Piezo1 activation using Yoda1 inhibits macropinocytosis in A431 human epidermoid carcinoma cells.

Macropinocytosis is a type of endocytosis accompanied by actin rearrangement-driven membrane deformation, such as lamellipodia formation and membrane ruffling, followed by the formation of large vesicles, macropinosomes. Ras-transformed cancer cells efficiently acquire exogenous amino acids for their survival through macropinocytosis. Thus, inhibition of macropinocytosis is a promising strategy for cancer therapy. To date, few specific agents that inhibit macropinocytosis have been developed. Here, focusing on the mechanosensitive ion channel Piezo1, we found that Yoda1, a Piezo1 agonist, potently inhibits macropinocytosis induced by epidermal growth factor (EGF). The inhibition of ruffle formation by Yoda1 was dependent on the extracellular Ca2+ influx through Piezo1 and on the activation of the calcium-activated potassium channel KCa3.1. This suggests that Ca2+ ions can regulate EGF-stimulated macropinocytosis. We propose the potential for macropinocytosis inhibition through the regulation of a mechanosensitive channel activity using chemical tools.

A Piezo1/KLF15/IL-6 axis mediates immobilization-induced muscle atrophy.

Although immobility is a common cause of muscle atrophy, the mechanism underlying this causality is unclear. We here show that Krüppel-like factor 15 (KLF15) and IL-6 are upregulated in skeletal muscle of limb-immobilized mice and that mice with KLF15 deficiency in skeletal muscle or with systemic IL-6 deficiency are protected from immobility-induced muscle atrophy. A newly developed Ca2+ bioimaging revealed that the cytosolic Ca2+ concentration ([Ca2+]i) of skeletal muscle is reduced to below the basal level by immobilization, which is associated with the downregulation of Piezo1. Acute disruption of Piezo1 in skeletal muscle induced Klf15 and Il6 expression as well as muscle atrophy, which was prevented by antibodies against IL-6. A role for the Piezo1/KLF15/IL-6 axis in immobility-induced muscle atrophy was validated in human samples. Our results thus uncover a paradigm for Ca2+ signaling in that a decrease in [Ca2+]i from the basal level triggers a defined biological event.

Cell-autonomous control of intracellular temperature by unsaturation of phospholipid acyl chains.

Intracellular temperature affects a wide range of cellular functions in living organisms. However, it remains unclear whether temperature in individual animal cells is controlled autonomously as a response to fluctuations in environmental temperature. Using two distinct intracellular thermometers, we find that the intracellular temperature of steady-state Drosophila S2 cells is maintained in a manner dependent on Δ9-fatty acid desaturase DESAT1, which introduces a double bond at the Δ9 position of the acyl moiety of acyl-CoA. The DESAT1-mediated increase of intracellular temperature is caused by the enhancement of F1Fo-ATPase-dependent mitochondrial respiration, which is coupled with thermogenesis. We also reveal that F1Fo-ATPase-dependent mitochondrial respiration is potentiated by cold exposure through the remodeling of mitochondrial cristae structures via DESAT1-dependent unsaturation of mitochondrial phospholipid acyl chains. Based on these findings, we propose a cell-autonomous mechanism for intracellular temperature control during environmental temperature changes.

Long-Term Observations of Thickness Changes of Each Retinal Layer following Macular Hole Surgery.

PURPOSE: To determine the long-term changes of the thickness of each retinal layer following macular hole (MH) surgery combined with internal limiting membrane (ILM) peeling. METHOD: The medical records of 42 eyes of 42 patients (41 to 86 years of age) who underwent MH surgery with ILM peeling between February 2016 and October 2018 were reviewed. A single surgeon operated on all patients, and all were followed for at least 24 months postoperatively. Spectral-domain optical coherence tomography (OCT) was performed to obtain retinal thickness maps of the parafoveal region corresponding approximately to the ILM peeled area. Each retinal layer was automatically segmented by the embedded software, and thickness maps were constructed for the total retinal layer (TRL), inner RL (IRL), middle RL (MRL), and outer RL (ORL). The averaged value of each retinal layer thickness was analyzed in the temporal/upper, temporal/lower, nasal/upper, and nasal lower quadrants. RESULTS: The TRL thickness was significantly decreased in the temporal areas postoperatively. The IRL thickness thinned progressively and significantly until 6 months without further thinning in the temporal quadrants. The MRL thickness of all areas was significantly thicker than the baseline values at 0.5 months and then gradually decreased in the temporal regions. However, the thickening in the nasal regions returned to the baseline values after 1.5 months. The ORL decreased transiently relative to the baseline values at 0.5 months in all areas. CONCLUSIONS: The ILM peeling does not affect only the thickness of the inner retina but also the middle and outer retinae in the parafoveal region. The chronological changes of the thickness after surgeries varied among the retinal layers and macular regions.

