Hiroaki Nakamura

Mechanisms involved in valvuloseptal endocardial cushion formation in early cardiogenesis: Roles of transforming growth factor (TGF)-β and bone morphogenetic protein (BMP)

Endothelial‐mesenchymal transformation (EMT) is a critical event in the generation of the endocardial cushion, the primordia of the valves and septa of the adult heart. This embryonic phenomenon occurs in the outflow tract (OT) and atrioventricular (AV) canal of the embryonic heart in a spatiotemporally restricted manner, and is initiated by putative myocardially derived inductive signals (adherons) which are transferred to the endocardium across the cardiac jelly. Abnormal development of endocardial cushion tissue is linked to many congenital heart diseases. At the onset of EMT in chick cardiogenesis, transforming growth factor (TGFβ)‐3 is expressed in transforming endothelial and invading mesenchymal cells, while bone morphogenetic protein (BMP)‐2 is expressed in the subjacent myocardium. Three‐dimensional collagen gel culture experiments of the AV endocardium show that 1) myocardially derived inductive signals upregulate the expression of AV endothelial TGFβ3 at the onset of EMT, 2) TGFβ3 needs to be expressed by these endothelial cells to trigger the initial phenotypic changes of EMT, and 3) myocardial BMP2 acts synergistically with TGFβ3 in the initiation of EMT. Anat Rec 258:119–127, 2000.

Expression of smooth muscle alpha-actin in mesenchymal cells during formation of avian endocardial cushion tissue: A role for transforming growth factor β3

During early cardiac morphogenesis, outflow tract (OT) and atrio‐ventricular (AV) endothelial cells differentiate into mesenchymal cells, which have characteristics of smooth muscle‐like myofibroblasts, and which form endocardial cushion tissue, the primordia of valves, and septa in the adult heart. During this embryonic event, transforming growth factor β3 (TGFβ3) is an essential element in the progression of endothelial‐transformation into mesenchyme. TGFβs are known to be a potent inducer for mesodermal differentiation and a promoter for differentiation of endothelial cells into smooth muscle‐like cells. Using a monoclonal antibody against smooth muscle‐specific alpha‐actin (SMA), we examined the immunohistochemical staining of this form of actin in avian endocardial cushion tissue formation. To determine whether TGFβ3 initiates the expression of SMA, the pre‐migratory AV endothelial monolayer was cultured with or without chicken recombinant TGFβ3 and the expression of SMA was examined immunochemically. Migrating mesenchymal cells expressed SMA beneath the cell surface membrane. These cells showed a reduction of endothelial specific marker antigen, QH1. Stationary endothelial cells did not express SMA. The deposition of SMA in the mesenchymal tissue persisted until the end of the fetal period. Pre‐migratory endothelial cells cultured in complete medium (CM199) that contained TGFβ3 expressed SMA, whereas cells cultured in CM199 alone did not. At the onset of the endothelial‐mesenchymal transformation, migrating mesenchymal cells express SMA and the expression of this form of actin is upregulated by TGFβ3. The induction of the expression of SMA by TGFβ3 is one of the initial events in the cytoskeletal reorganization in endothelial cells which separate from one another during the initial phenotypic change associated with the endothelial‐mesenchymal transformation. Dev. Dyn. 209:296–309, 1997.

Age-changes of brain synapses and synaptic plasticity in response to an enriched environment.

Numerical synaptic density and synaptic vesicle density in rat frontal cortex were examined by electron microscopy as a function of age. The density of axospinous synapses, a major population of synapses, was found to peak at age 1 month, and to gradually decrease with aging. The synaptic vesicle density in axospinous synapses was shown to rapidly increase to a peak during the first 3 weeks and then decrease to the adult level, which remained unchanged in senescence. The time course of synaptic changes in aging is presented in this study. In a previous report (Saito et al. [1994] J. Neurosci. Res. 39:57–62), we showed that enriched rearing conditions restored the age‐related decrease of synaptophysin contents. This might be due to increased numerical synaptic density or enhanced packing density of synaptic vesicles in synapses. The results of the present study support the latter explanation; that is, synaptic vesicle contents were increased without changes in synaptic density. Synaptic plasticity induced by environmental stimulation is shown to relate with synaptic strengthening, but not with the formation of new synapses. J. Neurosci. Res. 56:307–315, 1999.

