfunction, alterations in microvascular permeability, disseminated intravascular coagulation, and thrombosis, all of which can complicate cardiopulmonary bypass (CPB). We have measured the levels of circulating proinflammatory cytokines (IL-6, 8), polymorphonuclear leukocytes elastase (PMNL-E), and vascular endthelial factors (ET-1, TM, sICAM-1) in patients undergoing open heart surgery with CPB. Patients were divided into a control group and a ulinastatin group. We have examined the effects of ulinastatin on these humoral mediators and postoperative pulmonary function. Every factor except IL-8 increased after CBP in control group. IL-6 and PMNL-E declined sharply to normal level in a few hours, but it took several days after surgery for ET-1, TM, and sICAM-1 to return to preoperative levels. Ulinastatin significantly suppressed the elevation of PMNL-E after CPB, indirectly suppressing the increase of other factors. There was no significant relationship between levels of humoral mediators and postoperative pulmonary function between the two groups. Our results suggest that ulinastatin alleviates the damage of vascular endothelium due to CPB (first attack), and this may be beneficial to reduce excessive inflammatory reaction against secondary insults.

Immunohistochemical localization of low-affinity nerve growth factor receptor in the enteric nervous system of adult rats.

The localization of low-affinity nerve growth factor receptor in the enteric nervous system of adult rats has been studied by immunohistochemistry using a monoclonal antibody (clone 192) against the rat receptor. Cryostat and whole-mount sections were stained. By light and confocal microscopy, positive staining in neural structures was found in every part of the gut. In the ganglionic plexus, dense staining was detected in the neuropil surrounding neuronal cell bodies that were themselves devoid of immunoreactivity. Immunoelectron microscopy revealed deposition of reaction products on the outer plasma membranes of both perikarya and processes of neuronal as well as glial cells. Such a selective localization of the receptor in the plasma membrane, but not the cytoplasm, suggests that the mechanisms of receptor-ligand interaction in the gut may differ from those in the brain, where internalization of the receptor is observed in cholinergic cells. The present study provides the morphological basis for future studies designed to elucidate the functional significance of this enteric nervous system receptor. Since it is found in both neuronal and glial cells, it is probably under the influence of a number of trophic factors, including nerve growth factor.

Lack of toxicity of human neuromelanin to rat brain dopaminergic neurons.

Neuromelanin extracts from the substantia nigra of a parkinsonian and a non-parkinsonian case were injected into the substantia nigra and caudate-putamen of rat brains. Rats were sacrificed at periods ranging from 3 days to 8 months. Substantial amounts of neuromelanin remained at both injection sites even after 8 months, indicating a very slow rate of phagocytosis. No differences were seen in the reaction to control or parkinsonian melanin, and no neurotoxicity attributable to the melanin was observed. These data fail to support the hypothesis of neurotoxicity of melanin as a cause of Parkinson's disease.

Clusterin as a marker for ischaemic Purkinje cells in human brain.

Clusterin has been implicated in cell death both in peripheral tissues and in the central nervous system. In the present study, expression of clusterin in the cerebellar cortex was examined in two cases with hypoxic brain damage and in one case with cerebellar infarction. Intense staining of Purkinje cells was observed in each case, and these cells showed the shrunken and pyknotic appearance characteristic of irreversible ischaemic damage. In the cerebella of neurologically normal control cases, as well as in those of some other neurodegenerative diseases, no staining or only punctate staining of Purkinje cells was observed. The results provide additional evidence supporting an association of clusterin with dying neurons in human brain.

Expression of the complement membrane attack complex and its inhibitors in Pick disease brain.

The immunohistochemical localization of the complement membrane attack complex (MAC) was examined in Pick disease brain and compared with the distribution of three of its inhibitors, vitronectin, protectin and clusterin. Pick bodies were stained intensely for both the MAC and protectin, weakly for vitronectin, but negatively for clusterin. However, the clusterin antibody intensely stained some pyramidal neurons in affected cortical areas, including ballooned neurons. The present study indicates that a complement-mediated attack is associated with the formation of Pick bodies, and provides further suggestive evidence that clusterin may be a marker for active neuronal degeneration.

Projections of nitric oxide synthase-containing fibers from the sphenopalatine ganglion to cerebral arteries in the rat.

The origin and distribution of cerebral perivascular nerves containing nitric oxide, a short-acting messenger or neurotransmitter, have been studied in the rat by histochemistry for reduced nicotinamide adenine dinucleotide phosphate-diaphorase activity, a specific marker for neuronal nitric oxide synthase. Positively stained nerve fibers were distributed throughout the major vessels of the cerebral arteries, though the fiber density was higher in the anterior circulation, including the circle of Willis, than in the posterior arteries. Examination using axonal transport methods indicated that nitric oxide-containing neurons in the sphenopalatine ganglion innervate the cerebral arteries bilaterally. Nitric oxide synthase in these ganglionic cells often co-existed with vasoactive intestinal polypeptide. The anatomical information obtained is discussed in terms of non-adrenergic, non-cholinergic neuronal transmission in the cerebral arteries.

