Effects of early angiotensin-converting enzyme inhibition on cardiac gene expression after acute myocardial infarction.

BACKGROUND: ACE inhibition after myocardial infarction (MI) has been shown to have beneficial effects on cardiac anatomy and function. The purpose of this study was to examine the effects of ACE inhibition on cardiac gene expression after MI. METHODS AND RESULTS: Rats were randomized to receive captopril or no treatment 1 day after MI. Eight weeks later, cardiac function and hemodynamics were measured by use of indwelling catheters and perivascular flow probes. Myocardial gene expression was assessed with DNA microarrays and real-time reverse transcription-polymerase chain reaction. The ratios of heart and left ventricular weights to body weight were significantly increased by MI and normalized by captopril. Cardiac index and stroke volume index were lower in the untreated MI group than in sham controls but were normal in the MI+captopril group. Thirty-seven genes were found to be differentially expressed between the untreated MI group and sham controls; 31 were induced and 6 repressed. Captopril partially or completely inhibited changes in 10 of the genes. The 37 genes clustered into 11 functional groups, and 6 had >/=1 genes whose expression was modified by ACE inhibition. CONCLUSIONS: ACE inhibition after MI inhibits cardiac hypertrophy, preserves cardiac function, and attenuates changes in myocardial gene expression. Gene expression profiling reveals, however, that some elements of the pathophysiology may be unaffected by the treatment and be targets for new therapies.

Intervertebral disc degeneration associated with lumbosacral transitional vertebrae: a clinical and anatomical study.

We studied 52 patients, each with a lumbosacral transitional vertebra. Using MRI we found that the lumbar discs immediately above the transitional vertebra were significantly more degenerative and those between the transitional vertebrae and the sacrum were significantly less degenerative compared with discs at other levels. We also performed an anatomical study using 70 cadavers. We found that the iliolumbar ligament at the level immediately above the transitional vertebra was thinner and weaker than it was in cadavers without a lumbosacral transitional vertebra. Instability of the vertebral segment above the transitional vertebra because of a weak iliolumbar ligament could lead to subsequent disc degeneration which may occur earlier than at other disc levels. Some stability between the transitional vertebra and the sacrum could be preserved by the formation of either an articulation or by bony union between the vertebra and the sacrum through its transverse process. This may protect the disc from further degeneration in the long term.

Usefulness of quantitative susceptibility mapping for the diagnosis of Parkinson disease.

BACKGROUND AND PURPOSE: Quantitative susceptibility mapping allows overcoming several nonlocal restrictions of susceptibility-weighted and phase imaging and enables quantification of magnetic susceptibility. We compared the diagnostic accuracy of quantitative susceptibility mapping and R2* (1/T2*) mapping to discriminate between patients with Parkinson disease and controls. MATERIALS AND METHODS: For 21 patients with Parkinson disease and 21 age- and sex-matched controls, 2 radiologists measured the quantitative susceptibility mapping values and R2* values in 6 brain structures (the thalamus, putamen, caudate nucleus, pallidum, substantia nigra, and red nucleus). RESULTS: The quantitative susceptibility mapping values and R2* values of the substantia nigra were significantly higher in patients with Parkinson disease (P < .01); measurements in other brain regions did not differ significantly between patients and controls. For the discrimination of patients with Parkinson disease from controls, receiver operating characteristic analysis suggested that the optimal cutoff values for the substantia nigra, based on the Youden Index, were >0.210 for quantitative susceptibility mapping and >28.8 for R2*. The sensitivity, specificity, and accuracy of quantitative susceptibility mapping were 90% (19 of 21), 86% (18 of 21), and 88% (37 of 42), respectively; for R2* mapping, they were 81% (17 of 21), 52% (11 of 21), and 67% (28 of 42). Pair-wise comparisons showed that the areas under the receiver operating characteristic curves were significantly larger for quantitative susceptibility mapping than for R2* mapping (0.91 versus 0.69, P < .05). CONCLUSIONS: Quantitative susceptibility mapping showed higher diagnostic performance than R2* mapping for the discrimination between patients with Parkinson disease and controls.