Changes of focal macular and full-field electroretinograms after intravitreal aflibercept in patients with central retinal vein occlusion.

PURPOSE: To evaluate the physiology of the macular and whole retina after intravitreal aflibercept (IVAs) injections in patients with macular edema associated with a central retinal vein occlusion (CRVO) by electroretinography (ERG). METHODS: We studied 20 eyes of 20 patients with non-ischemic CRVO (72.0 ± 9.2 years). All patients were treated with monthly injections of IVA for the initial 3 months and then treated by the treat-and-extend (TAE) regimen for 12 months. The best-corrected visual acuity (BCVA), optical coherence tomographic images, focal macular ERGs (fmERGs), and full-field ERGs recorded before and after the treatment were compared. The fmERGs were elicited by a 15° white stimulus spot centered on the fovea. The full-field ERGs were recorded by a protocol recommended by International Society for Clinical Electrophysiology of Vision. The amplitudes and implicit times determined before and after the IVA were compared. RESULTS: The foveal thickness was significantly reduced accompanied by improvement of the BCVA after the treatments, and the improvements were maintained for at least 12 months. The amplitudes and implicit times of the fmERGs improved continuously for the 12 months. On the other hand, the reduced amplitudes of the full-field ERG, summed oscillatory potentials, and the photopic negative responses remained unchanged for the 12-month period. However, the implicit times of the maximum and cone responses were significantly shortened after the IVA. CONCLUSIONS: IVA injections by the TAE regimen led to a continuous improvement of the macular function in patients with ME associated with a CRVO. However, the function of the whole retina changed differently than the macula after the treatment.

Changes in cone-driven functions after intravitreal aflibercept injections in patients with age-related macular degeneration.

PURPOSE: To determine the changes in the cone-driven functions in patients with age-related macular degeneration (AMD) treated with intravitreal aflibercept. METHODS: We studied 44 eyes of 44 patients diagnosed with AMD whose mean age was 75 years. The contralateral unaffected eyes served as controls. All patients were initially treated with 3 consecutive monthly intravitreal aflibercept injections and thereafter with bimonthly injections for 12 months. Full-field cone electroretinograms (cone ERGs) were recorded at the baseline and at 3, 6, and 12 months after beginning the intravitreal aflibercept injections. The cone ERGs were elicited by red stimuli on a blue background. The focal macular ERGs (fmERGs) were elicited by 15 degrees white stimulus spot centered on the fovea. The amplitudes of the a- and b-waves, photopic negative response (PhNR), and sum of the oscillatory potentials (ΣOPs, sum of OP1-3 amplitudes) were analyzed. In addition, the implicit times of the a- and b-waves were also analyzed. RESULTS: The amplitudes and implicit times of all components of the fmERGs were significantly improved compared to the baseline at 3 months after beginning the intravitreal aflibercept injections (P < 0.0005-0.05). The amplitudes of the a-waves and PhNRs were further increased during the maintenance phase (P < 0.005-0.01). On the other hand, the amplitudes of the full-field a-waves and PhNR of the cone ERGs were significantly reduced at 6 and 12 months compared to the baseline. CONCLUSIONS: The macular function improved continuously during the maintenance phase of the intravitreal aflibercept injections. In contrast, the cone-driven functions of the more peripheral retina decreased with repeated injections suggesting adverse effects of the intravitreal aflibercept injections on the function of the more peripheral normal retina.

Hijacking of multiple phospholipid biosynthetic pathways and induction of membrane biogenesis by a picornaviral 3CD protein.