Accumulation of amyloid precursor protein in brain lesions of patients with Pick disease.

The expression of amyloid precursor protein (APP) was examined immunohistochemically in Pick disease brain using several antibodies to various segments of APP. Some neurons, including ballooned neurons, were intensely stained with antibodies to all but the N-terminal APP segment. However, Pick bodies were labeled with the antibody to that segment as well as the antibody to intermediate segment of APP. The pattern was similar to that previously observed for Alzheimer neurofibrillary tangles. These data provide additional evidence that Pick bodies and neurofibrillary tangles share some immunohistochemical characteristics.

Two types of dystrophic neurites in senile plaques of Alzheimer disease and elderly non-demented cases.

Senile plaques (SPs) occur profusely in brain tissue of Alzheimer disease (AD) cases, and sparsely in brain tissue of elderly normals. Two types of dystrophic neurites (DNs) have been identified in SPs. Type 1 are the classically described elongated forms. Type 2 are globular in shape. Type 1 DNs are stained immunohistochemically by antibodies to A68 protein, other forms of phosphorylated tau, and N-terminal epitopes of amyloid precursor protein (APP), as well as an antibody which preferentially recognized conjugated ubiquitin. Type 2 DNs are stained immunohistochemically by antibodies to chromogranin A and C-terminal epitopes of APP, as well as an antibody which preferentially recognizes free ubiquitin. SPs of AD cases usually contain a mixture of type 1 and type 2 DNs. However, in the neocortex of elderly normals, which have few neurofibrillary tangles and neuropil threads, the DNs in the SPs are restricted to type 2. These data suggest that SPs containing only type 2 DNs may be benign, and independent of neurofibrillary pathology.

Expression of chromogranin A in lesions in the central nervous system from patients with neurological diseases.

Expression of chromogranin A in various neurological diseases was examined immunohistochemically using purified anti-human chromogranin A antiserum. The antibody stained dystrophic neurites in senile plaques in Alzheimer disease brain, Pick bodies and ballooned neurons in Pick's disease brain, some Lewy bodies in the substantia nigra of Parkinson's disease, and axonal swellings in various neurological conditions including Parkinson's disease, striatonigral degeneration, Shy-Drager syndrome, amyotrophic lateral sclerosis and cerebral infarction. The present study shows that expression of chromogranin A is not an exclusive feature of Alzheimer disease or Pick's disease, and indicates that it could be a useful marker for various neurological diseases.

Histochemical localization of nitric oxide synthase in rat enteric nervous system.

The localization of nitric oxide synthase, the enzyme responsible for producing the short-acting messenger nitric oxide, has been determined in the digestive tract of the rat using histochemistry for reduced nicotinamide adenine dinucleotide phosphate-diaphorase activity, a specific marker for neuronal nitric oxide synthase. Positively stained neurons were found throughout the entire digestive tract from the esophagus to the rectum. Positive neuronal somata were very common in the myenteric ganglia. Dense positive fibers were distributed in internodal strands, the secondary plexus, the tertiary plexus, and were particularly abundant in the deep muscular plexus, while very few were observed in the submucosal ganglia. The density of these positive structures was higher in the small and large intestine than in the esophagus and stomach. The pattern of distribution suggested that some of these positive cells innervate gut muscles. Double-staining revealed that in these enteric neurons, nitric oxide synthase does not co-localize with acetylcholinesterase. Instead, vasoactive intestinal polypeptide almost always coexists with nitric oxide synthase in the myenteric plexus. Thus, nitric oxide and vasoactive intestinal polypeptide may be co-transmitters in a population of non-adrenergic, non-cholinergic neurons in the enteric nervous system.

Existence of renin in the endothelium of human artery.

OBJECTIVE: The existence and localization of renin in situ was examined in human arteries under non-pathological conditions. DESIGN: Biochemical and immunohistochemical procedures were adopted to examine the existence of renin in human gastroepiploic arteries using an antibody raised against human recombinant renin or a specific renin inhibitor. METHODS: (1) Renin activity (angiotensin I generating activity sensitive to antihuman recombinant renin antibody or a specific renin inhibitor) in the homogenate of human gastroepiploic arteries with and without endothelium was compared; (2) vascular renin was immunohistochemically stained using a modified avidin-biotin-peroxidase complex method. RESULTS: (1) Renin activity in human gastroepiploic arteries with endothelium was significantly higher than in those without endothelium; (2) immunoreactive staining selectively occurred in the endothelial cells of human gastroepiploic arteries. CONCLUSIONS: Renin is present in endothelial cells of human arteries under non-pathological conditions. Endothelial renin may play a role in the control of vascular tone through local production of angiotensin II.