Spatial and temporal gene expression for fibroblast growth factor type I receptor (FGFR1) during fracture healing in the rat.

Recent experiments have shown that exogenous basic fibroblast growth factor (bFGF) enlarges fracture callus and accelerates the healing of osteotomized long bones. The actions of bFGF are mediated by four different transmembrane receptors (FGFR1-4). Among them, FGFR1 has a high affinity for bFGF, and gain-of-function mutations of the FGFR1 gene cause craniosynostosis in humans. Gene expression for FGFR1 has been analyzed in embryogenesis; however, in skeletal repair, detailed expression of FGFR1 has not been fully established. In the present study, a rat model of closed femoral fracture healing was used to quantify mRNA encoding the FGFR1 and to characterize cells expressing FGFR1 by in situ hybridization. Gene expression for FGFR1 was rapidly upregulated after fracture; its mRNA level on day 1 was 3.4-fold higher than that of unfractured femora. At this stage, a moderate signal for FGFR1 was detected in periosteal osteoprogenitor cells, inflammatory cells near fracture sites, and cells among muscle layers. FGFR1 mRNA reached peak expression when callus remodeling actively progressed (6.8-fold on day 14), and remained elevated even in the later stages of healing (6.3-fold on day 28). During the intermediate stage of fracture healing, a strong signal for FGFR1 was diffusely distributed in mature osteoblasts in the hard callus, and mature osteoclasts also expressed a weak signal for FGFR1. These results suggest that FGF/FGFR1 signaling has multifunctional roles during fracture healing and may regulate both osteoblasts and osteoclasts, contributing to bone formation and callus remodeling.

Direct stimulation of osteoclastic bone resorption by bone morphogenetic protein (BMP)-2 and expression of BMP receptors in mature osteoclasts.

Bone morphogenetic proteins (BMPs) play an important role in various kinds of pattern formation and organogenesis during vertebrate development. In the skeleton, BMPs induce the differentiation of cells of chondrocytic and osteoblastic cell lineage and enhance their function. However, the action of BMPs on osteoclastic bone resorption, a process essential for pathophysiological bone development and regeneration, is still controversial. In this study, we examine the direct effect of BMPs on osteoclastic bone-resorbing activity in a culture of highly purified rabbit mature osteoclasts. BMP-2 caused a dose- and time-dependent increase in bone resorption pits excavated by the isolated osteoclasts. BMP-4 also stimulated osteoclastic bone resorption. The increase in osteoclastic bone resorption induced by BMP-2 was abolished by the simultaneous addition of follistatin, a BMP/activin binding protein that negates their biological activity. Just as it increased bone resorption, BMP-2 also elevated the messenger RNA expressions of cathepsin K and carbonic anhydrase II, which are key enzymes for the degradation of organic and inorganic bone matrices, respectively. Type IA and II BMP receptors (BMPRs), and their downstream signal transduction molecules, Smad1 and Smad5, were expressed in isolated osteoclasts as well as in osteoblastic cells, whereas type IB BMPR was undetectable. BMPs directly stimulate mature osteoclast function probably mediated by BMPR-IA and BMPR-II and their downstream molecules expressed in osteoclasts. The results presented here expand our understanding of the multifunctional roles of BMPs in bone development.

Novel biological response modifiers: phthalimides with tumor necrosis factor-alpha production-regulating activity.

Novel N-substituted phthalimides (2-substituted 1H-isoindole-1,3-diones) were prepared, and their effects on tumor necrosis factor-alpha (TNF-alpha) production by human leukemia cell line HL-60 stimulated with 12-O-tetradecanoylphorbol 13-acetate (TPA) or okadaic acid (OA) were examined. A structure-activity relationship study of the N-phenylphthalimides and N-benzylphthalimides revealed that their enhancing effect on TPA-induced TNF-alpha production by HL-60 cells and their inhibiting effect on OA-induced TNF-alpha production by HL-60 cells are only partially correlated.