Development of pharyngeal arch arteries in early mouse embryo

The formation and transformation of the pharyngeal arch arteries in the mouse embryo, from 8.5 to 13 days of gestation (DG), was observed using scanning electron microscopy of vascular casts and graphic reconstruction of 1‐µm serial epoxy‐resin sections. Late in 8.5–9DG (12 somites), the paired ventral aortae were connected to the dorsal aortae via a loop anterior to the foregut which we call the ‘primitive aortic arch’, as in the chick embryo. The primitive aortic arch extended cranio‐caudally to be transformed into the primitive internal carotid artery, which in turn gave rise to the primitive maxillary artery and the arteries supplying the brain. The second pharyngeal arch artery (PAA) appeared late in 9–9.5DG (16–17 somites), and the ventral aorta bent dorsolaterally to form the first PAA anterior to the first pharyngeal pouch by early in 9.5–10DG (21–23 somites). The third PAA appeared early in 9.5–10DG (21–23 somites), the fourth late in 9.5–10DG (27–29 somites), and the sixth at 10DG (31–34 somites). By 10.5DG (35–39 somites), the first and second PAAs had been transformed into other arteries, and the third, fourth and sixth PAAs had developed well, though the PAA system still exhibited bilateral symmetry. By 13DG, the right sixth PAA had disappeared, and the remaining PAAs formed an aortic‐arch system that was almost of the adult type.

Bone morphogenetic protein-2 acts synergistically with transforming growth factor-β3 during endothelial-mesenchymal transformation in the developing chick heart

In the early embryonic heart, endothelial cells in atrioventricular (AV) and outflow tract (OT) regions are transformed into the invasive mesenchymal cells that form endocardial cushion tissue (endothelial‐mesenchymal transformation). It has been reported that bone morphogenetic proteins (BMPs) are transcribed in the AV and OT regions of the embryonic mouse heart. We previously reported that transforming growth factor beta 3 (TGFβ3) triggers the initial phenotypic changes seen in endothelial‐mesenchymal transformation. We cloned BMP2 from embryonic chick hearts and examined its functional role during endocardial cushion tissue formation. In situ hybridization showed BMP2 transcripts in the myocardium of the AV and OT regions, but not in endothelial/mesenchymal cells. Antisense oligodeoxynucleotides to BMP2 inhibited mesenchyme formation in AV endocardium cocultured with associated myocardium. This inhibitory effect was reversed by the addition of recombinant BMP2. In cultured AV endothelial monolayers, recombinant BMP2 did not induce any cellular phenotypic changes characteristic of endothelial‐mesenchymal transformation. However, BMP2 enhanced the TGFβ‐induced initial phenotypic changes associated with endothelial‐mesenchymal transformation. These results suggest that BMP2 1) plays an important role in the formation of endocardial cushion tissue and 2) acts synergistically with TGFβ3 in the regulation of this developmental event. J. Cell. Physiol. 180:35–45, 1999.

Animal model of dementia induced by entorhinal synaptic damage and partial restoration of cognitive deficits by BDNF and carnitine.

A rat dementia model with cognitive deficits was generated by synapse‐specific lesions using botulinum neurotoxin (BoNTx) type B in the entorhinal cortex. To detect cognitive deficits, different tasks were needed depending upon the age of the model animals. Impaired learning and memory with lesions were observed in adult rats using the Hebb‐Williams maze, AKON‐1 maze and a continuous alternation task in T‐maze. Cognitive deficits in lesioned aged rats were detected by a continuous alternation and delayed non‐matching‐to‐sample tasks in T‐maze. Adenovirus‐mediated BDNF gene expression enhanced neuronal plasticity, as revealed by behavioral tests and LTP formation. Chronic administration of carnitine over time pre‐ and post‐lesions seemed to partially ameliorate the cognitive deficits caused by the synaptic lesion. The carnitine‐accelerated recovery from synaptic damage was observed by electron microscopy. These results demonstrate that the BoNTx‐lesioned rat can be used as a model for dementia and that cognitive deficits can be alleviated in part by BDNF gene transfer or carnitine administration.

