An affective disorder in zebrafish with mutation of the glucocorticoid receptor.

Upon binding of cortisol, the glucocorticoid receptor (GR) regulates the transcription of specific target genes, including those that encode the stress hormones corticotropin-releasing hormone (CRH) and adrenocorticotropic hormone. Dysregulation of the stress axis is a hallmark of major depression in human patients. However, it is still unclear how glucocorticoid signaling is linked to affective disorders. We identified an adult-viable zebrafish mutant in which the negative feedback on the stress response is disrupted, due to abolition of all transcriptional activity of GR. As a consequence, cortisol is elevated, but unable to signal through GR. When placed into an unfamiliar aquarium ('novel tank'), mutant fish become immobile ('freeze'), show reduced exploratory behavior and do not habituate to this stressor upon repeated exposure. Addition of the antidepressant fluoxetine to the holding water and social interactions restore normal behavior, followed by a delayed correction of cortisol levels. Fluoxetine does not affect the overall transcription of CRH, the mineralocorticoid receptor (MR), the serotonin transporter (Serta) or GR itself. Fluoxetine, however, suppresses the stress-induced upregulation of MR and Serta in both wild-type fish and mutants. Our studies show a conserved, protective function of glucocorticoid signaling in the regulation of emotional behavior and reveal novel molecular aspects of how chronic stress impacts vertebrate brain physiology and behavior. Importantly, the zebrafish model opens up the possibility of high-throughput drug screens in search of new classes of antidepressants.

The beta subunit of human granulocyte-macrophage colony-stimulating factor receptor forms a homodimer and is activated via association with the alpha subunit.

Human granulocyte-macrophage colony-stimulating factor (hGM-CSF) receptor (hGMR) consists of alpha and beta subunits, and the precise stoichiometry of these subunits has remained to be determined. In this work, oligomerization of the beta subunit was studied using a chemical cross-linker. In Ba/F3, a mouse interleukin-3-dependent cell line expressing both subunits of hGMR (Ba/F3-alpha,beta), a protein with a molecular mass corresponding to that of a homodimer of the beta subunit (beta homodimer) was detected only when cells were treated with the cross-linker. Dimerization of the beta subunit was confirmed by coimmunoprecipitation of a tagged beta subunit with the wild type beta subunit COS7 cells. The beta homodimer had already formed in the absence of hGM-CSF, whereas stimulation with the ligand brought both alpha and beta subunits into a complex, the result being tyrosine phosphorylation of the beta homodimer. Tyrosine phosphorylation of the subunit was impaired by deletion of the cytoplasmic domain of the alpha subunit without interfering with the association of both subunits. These results indicate that the beta homodimer, which alone is insufficient for signaling, forms the functional hGMR with the alpha subunit in response to hGM-CSF.

Glycogen synthase kinase-3: a new therapeutic target in renal cell carcinoma.

BACKGROUND: Renal cell carcinoma (RCC) is highly resistant to chemotherapy because of a high apoptotic threshold. Recent evidences suggest that GSK-3beta positively regulates human pancreatic cancer and leukaemia cell survival in part through regulation of nuclear factor (NF-kappaB)-mediated expression of anti-apoptotic molecules. Our objectives were to determine the expression pattern of GSK-3beta and to assess the anti-cancer effect of GSK-3beta inhibition in RCC. METHODS: Immunohistochemistry and nuclear/cytosolic fractionation were performed to determine the expression pattern of GSK-3beta in human RCCs. We used small molecule inhibitor, RNA interference, western blotting, quantitative RT-PCR, BrDU incorporation and MTS assays to study the effect of GSK-3beta inactivation on renal cancer cell proliferation and survival. RESULTS: We detected aberrant nuclear accumulation of GSK-3beta in RCC cell lines and in 68 out of 74 (91.89%) human RCCs. We found that pharmacological inhibition of GSK-3 led to a decrease in proliferation and survival of renal cancer cells. We observed that inhibition of GSK-3 results in decreased expression of NF-kappaB target genes Bcl-2 and XIAP and a subsequent increase in renal cancer cell apoptosis. Moreover, we show that GSK-3 inhibitor and Docetaxel synergistically suppress proliferation and survival of renal cancer cells. CONCLUSIONS: Our results show nuclear accumulation of GSK-3beta as a new marker of human RCC, identify that GSK-3 positively regulates RCC cell survival and proliferation and suggest inhibition of GSK-3 as a new promising approach in the treatment of human renal cancer.

