Biomedical subjects

Hiroshi Itoh

Publications and source records attributed to Hiroshi Itoh.

At least 19 recordsLinked to original sources

PubMed2003

Effects of long-term hypoxia and hypoxic exercise on brain monoamine levels in rat.

This study clarified the effects of long-term hypoxia and hypoxic exercise on monoamines in the whole brain, and in four specific regions of the rat brain. The male Wistar rat progenitors (P1 group) were randomly assigned to three groups: hypoxia (16.0% oxygen) and exercise (MHE-P1), hypoxia and sedentary (MHS-P1), normoxia and sedentary (MNS-P1). The male children of P1 (the first generation of hypoxic rats; F1) were randomly divided into two groups: hypoxia and exercise (MHE-F1) and hypoxic sedentary (MHS-F1). The monoamines of whole brain were measured in P1 males, and monoamines of cerebellum, frontal lobe, striatum and hippocampus were measured in F1 males. The monoamine levels of MHE-P1 were significantly lower than those of MHS-P1 and MNS-P1. No significant difference was found in monoamine levels between MHS-P1 and MNS-P1. Epinephrine, norepinephrine, and dopamine levels of the MHE-F1 group significantly decreased in the frontal lobe, cerebellum and striatum, compare with the other groups (hypoxic and sedentary; normoxic and sedentary, respectively). These monoamines in the hippocampus were not influenced by the hypoxia or hypoxic exercise conditions. This study suggests that long-term hypoxic exercise decreased monoamine levels in whole brain, and that sensitivity to hypoxia and hypoxic exercise differed according to brain region.

Aging↗

PubMed2003

Downregulation of stomach cancer-associated protein tyrosine phosphatase-1 (SAP-1) in advanced human hepatocellular carcinoma.

SAP-1 (stomach cancer-associated protein tyrosine phosphatase-1) is a transmembrane-type protein tyrosine phosphatase that has been implicated as a negative regulator of integrin-mediated signaling. The potential role of this enzyme in hepatocarcinogenesis has now been investigated by examining its expression in 32 surgically excised human hepatocellular carcinoma (HCC) specimens. Both immunohistochemical and immunoblot analyses revealed that normal liver tissue, as well as tissue affected by chronic hepatitis or cirrhosis, contained substantial amounts of SAP-1. The expression level of SAP-1 in 75% of well-differentiated HCCs was similar to or higher than that observed in the surrounding noncancerous tissue. In contrast, the abundance of SAP-1 in 85.7% of moderately differentiated HCCs and in all poorly differentiated HCCs was greatly reduced compared with that in the adjacent tissue. Indeed, SAP-1 was almost undetectable in 83.3% of poorly differentiated HCCs. Furthermore, expression of recombinant SAP-1 in two highly motile human HCC cell lines resulted in a change in morphology and a marked reduction in both migratory activity and growth rate. In conclusion, these results indicate that SAP-1 expression is downregulated during the dedifferentiation of human HCC, and that this downregulation may play a causal role in disease progression.

Adult↗

PubMed2003

Interaction of oxidative stress and inflammatory response in coronary plaque instability: important role of C-reactive protein.

OBJECTIVE: C-reactive protein (CRP), a predictor of cardiovascular events, localizes in atherosclerotic arteries and exerts proinflammatory effects on vascular cells. Reactive oxygen species (ROS) have been implicated in atherogenesis and plaque instability. METHODS AND RESULTS: Expressional pattern of CRP in directional coronary atherectomy specimens from 39 patients was examined. Characteristics of histological plaque instability and higher levels of serum CRP and fibrinogen were associated with the CRP immunoreactivity. In situ hybridization revealed the presence of CRP mRNA in coronary vasculature. Furthermore, the expression of CRP mRNA and protein was detected in cultured human coronary artery smooth muscle cells (CASMCs) by reverse transcriptase-polymerase chain reaction and Western blotting. In addition, CRP was frequently colocalized with p22phox, an essential component of NADH/NADPH oxidase, which is an important source of ROS in vasculature. Moreover, the incubation of cultured CASMCs with CRP resulted in the enhanced p22phox protein expression and in the generation of intracellular ROS. CONCLUSIONS: The expression of CRP in coronary arteries was associated with histological and clinical features of vulnerable plaque, and it had a prooxidative effect on cultured CASMCs, suggesting that it might play a crucial role in plaque instability and in the pathogenesis of acute coronary syndrome via its prooxidative effect.

Arteritis↗

PubMed2003

Adrenomedullin provokes endothelial Akt activation and promotes vascular regeneration both in vitro and in vivo.

We previously reported that adrenomedullin (AM), a vasodilating hormone secreted from blood vessels, promotes proliferation and migration of human umbilical vein endothelial cells (HUVECs). In this study, we examined the ability of AM to promote vascular regeneration. AM increased the phosphorylation of Akt in HUVECs and the effect was inhibited by the AM antagonists and the inhibitors for protein kinase A (PKA) or phosphatidylinositol 3-kinase (PI3K). AM promoted re-endothelialization in vitro of wounded monolayer of HUVECs and neo-vascularization in vivo in murine gel plugs. These effects were also inhibited by the AM antagonists and the inhibitors for PKA or PI3K. The findings suggest that AM plays significant roles in vascular regeneration, associated with PKA- and PI3K-dependent activation of Akt in endothelial cells, and possesses therapeutic potential for vascular injury and tissue ischemia.

Adrenomedullin↗

PubMed2003

Immunohistochemical localization of endothelial cell-derived lipase in atherosclerotic human coronary arteries.

OBJECTIVE: A novel lipoprotein lipase (LPL)-like gene, endothelial cell-derived lipase (EDL), was recently cloned from vascular endothelial cells. The presence of LPL in the vascular wall has been implicated in the progression of atherosclerosis through the bridging function between lipoprotein particles and matrix proteoglycans to enhance lipoprotein uptake into the vascular wall. The aim of this study was to investigate the local expression of EDL in human coronary arteries. METHODS AND RESULTS: Human coronary arterial specimens from 10 autopsied cases were examined by immunohistochemistry with polyclonal antibodies against specific synthetic EDL peptides. Immunohistochemical analysis revealed that EDL was expressed in endothelial cells and medial smooth muscle cells in non-atherosclerotic coronary arteries. In addition, EDL was expressed in infiltrating cells within atheromatous plaques as well as endothelial and smooth muscle cells. Double labeling immunofluorescence confirmed EDL positive-cells were endothelial cells, smooth muscle cells and macrophages. EDL immunoreactivity was also detected in neovasculature within atheromatous plaques in atherosclerotic coronary arteries. CONCLUSIONS: These results suggest that EDL may have unique functional roles in the pathogenesis of coronary artery diseases such as atherosclerosis as well as in lipid metabolism in the vessel wall.

Aged↗

PubMed2003

Src kinase regulates the activation of a novel FGD-1-related Cdc42 guanine nucleotide exchange factor in the signaling pathway from the endothelin A receptor to JNK.

Small GTPases act as binary switches by cycling between an inactive (GDP-bound) and an active (GTP-bound) state. Upon stimulation with extracellular signals, guanine-nucleotide exchange factors (GEFs) stimulate the exchange of GDP to GTP to shift toward the active forms of small GTPases, recognizing the downstream targets. Here we show that KIAA0793, containing substantial sequence homology with the catalytic Dbl homology domain of the faciogenital dysplasia gene product (FGD1), is a specific GEF for Cdc42. We, therefore, tentatively named it FRG (FGD1-related Cdc42-GEF). Src kinase directly phosphorylates and activates FRG, as Vav family GEFs. Additionally, FRG is involved in the signaling pathway from the endothelin A receptor to c-Jun N-terminal kinase, resulting in the inhibition of cell motility. These results suggest that FRG is a member of Cdc42-GEF and plays an important role in the signaling pathway downstream of G protein-coupled receptors.

Amino Acid Sequence↗

PubMed2003

Modification of GATA-2 transcriptional activity in endothelial cells by the SUMO E3 ligase PIASy.

GATA sequences are required for the optimal expression of endothelial cell-specific genes, including endothelin-1 (ET-1). We have identified PIASy in a search for new GATA-2 interacting proteins that can regulate GATA-2-mediated endothelial gene expression. Notably, among the cell populations comprising vascular walls, PIASy mRNA is selectively expressed in endothelial cells, and its expression can be regulated by angiogenic growth factors. We show that GATA-2 is covalently modified by small ubiquitin-like modifier (SUMO)-1 and -2 and that PIASy, through its E3 SUMO ligase activity, preferentially enhances the conjugation of SUMO-2 to GATA-2. Through a functional analysis, we demonstrate that PIASy potently suppresses the activity of the GATA-2-dependent human ET-1 promoter in endothelial cells. The suppressive effect of PIASy requires the GATA-binding site in the ET-1 promoter and depends on its interaction with GATA-2, which requires both N-terminal (amino acids 1-183) and C-terminal (amino acids 414-510) sequences in PIASy. We conclude that PIASy enhances the conjugation of SUMO-2 to GATA-2 and that the interaction of PIASy with GATA-2 can modulate GATA-mediated ET-1 transcription activity in endothelial cells through a RING-like domain-independent mechanism.

Animals↗

PubMed2003

A combinatorial G protein-coupled receptor reconstitution system on budded baculovirus. Evidence for Galpha and Galphao coupling to a human leukotriene B4 receptor.

To investigate the coupling selectivity of G proteins and G protein-coupled receptors (GPCRs), we developed a reconstitution system made up of GPCR and heterotrimeric G proteins on extracellular baculovirus particles (budded virus (BV)). BV released from Sf9 cells infected with a recombinant baculovirus coding for human leukotriene B4 receptor (BLT1) cDNA exhibited a high level of BLT1 expression (27.3 pmol/mg of protein) and specific [3H]leukotriene B4 binding activity (Kd = 3.67 nm). The apparent low affinity of the expressed BLT1 is thought to be due to relative non-availability of the Galphai isoform, which couples to BLT1, in BV. Co-infection of heterotrimeric G protein recombinant viruses led to co-expression of BLT1 and G protein subunits on BV. A guanosine-5'-(beta,gamma-imido)triphosphate-sensitive, high affinity ligand binding was observed in the BLT1 BV co-expressing Galphai1beta1gamma2 (Kd = 0.17 nm). A relatively large amount of high affinity receptor protein was recovered in the co-expressing BV fraction (6.81 pmol/mg of protein). A combination of BLT1 and Galphai1 without Gbeta1gamma2 did not exhibit high affinity ligand binding on BV, indicating the low background environment for the GPCR-G protein coupling in this BV reconstitution system. To test other G proteins for coupling, various Galpha subunits were combinatorially expressed in BV with BLT1 and Gbeta1gamma2. The BLT1 BV co-expressing GalphaoAbeta1gamma2 exhibited a comparably high affinity ligand binding as well as ligand-stimulated guanosine 5'-3-O-(thio)triphosphate binding to Galphai1beta1gamma2. Co-expression of other Galpha isoforms such as Galphas, Galpha11, Galpha14, Galpha16, Galpha12, or Galpha13 did not exhibit any significant effects on ligand binding affinity in this system. These results reveal that BLT1 and coupled trimeric G proteins were functionally reconstituted on BV and that Galphao as well as Galphai couples to BLT1. This expression system should prove highly useful for pharmacological characterization, biosensor chip applications, and also drug discovery directed at highly important targets of the membrane receptor proteins.

Baculoviridae↗

PubMed2003

Different differentiation kinetics of vascular progenitor cells in primate and mouse embryonic stem cells.

