摘要:

Angiopoietin-1 (Ang1) has been recently identified as the major physiological ligand for the tyrosine kinase receptor Tie2 and assigned responsibility for recruiting and sustaining periendothelial support cells. Angiopoietin-2 (Ang2) was found to disrupt blood vessel formation in the developing embryo by antagonizing the effects of Ang1 and Tie2 and was thus considered to represent a natural Ang1/Tie2 inhibitor. In vivo effects of either angiopoietin on postnatal neovascularization, however, have not been previously described. Accordingly, we used the cornea micropocket assay of neovascularization to investigate the impact of angiopoietins on neovascularization in vivo. Neither Ang1 nor Ang2 alone promoted neovascularization. Pellets containing vascular endothelial growth factor (VEGF) alone induced corneal neovascularity extending from the limbus across the cornea. Addition of Ang1 to VEGF (Ang1+VEGF) produced an increase in macroscopically evident perfusion of the corneal neovasculature without affecting macroscopic measurements of length (0.58 +/- 0.03 mm) or circumferential neovascularity (136 +/- 10[degree sign]). In contrast, pellets containing Ang2+VEGF promoted significantly longer (0.67 +/- 0.05 mm) and more circumferential (160 +/- 15[degree sign]) neovascularity than VEGF alone or Ang1+VEGF (P<0.05). Excess soluble Tie2 receptor (sTie2-Fc) precluded modulation of VEGF-induced neovascularization by both Ang2 and Ang1. Fluorescent microscopic findings demonstrated enhanced capillary density (fluorescence intensity, 2.55 +/- 0.23 e+9versus 1.23 +/- 0.17 e+9, P<0.01) and increased luminal diameter of the basal limbus artery (39.0 +/- 2.8 versus 27.9 +/- 1.3 [micro sign]m, P<0.01) for Ang1+VEGF compared with VEGF alone. In contrast to Ang1+VEGF, Ang2+VEGF produced longer vessels and, at the tip of the developing capillaries, frequent isolated sprouting cells. In the case of Ang2+VEGF, however, luminal diameter of the basal limbus artery was not increased (26.7 +/- 1.9 versus 27.9 +/- 1.3, P=NS). These findings constitute what is to our knowledge the first direct demonstration of postnatal bioactivity associated with either angiopoietin. In particular, these results indicate that angiopoietins may potentiate the effects of other angiogenic cytokines. Moreover, these findings provide in vivo evidence that Ang1 promotes vascular network maturation, whereas Ang2 works to initiate neovascularization. (Circ Res. 1998;83:233-240.)

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

The degree of lumen narrowing in advanced lesions correlates poorly with the amount of intimal mass accumulated in the atherosclerotic plaque. As an alternate mechanism of stenosis, we propose that human smooth muscle cells bind to fibrin deposited in the matrix and exert contractile forces to cause a narrowing of the lumen. In the present study we demonstrated in vitro that human newborn aortic smooth muscle cell lines can contract and adhere to fibrin clots composed of either fibronectin-depleted plasma ("plasma") or recombinant fibrin. By using neutralizing antibodies and RGD peptides, we showed that members of the integrin family mediated the interaction between human newborn smooth muscle cells and fibrin. Neutralizing antibodies against the integrin alpha v beta 3 (c7E3 Fab and LM609) did not inhibit either plasma clot contraction or recombinant fibrin clot contraction by human newborn smooth muscle cells. In contrast, antibodies against alpha 5, beta 1, and alpha 5 beta 1 inhibited contraction of clots composed of either plasma or recombinant fibrin. Anti-alpha v beta 3, anti-alpha v, anti-alpha 5, anti-beta 1, and anti-alpha 5 beta 1 antibodies inhibited human newborn smooth muscle cell adhesion to plasma clots; however, only anti-alpha 5, anti-beta 1, and anti-alpha 5 beta 1 antibodies significantly inhibited adhesion to recombinant fibrin. While the linear RGD peptides had no effect, the cyclic peptide penRGD inhibited adhesion to plasma clots and recombinant fibrin. However, it did not block contraction of recombinant fibrin clots. These results suggest that during the interaction of human newborn smooth muscle cell lines with fibrin, alpha 5 beta 1 plays a significant role. This interaction is of potential interest as a target for efforts to block vascular contraction. (Circ Res. 1998;83:241-251.)