Effects of nifedipine and TMB-8 on renal vasoconstriction induced by hypertonic saline in dogs.

Intrarenal arterial infusion of hypertonic saline (+30 mEq/l NaCl in renal plasma) reduced renal blood flow and glomerular filtration rate with little change in filtration fraction in anesthetized dogs. The blood flow and the filtration rate responses were suppressed during infusion of nifedipine (0.1 microgram/kg per min). TMB-8 (50 micrograms/kg per min) also suppressed the blood flow response but not the filtration rate response. These results suggest that preglomerular vasoconstriction during hypertonic saline infusion requires Ca2+ influx via voltage-dependent Ca2+ channels and that hypertonic saline slightly contracts postglomerular vessels by activating a TMB-8-sensitive Ca2+ movement pathway.

Spatial and temporal gene expression in chondrogenesis during fracture healing and the effects of basic fibroblast growth factor.

Chondrogenesis is an essential component of endochondral fracture healing, though the molecular and cellular events by which it is regulated have not been fully elucidated. In this study, we used a rat model of closed fracture healing to determine the spatial and temporal expression of genes for cartilage-specific collagens. Furthermore, to determine the effects of basic fibroblast growth factor (bFGF) on chondrogenesis in fracture healing, we injected 100 microg recombinant human bFGF into the fracture site immediately after fracture. In normal calluses, pro-alpha1(II) collagen mRNA (COL2A1) was detected in proliferative chondrocytes beginning on day 4 after the fracture, and pro-alpha1(X) collagen mRNA (COL10A1) in hypertrophic chondrocytes beginning on day 7. In FGF-injected calluses, the cartilage enlarged in size significantly. On day 14, both COL2A1- and COL10A1-expressing cells were more widely distributed, and the amounts of COL2A1 and COL10A1 mRNAs were both approximately 2-fold increased when compared with uninjected fractures. Temporal patterns of expression for these genes were, however, identical to those found in normal calluses. The number of proliferating cell nuclear antigen-positive cells was increased in the non-cartilaginous area in the bFGF-injected calluses by day 4. The present molecular analyses demonstrate that a single injection of bFGF enhances the proliferation of chondroprogenitor cells in fracture callus, and thus contributes to the formation of a larger cartilage. However, maturation of chondrocytes and replacement of the cartilage by osseous tissue are not enhanced by exogenous bFGF, and this results in the prolonged cartilaginous callus phase. We conclude that, in the healing of closed fractures of long bones, exogenous bFGF has a capacity to enlarge the cartilaginous calluses, but not to induce more rapid healing.

Spatial and temporal distribution of CD44 and osteopontin in fracture callus.

The multifunctional adhesion molecule CD44 is a major cell-surface receptor for hyaluronic acid (HUA). Recent data suggest that it may also bind the ubiquitous bone-matrix protein, osteopontin (OPN). Because OPN has been shown to be a potentially important protein in bone remodelling, we investigated the hypothesis that OPN interactions with the CD44 receptor on bone cells participate in the regulation of the healing of fractures. We examined the spatial and temporal patterns of expression of OPN and CD44 in healing fractures of rat femora by in situ hybridisation and immunohistochemistry. We also localised HUA in the fracture callus using biotinylated HUA-binding protein. OPN was expressed in remodelling areas of the hard callus and was found in osteocytes, osteoclasts and osteoprogenitor cells, but not in cuboidal osteoblasts which were otherwise shown to express osteocalcin. The OPN signal in osteocytes was not uniformly distributed, but was restricted to specific regions near sites where OPN mRNA-positive osteoclasts were attached to bone surfaces. In the remodelling callus, intense immunostaining for CD44 was detected in osteocyte lacunae, along canaliculi, and on the basolateral plasma membrane of osteoclasts, but not in the cuboidal osteoblasts. HUA staining was detected in fibrous tissues but little was observed in areas of hard callus where bone remodelling was progressing. Our findings suggest that OPN, rather than HUA, is the major ligand for CD44 on bone cells in the remodelling phase of healing of fractures. They also raise the possibility that such interactions may be involved in the communication of osteocytes with each other and with osteoclasts on bone surfaces. The interactions between CD44 and OPN may have important clinical implications in the repair of skeletal tissues.