Calcium waves along the cleavage furrows in cleavage-stage Xenopus embryos and its inhibition by heparin.

Calcium signaling is known to be associated with cytokinesis; however, the detailed spatio-temporal pattern of calcium dynamics has remained unclear. We have studied changes of intracellular free calcium in cleavage-stage Xenopus embryos using fluorescent calcium indicator dyes, mainly Calcium Green-1. Cleavage formation was followed by calcium transients that localized to cleavage furrows and propagated along the furrows as calcium waves. The calcium transients at the cleavage furrows were observed at each cleavage furrow at least until blastula stage. The velocity of the calcium waves at the first cleavage furrow was approximately 3 microns/s, which was much slower than that associated with fertilization/egg activation. These calcium waves traveled only along the cleavage furrows and not in the direction orthogonal to the furrows. These observations imply that there exists an intracellular calcium-releasing activity specifically associated with cleavage furrows. The calcium waves occurred in the absence of extracellular calcium and were inhibited in embryos injected with heparin an inositol 1,4,5-trisphosphate (InsP3) receptor antagonist. These results suggest that InsP3 receptor-mediated calcium mobilization plays an essential role in calcium wave formation at the cleavage furrows.

Role of inositol 1,4,5-trisphosphate receptor in ventral signaling in Xenopus embryos.

The inositol 1,4,5-trisphosphate (IP3) receptor is a calcium ion channel involved in the release of free Ca2+ from intracellular stores. For analysis of the role of IP3-induced Ca2+ release (IICR) on patterning of the embryonic body, monoclonal antibodies that inhibit IICR were produced. Injection of these blocking antibodies into the ventral part of early Xenopus embryos induced modest dorsal differentiation. A close correlation between IICR blocking potencies and ectopic dorsal axis induction frequency suggests that an active IP3-Ca2+ signal may participate in the modulation of ventral differentiation.

Hybrid surgical and endovascular therapy in multifocal peripheral TASC D lesions: up to three-year follow-up.

AIM: The authors described their three-year experience with hybrid surgical and endovascular therapy for multifocal peripheral TASC D lesions, involving both the aortoiliac and/or superficial femoral and common femoral arteries. METHODS: From February 2005 to March 2008, 21 lower limbs in 20 patients with multifocal peripheral artery disease, involving the aortoiliac and/or superficial femoral as well as common femoral arteries, were treated by hybrid surgical and endovascular therapy, such as aortoiliac and/or superficial femoral artery stenting as an adjunct to common femoral artery endarterectomy. Technical and hemodynamic success as well as primary and primary assisted patency and limb salvage rates were determined in concordance with the Society for Vascular Surgery guidelines. RESULTS: All lower limbs successfully underwent successful hybrid surgical and endovascular therapy. The average ABPI before and after hybrid therapy significantly increased from 0.50 +/- 0.32 to 0.79 +/- 0.24 (P = 0.0022). The mean duration of follow-up was 357 days (range, 4 to 1400 days). Over all, the primary patency rates were 94%, 70% and 70% at 6, 12, and 24 months, respectively, and the primary assisted patency rates were 94% at 24 months. The limb salvage rate was 100% at 24 months. The survival rates were 95%, 88%, and 88% at 6, 12, and 24 months, respectively. The primary patency rate for intermittent claudication was significantly higher that that for critical limb ischemia, while no significant difference was found in the assisted primary patency and survival rates between intermittent claudication and critical limb ischemia. CONCLUSION: Hybrid surgical and endovascular therapy, such as aortoiliac and/or superficial femoral artery stenting as an adjunct to common femoral artery endarterectomy, can provide a less invasive yet effective and durable option to patients with multifocal peripheral artery disease.

Fate of varicose veins after great saphenous vein stripping alone.

AIM: The aim of this study was to observe prospectively the clinical sequelae of varicose veins after great saphenous vein (GSV) stripping alone, and to examine whether spontaneous varicose vein regression or disappearance continued for a long period (>3 years). METHODS: Thirty-nine consecutive patients (20 males and 19 females; mean age 57.2), who underwent GSV stripping in Fujita Health University (55 limbs) between November 1, 2002 and December 31, 2003 were enrolled. RESULTS: At four to six weeks, varicose veins spontaneously resolved in 50 limbs (91%), in which subsequent sclerotherapy was not necessary. Five limbs subsequently underwent sclerotherapy for residual varicose veins (5%). At more than three years, 49 limbs (89%) completed the follow-up study. The recurrence after GSV stripping alone occurred in four of the 45 limbs (9%), while those of GSV stripping with sclerotherapy was one of the four limbs (25%). CONCLUSIONS: This study definitely demonstrated that spontaneous varicose vein resolution can continue for more than three years after GSV stripping alone, suggesting that varicectomy can be deferred or avoided in many patients.