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

In previous studies, we showed that induction of pulmonary artery (PA) smooth muscle cell (SMC) elastase activity by serum-treated elastin (STE) requires DNA transcription. We therefore used differential mRNA display to identify transcripts expressed coincident with elastase induction. Twenty-four individual transcripts were differentially expressed from a screen of [approximate]2000 mRNA sequences. An mRNA with sequence homology to the human transcription factor AML1 was identified and subsequently cloned from ovine PA SMCs. Since AML1 binds to a consensus sequence in the promoter of neutrophil elastase, we pursued the possibility that AML1 is a candidate transcription factor for SMC elastase. We documented by immunohistochemistry that serum stimulation induces increased expression of AML1 in the nucleus of PA SMCs. We also showed that STE induction of elastase activity is associated with early expression of AML1 mRNA and protein and that AML1 consensus sequence DNA binding activity is increased in nuclear extracts of STE-treated cells. In addition, AML1 antisense oligonucleotides reduced serum induction of elastase activity. Our study thus provides the first functional evidence of AML1 transcriptional activity related to elastase genes and offers novel insights into the broader biological significance of AML1 in nonmyeloid cells. (Circ Res. 1998;83:252-263.)

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

During the development of atherosclerotic lesions, lipoprotein(a) [Lp(a)], a highly atherogenic lipoprotein, accumulates within fibrin clots attached to blood vessel walls. As Lp(a) accumulates within the fibrin clot with time, fatty streaks are formed that develop into occlusive atherosclerotic plaques. It is not understood, however, which mechanisms are involved in the binding of Lp(a) to fibrin and, hence, the stable incorporation of Lp(a) into the fibrin clot. The results of the present study demonstrate that factor XIIIa, a transglutaminase that catalyzes the formation of amide bonds between endo-gamma-glutaminyl and endo-epsilon-lysyl residues of proteins, is capable of cross-linking Lp(a) to fibrinogen, the soluble precursor of fibrin. Biochemical assays were conducted to demonstrate that factor XIIIa cross-links Lp(a) with fibrinogen in a time- and concentration-dependent manner. Additionally, immunohistochemical studies revealed that factor XIII protein expression colocalizes with Lp(a) expression in human atherosclerotic plaques. It is proposed that factor XIIIa-mediated cross-linking of Lp(a) to fibrin effectively increases the local concentration of Lp(a) within a fibrin clot. The accumulation of Lp(a) within the blood vessel promotes an antifibrinolytic environment, foam cell formation, the generation of a fatty streak, and an increase in smooth muscle cell content, all of which may contribute to the pathogenesis of atherosclerosis. (Circ Res. 1998;83:264-269.)

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

Atherosclerotic plaque stability depends on the structural integrity of its extracellular matrix skeleton. The balance between degradation by matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) may regulate plaque stability. Although MMP expression in atheroma is well documented, localization and control of expression of TIMPs in these lesions is incomplete. Extracts of atheroma (n=14) had 5-fold higher levels of TIMP-3 than nonatherosclerotic tissue (n=10). Plaques (n=24) contained abundant TIMP-1, -2, and -3 in macrophages in plaque shoulders, intimal-medial borders, and areas overlying the lipid core, as well as in medial smooth muscle cells, albeit in lesser amounts. These observations suggested that macrophages, a cell type not heretofore known to express TIMP-3, did so in atheroma in vivo. Further studies in vitro established the human macrophage as a novel source of TIMP-3 mRNA and protein. Human smooth muscle cells constitutively expressed TIMP-1, -2 and -3 proteins; platelet-derived growth factor and transforming growth factor-beta augmented levels of TIMP-1 and TIMP-3 but not TIMP-2. These findings suggest that regulated expression of TIMP-3, in addition to the presence of TIMP-1 and TIMP-2, counteracts MMP activity in atheroma and hence influences plaque stability. (Circ Res. 1998;83:270-278.)

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

Two NO synthase (NOS) isoforms have been described in vessels, an endothelial constitutive NOS (eNOS) and an inducible NOS (iNOS). The purpose of the present study was to examine the endothelium-dependent and endothelium-independent hypotensive response in aging rats, analyzing the ability of their vessels to produce NO. The studies were performed in 2 groups of euvolemic, conscious, male Wistar rats: aging rats (n=20, 18 months old) and young rats (n=20, 5 months old). The hypotensive responses to acetylcholine, bradykinin, and sodium nitroprusside were determined. Furthermore, the expression of the NOS isoforms by Western blot and the eNOS and iNOS activities, defined as Ca2+-dependentand Ca2+-independentconversion of [(14) C]L-arginine into [(14) C]L-citrulline, respectively, were also determined. In the aging rats, we found an impaired hypotensive response to acetylcholine and bradykinin (2 NO-and endothelium-dependent hypotensive agents) that was accompanied by a preserved hypotensive response to sodium nitroprusside. Aging rats also demonstrated an enhanced sensitivity response to the pressor effect of the L-arginine antagonist L-Nomega-nitro-L-arginineand a reduced vasoconstrictor response to angiotensin II. The inhibition of NO synthesis normalized the pressor effect of angiotensin II in the aging animals. Nitrite plus nitrate plasma levels were increased in aging rats. Furthermore, cGMP content was also higher in the aging vessels. In the aging aortas, the expression of both eNOS and iNOS isoforms was enhanced. However, in aging rats, the activity of the eNOS isoform was markedly reduced, a finding that was accompanied by the presence of iNOS activity. The vessel wall of aging rats showed an enhanced expression of eNOS and iNOS isoforms. However, eNOS activity was reduced in the aging animals. These findings could explain the impaired endothelium-dependent hypotensive response associated with aging. (Circ Res. 1998;83:279-286.)

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

80%). No such defect was seen in intercellular adhesion molecule-1 (ICAM-1)-deficient mice. E-Selectin-null mice showed more rapid leukocyte rolling than wild-type or ICAM-1-deficient mice, resulting in significantly shortened leukocyte transit times through venules. Topical application of fMLP onto the whole cremaster muscle generated the same number of adherent leukocytes in wild-type and E-selectin-deficient mice. We conclude that slow leukocyte rolling through E-selectin results in long transit times, which are essential for efficient leukocyte adhesion in response to a local chemotactic stimulus. (Circ Res. 1998;83:287-294.)

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

Cyclooxygenases catalyze a rate-limiting step in the synthesis of vascular endothelial prostaglandins. Expression of the inducible cyclooxygenase-2 (COX-2) gene is increased by hypoxia in human vascular endothelial cells via the nuclear factor (NF)-kappa B p65 transcription factor, which is necessary but not sufficient to fully induce COX-2 transcription in response to hypoxia. After finding that cytoplasmic NF-kappa B p65 and I kappa B alpha (an inhibitory protein that binds NF-kappa B p65 precursors) levels are not changed by hypoxia, we hypothesized that other factors might play a role in regulating the COX-2 promoter, like the high-mobility-group (HMG) I(Y) family of proteins, which features multiple A[center dot]T hooks and is associated with NF-kappa B-mediated transactivation. Nuclear protein obtained from human umbilical vein endothelial cells (HUVECs) was supplemented with HMG I(Y) during electrophoretic mobility shift assays using an NF-kappa B-3[prime] element probe. These data suggested that HMG I(Y) proteins interact with NF-kappa B p65 to induce COX-2 promoter activity. We also found that TATA-box DNA demonstrated increased electrophoretic shifting indicative of DNA binding after incubation with either hypoxic HUVEC nuclear protein or normoxic nuclear protein supplemented with HMG I(Y). Transfection of HUVECs with an expression vector containing the COX-2 promoter ligated to HMG I(Y) cDNA demonstrated positive feedback on COX-2 promoter activity in hypoxia. We confirmed that COX-2 is transcriptionally regulated by hypoxia using a nuclear runoff assay. Hypoxia increased steady-state cellular levels of HMG I(Y) mRNA as an early event, corresponding with increases in HMG I(Y) protein. Overexpression of HMG I(Y) was associated in a dose-response relationship with increasing prevalence of the COX-2 protein in hypoxic HUVECs. Furthermore, sense (and antisense) HMG I(Y) overexpression caused stimulation (or inhibition) of COX-2 promoter activity as measured by luciferase reporter gene expression. The physiological significance of these findings was demonstrated by cyclooxygenase-dependent release of prostaglandin E2by HUVECs in hypoxia. We concluded that hypoxia increases expression of HMG I(Y) proteins while facilitating transactivation of the COX-2 promoter. The HMG I(Y) family of proteins may therefore function as part of a hypoxia-induced enhanceosome that helps to promote transcription of COX-2. (Circ Res. 1998;83:295-304.)