Downward shift in IQ in persons with Duchenne muscular dystrophy compared to those with spinal muscular atrophy.

Patients with Duchenne progressive muscular dystrophy (DMD) and those with spinal progressive muscular atrophy (SPMA) were compared on the basis of memory and IQ. The DMD group was inferior to the SPMA group in memory tests and Full Scale IQ. The DMD group showed poorer scores, despite the fact that functional disabilities and social environments were similar for the two groups. Results thus support the current view that lower IQ is one of the primary manifestations of DMD.

Similarity of IQs of siblings with Duchenne progressive muscular dystrophy.

The similarity of IQs of siblings with Duchenne progressive muscular dystrophy (DMD) was examined to determine whether the view that downward shift in IQ of patients with DMD is a genetically determined primary manifestation of the disease. The mean IQ difference was smaller and the IQ correlation was higher for siblings with DMD than for siblings without the disease. Moreover, these two indices were closer to those for healthy monozygotic twins reared together. These results suggest that the lowered IQ in patients with DMD has a genetic background.

Estimation of age from teeth by amino acid racemization: influence of fixative.

To determine the age of a subject from teeth accurately utilizing the racemization rates of amino acids, standard samples of the same tooth species from the same jaw are necessary as controls, as well as data for identification. However, standard teeth are generally stored in fixatives such as ethanol and formalin. We investigated and compared the degree of progression of racemization of dentinal aspartic acid in teeth stored in 95% ethanol, 10% formalin, or 10% neutral formalin fixatives. The racemization rate of dentinal aspartic acid in teeth stored in 10% neutral formalin was the highest, followed by that for teeth stored in 10% formalin then that for teeth stored in 95% ethanol. Teeth stored in these fixatives at 15 degrees C showed almost no progression of racemization. The racemization ratio (D/L ratio) in teeth extracted 10 years previously was almost unchanged from that at the time of extraction, and allowed an accurate evaluation of the subjects age at tooth extraction.

Omega-3 fatty acids in Hawaii seafood.

Omega-3 fatty acids, the most potent of which are found in seafood, are of interest because of their effects on cardiovascular and inflammatory diseases and their possible effects on cancer. However, consumers in Hawaii wishing to increase their dietary omega-3 to omega-6 fatty acid ratio are faced with the difficulty that several types of seafood popular in Hawaii and aquacultured seafood new in the marketplace have unknown omega-3 fatty acid levels. The purpose of this work is to determine omega-3 fatty acid levels of selected seafood and fish oil capsules. Several seafoods and some over-the-counter fish oil capsules were sampled and analyzed. Aku eggs, aquacultured hamachi (yellowtail jack from Japan), one sample of turbot, and EPA Plus, Promega, and Omega-3 Super EPA capsules were found to contain high levels of omega-3 fatty acids. Levels were comparable to those in the fatty fishes such as salmon and mackerel. Butterfish, mahimahi eggs; other fish oil capsules (ProEPA and Omega 3) contained moderate levels of omega-3 fatty acids. Fish cakes, seaweed, several lean fishes, and cod liver oil capsules had small quantities of omega-3 fatty acids. It appeared that the omega-3 fatty acid content of aquacultured species studied was significantly higher than in wild caught species. There was a substantial difference between claimed and actual omega-3 fatty acid levels in commercially available fish oil capsules. These findings can help consumers when selecting types of seafood for their diet that are good sources of omega-3 fatty acids.