Stent placement in the superficial femoral artery for patients on chronic hemodialysis with peripheral artery disease.

AIM: Chronic hemodialysis is associated with a high prevalence of peripheral artery disease (PAD), and patients on chronic hemodialysis with PAD have an increased risk of critical limb ischemia. The present study assessed the hemodynamic and clinical outcomes of stent placement in the superficial femoral artery (SFA) for patients on chronic hemodialysis. METHODS: Between February 2005 to August 2008, 43 consecutive lower limbs in 42 patients with SFA lesions that were successfully treated by primary stent placement were included in this study. Those were divided into a dialysis group (18 limbs) and a nondialysis group (25 limbs). Outcome measures included primary patency, assisted primary patency, limb salvage, and survival. RESULTS: Patients were significantly younger and presented with significantly more symptomatic limb ischemia in the dialysis group compared to the nondialysis group, despite comparable TransAtlantic Inter-Society Consensus (TASC) classification scores of SFA lesions between the two groups. The primary patency, primary assisted patency, limb salvage, and survival rates of the dialysis group were similar to those of the nondialysis group. CONCLUSIONS: Stent placement in the SFA is a feasible, safe, and effective procedure in patients on chronic hemodialysis with PAD, and may be offered as a first-choice therapeutic option for these patients.

A bacterial RNA that functions as both a tRNA and an mRNA.

Bacterial tmRNA (transfer-messenger RNA, also known as 10Sa RNA) contains a tRNA-like structure in the 5'- and 3'-end sequences and an internal reading frame encoding a 'tag' peptide. The dual function of this molecule as both a tRNA and an mRNA facilitates a trans-translation reaction, in which a ribosome can switch between translation of a truncated mRNA and the tmRNA's tag sequence. The result is a chimeric protein with the tag peptide attached to the C-terminus of the truncated peptide.

Protective effect of sivelestat sodium (Eraspol) on postoperative lung dysfunction in patients with type A acute aortic dissection: a pilot study.

AIM: The authors evaluated the protective effect of sivelestat sodium on postoperative lung dysfunction in patients with type A acute aortic dissection who underwent aortic arch surgery with cardiopulmonary bypass (CPB) under deep hypothermia with circulatory arrest (DHCA). METHODS: Twelve patients with type A acute aortic dissection who underwent aortic arch replacement under CPB with DHCA and were pretreated with or without sivelestat sodium (sivelestat group, N.=7 patients; control group, N.=5 patients) were observed. The ratio of arterial oxygen tension to inspired oxygen fraction (P/F ratio) was measured as a parameter of pulmonary function before and after operation. The number of white blood cells was also counted as an index of inflammatory reaction before and after the operation. RESULTS: The P/F ratio decreased significantly after operation in the control group. However, the P/F ratio was unchanged between before and after operation in the sivelestat group. The number of white blood cells tended to increase after operation in the control group, whereas it decreased significantly after operation in the sivelestat group. CONCLUSION: The present study demonstrated the protective effect of sivelestat sodium on postoperative lung injury in patients with acute type A aortic dissection undergoing aortic arch surgery under CPB with DHCA.

Relationship between parameters of air plethysmography and types of superficial venous reflux in patients with primary varicose veins.

AIM: We investigated whether parameters of air plethysmography (APG) were correlated with types of superficial venous reflux as categorized by ascending venography in patients with primary varicose veins. METHODS: Two hundred and eight limbs with primary varicose veins in 135 patients were evaluated by both APG and ascending venography. Venous hemodynamics was assessed with APG. The location of incompetent vein segments was determined based on the results of ascending venography. RESULTS: Seventy-seven limbs had incompetence of the greater saphenous vein (GSV, G group), 36 had incompetence of the lesser saphenous vein (LSV, L group), and 77 had incompetence of the GSV and LSV (GL group). Twenty-five limbs did not have incompetence of the GSV or LSV (N group). The venous filling index (VFI) differed significantly between the N and the G and GL groups, the L group and the G and GL groups, and the G and GL groups. No significant difference was found between the N and L groups. The venous volume, ejection fraction, and residual volume fraction did not differ significantly among all four groups. CONCLUSION: The VFI as measured by APG discriminates well between limbs with incompetence of the GSV and those without incompetence of the GSV or LSV, and between limbs with incompetence of the GSV and those with the LSV in patients with primary varicose veins, suggesting that the hemodynamic severity of superficial venous reflux progresses with involvement from the LSV to the GSV to both saphenous veins.