Deficiency of triad formation in developing skeletal muscle cells lacking junctophilin type 1

Junctophilins (JP‐1, JP‐2, and JP‐3) are transmembrane proteins expressed in the junctional membrane complexes in excitable cells. Both JP‐1 and JP‐2 are co‐expressed in the triads of skeletal muscle, but only JP‐2 is expressed in cardiac muscle. We analyzed the roles played by JP‐1 and JP‐2 in triad formation in skeletal muscle by comparing developing skeletal muscles in wild‐type and JP‐1‐knockout (KO) mice (both before and after birth). In the skeletal muscles of embryos, most of the couplings between sarcoplasmic reticulum (SR) and transverse tubule (T‐tubule) were diads, with triads being very scarce. The number of triads increased markedly after birth in wild‐type mice. However, there was no increase in the number of triads in the neonates of JP‐1‐KO mice, and they died within 1 day after birth. JP‐2 expression was constant before and after birth, while expression of JP‐1 increased with birth. Quantitative and morphological differences were not seen between wild‐type and JP‐1‐KO mice in the formation of diads in the period just before the JP‐1‐KO mice died. The SR swelled and developed large vacuoles in skeletal muscle cells just before the JP‐1‐KO mice died. The present results strongly suggest that JP‐1 and JP‐2 play important roles in the formation of triads and diads, respectively, during the development of skeletal muscle in mouse.

Localization of oxidized HDL in atheromatous plaques and oxidized HDL binding sites on human aortic endothelial cells

We examined the localization of oxidized high-density lipoprotein (HDL) in atheromatous plaques and the oxidized HDL binding sites on endothelial cells. Histochemical analysis using CuSO4-oxidized HDL-specific 9F5-3a antibody indicated the presence of oxidized HDL in the intima of atheromatous plaques in human abdominal aortae. The cell surface binding of 125I-oxidized HDL to cultured human aortic endothelial cells (HAEC) was saturable, with an apparent dissociation constant (K d) of 1.43 μmol/L. Competition for 125I-oxidized HDL binding was strong for oxidized HDL, moderate for native HDL and low for acetylated low-density lipoprotein (LDL) or oxidized LDL. Using oxidized HDL as a ligand for blotting, a major 130-kDa band was detected in HAEC. These results suggest that oxidized HDL and its putative binding protein are present in atheromatous plaques and endothelial cells, respectively.

Age-associated ultrastructural changes in the aortic intima of rats with diet-induced hypercholesterolemia

The ultrastructure of the aortic intima and serum lipid levels in Fischer 344 rats were examined at the ages of 12, 18 and 24 months after feeding the animals an atherogenic diet (2% cholesterol, 0.25% sodium cholate, 5% beef fat) for 6 months. Structural changes in the intima were noticeable only at the age of 24 months. In control rats, the endothelial cells were irregular in shape and each had a well-developed Golgi complex and a few lipid droplets. Simultaneously, reticular, basal lamina-like material and electron-dense granules of extracellular liposomes accumulated in the subendothelium. In fat-fed rats, these structural changes were more conspicuous in association with hypercholesterolemia, and numerous monocytes with lipid droplets were attached to the endothelium, occasionally invading into the subendothelium. Slight foam cell lesions were evident in the intima. The finding that older rats were more susceptible to the atherogenic diet suggests that atherogenesis in the rat is promoted by intrinsic age-associated changes in the aortic intima.

An ultrastructural study of Porphyromonas gingivalis-induced platelet aggregation

One of the major pathogens of periodontitis, Porphyromonas gingivalis (P. gingivalis), has the ability to aggregate human platelets. To investigate the interaction between P. gingivalis and human platelets in platelet rich plasma (PRP), platelet aggregation was measured by an aggregometer based on laser light scattering (LS) methods, and an ultrastructural study was performed using electron microscopy. A sharp and rapid increase of small-sized platelet aggregates was observed immediately after the addition of P. gingivalis to PRP, followed by the formation of medium- and large-sized aggregates in 2-3 min. In contrast, when Staphylococcus aureus (S. aureus) was used in the control experiment, only a slight increase in small-sized aggregates was detected. By electron microscopy, discoid-shaped platelets were observed prior to adding P. gingivalis. By 5 min after the addition of the bacteria, enormous platelet aggregates were observable. Most of the P. gingivalis were present between the adherent platelets, while some were internalized in platelet engulfment vacuoles. In contrast, when washed platelets were incubated with the bacteria under a non-stirring condition to prevent platelet aggregation, and stained with ruthenium red (RR) as an electron dense tracer of the cell surface including the open canalicular system (OCS), both RR-positive and -negative vacuoles containing P. gingivalis were identified in the activated platelets. Thus, this observation suggests that P. gingivalis residing in the RR-negative vacuoles is incorporated into the platelet cytoplasm by phagocytosis.