[A case of mediastinal schwannoma originating from the intrathoracic vagal nerve].

A 46-years-old man was admitted because of an abnormal shadow on X-ray. The operation underwent under the diagnosis of benign tumor in the antero-superior mediastinum. At the operation, the tumor arise from the right intrathoracic vagal nerve and it was resected with the transection of vagal nerve. The histological diagnosis was schwannoma. Postoperative course was uneventful except the slight hoarseness. Intrathoracic vagal tumor is rare and we have found 19 reported cases among Japanese literatures. This case is considered to be the 20th case originated from the intrathoracic vagal nerve in Japan.

[A case of primary pulmonary cryptococcosis].

A 53-year-old male was admitted because of an abnormal chest roentgenogram showing a solitary tumor shadow in the right upper lung field. Laboratory data was normal. Although examination with the bronchoscope and sputum cytology showed no abnormal findings, a partial resection of the right upper lobe was performed. Microscopic examination of the lesion was cryptococcosis. As another focus was not found by systemic examination, the lesion was disclosed primary pulmonary cryptococcosis. Cryptococcosis is rare but we must think of it when we meet a solitary tumor shadow on the chest roentgenogram.

[Antiangiogenic and antimetastatic effects of toremifene citrate].

In this study, we demonstrated that toremifene citrate (TOR) inhibited the tube formation and migration of human umbilical vein endothelial cells (HUVEC) in vitro. Moreover, TOR suppressed angiogenesis in rabbit cornea and lung metastasis of human fibrosarcoma HT-1080 cells in nude mice. The antiangiogenic activity in vitro was apparent at the concentration of 5 microM which is clinically achievable by oral administration of 120 mg/kg of TOR. These results suggest that clinical treatment with 120 mg/day of TOR might be expected to exhibit antiangiogenesis and antimetastasis effects, in addition to inhibition of estrogen-dependent tumor cell growth.

[Estimation of age from teeth using the racemization of aspartic acid (racemization method)].

We report a method for estimation of age from teeth using the racemization of amino acids (racemization method). This method is based on the characteristics of the constant age-related increase in the amount of D-aspartic acid in dentin. We estimated age by measuring the ratio of D-aspartic acid to L-aspartic acid, i.e. the ratio of racemization ¿ratio of D/L, ln[(1 + D/L)/(1 - D/L)]¿. Because different D/L ratios have been obtained from different teeth in the same individuals and from different sites of dentin in the same tooth, we usually prepare bucco-lingual longitudinal sections at the central part of each tooth, and prepare samples of powdered whole dentin. This powder is then mixed and used to measure the D/L ratio in the dentin. To accurately estimate age from forensic specimens, we simultaneously measured the D/L ratios in more than four control teeth of the same type obtained from subjects of known age. Use of control teeth is necessary because it is sometimes difficult to maintain constant running conditions for gas chromatography to obtain reproducible values in different runs. Therefore, for every measurement, we determined an equation for calculating age from the D/L ratios of control teeth, and estimated the age of the specimen tooth by substituting in its D/L ratio. The most reliable results were obtained using samples of lower incisors or premolars, which are single-rooted teeth with a relatively small volume of dentin. Thus sampling of the dentin is easier than for other teeth. It is better to keep control teeth desiccated because racemization does not proceed readily under such conditions. The deviation from the actual age in the cases we examined was less than 3 years. Thus, racemization of amino acids can be used for accurate estimation of age from teeth.

[Recent progress in studies on osteocytes--osteocytes and mechanical stress].