Reconstituted human granulocyte-macrophage colony-stimulating factor receptor transduces growth-promoting signals in mouse NIH 3T3 cells: comparison with signalling in BA/F3 pro-B cells.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) plays a critical role in growth and differentiation of myeloid cells. We previously reconstituted high-affinity human GM-CSF receptor (hGM-CSFR) in a pro-B cell line, BA/F3, by cotransfecting alpha- and beta-chain cDNA clones and showed that the reconstituted receptor could transduce growth-promoting signals. The high-affinity hGM-CSFR was also reconstituted in mouse NIH 3T3 cells, but its ability to transduce signals in fibroblasts remained undetermined. In the present study, we further characterized signal transduction by the reconstituted hGM-CSFR in both NIH 3T3 cells and BA/F3 cells. We found that the reconstituted hGM-CSFR transduces signals in NIH 3T3 fibroblasts and BA/F3 cells in response to hGM-CSF to activate transcription of the c-fos, c-jun, and c-myc proto-oncogenes. hGM-CSF also induces protein tyrosine phosphorylation and DNA synthesis in both cell types. These results indicated that hGM-CSFR is functional in fibroblasts, that signal transduction via hGM-CSFR in fibroblasts involves tyrosine kinase(s), and that association of hGM-CSFR with a factor(s) specific to hematopoietic cell lineage is not essential to transduce growth-promoting signals.

A combinatorial code for gene expression generated by transcription factor Bach2 and MAZR (MAZ-related factor) through the BTB/POZ domain.

Bach2 is a B-cell- and neuron-specific transcription repressor that forms heterodimers with the Maf-related oncoproteins. We show here that Bach2 activates transcription by interacting with its novel partner MAZR. MAZR was isolated by the yeast two-hybrid screen using the BTB/POZ domain of Bach2 as bait. Besides the BTB/POZ domain, MAZR possesses Zn finger motifs that are closely related to those of the Myc-associated Zn finger (MAZ) protein. MAZR mRNA was coexpressed with Bach2 in B cells among hematopoietic cells and in developing mouse limb buds, suggesting a cooperative role for MAZR and Bach2 in these cells. MAZR forms homo- and hetero-oligomers with Bach2 through the BTB domain, which oligomers bind to guanine-rich sequences. Unlike MAZ, MAZR functioned as a strong activator of the c-myc promoter in transfection assays with B cells. However, it does not possess a typical activation domain, suggesting a role for it as an unusual type of transactivator. The fgf4 gene, which regulates morphogenesis of limb buds, contains both guanine-rich sequences and a Bach2 binding site in its regulatory region. In transfection assays using fibroblast cells, the fgf4 gene was upregulated in the presence of both MAZR and Bach2 in a BTB/POZ domain-dependent manner. The results provide a new perspective on the function of BTB/POZ domain factors and indicate that BTB/POZ domain-mediated oligomers of transcription factors may serve as combinatorial codes for gene expression.

Laser Doppler skin perfusion pressure in the diagnosis of limb ischemia in patients with diabetes mellitus and/or hemodialysis.

AIM: We investigated whether measurement of skin perfusion pressure (SPP), as measured by laser Doppler, can be used to evaluate the severity of limb ischemia in diabetes mellitus (DM) and/or hemodialysis (HD) patients. METHODS: From April 2004 to March 2005, the ankle brachial pressure index (ABPI) and SPP were evaluated in 44 consecutive lower limbs with peripheral artery disease (PAD) and in 24 patients (21 males and 3 females, aged from 45 to 84 years, with a mean age of 69.3 years) with DM and/or HD. Twelve limbs were categorized as Fontaine stage II, 19 as Fontaine stage III and 24 as Fontaine stage IV. RESULTS: The SPP did not differ significantly between limbs at Fontaine stage II and those at Fontaine stage III, but it was significantly lower in limbs at Fontaine stage IV than in those at Fontaine stage II or III. The ABPI did not differ significantly among limbs at Fontaine stages II, III and IV. CONCLUSION: The SPP, as measured by the laser Doppler technique, may be used as a standard for classifying the severity of PAD in patients with DM and/or HD.

