摘要:

&NA;Manipulation of the mouse genome by traditional transgenic approaches has facilitated studies of gene function within the context of the intact organism and allowed for the creation of useful animal models of human disease. However, the timing of gene activation or repression is a critical determinant of phenotype, and the ability to regulate the temporal profile of transgene expression remains an important experimental goal. In this Mini Review, we describe the current status of systems to tightly regulate target gene expression in vivo, focusing on binary systems using chimeric transcription factors. Although experimental difficulties persist, regulated expression systems are beginning to produce conditional phenotypes with exciting experimental implications. We review the experience to date and examine the potential utility of these approaches within the context of cardiovascular medicine. (Circ Res. 1998;82:837‐844.)

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

&NA;We identified the cell surface glycoprotein Thy‐1 on the endothelium of newly formed blood vessels in four models of angiogenesis in adult rats. Anti‐Thy‐1 staining showed that Thy‐1 was upregulated in adventitial blood vessels after balloon injury to the carotid artery. Preabsorption with a rat Thy‐1‐Ig fusion construct eliminated all immunoreactivity and thus confirmed the specificity of the Thy‐1 staining. Thy‐1 was also expressed in the endothelium of small blood vessels formed after tumor implantation in the cornea, in periureteral vessels formed after ligation of the renal artery, and in small blood vessels of the uterus formed during pregnancy. In contrast with its expression during adult angiogenesis, Thy‐1 was not expressed in the endothelium of blood vessels during embryonic angiogenesis. In vitro, the inflammatory cytokines interleukin‐1 beta and tumor necrosis factor‐alpha upregulated Thy‐1 mRNA by 8‐ and 14‐fold, respectively. Vascular endothelial growth factor, basic fibroblast growth factor, transforming growth factor‐beta, and platelet‐derived growth factor‐BB had no effect on Thy‐1 mRNA. Thus, Thy‐1 appears to be a marker of adult but not embryonic angiogenesis. The upregulation of Thy‐1 by cytokines but not growth factors indicates the importance of inflammation in the pathogenesis of adult angiogenesis. (Circ Res. 1998;82:845‐851.)

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

&NA;Cyclic nucleotide phosphodiesterases (PDEs) hydrolyze cAMP or cGMP and terminate their signaling. Two important families of PDEs that regulate cAMP signaling in cardiovascular tissues are the cGMP‐inhibited PDEs (PDE3) and the cAMP‐specific PDEs (PDE4). In this study, we have used a combination of an in vitro motility assay and a sensitive method for the measurement of cAMP in order to determine the relative roles of PDE3 and of PDE4 in the regulation of cAMP‐mediated inhibition of VSMC migration. Our data demonstrate that forskolin, an activator of adenylyl cyclases, causes concentration‐dependent inhibition of platelet‐derived growth factor‐induced VSMC migration. Incubation of cultured VSMCs with a PDE4‐selective inhibitor, Ro 20‐1724, markedly potentiated both the antimigratory effect and the increase in cAMP caused by forskolin. Cilostamide, a PDE3‐selective compound, did not affect either the antimigratory activity of forskolin or its ability to increase cAMP. Cilostamide and Ro 20‐1724 interacted synergistically to potentiate the inhibition of VSMC migration by forskolin and caused a supra‐additive increase in cAMP. These data are consistent with an important role for both PDE3 and PDE4 in the regulation of cAMP‐mediated inhibition of VSMC migration. (Circ Res. 1998;82:852‐861.)