Although osteocytes are of the most abundant cells in bone, our knowledge about the role of osteocytes in bone metabolism is still poor compared with that about osteoblasts and osteoclasts, both being on the surface of bone. Osteocytes are terminally differentiated bone-forming cells. During bone formation, some of the osteoblasts lining the surface of bone are incorporated into the newly formed osteoid matrix and become osteocytes, while the other osteoblasts remain on the surface as lining cells. During this transition from osteoblasts to osteocytes, the cells lose numerous osteoblastic phenotypes and acquire osteocytic characteristics such as high expression of osteocalcin and particularly their specific morphology. Osteocytes are connected with each other in bone and with osteoblasts on the bone surface through canaliculi, forming cellular networks; and gap-junctions present at the contact sites mediate their intercellular communication. Several roles of osteocytes in bone have been proposed so far. Of them, based on the morphological characteristics of osteocytes, sensation of mechanical stress loaded onto bone is suspected to be one of their functions. One of the mechanical stresses on bone is fluid shear stress. Between the osteocyte's plasma membrane and the bone matrix is the periosteocytic space. This space exists both in the lacunae and in the canaliculi, and it is filled with extracellular fluid (ECF). Application of mechanical stress to bone locally deforms the tissue. This periodical deformation subsequently causes an increase in the flow of ECF in the periosteocytic space, resulting in shear stress on the surface of the osteocytes. Experimental studies demonstrated that bone cells were equivalently or more sensitive to the fluid shear stress than epithelial cells. Osteocytic cells cultured enhanced expression of prostaglandin (PG) G/H synthase-2 (COX-2) mRNA in response to shear stress. PGE2 is a potent regulator of proliferation and function of osteoblasts and osteoclasts. Therefore, a metabolic response by osteoblasts and osteoclasts lining the bone surface may be caused by PGE2 produced by osteocytes in response to shear stress when the prostanoid reaches the surface through the canaliculi. In conclusion, osteocytes play an important role in sensing extracellular mechanical stress, and the mechanical signals mediated by osteocytes may regulate the overall metabolism of cells in bone tissue.

An intracellular calcium release inhibitor TMB-8 suppresses renal nerve stimulation-induced antinatriuresis in dogs.

Effects of nifedipine and TMB-8 on antinatriuresis induced by renal nerve stimulation (RNS) were examined in pentobarbital-anesthetized dogs. RNS (1 Hz) decreased urine flow rate, urinary sodium excretion rate and fractional excretion of sodium and increased renal norepinephrine efflux and renal venous plasma renin activity with little changes in renal hemodynamics. Intrarenal arterial infusion of nifedipine (0.1 microgram/kg/min) or TMB-8 (50 and 100 micrograms/kg/min) increased basal urine flow rate, urinary sodium excretion rate and fractional excretion of sodium without affecting renal venous plasma norepinephrine concentration or plasma renin activity. Neither nifedipine nor TMB-8 affected the RNS-induced increases in norepinephrine efflux and plasmin renin activity. The RNS-induced decreases in urinary sodium excretion rate and fractional excretion of sodium were suppressed during the TMB-8 infusion, whereas nifedipine failed to affect these urinary responses. These results raise the possibility that the release of intracellular calcium from TMB-8-sensitive stores, but not the influx of extracellular calcium through dihydropyridine-sensitive calcium channels, participates in neural control of tubular sodium reabsorption in the dog kidney.

Cromakalim suppresses hypertonic saline-induced renal vasoconstriction in anaesthetized dogs.

1. Intrarenal arterial infusion of hypertonic saline (HS) transiently increased and then gradually reduced renal blood flow (RBF) in anaesthetized dogs. Glomerular filtration rate (GFR) but not filtration fraction decreased at the end of the infusion. 2. In the presence of a potassium channel opener cromakalim (0.3 microgram/kg per min), HS infusion failed to reduce RBF; the initial increase in RBF was maintained throughout the infusion. Since cromakalim also prevented the decrease in GFR, HS infusion lowered filtration fraction. 3. The results suggest that cromakalim inhibits both pre-and postglomerular vasoconstriction induced by HS infusion.