RNA viruses induce specialized membranous structures for use in genome replication. These structures are often referred to as replication organelles (ROs). ROs exhibit distinct lipid composition relative to other cellular membranes. In many picornaviruses, phosphatidylinositol-4-phosphate (PI4P) is a marker of the RO. Studies to date indicate that the viral 3A protein hijacks a PI4 kinase to induce PI4P by a mechanism unrelated to the cellular pathway, which requires Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1, GBF1, and ADP ribosylation factor 1, Arf1. Here we show that a picornaviral 3CD protein is sufficient to induce synthesis of not only PI4P but also phosphatidylinositol-4,5-bisphosphate (PIP2) and phosphatidylcholine (PC). Synthesis of PI4P requires GBF1 and Arf1. We identified 3CD derivatives: 3CDm and 3CmD, that we used to show that distinct domains of 3CD function upstream of GBF1 and downstream of Arf1 activation. These same 3CD derivatives still supported induction of PIP2 and PC, suggesting that pathways and corresponding mechanisms used to induce these phospholipids are distinct. Phospholipid induction by 3CD is localized to the perinuclear region of the cell, the outcome of which is the proliferation of membranes in this area of the cell. We conclude that a single viral protein can serve as a master regulator of cellular phospholipid and membrane biogenesis, likely by commandeering normal cellular pathways.

An N-terminal di-proline motif is essential for fatty acid-dependent degradation of Δ9-desaturase in Drosophila.

The Δ9-fatty acid desaturase introduces a double bond at the Δ9 position of the acyl moiety of acyl-CoA and regulates the cellular levels of unsaturated fatty acids. However, it is unclear how Δ9-desaturase expression is regulated in response to changes in the levels of fatty acid desaturation. In this study, we found that the degradation of DESAT1, the sole Δ9-desaturase in the Drosophila cell line S2, was significantly enhanced when the amounts of unsaturated acyl chains of membrane phospholipids were increased by supplementation with unsaturated fatty acids, such as oleic and linoleic acids. In contrast, inhibition of DESAT1 activity remarkably suppressed its degradation. Of note, removal of the DESAT1 N-terminal domain abolished the responsiveness of DESAT1 degradation to the level of fatty acid unsaturation. Further truncation and amino acid replacement analyses revealed that two sequential prolines, the second and third residues of DESAT1, were responsible for the unsaturated fatty acid-dependent degradation. Although degradation of mouse stearoyl-CoA desaturase 1 (SCD1) was unaffected by changes in fatty acid unsaturation, introduction of the N-terminal sequential proline residues into SCD1 conferred responsiveness to unsaturated fatty acid-dependent degradation. Furthermore, we also found that the Ca2+-dependent cysteine protease calpain is involved in the sequential proline-dependent degradation of DESAT1. In light of these findings, we designated the sequential prolines at the second and third positions of DESAT1 as a "di-proline motif," which plays a crucial role in the regulation of Δ9-desaturase expression in response to changes in the level of cellular unsaturated fatty acids.

Tubular aggregate myopathy caused by a novel mutation in the cytoplasmic domain of STIM1.

OBJECTIVE: To identify the gene mutation of tubular aggregate myopathy (TAM) and gain mechanistic insight into the pathogenesis of the disorder. METHODS: We described a family affected by autosomal dominant TAM and performed exome and Sanger sequencing to identify mutations. We further analyzed the functional significance of the identified mutation by expression studies and intracellular Ca(2+) measurements. RESULTS: A 42-year-old man presented with slowly progressive muscle weakness and atrophy in all 4 limbs and the trunk. Muscle biopsy and microscopic examination revealed tubular aggregates in his skeletal muscle. Genetic analysis of this family identified a novel heterozygous mutation, c.1450_1451insGA (p.Ile484ArgfsX21), in stromal interaction molecule 1 (STIM1), a Ca(2+) sensor in sarcoplasmic reticulum. We transfected cultured cells with STIM1 and demonstrated that the mutant STIM1 exhibited aggregation-like appearance in shrunk cytoplasm. Furthermore, we revealed that the intracellular Ca(2+) influx is decreased by the mutant STIM1. CONCLUSIONS: The novel mutation p.Ile484ArgfsX21 is located in the cytoplasmic C-terminal inhibitory domain (CTID) of STIM1. However, all mutations reported so far in TAM reside in the luminal N-terminal EF hand region. The aggregation-like appearance of STIM1 and the decreased intracellular Ca(2+) influx in cells transfected with CTID mutant are in sharp contrast to these previous reports. Taken together, these findings indicate that mutations of STIM1 cause TAM through the dysregulation of Ca(2+) homeostasis.

Development of a Novel Tetravalent Synthetic Peptide That Binds to Phosphatidic Acid.