Differential regulation of early response genes and cell proliferation through the human granulocyte macrophage colony-stimulating factor receptor: selective activation of the c-fos promoter by genistein.

Granulocyte macrophage colony-stimulating factor (GM-CSF) binds to the high-affinity GM-CSF receptor (GMR) consisting of alpha and beta subunits and induces rapid tyrosine phosphorylation, activation of early response genes, and proliferation of hematopoietic cells. The alpha subunit is the primary cytokine binding component and the beta subunit is required for high-affinity binding as well as for signal transduction. Using tyrosine kinase inhibitors and cytoplasmic deletion mutants of the beta subunit, we obtained evidence that there are at least two distinct pathways downstream of the GMR in BA/F3 cell, one which is essential for proliferation, leads to the c-myc gene activation, and is sensitive to herbimycin and genistein. Activation of this pathway depends on the cytoplasmic region between amino acid positions 455 and 517 of the beta subunit. The second pathway, which leads to activation of c-fos and c-jun genes, is only partially sensitive to herbimycin, is resistant to genistein and depends on the region between amino acid positions 626 and 763 of the beta subunit. Unexpectedly, the c-fos mRNA induction was augmented by genistein. The enhanced expression of c-fos mRNA by genistein also occurred with stimulation with cAMP, PMA, or EGF in NIH3T3 cells. It thus seems likely that genistein affects a common pathway downstream of these signals.

Pyrolysis of waste electrical and electronic equipment: effect of antinomy trioxide on the pyrolysis of styrenic polymers.

This work has investigated the effect that antimony trioxide has on the pyrolysis of styrenic polymers and the effect that different types of brominated flame retardants used in plastics have on the composition of the pyrolysis products. Brominated high impact polystyrene (Br-HIPS) which contained either 5% or 0% antimony trioxide and either decabromodiphenyl oxide (DDO) or decabromodiphenyl ethane (DDE) was pyrolysed in a fixed bed reactor at 430 degrees C. Some experiments on the fixed bed reactor involved mixing the Br-HIPS with polystyrene. The gaseous products were analysed by GC-FID and GC-TCD and it was found that antimony trioxide caused an increase in the proportion of ethane and ethene and suppressed the proportion of butane and butene. When DDE was the flame retardant increased proportions of ethane and ethene were found in the pyrolysis gas compared to when DDO used. When polystyrene was mixed with the Br-HIPS it suppressed the trends observed in the gas composition during the pyrolysis of Br-HIPS. The pyrolysis oils were characterised using FT-IR, GC-MS, GC-FID, and GC-ECD. It was found that the plastic which did not contain antimony trioxide pyrolysed to form mainly toluene, ethylbenzene, styrene, cumene, and alpha-methylstyrene. The oils produced from the pyrolysis of the plastic that contained antimony trioxide did not contain any styrene or alpha-methylstyrene, but instead contained greater concentrations of ethylbenzene and cumene. The absence of styrene and alpha-methylstyrene from the pyrolysis oil occurred even when the Br-HIPS was mixed with polystyrene. GC-ECD analysis of the oils showed that the plastics which did not contain antimony trioxide pyrolysed to form (1-bromoethyl)benzene, which was totally absent from the pyrolysis oils when antimony trioxide was present in the plastic.

An NMR and mutational analysis of an RNA pseudoknot of Escherichia coli tmRNA involved in trans-translation.

Transfer-messenger RNA (tmRNA) is a unique molecule that combines properties from both tRNA and mRNA, and facilitates a novel translation reaction termed trans -translation. According to phylogenetic sequence analysis among various bacteria and chemical probing analysis, the secondary structure of the 350-400 nt RNA is commonly characterized by a tRNA-like structure, and four pseudoknots with different sizes. A mutational analysis using a number of Escherichia coli tmRNA variants as well as a chemical probing analysis has recently demonstrated not only the presence of the smallest pseudoknot, PK1, upstream of the internal coding region, but also its direct implication in trans -translation. Here, NMR methods were used to investigate the structure of the 31 nt pseudoknot PK1 and its 11 mutants in which nucleotide substitutions are introduced into each of two stems or the linking loops. NMR results provide evidence that the PK1 RNA is folded into a pseudoknot structure in the presence of Mg(2+). Imino proton resonances were observed consistent with formation of two helical stem regions and these stems stacked to each other as often seen in pseudoknot structures, in spite of the existence of three intervening nucleo-tides, loop 3, between the stems. Structural instability of the pseudoknot structure, even in the presence of Mg(2+), was found in the PK1 mutants except in the loop 3 mutants which still maintained the pseudoknot folding. These results together with their biological activities indicate that trans -translation requires the pseudoknot structure stabilized by Mg(2+)and specific residues G61 and G62 in loop 3.

