Gene transfer in utero biologically engineers a patent ductus arteriosus in lambs by arresting fibronectin-dependent neointimal formation

Closure of the ductus arteriosus requires prenatal formation of intimal cushions, which occlude the vessel lumen at birth. Survival of newborns with severe congenital heart defects, however, depends on ductal patency. We used a gene transfer approach to create a patent ductus arteriosus by targeting the fibronectin-dependent smooth muscle cell migration required for intimal cushion formation. Fetal lamb ductus arteriosus was transfected in utero with hemagglutinating virus of Japan liposomes containing plasmid encoding 'decoy' RNA to sequester the fibronectin mRNA binding protein. Fibronectin translation was inhibited and intimal cushion formation was prevented. We thus established the essential role of fibronectin-dependent smooth muscle cell migration in intimal cushion formation in the intact animal and the feasibility of incorporating biological engineering in the management of congenital heart disease.     

Aortic carboxypeptidase-like protein, a novel protein with discoidin and carboxypeptidase-like domains, is up-regulated during vascular smooth muscle cell differentiation

Phenotypic modulation of vascular smooth muscle cells plays an important role in the pathogenesis of arteriosclerosis. In a screen of proteins expressed in human aortic smooth muscle cells, we identified a novel gene product designated aortic carboxypeptidase-like protein (ACLP). The approximately 4-kilobase human cDNA and its mouse homologue encode 1158 and 1128 amino acid proteins, respectively, that are 85% identical. ACLP is a nonnuclear protein that contains a signal peptide, a lysine- and proline-rich 11-amino acid repeating motif, a discoidin-like domain, and a C-terminal domain with 39% identity to carboxypeptidase E. By Western blot analysis and in situ hybridization, we detected abundant ACLP expression in the adult aorta. ACLP was expressed predominantly in the smooth muscle cells of the adult mouse aorta but not in the adventitia or in several other tissues. In cultured mouse aortic smooth muscle cells, ACLP mRNA and protein were up-regulated 2-3-fold after serum starvation. Using a recently developed neural crest cell to smooth muscle cell in vitro differentiation system, we found that ACLP mRNA and protein were not expressed in neural crest cells but were up-regulated dramatically with the differentiation of these cells. These results indicate that ACLP may play a role in differentiated vascular smooth muscle cells.     

Molecular mechanism of regulation of pentylenetetrazol-induced calcium entry by 3'-untranslated region of a seizure-related cDNA, PTZ-17, in Xenopus oocytes

To determine the molecular mechanism of regulation of pentylenetetrazol (PTZ)-induced calcium entry by the seizure-related gene, PTZ-17, the role of the 3'-untranslated region (3'UTR) and also interaction between 3'UTR and intracellular factors were investigated. PTZ-induced calcium inward current in Xenopus oocytes injected with PTZ-17 RNA varied in magnitude among strains of mice: RNA derived from the DBA/2 mouse, which has a high susceptibility to convulsions, showed the largest current and that from the BALB/c mouse with a low susceptibility to convulsions showed no PTZ response. The sequence of 3'UTR showed alterations among mouse strains: 3'UTR of BALB/c showed a sequence alteration from T to G and that of DBA/2 showed a GTG insertion compared with that of B6. The 3'UTR also regulated the translation of chloramphenicol acetyltransferase (CAT) RNA depending on its sequence. A particular region within the 3'UTR demonstrated interaction with 60- and 47-kDa proteins. Sequence alterations in this region corresponded to disappearance or increase in PTZ-induced calcium entry. These findings suggest that a particular region within 3'UTR of the seizure-related gene, PTZ-17, is involved in PTZ-induced calcium entry via interaction between mRNA and specific RNA-binding proteins.     

Transitions in cell organization and in expression of contractile and extracellular matrix proteins during development of chicken aortic smooth muscle: evidence for a complex spatial and temporal differentiation program

Whereas the understanding of the mechanisms underlying skeletal and cardiac muscle development has been increased dramatically in recent years, the understanding of smooth muscle development is still in its infancy. This paper summarizes studies on the ontogeny of chicken smooth muscle cells in the wall of the aorta and aortic arch-derived arteries. Employing immunocytochemistry with antibodies against smooth muscle contractile and extracellular matrix proteins we trace smooth muscle cell patterning from early development throughout adulthood. Comparing late stage embryos to young and adult chickens we demonstrate, for all the stages analyzed, that the cells in the media of aortic arch-derived arteries and of the thoracic aorta are organized in alternating lamellae. The lamellar cells, but not the interlamellar cells, express smooth muscle specific contractile proteins and are surrounded by basement membrane proteins. This smooth muscle cell organization of lamellar and interlamellar cells is fully acquired by embryonic day 11 (ED 11). We further show that, during earlier stages of embryogenesis (ED3 through ED7), cells expressing smooth muscle proteins appear only in the peri-endothelial region of the aortic and aortic arch wall and are organized as a narrow band of cells that does not demonstrate the lamellar-interlamellar pattern. On ED9, infrequent cells organized in lamellar-interlamellar organization can be detected and their frequency increases by ED10. In addition to changes in cell organization, we show that there is a characteristic sequence of contractile and extracellular matrix protein expression during development of the aortic wall. At ED3 the peri-endothelial band of differentiated smooth muscle cells is already positive for smooth muscle alpha actin (alphaSM-actin) and fibronectin. By the next embryonic day the peri-endothelial cell layer is also positive for smooth muscle myosin light chain kinase (SM-MLCK). Subsequently, by ED5 this peri-endothelial band of differentiated smooth muscle cells is positive for alphaSM-actin, SM-MLCK, SM-calponin, fibronectin, and collagen type IV. However, laminin and desmin (characteristic basement membrane and contractile proteins of smooth muscle) are first seen only at the onset of the lamellar-interlamellar cell organization (ED9 to ED10). We conclude that the development of chicken aortic smooth muscle involves transitions in cell organization and in expression of smooth muscle proteins until the adult-like phenotype is achieved by mid-embryogenesis. This detailed analysis of the ontogeny of chick aortic smooth muscle should provide a sound basis for future studies on the regulatory mechanisms underlying vascular smooth muscle development.     

Insulin-like growth factor binding protein production by bovine and human vascular smooth muscle cells: production of insulin-like growth factor binding protein-6 by human smooth muscle

Insulin-like growth factor binding protein (IGFBP) secretory profiles were determined for vascular smooth muscle cells (VSMC) derived from bovine aorta and human aorta, pulmonary artery, and coronary artery. The bovine cells produced IGFBP-4, IGFBP-3, and an IGFBP-3 protease. IGF-I stimulated messenger RNA (mRNA) and media levels of IGFBP-3. The human cells produced IGFBP-3, IGFBP-4, and IGFBP-3 and IGFBP-4 proteases. The three human cells also produced a 30K IGFBP, shown to be IGFBP-6, based on increased affinity for IGF-II vs. IGF-I, size decrease when treated with O-glycanase, but not N-glycanase, reactivity with IGFBP-6 antiserum, presence of a 1.3-kilobase pair mRNA that hybridized to IGFBP-6 specific complementary DNA, and N-terminal amino acid sequence corresponding to IGFBP-6. In the human cells, IGF-I increased media levels of IGFBP-3 through stimulation of IGFBP-3 mRNA and dissociation of cell bound IGFBP-3, and decreased IGFBP-4 via potentiation of IGFBP-4 proteolysis. Neither the bovine nor the human aorta VSMC produced sufficient IGFBP-2 or IGFBP-2 mRNA to be detected by ligand blot and Northern analysis, as previously reported for porcine and rat aorta smooth muscle cells. The variable expression of IGFBPs and IGFBP proteases by VSMC are likely to contribute to differential vascular reactivity to the IGFs in larger arterial blood vessels.     

Autoxidation of pyridoxalated hemoglobin polyoxyethylene conjugate

Vascular endothelial growth factor (VEGF) is an endothelial cell mitogen which stimulates angiogenesis. VEGF is regulated by multiple factors such as hypoxia, phorbol esters, and growth factors. However, data concerning the expression of VEGF in the different vascular cell types and its regulation by cAMP are not available. In the present study, we have investigated the effect of adenylate cyclase activation on VEGF mRNA expression in rat vascular cells in primary culture. Basal VEGF expression is greater in smooth muscle cells than in endothelial cells and fibroblasts. A 4-h treatment with forskolin (10(-5) M) induced a 2-fold stimulation of VEGF mRNA expression in smooth muscle cells and fibroblasts, but, in contrast, did not affect VEGF expression in endothelial cells. In smooth muscle cells, a pharmacologically induced increase in intracellular cAMP levels using iloprost or isoprenaline led to a rise in VEGF mRNA expression comparable to that induced by forskolin. Adenosine, which increases cAMP levels in smooth muscle cells, also increases VEGF expression. Moreover, the 2.2-fold stimulation of VEGF expression by adenosine was enhanced following a cotreatment with cobalt chloride (a hypoxia miming agent). The observed additive effect (4.3-fold increase) suggests that these two factors, hypoxia and adenosine, regulate VEGF mRNA expression in smooth muscle cells by independent mechanisms.     

Specific determination of PAH and its N-acetyl metabolite by HPLC increases the accuracy and precision of PAH clearance measurements

Alpha2-adrenergic receptors (alpha2-ARs) in vascular smooth muscle cells are known to mediate vasoconstriction; however, it is unknown which of the 3 subtypes of alpha2-AR (alpha2A, alpha2B, or alpha2C) is expressed in vascular tissue. We have used subtype-specific probes in in situ hybridization and RNase protection assays to analyze the expression of alpha2-AR in the thoracic aorta of New Zealand White (NZW) and Watanabe heritable hyperlipidemic (WHHL) rabbits, a model for atherosclerosis. We found that the alpha2A-AR mRNA was in endothelial and smooth muscle cells in both NZW and WHHL aorta. In addition, the shoulders and subendothelial regions of the atherosclerotic lesions in WHHL aorta showed abundant expression of alpha2A-AR mRNA. Antibodies to macrophage (RAM-11) and smooth muscle cell (HHF-35) antigens were used to localize macrophage and smooth muscle cells in aortic sections from WHHL rabbits. The expression of alpha2A-AR mRNA within the lesions of WHHL rabbits correlated with the presence of infiltrating macrophages. We discuss the potential role of alpha2A-ARs in macrophage function and in promoting atherosclerosis.     

Serotonin stimulates protein tyrosyl phosphorylation and vascular contraction via tyrosine kinase

Serotonin (5-HT, 5-hydroxytryptamine) is a mitogen in vascular smooth muscle and vascular reactivity to 5-HT is significantly enhanced in hypertension and atherosclerosis. We have tested the hypothesis that tyrosine kinases, enzymes important for mitogenesis, may play a role in 5-HT-induced vascular smooth muscle contractility. Helical strips of rat carotid artery and aorta denuded of endothelium were mounted in tissue baths for measurement of contractile force. The tyrosine kinase inhibitor genistein (5 x 10(-6) M) decreased the potency of 5-HT approximately 4-fold and reduced maximal contraction to 5-HT in carotid arterial strips denuded of endothelium (58% control). Genistein's inactive congener daidzein (5 x 10(-6) M) did not reduce maximal contraction to 5-HT in carotid arteries but did shift the 5-HT concentration response curve 3-fold to the right. Tyrphostin 23 (5 x 10(-5) M), another tyrosine kinase inhibitor, decreased the potency of 5-HT 4-fold and reduced the maximal contraction to 5-HT in the carotid artery (10% control). Contractions induced by phorbol-12,13-dibutyrate (10(-9) to 10(-5) M) were not reduced or shifted by either tyrosine kinase inhibitor, indicating that phorbolester-sensitive protein kinase C isoforms were not affected. KCl-induced contraction was shifted 2-fold and the maximum significantly inhibited by tyrphostin 23 (38.6% control) but not genistein or daidzein, indicating that tyrphostin 23 but not genistein may inhibit voltage-gated calcium channels to reduce contractility. Western blot analysis using antiphosphotyrosine antibody confirmed that 5-HT produced a time- and concentration-dependent increase in the phosphotyrosine immunoreactivity of a 42-kD protein in cultured aortic smooth muscle cells. Lysate immunoprecipitation with an antimitogen-activated-protein (MAP)-kinase antibody indicated that the 42-kD protein was most likely a MAP kinase. 5-HT (10(-5) M) stimulated contraction and increased antiphosphotyrosine immunoreactivity in whole aorta mounted in tissue baths. Importantly, aortic contraction to 5-HT was shifted (5-fold rightward) and reduced (69% control) by genistein but not daidzein. These findings demonstrate that (1) tyrosine kinase activation may partially mediate contractility to 5-HT in arterial smooth muscle, (2) tyrphostin 23 is somewhat nonselective and (3) 5-HT stimulates tyrosine kinase as documented by increased tyrosyl phosphorylation of proteins in cultured aortic smooth muscle cells and aortic tissue in active contraction of 5-HT. These findings have significant implications not only in understanding a novel pathway of 5-HT signal transduction but also in vascular diseases in which growth and/or contractility to 5-HT is increased (e.g. hypertension, atherosclerosis).     

HSP101 functions as a specific translational regulatory protein whose activity is regulated by nutrient status

The 5' leader (Omega) of tobacco mosaic viral RNA functions as a translational enhancer. Sequence analysis of a 102-kD protein, identified previously as a specific Omega RNA-binding protein, revealed homology to the HSP101/HSP104/ClpB family of heat shock proteins and its expression in yeast complemented a thermotolerance defect caused by a deletion of the HSP104 gene. Up to a 50-fold increase in the translation of Omega-luc, but not luc mRNA was observed in yeast expressing the tobacco HSP101 whereas Omega failed to enhance translation in the absence of HSP101. Therefore, HSP101 and Omega comprise a two-component translational regulatory mechanism that can be recapitulated in yeast. Analysis of HSP101 function in yeast translation mutants suggested that the initiation factor (eIF) 3 and specifically one (TIF4632) of the two eIF4G proteins were required for the HSP101-mediated enhancement. The RNA-binding and translational regulatory activities of HSP101 were inactive in respiring cells or in cells subject to nutrient limitation, but its thermotolerance function remained unaffected. This is the first identification of a protein required for specific translational enhancement of capped mRNAs, the first report of a translational regulatory function for any heat-shock protein, and the first functional distinction between the two eIF4G proteins present in eukaryotes.     

Inhibition of vascular smooth muscle cell growth by inhibition of fibronectin matrix assembly

The regulation of vascular smooth muscle cell (VSMC) proliferation by the fibronectin matrix was tested by treating human umbilical artery smooth muscle cells (HUASMCs) with a recombinant fragment of fibronectin (protein III1-C) that has previously been shown to modulate fibronectin matrix assembly. III1-C inhibited HUASMC proliferation by 75% to 90%. The inhibition of growth was time dependent; III1-C had no effect on DNA synthesis after 0 to 5 hours of treatment but did have an effect at 24 hours and beyond. III1-C did not stimulate apoptosis in these cells, indicating that the inhibition of proliferation was not due to an induction of programmed cell death. The effects of III1-C on cell growth were only specific for normal diploid smooth muscle cells. III1-C had no effect on the proliferation of IMR-90 fibroblasts, endothelial cells, NIH 3T3 cells, or the rat aortic smooth muscle cell line A7r5. However, III1-C did inhibit proliferation by primary rat aortic smooth muscle cells. An analysis of HUASMC fibronectin receptor (integrin alpha5beta1) distribution revealed that III1-C did not inhibit alpha5beta1 localization to focal contacts. Moreover, III1-C had no effect on the relative expression levels of seven different integrin subunits on HUASMCs. However, III1-C did inhibit fibronectin matrix assembly by rat aortic smooth muscle cells, HUASMCs, A7r5 cells, IMR-90 cells, and endothelial cells. An analysis of fibronectin synthesis indicated that the inhibition of fibronectin matrix assembly by III1-C was not due solely to a decrease in fibronectin synthesis. Finally, treatment of HUASMCs with anti-fibronectin monoclonal antibody L8 (which is known to inhibit fibronectin matrix assembly) also decreased the rate of HUASMC DNA synthesis. These results demonstrate that III1-C inhibits VSMC proliferation and suggest that this effect may be mediated by the inhibition of fibronectin matrix assembly.