We employed a multivalent peptide-library screening technique to identify a peptide motif that binds to phosphatidic acid (PA), but not to other phospholipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS). A tetravalent peptide with the sequence motif of MARWHRHHH, designated as PAB-TP (phosphatidic acid-binding tetravalent peptide), was shown to bind as low as 1 mol% of PA in the bilayer membrane composed of PC and cholesterol. Kinetic analysis of the interaction between PAB-TP and the membranes containing 10 mol% of PA showed that PAB-TP associated with PA with a low dissociation constant of KD = 38 ± 5 nM. Coexistence of cholesterol or PE with PA in the membrane enhanced the PAB-TP binding to PA by increasing the ionization of the phosphomonoester head group as well as by changing the microenvironment of PA molecules in the membrane. Amino acid replacement analysis demonstrated that the tryptophan residue at position 4 of PAB-TP was involved in the interaction with PA. Furthermore, a series of amino acid substitutions at positions 5 to 9 of PAB-TP revealed the involvement of consecutive histidine and arginine residues in recognition of the phosphomonoester head group of PA. Our results demonstrate that the recognition of PA by PAB-TP is achieved by a combination of hydrophobic, electrostatic and hydrogen-bond interactions, and that the tetravalent structure of PAB-TP contributes to the high affinity binding to PA in the membrane. The novel PA-binding tetravalent peptide PAB-TP will provide insight into the molecular mechanism underlying the recognition of PA by PA-binding proteins that are involved in various cellular events.

Dystroglycan function requires xylosyl- and glucuronyltransferase activities of LARGE.

Posttranslational modification of alpha-dystroglycan (α-DG) by the like-acetylglucosaminyltransferase (LARGE) is required for it to function as an extracellular matrix (ECM) receptor. Mutations in the LARGE gene have been identified in congenital muscular dystrophy patients with brain abnormalities. However, the precise function of LARGE remains unclear. Here we found that LARGE could act as a bifunctional glycosyltransferase, with both xylosyltransferase and glucuronyltransferase activities, which produced repeating units of [-3-xylose-α1,3-glucuronic acid-ß1-]. This modification allowed α-DG to bind laminin-G domain-containing ECM ligands.

The juvenile myoclonic epilepsy-related protein EFHC1 interacts with the redox-sensitive TRPM2 channel linked to cell death.

The transient receptor potential M2 channel (TRPM2) is the Ca(2+)-permeable cation channel controlled by cellular redox status via ß-NAD(+) and ADP-ribose (ADPR). TRPM2 activity has been reported to underlie susceptibility to cell death and biological processes such as inflammatory cell migration and insulin secretion. However, little is known about the intracellular mechanisms that regulate oxidative stress-induced cell death via TRPM2. We report here a molecular and functional interaction between the TRPM2 channel and EF-hand motif-containing protein EFHC1, whose mutation causes juvenile myoclonic epilepsy (JME) via mechanisms including neuronal apoptosis. In situ hybridization analysis demonstrates TRPM2 and EFHC1 are coexpressed in hippocampal neurons and ventricle cells, while immunoprecipitation analysis demonstrates physical interaction of the N- and C-terminal cytoplasmic regions of TRPM2 with the EFHC1 protein. Coexpression of EFHC1 significantly potentiates hydrogen peroxide (H(2)O(2))- and ADPR-induced Ca(2+) responses and cationic currents via recombinant TRPM2 in HEK293 cells. Furthermore, EFHC1 enhances TRPM2-conferred susceptibility of HEK293 cells to H(2)O(2)-induced cell death, which is reversed by JME mutations. These results reveal a positive regulatory action of EFHC1 on TRPM2 activity, suggesting that TRPM2 contributes to the expression of JME phenotypes by mediating disruptive effects of JME mutations of EFHC1 on biological processes including cell death.

RIM1 confers sustained activity and neurotransmitter vesicle anchoring to presynaptic Ca2+ channels.