Importance of the conserved nucleotides around the tRNA-like structure of Escherichia coli transfer-messenger RNA for protein tagging.

A bacterial RNA functioning as both tRNA and mRNA, transfer-messenger RNA (tmRNA) rescues stalled ribosomes and clears the cell of incomplete polypeptides. For function, Escherichia coli tmRNA requires an elaborate interplay between a tRNA-like structure and an internal mRNA domain that are connected by a 295 nt long compact secondary structure. The tRNA-like structure is surrounded by 16 unpaired nt, including 10 residues that are >95% conserved among the known 140 tmRNA sequences. All these residues were mutated to define their putative role(s) in trans-translation. Both the extent of aminoacylation and the alanine incorporation into the tag sequence, reflecting the two functions of tmRNA, were measured in vitro for all variants. As anticipated from the low sequence conservation, mutating positions 8-12 and position 15 affects neither aminoacylation nor protein tagging. Mutating a set of two conserved positions 13 and 14 abolishes both functions. Probing the solution conformation indicates that this defective mutant adopts an alternate conformation of its acceptor stem that is no more aminoacylatable, and thus inactive in protein tagging. Selected point mutations at the conserved nucleotide stretches 16-20 and 333-335 seriously impair protein tagging with only minor changes in their solution conformations and aminoacylation. Point mutations at conserved positions 19 and 334 abolish trans-translation and 70S ribosome binding, although retaining nearly normal aminoacylation capacities. Two proteins that are known to interact with tmRNA were purified, and their interactions with the defective RNA variants were examined in vitro. Based on phylogenetic and functional data, an additional structural motif consisting of a quartet composed of non-Watson-Crick base pairs 5'-YGAC-3':5'-GGAC-3' involving some of the conserved nucleotides next to the tRNA-like portion is proposed. Overall, the highly conserved nucleotides around the tRNA-like portion are maintained for both structural and functional requirements during evolution.

False aneurysm formation at proximal and distal ends of a stent graft: a rare complication in the repair of endovascular thoracic aortic aneurysm.

This case report demonstrates a rare complication of false aneurysm formation at the proximal and distal ends of a stent graft that was placed in the descending thoracic aorta to repair an atherosclerotic aneurysm with a fibrotic, solid aortic wall. This complication can develop not only in penetrating aortic ulcers and aortic dissections but also in atherosclerotic aneurysms.

Presence of prostacyclin receptor in arteriosclerotic human femoral artery.

AIM: Prostacyclin, which is mainly synthesized by vascular endothelial cells, exerts antiplatelet and smooth-muscle-relaxant effects, thereby maintaining cardiovascular homeostasis. Prostacyclin analogues have been clinically proven to improve ischemic symptoms and prevent the occurrence of vascular events in the lower extremities of patients with arteriosclerosis obliterans. We examined the presence of prostacyclin receptor (IP receptor) in an arteriosclerotic human femoral artery. METHODS: Specimens of the femoral artery were obtained at the time of limb amputation from an 83-year-old woman. Atherosclerotic lesions and associated changes such as calcification were evident. The specimens were stained with hematoxylin and eosin, and processed for immunohistochemistry. RESULTS: A monolayer of cells was observed on the luminal side of the femoral artery. Single immunohistochemistry showed the presence of the IP receptors on cells of the luminal side of the femoral artery. Triple-immunofluorescence staining revealed colocalization of IP-receptor-positive cells and cells positive for von Willebrand factor, a marker of vascular endothelial cells. CONCLUSIONS: We investigated the presence of the IP receptor in the human femoral artery immunohistochemically, and demonstrated their strong expression in endothelial cells. This finding suggests that prostacyclin or prostacyclin analogues may act on their receptors on endothelial cells in patients with arteriosclerosis obliterans.