BACKGROUND: We demonstrated that vascular endothelial growth factor receptor 2 (VEGF-R2)-positive cells derived from mouse embryonic stem (ES) cells can differentiate into both endothelial cells and mural cells to suffice as vascular progenitor cells (VPCs). Here we examined whether VPCs occur in primate ES cells and investigated the differences in VPC differentiation kinetics between primate and mouse ES cells. METHODS AND RESULTS: In contrast to mouse ES cells, undifferentiated monkey ES cells expressed VEGF-R2. By culturing these undifferentiated ES cells for 4 days on OP9 feeder layer, VEGF-R2 expression disappeared, and then reappeared after 8 days of differentiation. We then isolated these VEGF-R2-positive and vascular endothelial cadherin (VEcadherin)-negative cells by flow cytometry sorting. Additional 5-day reculture of these VEGF-R2+ VEcadherin- cells on OP9 feeder layer resulted in the appearance of platelet endothelial cell adhesion molecule-1 (PECAM1)-positive, VEcadherin-positive, endothelial nitric oxide synthase (eNOS)-positive endothelial cells. On a collagen IV-coated dish in the presence of serum, these cells differentiated into smooth muscle actin (SMA)-positive and calponin-positive mural cells (pericytes or vascular smooth muscle cells). Addition of 50 ng/mL VEGF to the culture on a collagen IV-coated dish resulted in the appearance of PECAM1+ cells surrounded by SMA+ cells. In addition, these differentiated VEGF-R2+ cells can form tube-like structures in a 3-dimensional culture. CONCLUSIONS: Our findings indicate that differentiation kinetics of VPCs derived from primate and mouse ES cells were different. Differentiated VEGF-R2+ VEcadherin- cells can act as VPCs in primates. To seek the clinical potential of VPCs for vascular regeneration, investigations of primate ES cells are indispensable.

Animals↗

PubMed2003

Suppression of invasion and peritoneal carcinomatosis of ovarian cancer cell line by overexpression of bikunin.

Bikunin (bik), a Kunitz-type protease inhibitor, also known as urinary trypsin inhibitor, is proposed as a main participant in the inhibition of tumor cell invasion and metastasis, possibly through the direct inhibition of cell-associated plasmin activity and suppression of urokinase-type plasminogen activator (uPA) mRNA expression. In the present study, we transfected the human ovarian carcinoma cell line HRA, highly invasive cells, with an expression vector harboring a cDNA encoding for human bik. Our study was designed to investigate the effect of bik overexpression and changes in tumor cell phenotype and invasiveness in the stably transfected clones. Bik gene transfection of HRA gave the following results: 1) transfection of HRA with the bik cDNA resulted in 5 variants stably expressing functional bik; 2) bik(+) clones exhibited a significantly reduced uPA mRNA expression as compared to the parental cells; 3) bikunin negatively regulates the ERK1/2 activity; 4) secretion-blocking treatments of bik(+) clones abrogated bik-mediated suppression of ERK1/2 activation and uPA expression; 5) the regulation of invasion seen in the HRA cells is mainly mediated by the uPA-plasmin-MMP-2 system; 6) transfection of HRA with the bik gene significantly reduced invasion, but not proliferation, adhesion, or migration relative to the parental cells; and 7) animals with bik(+) clones induced reduced peritoneal dissemination and long term survival. We conclude that transfection of HRA cells with the bik cDNA constitutively suppresses ERK1/2 activation, which results in inhibition of uPA expression and subsequently reduces dissemination of bik(+) clones.

Animals↗

PubMed2003

Shear stress attenuates endothelin and endothelin-converting enzyme expression through oxidative stress.

Shear stress is known to dilate blood vessels and exert an antiproliferative effect on vascular walls. These effects have partly been ascribed to shear stress-induced regulation of the secretion of endothelium-derived vasoactive substances. In this study, to elucidate the role of shear stress in endothelin production by endothelial cells, we examined the effect of physiological shear stress on the mRNA expression of endothelin-converting enzyme-1 (ECE-1) as well as endothelin-1 (ET-1) in cultured bovine carotid artery endothelial cells (BAECs) and human umbilical vein endothelial cells (HUVECs), using a parallel plate-type flow chamber. ECE-1 mRNA expression was significantly down-regulated by shear stress in an intensity- and time-dependent manner within the physiological range (1.5 to 15 dyn/cm(2)). ET-1 mRNA expression decreased together with ECE-1 mRNA expression. Shear stress at 15 dyn/cm(2) for 30 min induced a significant increase in the intracellular peroxide concentration, and the down-regulation of ECE-1 and ET-1 mRNA expression by shear stress was attenuated almost completely on treatment with N-acetyl cysteine (NAC), an antioxidant (20 mM). Furthermore, when H(2)O(2) (0.5 to 2 mM) was added to BAECs in static culture, the ECE-1 as well as ET-1 mRNA expression was attenuated in proportion to the concentration of H(2)O(2). It is suggested that endothelial cells sense shear stress as oxidative stress and transduce signal for the regulation of the gene expression of ECE as well as ET to attenuate vascular tone and inhibit the proliferation of vascular smooth muscle cells.

Acetylcysteine↗

PubMed2003

Characterization of transcripts generated from mouse hepatocyte growth factor activator inhibitor type 2 (HAI-2) and HAI-2-related small peptide (H2RSP) genes: chimeric mRNA transcribed from both HAI-2 and H2RSP genes is detected in human but not in mouse.

We previously reported a novel small gene, designated hepatocyte growth factor activator inhibitor type 2 (HAI-2) related small peptide (H2RSP), in the process of the search for splicing variant forms of HAI-2 by 3(')-rapid amplification of cDNA ends method [Biochem. Biophys. Res. Commun. 288 (2001) 390]. Human H2RSP gene consisted of four exons spanning approximately 1kbp and was located in 11kbp downstream of HAI-2 gene. In this study, we cloned and characterized the mouse counterpart of H2RSP gene, which was located in 6.6kbp downstream of mouse HAI-2 gene, and analyzed the transcripts generated from both genes. Similar to human, mouse H2RSP mRNA (0.5kb) was detected abundantly in various tissues including the gastrointestinal tract, and has nuclear localization signal (NLS) in the lysine-rich region (exon 4), which was well-conserved between human and mouse genes. However, chimeric mRNA transcribed from both HAI-2 (exons 1-7) and H2RSP (exons 2-4) genes, which was found in the kidney, prostate, and placenta of human by Northern blot analysis, was not detected in mouse tissue even by a reverse transcription-polymerase chain reaction (RT-PCR). Instead of the chimeric mRNA, a novel splicing variant lacking putative transmembrane domain of HAI-2 was found in mouse but not in human as a putative secrete form of HAI-2. These results suggest that the organization of H2RSP and HAI-2 gene complex is well-conserved, but the usage of these genes was quite different between human and mouse.

Alternative Splicing↗

PubMed2003

Significance and therapeutic potential of the natriuretic peptides/cGMP/cGMP-dependent protein kinase pathway in vascular regeneration.

Natriuretic peptides (NPs), which consist of atrial, brain, and C-type natriuretic peptides (ANP, BNP, and CNP, respectively), are characterized as cardiac or vascular hormones that elicit their biological effects by activation of the cGMPcGMP-dependent protein kinase (cGK) pathway. We recently reported that adenoviral gene transfer of CNP into rabbit blood vessels not only suppressed neointimal formation but also accelerated reendothelialization, a required step for endothelium-dependent vasorelaxation and antithrombogenicity. Accordingly, we investigated the therapeutic potential of the NPscGMPcGK pathway for vascular regeneration. In transgenic (Tg) mice that overexpress BNP in response to hindlimb ischemia, neovascularization with appropriate mural cell coating was accelerated without edema or bleeding, and impaired angiogenesis by the suppression of nitric oxide production was effectively rescued. Furthermore, in BNP-Tg mice, inflammatory cell infiltration in ischemic tissue and vascular superoxide production were suppressed compared with control mice. Ischemia-induced angiogenesis was also significantly potentiated in cGK type I Tg mice, but attenuated in cGK type I knockout mice. NPs significantly stimulated capillary network formation of cultured endothelial cells by cGK stimulation and subsequent Erk12 activation. Furthermore, gene transfer of CNP into ischemic muscles effectively accelerated angiogenesis. These findings reveal an action of the NPscGMPcGK pathway to exert multiple vasculoprotective and regenerative actions in the absence of apparent adverse effects, and therefore suggest that NPs as the endogenous cardiovascular hormone can be used as a strategy of therapeutic angiogenesis in patients with tissue ischemia.

Animals↗

PubMed2003

Coactivation in arm and shoulder muscles during voluntary fixation of a single joint.

The objective of this study was to determine the organization of coactivation in the arm and shoulder muscles. Normal human subjects made alternate movements of a joint in the horizontal plane, either adduction-abduction of the second finger and shoulder, ulnar-radial deviation of the wrist, or extension-flexion of the elbow, during which they fixed a focal joint while decreasing the movement amplitude and increasing the fixation strength. They varied the fixation strength at four different levels up to the maximum. The focal-joint angle, and surface electromyograms (EMGs) from the intrinsic hand, antebrachial, upper-arm, and shoulder muscles were recorded. EMGs in the phase of fixation were quantified by integration after rectification. The degree of coactivation among the muscles was evaluated by calculating correlation coefficients across the integrated EMGs. There were correlations in the integrated EMGs among focal-joint muscles (FJMs), and also between one of the FJMs and the muscles distal and/or proximal to the FJMs: in the finger fixation between the hand and antebrachial muscles, in the wrist fixation between the antebrachial and hand/upper-arm muscles, in the elbow fixation between the upper-arm and antebrachial/shoulder muscles, and in the shoulder fixation between the shoulder and upper-arm muscles. Moderate or slight correlations were seen in muscles more distant from FJMs. Our results indicate that the longitudinal distance from FJMs in the shoulder and arm muscles is an important factor in determining levels of coactivation. This is discussed in relation to the fact that neighboring muscles share joints with FJMs.

Adolescent↗

PubMed2003

Bikunin target genes in ovarian cancer cells identified by microarray analysis.

Bikunin, a Kunitz-type protease inhibitor, could potentially suppress tumor cell invasion and metastasis. Our previous study revealed that overexpression of bikunin in a human ovarian cancer cell line, HRA, resulted in a down-regulation in uPA and uPAR gene expression. For identifying the full repertoire of bikunin-regulated genes, a cDNA microarray hybridization screening was conducted using mRNA from bikunin-treated or bikunin-transfected HRA cells. A number of bikunin-regulated genes were identified, and their regulation was confirmed by Northern blot analysis. Our screen identified 11 bikunin-stimulated genes and 29 bikunin-repressed genes. The identified genes can indeed be classified into distinct subsets. These include transcriptional regulators, oncogenes/tumor suppressor genes, signaling molecules, growth/cell cycle, invasion/metastasis, cytokines, apoptosis, ion channels, extracellular matrix proteins, as well as some proteases. This screen identified suppression of several genes such as CDC-like kinase, LIM domain binding, Ets domain transcription factor, Rho GTPase-activating protein, tyrosine phosphorylation-regulated kinase, hyaluronan-binding protein, matriptase, and pregnancy-associated plasma protein-A (PAPP-A), which have previously been implicated in enhancing tumor promotion. Northern blot analysis confirmed that several genes including matriptase and PAPP-A were down-regulated by bikunin by approximately 9-fold. Further, genetic inhibition of matriptase or PAPP-A could lead to diminished invasion. These results show that bikunin alters the pattern of gene expression in HRA cells leading to a block in cell invasion.

Cell Movement↗

PubMed2003

Insulin lispro reduces insulin antibodies in a patient with type 2 diabetes with immunological insulin resistance.

We describe a 54-year-old Japanese female with type 2 diabetes admitted to our hospital with poor metabolic control. On admission the patient's HbA1c was 9.1% despite having taken over 60 U of human insulin per day for the previous 10 years. A high titer of antibodies to insulin was detected in the serum, and therefore, we decided to introduce insulin lispro with the aim of minimizing immunogenicity. Over the next 2 months, the dosage of insulin required to achieve reasonable blood glucose control was reduced, with the HbA1c level decreasing significantly to 6.8%. The patient was subsequently withdrawn from insulin and discharged on diet therapy only. This case demonstrates that insulin lispro may ameliorate resistance to insulin therapy even in the presence of human insulin antibodies.

Autoantibodies↗

PubMed2003

Role of cancer cell-stroma interaction in invasive growth of cancer cells.

Invasive growth is one of the hallmarks of cancer malignancy. To date, a significant body of evidence is accumulating in favor of the notion that invasive growth results from the cross-talk between cancer cells and the host stromal cells, comprising fibroblasts (myofibroblasts), endothelial cells, and leukocytes, all of which are themselves invasive. In this review we describe cross-talk between invasive cancer cells and host stromal fibroblasts and an impact of pericellular microenvironment on the invasive phenotype of cancer cells, focusing on two molecules, extracellular matrix metalloproteinase inducer (EMMPRIN, also known as tumor cell-derived collagenase stimulatory factor, basigin, CD147) and hepatocyte growth factor (HGF, also known as scatter factor). Both molecules are deeply involved in the regulation of invasion-associated cellular activities, such as pericellular proteolysis, migration and ectopic survival of cancer cells.

Antigens, CD↗

PubMed2003

Adrenomedullin promotes proliferation and migration of cultured endothelial cells.

Adrenomedullin (AM) is a vasoactive hormone which exerts its action through cyclic adenosine monophosphate(cAMP) /cAMP-dependent protein kinase (PKA) cascade and intracellular Ca2+ mobilization. Recently, evidence has accumulated that AM plays a critical role in the regulation of vascular tone, remodeling and morphogenesis. And although numerous reports have examined the action of AM on cultured vascular cells, the results have not been consistent and have depended on the experimental conditions used. Accordingly, the purpose of this study was to clarify the effect of AM on the proliferation and migration of cultured endothelial cells. Our results revealed that AM promoted the growth and migration of endothelial cells (ECs). AM significantly promoted the proliferation of human umbilical vein endothelial cells (HUVECs) (56.0 +/- 8.7% over the controls at 10(-9) mol/l) and this stimulative effect was inhibited by two AM antagonists, AM(22-52) and calcitonin gene-related peptide (CGRP) (8-37). The number of HUVECs migrated to the lower surface of the transwell apparatus was also increased dose-dependently in the AM group (30.4 +/- 4.2% over the controls at 10(-7) mol/l), and this increase was suppressed by the two AM antagonists and by two PKA antagonists, adenosine 3'5'-cyclic monophosphorothioate Rp-isomer and myristoylated protein kinase A inhibitor amide 14-22. The promoting action of AM on endothelial migration was also suppressed by LY294002, an inhibitor for phosphatidylinositol 3-kinase, but not by N(G)-nitro-L-arginine-methyl ester (L-NAME), an antagonist for nitric oxide synthase (NOS). These results indicate that AM promotes proliferation and migration of ECs via a cAMP/PKA dependent pathway and lend support to the idea that AM exerts beneficial effects on vascular regeneration and might be used as a novel therapeutic strategy for patients with vascular disease.

Adrenomedullin↗