The molecular organization of presynaptic active zones is important for the neurotransmitter release that is triggered by depolarization-induced Ca2+ influx. Here, we demonstrate a previously unknown interaction between two components of the presynaptic active zone, RIM1 and voltage-dependent Ca2+ channels (VDCCs), that controls neurotransmitter release in mammalian neurons. RIM1 associated with VDCC beta-subunits via its C terminus to markedly suppress voltage-dependent inactivation among different neuronal VDCCs. Consistently, in pheochromocytoma neuroendocrine PC12 cells, acetylcholine release was significantly potentiated by the full-length and C-terminal RIM1 constructs, but membrane docking of vesicles was enhanced only by the full-length RIM1. The beta construct beta-AID dominant negative, which disrupts the RIM1-beta association, accelerated the inactivation of native VDCC currents, suppressed vesicle docking and acetylcholine release in PC12 cells, and inhibited glutamate release in cultured cerebellar neurons. Thus, RIM1 association with beta in the presynaptic active zone supports release via two distinct mechanisms: sustaining Ca2+ influx through inhibition of channel inactivation, and anchoring neurotransmitter-containing vesicles in the vicinity of VDCCs.

Nitric oxide activates TRP channels by cysteine S-nitrosylation.

Transient receptor potential (TRP) proteins form plasma-membrane cation channels that act as sensors for diverse cellular stimuli. Here, we report a novel activation mechanism mediated by cysteine S-nitrosylation in TRP channels. Recombinant TRPC1, TRPC4, TRPC5, TRPV1, TRPV3 and TRPV4 of the TRPC and TRPV families, which are commonly classified as receptor-activated channels and thermosensor channels, induce entry of Ca(2+) into cells in response to nitric oxide (NO). Labeling and functional assays using cysteine mutants, together with membrane sidedness in activating reactive disulfides, show that cytoplasmically accessible Cys553 and nearby Cys558 are nitrosylation sites mediating NO sensitivity in TRPC5. The responsive TRP proteins have conserved cysteines on the same N-terminal side of the pore region. Notably, nitrosylation of native TRPC5 upon G protein-coupled ATP receptor stimulation elicits entry of Ca(2+) into endothelial cells. These findings reveal the structural motif for the NO-sensitive activation gate in TRP channels and indicate that NO sensors are a new functional category of cellular receptors extending over different TRP families.

TRPM2 activation by cyclic ADP-ribose at body temperature is involved in insulin secretion.

There are eight thermosensitive TRP (transient receptor potential) channels in mammals, and there might be other TRP channels sensitive to temperature stimuli. Here, we demonstrate that TRPM2 can be activated by exposure to warm temperatures (>35 degrees C) apparently via direct heat-evoked channel gating. beta-NAD(+)- or ADP-ribose-evoked TRPM2 activity is robustly potentiated at elevated temperatures. We also show that, even though cyclic ADP-ribose (cADPR) does not activate TRPM2 at 25 degrees C, co-application of heat and intracellular cADPR dramatically potentiates TRPM2 activity. Heat and cADPR evoke similar responses in rat insulinoma RIN-5F cells, which express TRPM2 endogenously. In pancreatic islets, TRPM2 is coexpressed with insulin, and mild heating of these cells evokes increases in both cytosolic Ca(2+) and insulin release, which is K(ATP) channel-independent and protein kinase A-mediated. Heat-evoked responses in both RIN-5F cells and pancreatic islets are significantly diminished by treatment with TRPM2-specific siRNA. These results identify TRPM2 as a potential molecular target for cADPR, and suggest that TRPM2 regulates Ca(2+) entry into pancreatic beta-cells at body temperature depending on the production of cADPR-related molecules, thereby regulating insulin secretion.

Intracellular-produced hydroxyl radical mediates H2O2-induced Ca2+ influx and cell death in rat beta-cell line RIN-5F.

The melastatin-related transient receptor potential channel TRPM2 is a Ca(2+)-permeable channel that is activated by H(2)O(2), and the Ca(2+) influx through TRPM2 mediates cell death. However, the responsible oxidants for TRPM2 activation remain to be identified. In the present study, we investigated the involvement of hydroxyl radical on TRPM2 activation in TRPM2-expressing HEK293 cells and the rat beta-cell line RIN-5F. In both cell types, H(2)O(2) induced Ca(2+) influx in a concentration-dependent manner. However, the addition of hydroxyl radical, which was produced by mixing FeSO(4) and H(2)O(2), to the cells, did not increase intracellular Ca(2+) concentration. Interestingly, when H(2)O(2) was added to the cells under intracellular Fe(2+)-accumulated conditions, Ca(2+) influx was markedly enhanced compared to H(2)O(2) alone. In addition, the H(2)O(2)-induced Ca(2+) influx was reduced by hydroxyl radical scavengers and an iron chelator. Under intracellular Fe(2+)-accumulated conditions, H(2)O(2)-induced RIN-5F cell death through TRPM2 activation was also markedly enhanced. Hydroxyl radical scavengers and an iron chelator suppressed the RIN-5F cell death by H(2)O(2). These results strongly suggest that the intracellular hydroxyl radical plays a key role in the activation of TRPM2 during H(2)O(2) treatment, and TRPM2 activation mediated by hydroxyl radical is implicated in H(2)O(2)-induced cell death in the beta-cell line RIN-5F.