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

Migration of vascular smooth muscle cells (SMCs) is a key step in vascular remodeling and formation of pathological lesions in diseased arteries and may be controlled by extracellular matrix (ECM) and by factors that regulate ECM composition, such as platelet-derived growth factor (PDGF). In culture, PDGF-AB and -BB enhance but PDGF-AA (although having no effect alone) suppresses SMC migration stimulated by other PDGF isoforms. To determine whether the migration-inhibitory mechanism of PDGF-AA was mediated by ECM composition, we examined baboon SMC migration in a Boyden chamber assay using filters coated with different ECM proteins. PDGF-AA suppressed the PDGF-BB-induced migration of baboon SMCs on a filter coated with basement membrane proteins (Matrigel) and fibronectin but failed to inhibit cell migration on a type I collagen (Vitrogen)-coated filter. Fibronectin and fibronectin fragments that contain heparin-binding domains permitted PDGF-AA inhibition of cell migration, but a fragment lacking heparin-binding domains did not. Treatment of SMCs with heparin lyases II and III, but not with chondroitin ABC lyase, diminished the PDGF-AA-mediated inhibition of migration. PDGF-AA stimulated accumulation of proteoglycan (PG) in the cell layer more potently than did PDGF-BB, whereas the turnover of cell layer PG was unaffected by either PDGF-AA or -BB. Northern blot analysis revealed that PDGF-AA increased syndecan-1 mRNA expression more than did PDGF-BB, whereas both PDGF isoforms decreased perlecan expression. The changes in cell migration and PG synthesis induced by PDGF-AA were accompanied by changes in the morphology of SMCs. PDGF-AA dramatically induced the spreading of SMCs, whereas the heparin lyase treatment of PDGF-AA-stimulated cultures diminished cell spreading. The data suggest that PDGF-AA selectively modifies heparan sulfate PG accumulation on SMCs and thereby influences the interactions of SMCs with heparin-binding ECM proteins. These interactions, in turn, generate signals that suppress SMC migration. (Circ Res. 1998;83:305-313.)

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

We have previously shown endothelin (ET)-like immunoreactive staining in Weibel-Palade bodies, storage granules that are an integral component of the regulated secretory pathway in endothelial cells. These structures degranulate after chemical or mechanical stimuli that result in cytosolic calcium influx. We therefore investigated whether the regulated pathway might be an intracellular site involved in the cleavage of big ET-1 to the biologically active peptide ET-1 by determining the ultrastructural localization of endothelin-converting enzyme (ECE)-1. A low level of ECE-like immunoreactivity was detected on the cell surface of human umbilical vein and coronary artery endothelial cells by scanning electron microscopy. Exogenous big ET-1 was added to permeabilized and nonpermeabilized cultured human umbilical vein endothelial cells, and ECE activity was measured by the detection of ET-like immunoreactivity in the culture supernatant. A marked increase in ECE activity was observed in permeabilized cells, indicating that ECE may also be expressed in intracellular compartments. Confocal microscopy revealed intense immunofluorescence staining for big ET-1 and the 2 isoforms of ECE-1 (ECE-1 alpha and ECE-1 beta) in the perinuclear region and in Weibel-Palade bodies of the human umbilical vein endothelial cells. Stimulated degranulation of storage granules by the calcium ionophore A23187 caused release of ET into the culture supernatants. The findings of this study indicate that big ET-1 is processed to the mature vasoactive peptide by ECEs located within endothelial storage granules. We hypothesize that this activity may be important in the regulated mobilization of ET in human endothelial cells. (Circ Res. 1998;83:314-321.)

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID