Biomedical subjects

K N Koh

Publications and source records attributed to K N Koh.

8 recordsLinked to original sources

PubMed2000

Angiopoietin-1 inhibits irradiation- and mannitol-induced apoptosis in endothelial cells.

BACKGROUND AND PURPOSE: Angiopoietin-1 (Ang1) is a vasculogenic factor that signals through the endothelial cell-specific Tie2 receptor tyrosine kinase. We recently reported that Ang1 prevented apoptosis induced by serum deprivation in endothelial cells. In this study, we examined whether Ang1 prevents apoptosis in endothelial cells treated with irradiation or clinical concentrations of mannitol. METHODS AND RESULTS: ++Ang1 prevented irradiation- and mannitol-induced apoptosis in human umbilical vein endothelial cells in a dose-dependent manner. Pretreatment with soluble Tie2 receptor, but not Tie1 receptor, blocked the antiapoptotic effect of Ang1. Two phosphatidylinositol 3'-kinase (PI3-kinase)-specific inhibitors, wortmannin and LY294002, blocked the Ang1-induced antiapoptotic effect. The antiapoptotic potency of Ang1 was similar to or greater than that of vascular endothelial growth factor, basic fibroblast growth factor, and endothelin-1. Ang1 also prevented apoptosis in cultured endothelial cells from porcine pulmonary and coronary arteries and in endothelial cells of explanted rat aorta. CONCLUSIONS: Ang1 promotes the survival of endothelial cells in irradiation- and mannitol-induced apoptosis through Tie2 receptor binding and PI3-kinase activation. Pretreatment with Ang1 could be beneficial in maintaining normal endothelial cell integrity during intracoronary irradiation or systemic mannitol therapy.

Angiopoietin-1↗

PubMed2000

Angiopoietin-1 induces endothelial cell sprouting through the activation of focal adhesion kinase and plasmin secretion.

Angiopoietin-1 (Ang1) is a strong inducer of endothelial cell sprouting, which is a first step in both angiogenesis and neovascularization. We examined the mechanisms underlying Ang1-induced cell sprouting using porcine pulmonary artery endothelial cells. Ang1 induced the nondirectional and directional migration of endothelial cells mediated through the Tie2 but not the Tie1 receptor. Ang1 induced tyrosine phosphorylation of p125(FAK), and this phosphorylation was dependent on phosphatidylinositol (PI) 3'-kinase activity. Ang1 induced the secretion of plasmin and matrix metalloproteinase-2 (MMP-2), which is inhibited by PI 3'-kinase inhibitors. Ang1 also induced the secretion of small amounts of proMMP-3 and proMMP-9 but not proMMP-1. Ang1 suppressed the secretion of tissue inhibitor of metalloproteinase-2 (TIMP-2), but not of TIMP-1. Addition of alpha(2)-antiplasmin, a combination of TIMP-1 and TIMP-2, or PI 3'-kinase inhibitors inhibited Ang1-induced sprouting activity. Therefore, Ang1-induced sprouting activity in endothelial cells may be accomplished by cytoskeletal changes and secretion of proteinases and may be largely mediated through intracellular PI 3'-kinase activation.

Angiopoietin-1↗

PubMed1999

Molecular cloning, expression, and characterization of angiopoietin-related protein. angiopoietin-related protein induces endothelial cell sprouting.

Using degenerate polymerase chain reaction, we isolated a cDNA encoding a novel 493-amino acid protein from human and mouse adult heart cDNAs and have designated it angiopoietin-related protein-2 (ARP2). The NH(2)-terminal and COOH-terminal portions of ARP2 contain the characteristic coiled-coil domain and fibrinogen-like domain that are conserved in angiopoietins. ARP2 has two consensus glycosylation sites and a highly hydrophobic region at the NH(2) terminus that is typical of a secretory signal sequence. Recombinant ARP2 expressed in COS cells is secreted and glycosylated. In human adult tissues, ARP2 mRNA is most abundant in heart, small intestine, spleen, and stomach. In rat embryos, ARP2 mRNA is most abundant in the blood vessels and skeletal muscles. Endothelial and vascular smooth muscle cells also contain ARP2 mRNA. Recombinant ARP2 protein induces sprouting in vascular endothelial cells but does not bind to the Tie1 or Tie2 receptor. These results suggest that ARP2 may exert a function on endothelial cells through autocrine or paracrine action.

Amino Acid Sequence↗

PubMed1999

Molecular cloning and characterization of a novel angiopoietin family protein, angiopoietin-3.

Using homology-based PCR, we have isolated cDNA encoding a novel member (491 amino acids) of the angiopoietin (Ang) family from human adult heart cDNA and have designated it angiopoietin-3 (Ang3). The NH2-terminal and COOH-terminal portions of Ang-3 contain the characteristic coiled-coil domain and fibrinogen-like domain that are conserved in other known Angs. Ang3 has a highly hydrophobic region at the N-terminus (approximately 21 amino acids) that is typical of a signal sequence for protein secretion. Ang3 mRNA is most abundant in adrenal gland, placenta, thyroid gland, heart and small intestine in human adult tissues. Additionally, Ang3 is a secretory protein, but is not a mitogen in endothelial cells.

Adult↗

PubMed1998

Programmed changes of cell cycle regulators by serum deprivation regardless of skeletal myocyte differentiation.

Permanent withdrawal of skeletal myoblasts from the cell cycle precedes differentiation. We examined the changes of protein levels of cell cycle regulators and the activities of CDKs in differentiating (by serum deprivation) or in differentiation-inhibited (by serum deprivation + TGF-beta1 treated) C2C12 skeletal myocytes. Regardless of differentiation, protein levels of most cyclins declined over time while this effect was delayed slightly by TGF-beta1 for cyclins D1 and F. Although the protein levels of CDKs gradually decreased over time, the activities of CDK2 and cdc2 decreased dramatically between 0 and 12 h after serum deprivation in both groups. Decreased CDK2 and cdc2 activities were temporally related to decreased DNA synthesis. While the p27KIP1 protein increased in both groups, the p21CIP1 protein increased only in differentiating cells. In vivo, protein expressions of cyclins, CDKs, and p21CIP1 were high in fetal, but almost undetectable in adult skeletal muscle. In contrast, the levels of p27KIP1 protein in skeletal muscle were high throughout development. Thus, changes of cell cycle regulators in differentiating C2C12 myocytes paralleled those during skeletal muscle development of mice. These results suggest that the rapid reduction of DNA synthesis and activities of CDKs slow reduction of protein levels of cyclins and CDKs, and that the increase of p27KIP1 are programmed mechanisms upon mitogen deprivation regardless of differentiation in skeletal myocytes.

Animals↗

PubMed1998

Persistent and heterogenous expression of the cyclin-dependent kinase inhibitor, p27KIP1, in rat hearts during development.

We have previously shown that there were differential and dramatic decreases of cyclin and cyclin-dependent kinase (CDK) activities in cardiomyocytes during the neonatal period. The activity of CDKs control cell cycle progression, and this activity is regulated positively and negatively by association of CDKs with cyclins and cyclin-dependent kinase inhibitors (CKIs), respectively. While the INK family (p15(INK4B)/p16(INK4A)/p18(INK4C)/p19(INK4D)) of CKIs is not detectable in hearts, the KIP/CIP family (p21(CIP1), p27(KIP1) and p57(KIP2)) of CKIs is detectable in most organs including the heart. Differential and dramatic changes of the KIP/CIP family (p21(CIP1), p27(KIP1) and p57(KIP2)) of CKIs were detected in rat hearts during development. The mRNA and protein levels of p21(CIP1) and p57(KIP2) were readily detectable in hearts at gestational and early postnatal periods and decreased thereafter. The mRNA levels of p27(KIP1) in ventricles were high during the gestational period, and did not change until day 30 postnatal, then were decreased slightly in 90-day-old rats. The protein levels of p27(KIP1) increased significantly in the early postnatal period, then were expressed persistently, although levels decreased slightly in the adult period. However, protein levels of p27(KIP1) in atria did not change during development. Variable immuno-staining patterns of p27(KIP1) were observed at different periods of development and in various locations in myocardium. During the gestational period, approximately 35-50% of myocardial cells in the cardiac wall were p27(KIP1) immuno-positive and were distributed diffusely. These p27(KIP1) immunopositive cells increased predominantly in endocardial and mid-portion areas of ventricular myocardium at the early postnatal period. This heterogenous pattern of p27(KIP1) protein expression persisted to adult hearts though the percentage of p27(KIP1) immuno-positive cells decreased slightly. High magnification revealed that more than 50% of adult cardiomyocytes were p27(KIP1) immuno-positive and that p27(KIP1) was located solely in nuclei. These results indicate that p27(KIP1) may be an important inhibitor of CDK activities in cardiomyocytes during early postnatal development and may block the re-entrance of adult cardiomyocytes into the cell cycle after injury.

Animals↗

PubMed1998

Cell cycle regulators during human atrial development.

OBJECTIVES: The molecular mechanisms that regulate cardiomyocyte cell cycle and terminal differentiation in humans remain largely unknown. To determine which cyclins, cyclin dependent kinases (CDKs) and cyclin kinase inhibitors (CKIs) are important for cardiomyocyte proliferation, we have examined protein levels of cyclins, CDKs and CKIs during normal atrial development in humans. METHODS: Atrial tissues were obtained in the fetus from inevitable abortion and in the adult during surgery. Cyclin and CDK proteins were determined by Western blot analysis. CDK activities were determined by phosphorylation amount using specific substrate. RESULTS: Most cyclins and CDKs were high during the fetal period and their levels decreased at different rates during the adult period. While the protein levels of cyclin D1, cyclin D3, CDK4, CDK6 and CDK2 were still detectable in adult atria, the protein levels of cyclin E, cyclin A, cyclin B, cdc2 and PCNA were not detectable. Interestingly, p27KIP1 protein increased markedly in the adult period, while p21CIP1 protein in atria was detectable only in the fetal period. While the activities of CDK6, CDK2 and cdc2 decreased markedly, the activity of CDK4 did not change from the fetal period to the adult period. CONCLUSION: These findings indicate that marked reduction of protein levels and activities of cyclins and CDKs, and marked induction of p27KIP1 in atria, are associated with the withdrawal of cardiac cell cycle in adult humans.

Adult↗

PubMed1997

Cyclins and cyclin dependent kinases during cardiac development.

The molecular mechanisms that regulate the cardiomyocyte cell cycle and its terminal differentiation remain largely unknown. To determine which cyclins or cyclin dependent kinases (CDKs) are important for cardiomyocyte proliferation, we examined the expression of cyclins and CDKs during normal cardiac development. All cyclins and CDKs were highly expressed during embryonic cardiac development, then they decreased at different rates after birth. The mRNAs and proteins of cyclins A and B (G2 and M phase cyclins) were found in embryonic and neonatal hearts, but were not detected in young or adult hearts. In contrast, while the mRNAs of cyclins D1, D2, D3, and E (G1 and S phase cyclins) were observed during all stages of development, the proteins of cyclins D1, D3, and E were observed in hearts at the young growth stage, although the levels decreased differently. Reverse transcriptase-polymerase chain reaction (RT-PCR) using specific cyclin B and D3 primers revealed that cyclins B and D3 originated from cardiomyocytes and noncardiomyocytes. The CDKs (cdc2, CDK2, and CDK4) were highly expressed during embryonic cardiac development and maintained almost constant levels during neonatal periods. However, they were expressed at very low levels at the young and adult stages. The pattern of proliferating cell nuclear antigen (PCNA) expression during cardiac development was similar to the expression of CDKs. These findings suggest that all cyclins and CDKs are involved in the cardiac cell cycle, and that marked and rapid reduction of mitotic cyclins may be associated with the withdrawal of the cardiac cell cycle after birth.

Animals↗