Serum markers of angiogenesis and myocardial ultrasonic tissue characterization in patients with dilated cardiomyopathy.

BACKGROUND AND AIMS: It has been proven that a disturbance in angiogenesis contributes to the progression of myocardial interstitial fibrosis in idiopathic dilated cardiomyopathy (DCM). This study was designed to evaluate the relationship between serum activity of angiogenic factors and myocardial ultrasonic tissue characterization in patients with DCM. METHODS AND RESULTS: We studied 30 patients with DCM and 15 healthy control subjects. Serum levels of vascular endothelial growth factor (VEGF), interleukin (IL)-4 and IL-13 were measured using enzyme-linked immunosorbent assay. We determined calibrated myocardial integrated backscatter (IB) as the value of myocardial interstitial fibrosis using ultrasonic tissue characterization and also quantified the magnitude of cyclic variations in IB (CV-IB). Serum levels of VEGF and IL-13 were significantly higher in patients with DCM than in control subjects (both P<0.05). Calibrated IB was significantly higher and CV-IB was markedly lower in patients with DCM than in control subjects (both P<0.01). In patients with DCM, the levels of IL-13 significantly correlated with calibrated IB (r=0.520, P=0.018). In addition, there was a significant negative correlation between levels of VEGF and CV-IB (r=-0.611, P=0.007). CONCLUSION: The increase in serum VEGF and IL-13 may be closely related to alterations in myocardial texture in DCM.

Endothelial nitric oxide synthase gene polymorphism (Glu298Asp) in patients with coexistent hypertrophic cardiomyopathy and coronary spastic angina.

Coronary vasospasm appears to play a significant role in the etiology of myocardial ischemia in patients with hypertrophic cardiomyopathy (HCM). Furthermore, the management of patients with coexistent HCM and coronary spastic angina (CSA) presents a therapeutic challenge. The purpose of this study was to examine the Glu298Asp variant of the endothelial nitric oxide synthase (eNOS) gene to determine whether this polymorphism was associated with susceptibility to CSA in patients with HCM. The eNOS gene polymorphism (Glu298Asp) was genotyped in 150 HCM patients by the TaqMan chemical method. Patients were classified into group A (n=12) if they had CSA provoked by intracoronary acetylcholine, and group B (n=138) if they did not. In group A, the frequency of Glu/Glu, Glu/Asp, and Asp/Asp genotypes was 5 (41.7%), 6 (50%), and 1 (8.3%), respectively. In group B, it was 119 (86.2%), 17 (12.3%), and 2 (1.5%), respectively. The frequency of the Asp298 variant was significantly higher in group A than in group B (P<0.001). Multivariate logistic regression analysis showed that the Asp298 variant was a significant risk factor for CSA (odds ratio 11.8; P<0.001) that was independent of age, gender, smoking status or body mass index. Significantly more drugs were used by the patients in group A than those in group B and the patients with the Asp298 variant were treated with significantly more drugs than those without it. In conclusion, the Asp298 variant of the eNOS gene may be associated with CSA in HCM patients. HCM patients with CSA or the Asp298 variant may need more drugs to relieve their symptoms.

[A 73-year-old man with primary pulmonary hypertension].

A rare case of primary pulmonary hypertension was observed in a 73-year-old man. Angina pectoris was diagnosed at the age of 67 and he received several percutaneous coronary interventions because of refractory restenosis. He also had coronary artery bypass surgery at the age of 69. After 4 years, he again suffered from dyspnea and chest pain upon physical exertion. On admission to our hospital, a chest radiograph showed dilatation of bilateral pulmonary arteries. Moreover, echocardiography showed right ventricular dilatation and tricuspid regurgitation. Continuous wave Doppler imaging revealed a pressure gradient of 82.1 mmHg. Pulmonary capillary wedge pressure was normal, but pulmonary artery pressure was elevated upon cardiac catheterization. Because there was no apparent etiology of pulmonary hypertension, primary pulmonary hypertension was diagnosed and appropriate conventional therapy was started.