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

&NA;Endothelial cells in normal blood vessels might prevent the unscheduled proliferation of smooth muscle cells (SMCs) by the expression of cell migration and growth inhibitors. NO, a potent vasodilator, generated by endothelium‐specific constitutive NO synthase (ecNOS) might be such an inhibitor. To test this hypothesis, we overexpressed human ecNOS in syngeneic rat arterial SMCs using retrovirus‐mediated gene transfer. Compared with SMCs transduced with vector alone (LXSN SMCs), DNA synthesis and cell proliferation were inhibited in the ecNOS‐expressing SMCs (LCNSN SMCs). Basal and stimulated (by the calcium ionophore A23187) secretion of NO and intracellular cGMP were increased in LCNSN SMCs. Nomega‐Nitro‐L‐arginine(L‐NA), an inhibitor of NO synthesis, enhanced the proliferation of LCNSN SMCs but had no effect on LXSN SMCs. LCNSN SMCs seeded onto the luminal surface of balloon‐injured rat carotid arteries inhibited neointimal formation by 37% and induced marked dilatation (3‐fold increase in vessel diameter) at 2 weeks compared with LXSN SMC‐seeded arteries. Orally administered L‐NA blocked these changes. Phosphorylation of vasodilator‐stimulated phosphoprotein, which is regulated in part by NO, was elevated in LCNSN SMCs and in LCNSN SMC‐seeded arteries. This study demonstrates that NO generation by ecNOS inhibits SMC proliferation in vitro and modulates vascular tone locally in vivo. (Circ Res. 1998;82:862‐870.)

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

&NA;To study the role of vascular cell adhesion molecule‐1 (VCAM‐1) in monocyte recruitment and atherogenesis, we constructed a recombinant adenovirus, AdRSVrVCAM‐1, carrying the rabbit VCAM‐1 cDNA. We have previously shown that AdRSVrVCAM‐1‐transduced human umbilical vein endothelial cells (HUVECs) support the adhesion of CD4+CD45RO+memory T lymphocytes under laminar flow conditions. We now demonstrate that AdRSVrVCAM‐1‐transduced HUVECs support the adhesion of peripheral blood monocytes at a shear stress of <or=to1.5 dyne/cm2. Although VCAM‐1 supported only firm adhesion of lymphocytes, it was able to mediate monocyte rolling, firm adhesion, and transmigration when expressed in the context of otherwise unactivated vascular endothelium. VCAM‐1‐transduced HUVECs supported the adhesion of as many as 4‐fold more monocytes than T cells under laminar flow. The greater monocyte adhesion was explained at least in part by leukocyte‐leukocyte interactions (secondary adhesions), which were not seen with T cells. These secondary monocyte interactions were specifically blocked by monoclonal antibodies to L‐selectin and P‐selectin glycoprotein ligand‐1. These data demonstrate that VCAM‐1 expressed in the context of unactivated vascular endothelium supports the adhesion of the leukocyte populations present in atherosclerotic plaque and may contribute to the predominance of monocytes over lymphocytes. (Circ Res. 1998;82:871‐878.)

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

&NA;Spontaneous rupture of the internal elastic lamina (IEL) occurs in some arteries of the rat during growth and aging. Inbred, normotensive, Brown Norway (BN) rats are particularly susceptible to rupture of the IEL, especially in the abdominal aorta (AA). Preliminary experiments showed that different angiotensin‐converting enzyme (ACE) inhibitors protect against rupture of the IEL in the BN rat to a greater extent than hydralazine, suggesting a role of the renin‐angiotensin system (RAS) in this phenomenon. To explore this possibility, we have treated male BN rats from 4.5 to 14 weeks of age with either enalapril or losartan (both at 1, 3, and 10 mg [center dot] kg‐1[center dot] d‐1) or with the calcium antagonists mibefradil (at 3, 10, 30, and 45 mg [center dot] kg‐1[center dot] d‐1) and amlodipine (at 30 mg [center dot] kg‐1[center dot] d‐1). Systolic blood pressure (SBP) was measured weekly, and at the end of treatment we (1) recorded body and heart weights, (2) measured various parameters of the RAS in plasma, (3) quantified interruptions in the IEL on “en face” preparations of AA, and (4) quantified elastin, collagen, and cell proteins in the media of the thoracic aorta. Results showed that enalapril and losartan similarly decrease SBP and rupture of the IEL in the AA, suggesting that enalapril inhibits the latter via a decrease in the production of angiotensin II (Ang II) and not via another effect on ACE. The decrease in IEL rupture and in SBP, as well as the modifications in the parameters of the RAS, were all dose dependent. Mibefradil had little effect on the RAS and, at the highest doses, decreased SBP to an extent similar to that for enalapril at 3 mg [center dot] kg‐1[center dot] d‐1but did not significantly inhibit IEL rupture. Amlodipine decreased SBP, increased plasma renin concentration, and was without effect on IEL rupture. All treatments at the highest doses had a hypotrophic effect on the aortic media but differed in their effects on the heart, with enalapril and losartan decreasing and mibefradil and amlodipine increasing heart weight, suggesting that the inhibition of IEL rupture may be related to a cardiac hypotrophic effect. All these results, taken together, suggest that Ang II plays a role in the rupture of the IEL that is, in part, independent of SBP. (Circ Res. 1998;82:879‐890.)

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

&NA;This study was designed to investigate the potential role of endogenous peroxynitrite (ONOO‐) formation in the inhibition of cardiac muscle contractility and mitochondrial respiration during posthypoxic reoxygenation. Isometric contraction of isolated rat left ventricular posterior papillary muscle was virtually eliminated at the end of an exposure to 15 minutes of hypoxia and remained 40 +/‐ 5% depressed an hour after the reintroduction of O2. O2uptake by rat left ventricular cardiac muscle, measured by a Clark‐type O2electrode, was also inhibited by 24 +/‐ 2% at 10 minutes after reoxygenation. The inhibition of contractility and respiration during posthypoxic reoxygenation was markedly attenuated by the NO synthase inhibitor nitro‐L‐arginine, exogenous superoxide dismutase, and the ONOO‐scavenger urate but not by the hydroxyl radical scavenger mannitol. Generation of ONOO‐with the NO donor S‐nitroso‐N‐acetylpenicillamine (SNAP) plus the superoxide‐releasing agent pyrogallol caused an irreversible inhibition of cardiac contractile and respiratory function. Unlike ONOO‐, exogenous (SNAP) and endogenous (bradykinin) sources of NO inhibited contractility in a reversible manner. Under conditions of comparable amounts of respiratory inhibition in unstimulated incubated muscle, the NO‐dependent agents and the mitochondrial antagonist NaCN produced a smaller degree of suppression of contractility compared with ONOO‐and posthypoxic reoxygenation. These results are consistent with a contributing role for endogenous ONOO‐formation in the inhibition of cardiac muscle contractility and mitochondrial respiration during posthypoxic reoxygenation. (Circ Res. 1998;82:891‐897.)

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

&NA;Our purpose was to determine whether hearts from mice bioengineered to lack either the M isoform of creatine kinase (MCK‐/‐ mice) or both the M and mitochondrial isoforms (M/MtCK‐/‐ mice) have deficits in cardiac contractile function and energetics, which have previously been reported in skeletal muscle from these mice. The phenotype of hearts with deleted creatine kinase (CK) genes is of clinical interest, since heart failure is associated with decreased total CK activity and changes in the relative amounts of the CK isoforms in the heart. We measured isovolumic contractile performance in isolated perfused hearts from wild‐type, MCK (‐)/‐, and M/MtCK‐/‐ mice simultaneously with cardiac energetics (31) P‐nuclear magnetic resonance spectroscopy) at baseline, during increased cardiac work, and during recovery. Hearts from wild‐type, MCK‐/‐, and M/MtCK‐/‐ mice had comparable baseline function and responded to 10 minutes of increased heart rate and perfusate Ca2+with similar increases in rate‐pressure product (48 +/‐ 5%, 42 +/‐ 6%, and 51 +/‐ 6%, respectively). Despite a similar contractile response, M/MtCK‐/‐ hearts increased [ADP] by 95%, whereas wild‐type and MCK‐/‐ hearts maintained [ADP] at baseline levels. The free energy released from ATP hydrolysis decreased by 3.6 kJ/mol in M/MtCK‐/‐ hearts during increased cardiac work but only slightly in wild‐type (1.7 kJ/mol) and MCK‐/‐ (1.5 kJ/mol) hearts. In contrast to what has been reported in skeletal muscle, M/MtCK‐/‐ hearts were able to hydrolyze and resynthesize phosphocreatine. Taken together, our results demonstrate that when CK activity is lowered below a certain level, increases in cardiac work become more “energetically costly” in terms of high‐energy phosphate use, accumulation of ADP, and decreases in free energy released from ATP hydrolysis, but not in terms of myocardial oxygen consumption. (Circ Res. 1998;82:898‐907.)

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

&NA;The smooth muscle myosin heavy chain (SM‐MHC) gene encodes a major contractile protein whose expression exclusively marks the smooth muscle cell (SMC) lineage. To better understand smooth muscle differentiation at the transcriptional level, we have initiated studies to identify those DNA sequences critical for expression of the SM‐MHC gene. Here we report the identification of an SM‐MHC promoter‐intronic DNA fragment that directs smooth muscle‐specific expression in transgenic mice. Transgenic mice harboring an SM‐MHC‐lacZ reporter construct containing [approximate]16 kb of the SM‐MHC genomic region from ‐4.2 to +11.6 kb (within the first intron) expressed the lacZ transgene in all smooth muscle tissue types. The inclusion of the intronic sequence was required for transgene expression, since 4.2 kb of the 5[prime]‐flanking region alone was not sufficient for expression. In the adult mouse, transgene expression was observed in both arterial and venous smooth muscle, in airway smooth muscle of the trachea and bronchi, and in the smooth muscle layers of all abdominal organs, including the stomach, intestine, ureters, and bladder. During development, transgene expression was first detected in airway SMCs at embryonic day 12.5 and in vascular and visceral SMC tissues by embryonic day 14.5. Of interest, expression of the SM‐MHC transgene was markedly reduced or absent in some SMC tissues, including the pulmonary circulation. Moreover, the transgene exhibited a heterogeneous pattern between individual SMCs within a given tissue, suggesting the possibility of the existence of different SM‐MHC gene regulatory programs between SMC subpopulations and/or of episodic rather than continuous expression of the SM‐MHC gene. To our knowledge, results of these studies are the first to identify a promoter region that confers complete SMC specificity in vivo, thus providing a system with which to define SMC‐specific transcriptional regulatory mechanisms and to design vectors for SMC‐specific gene targeting. (Circ Res. 1998;82:908‐917.)

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

&NA;Delivery of a strong electric shock to the heart remains the only effective therapy against ventricular fibrillation. Despite significant improvements in implantable cardioverter defibrillator (ICD) therapy, the fundamental mechanisms of defibrillation remain poorly understood. We have recently demonstrated that a monophasic defibrillation shock produces a highly nonuniform epicardial polarization pattern, referred to as a virtual electrode pattern (VEP). The VEP consists of large adjacent areas of strong positive and negative polarization. We sought to determine whether the VEP may be responsible for defibrillation failure by creating dispersion of postshock repolarization and reentry. Truncated exponential biphasic and monophasic shocks were delivered from a bipolar ICD lead in Langendorff‐perfused rabbit hearts. Epicardial electrical activity was mapped during and after defibrillation shocks and shocks applied at the plateau phase of a normal action potential produced by ventricular pacing. A high‐resolution fluorescence mapping system with 256 recording sites and a voltage‐sensitive dye were used. Biphasic shocks with a weak second phase (<20% leading‐edge voltage of the second phase with respect to the leading‐edge voltage of the first phase) produced VEPs similar to monophasic shocks. Biphasic shocks with a strong second phase (>70%) produced VEPs of reversed polarity. Both of these waveforms resulted in extra beats and arrhythmias. However, biphasic waveforms with intermediate second‐phase voltages (20% to 70% of first‐phase voltage) produced no VEP, because of an asymmetric reversal of the first‐phase polarization. Therefore, there was no substrate for postshock dispersion of repolarization. Shocks producing strong VEPs resulted in postshock reentrant arrhythmias via a mechanism of phase singularity. Points of phase singularity were created by the shock in the intersection of areas of positive, negative, and no polarization, which were set by the shock to excited, excitable, and refractory states, respectively. Shock‐induced VEPs may reinduce arrhythmias via a phase‐singularity mechanism. Strong shocks may overcome the preshock electrical activity and create phase singularities, regardless of the preshock phase distribution. Optimal defibrillation waveforms did not produce VEPs because of an asymmetric effect of phase reversal on membrane polarization. (Circ Res. 1998;82:918‐925.)

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID