ISSN:0009-7330    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

We describe a straightforward gene-targeting technique to achieve uniform, stable, and genetically invariant expression of a transgene in the vascular endothelium of mice. To demonstrate the feasibility of this approach, the reporter gene bacterial beta-galactosidase was inserted via homologous recombination into the intronless thrombomodulin locus of murine embryonic stem cells. In this fashion, the lacZ gene is placed under the regulatory control of the endogenous thrombomodulin promoter. The expression of the transgene in adult mice recapitulated the widespread, stable, and high-level expression of the thrombomodulin gene in vascular endothelium. These data indicate that targeting of cDNAs into the thrombomodulin locus serves as a viable strategy to express transgenes in endothelial cells. Analysis of reporter gene expression revealed a heterogeneous pattern of thrombomodulin gene activity in the endothelium of the aorta and its tributaries. We also show that embryonic stem cells with a targeted thrombomodulin locus contribute in a mosaic fashion to the vascular endothelium of chimeric mice. This method for generating animals with a functionally heterogeneous cardiovascular system should provide an experimental technique for studying how localized genetic abnormalities in endothelial cell function lead to the development of vascular diseases.(Circ Res. 1996;78:180-187.)

ISSN:0009-7330    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

SM22 alpha is a calponin-related protein that is expressed specifically in adult smooth muscle. To begin to define the mechanisms that regulate the establishment of the smooth muscle lineage, we analyzed the expression pattern of the SM22 alpha gene during mouse embryogenesis. In situ hybridization demonstrated that SM22 alpha transcripts were first expressed in vascular smooth muscle cells at about embryonic day (E) 9.5 and thereafter continued to be expressed in all smooth muscle cells into adulthood. In contrast to its smooth muscle specificity in adult tissues, SM22 alpha was expressed transiently in the heart between E8.0 and E12.5 and in skeletal muscle cells in the myotomal compartment of the somites between E9.5 and E12.5. The expression of SM22 alpha in smooth muscle cells, as well as early cardiac and skeletal muscle cells, suggests that there may be commonalities between the regulatory programs that direct muscle-specific gene expression in these three myogenic cell types.(Circ Res. 1996;78:188-195.)

ISSN:0009-7330    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

Proliferation and phenotypic modulation of smooth muscle cells (SMCs) are major components of the vessel's response to injury in experimental models of restenosis. Some of the growth factors involved in restenosis have been identified, but to date little is known about the transcription factors that ultimately regulate this process. We examined the expression of the four members of the myocyte enhancer binding factor-2 (MEF2) family of transcription factors in cultured rat aortic SMCs (RASMCs) and a rat model of restenosis because of their known importance in regulating the differentiated phenotype of skeletal and cardiac muscle. In skeletal and cardiac muscle, the MEF2s are believed to be important for activating the expression of contractile protein and other muscle-specific genes. Therefore, we anticipated that the MEF2s would be expressed at high levels in medial SMCs that are producing contractile proteins and that they would be downregulated along with the contractile protein genes in neointimal SMCs. On the contrary, we observe that MEF2A, MEF2B, and MEF2D mRNAs are upregulated in the neointima, with the highest levels in the layer of cells nearest to the lumen, whereas MEF2C mRNA levels do not appreciably increase. Moreover, few cells in the media are making MEF2 proteins detectable by immunohistochemistry, whereas large numbers of neointimal cells are positive for all four MEF2s. These data suggest that the MEF2s are involved in the activated smooth muscle phenotype and not in the maintenance of contractile protein gene expression.(Circ Res. 1996;78:196-204.)

ISSN:0009-7330    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

The QCE-6 cell line was derived from precardiac mesoderm of the Japanese quail. As previously reported, these cells are able to differentiate into two distinct cardiac cell types with myocardial or endocardial endothelial cell properties. This present communication describes in detail the derivation of this cell line and further characterizes the nontreated and induced myocardial and endothelial phenotypes of these cells. The QCE-6 cells exhibit an epithelial morphology, as well as the pattern of protein expression, that is characteristic of precardiac mesoderm. Treatment with retinoic acid, basic fibroblast growth factor (bFGF), transforming growth factor (TGF)-beta 2, and TGF-beta 3 induces these cells to differentiate and produce mixed cultures of epithelial and mesenchymal cells. The epithelial cells express myosin, desmin, and cardiac troponin I in a punctate pattern throughout the cytoplasm. These sarcomeric proteins become organized in a premyofibrillar pattern when TGF-beta 1, platelet-derived growth factor (PDGF)-BB, and insulin-like growth factor (IGF) II are added in combination along with retinoic acid, bFGF, TGF-beta 2, and TGF-beta 3, Also, these treatments induce Naplus, Kplus-ATPase expression. When the QCE-6 cells are cultured on collagen type I, the mesenchymal cells that are promoted by retinoic acid, bFGF, TGF-beta 2, and TGF-beta 3 will invade the gel. These mesenchymal cells are positive for QH1 and JB3, which are both markers for presumptive endocardial cells within the early cardiogenic mesoderm. The addition of both PDGF-BB and IGF II to QCE-6 cell cultures will inhibit the ability of retinoic acid, bFGF, TGF-beta 2, and TGF-beta 3 to induce both the mesenchymal morphology and QH1 and JB3 expression. Collectively, these results suggest that the process of cardiac cell differentiation is regulated by multiple signals and that early cardiogenic mesoderm contains a bipotential stem cell that can give rise to both the myocardial and endocardial lineages. More important, since the QCE-6 cells are representative of early cardiogenic cells, this cell line offers a unique model system to study cardiac cell differentiation.(Circ Res. 1996;78:205-216.)

ISSN:0009-7330    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

Cardiac myocytes have recently been shown to express a constitutive Ca sup 2 plus-sensitive isoform of NO synthase (NOS3), although the mechanism(s) responsible for activation of NOS3 and its physiological function remain to be determined. Since the activity of NOS3 is known to be regulated in part by the intracellular Ca2plus activity ([Ca2plus]i) in endothelial cells, we determined whether increasing myocyte [Ca2plus]iby uniform electric field pacing was accompanied by an increase in NOS3 activity, detected as nitrite accumulation in the medium. A higher [Ca2plus]iwith increasing pacing frequencies was shown to be accompanied by a time-dependent accumulation of nitrite in medium that bathed adult rat ventricular myocytes stimulated at 3 Hz. Nitrite release by paced cells was significantly attenuated by treatment with either the NO synthase inhibitor nitro-L-arginine (L-NA, 1 mmol/L) or the intracellular Ca2plus chelator BAPTA-AM (20 mu mol/L). Paced myocytes also exhibited a frequency- and time-dependent increase in intracellular cGMP content that could be inhibited significantly by either L-NA or the soluble guanylate cyclase inhibitor LY83583 (5 mu mol/L). To determine whether the increase in NOS3 activity with pacing affected contractile function, myocytes were sequentially paced at frequencies from 0.5 to 3 Hz. Methylene blue, L-NA, and LY83583 all increased the amplitude of shortening of myocytes paced at 3 Hz. Furthermore, a significantly greater positive inotropic response to high extracellular Ca2plus (3 mmol/L) was demonstrated by myocytes pretreated with L-NA compared with control cells. These data indicate that myocyte NOS3 activity is regulated in part by [Ca2plus] sub i, whether induced by changes in pacing frequency or [Ca2plus]o, and depresses myocyte contractile responsiveness to higher stimulation frequencies.(Circ Res. 1996;78:217-224.)

ISSN:0009-7330    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

Augmentation of nitric oxide (NO) production in vivo decreases lesions in a variety of models of arterial injury, and inhibition of NO synthase exacerbates experimental intimal lesions. Both vascular smooth muscle cell (VSMC) prolifcration and migration contribute to lesion formation. Although NO inhibits VSMC proliferation, its effects on VSMC migration are unknown. To test the hypothesis that NO inhibits VSMC migration independent of inhibition of proliferation, we examined migration of rat aortic VSMCs after wounding of a confluent culture in the presence of chemical donors of NO. Hydroxyurea was used to eliminate any confounding effect of NO on proliferation. Three NO donors, diethylamine NONOate, spermine NONOate, and S-nitrosoglutathione, exhibited concentration-dependent inhibition of both number of migrating VSMCs and maximal distance migrated. Inhibition of migration was also seen with 8-Br-cGMP, suggesting that activation of guanylate cyclase may play a role in mediating the antimigratory effects of NO. Migration resumed after removal of NO donors, as evidenced by an increase in distance migrated. Measurement of VSMC protein synthesis and mitochondrial respiration indicated that inhibition of migration by NO donors was not due to metabolic cytostasis. These findings indicate that NO reversibly inhibits VSMC migration independent of proliferation or cytotoxicity, a novel mechanism by which both endogenous and pharmacological NO may alter vascular pathology.(Circ Res. 1996;78:225-230.)

ISSN:0009-7330    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

The villi of the small intestine maintain a hypertonic interstitium at all times, and the submucosal glands constantly secrete ions and accompanying water into the lumen. Generation of the 400- to 600-mOsm interstitial fluid in the villus and secretion by glands may require a large expenditure of energy and, consequently, have major effects on intestinal vascular regulation to supply oxygen and nutrients. Blood flow and oxygen consumption were measured in the ileum of anesthetized rats during natural resting conditions with physiological sodium chloride in the bathing fluid and during isosmotic replacement of sodium chloride with mannitol. Microvascular pressures and blood flow were used to determine the changes in resistance of the major arterioles and the terminal vasculature. When mannitol replaced sodium chloride in contact with the villi, intestinal blood flow decreased to 58.6 plus minus 2.8% of control, and oxygen consumption was 54.2 plus minus 3.4% of control. Resistance of the major arterioles increased 101.7 plus minus 9.9%, and that of the terminal vasculature increased 40.4 plus minus 6.2%. The increased resistance appeared to be caused by suppression of a nitric oxide mechanism. Local application of 10minus4 mol/L NG-nitro-L-arginine methyl ester caused about the same reduction in flow and increases in regional vascular resistance as during replacement of sodium but did not alter the oxygen consumption. These data indicate that about half of the intestinal metabolic rate during natural resting conditions is devoted to sodium secretion/absorption. Large resistance vessels are dilated to maintain a high blood flow through release of nitric oxide. We propose that dilation of the terminal vasculature in the metabolically active tissues increased flow velocity sufficiently in the major resistance vessels to cause a flow-mediated release of nitric oxide.(Circ Res. 1996;78:231-237.)

ISSN:0009-7330    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

The effect of an opener (levcromakalim) and a blocker (glibenclamide) of ATP-sensitive Kplus(KATP) channels was investigated in the vertebrobasilar system of the rabbit. Arterial tension and membrane potential were measured by the isometric tension recording method and the microelectrode technique, respectively. Glibenclamide (10minus6 mol/L) depolarized the membrane and potentiated the contraction to histamine in vertebral arteries. The sensitivity to the relaxant effects of levcromakalim was in the following descending order: vertebral more than proximal basilar more than distal basilar more than superior cerebellar arteries. Vertebral arteries were approximate equals 50 times more sensitive to levcromakalim than were superior cerebellar arteries. The relaxation to levcromakalim was abolished by glibenclamide (10minus6 mol/L). Glibenclamide attenuated vasorelaxation to adenosine in proximal arteries (vertebral and proximal basilar) but not in superior cerebellar arteries. Levcromakalim (7 times 10minus8 mol/L) and adenosine (10minus5 mol/L) induced glibenclamide-sensitive membrane hyperpolarization in vertebral arteries but not in distal basilar arteries. These results suggest that KATPchannels contribute to the determination of resting membrane potential and resting tone in vertebral arteries. Furthermore, there is a marked heterogeneity in the sensitivity to an opener of KATPchannels, and the heterogeneity has a functional link to the mechanism underlying vasorelaxation to adenosine in the vertebrobasilar system of the rabbit.(Circ Res. 1996;78:238-243.)

ISSN:0009-7330    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

Voltage-gated Napluschannels are essential for the normal electrical excitability of neuronal and striated muscle membranes. Distinct isoforms of the Napluschannel alpha-subunit have been identified by molecular cloning, and their functional attributes have been defined by heterologous expression coupled with electrophysiological recording. Two closely related Napluschannel alpha-subunit isoforms, hH1 (human heart) and hSkM1 (human skeletal muscle), exhibit differences in their inactivation properties and in their response to the coexpressed beta1-subunit. To localize regions that contribute to inactivation and to beta1-subunit response, we have exploited these functional differences by studying chimeric channels composed of segments from both hH1 and hSkM1. Chimeras in which one or more of the cytoplasmic interdomain regions (ID1-2, ID2-3, and ID3-4) were exchanged between hH1 and hSkM1 exhibit inactivation properties identical with the background channel isoform, suggesting that these regions are not sufficient to cause gating differences. In contrast, inactivation properties of chimeras composed of approximately equal halves of the two channel isoforms were intermediate between hH1 and hSkM1. Furthermore, the response to the coexpressed beta1-subunit was dependent on structures located in the carboxy-terminal half of the alpha-subunit, although domains D3, D4, and the carboxy terminal are not singularly responsible for this effect. These data indicate that inactivation differences between hH1 and hSkM1 are determined by multiple alpha-subunit domains.(Circ Res. 1996;78:244-252.)

ISSN:0009-7330    

出版商:OVID    

年代:1996

数据来源: OVID