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To determine whether glucocorticoids and beta-adrenoceptor agonists act independently to inhibit airway smooth muscle (ASM) proliferation, the present study investigated the effects of methylprednisolone (MP) and isoproterenol (ISO) alone and in combination on leukotriene D4-induced ASM proliferation. MP and ISO had no effect on unstimulated ASM cell growth. In contrast, MP and ISO demonstrated dose-dependent inhibition of LTD4-induced proliferation, and the inhibitory effect was additive for combinations of MP and ISO. The competitive cAMP receptor antagonist, Rp-cAMPS, ablated the ISO-induced inhibition but had no affect on the inhibitory response to MP. In cells exposed to both ISO and MP, Rp-cAMPS attenuated the growth inhibition to levels achieved by MP alone. Accordingly, these findings demonstrate that glucocorticoids and beta-adrenergic agonists inhibit LTD4-induced ASM proliferation, and that their inhibitory effects are mediated by different signaling pathways.

The tachykinins, substance P (SP) and neurokinins A (NKA) and B (NKB), have been identified in the respiratory tract and implicated in mediating neurogenic inflammation of the airways. To the extent that these neuropeptides may be involved in the pathogenesis of asthma, a condition associated with hyperplasia of airway smooth muscle (ASM), we examined the mitogenic effects and mechanisms of action of tachykinins in cultured rabbit ASM cells. SP was found to elicit dose-dependent (10(-14) to 10(-4) M) stimulation of ASM cell proliferation, with a mean (+/- SE) maximal increase in cell number of 169 +/- 6.1% of control. In contrast, NKA and NKB had little and no effect on ASM cell growth, respectively. Because SP is nonselective in its binding to the tachykinin receptors, to identify the specific NK receptor subtype(s) mediating the promitogenic action of SP, in separate studies we found that 1) the NK1-receptor-specific agonist, [beta-Ala4, Sar9, Met(O2)11]SP-(4-11) induced stimulation of ASM cell growth similar in magnitude to that elicited by SP; 2) in contrast, neither the NK1- nor NK2-receptor-specific agonists, [beta-Ala8]NKA-(4-10) and [MePhe7]NKB, respectively, had any effect on ASM cell growth; and 3) the promitogenic action of SP was inhibited by the NK1-receptor antagonist, GR-82,334. Moreover, in extended experiments, we found that the phospholipase C and phospholipase A2 inhibitors, neomycin and quinacrine, respectively, each inhibited SP-induced ASM cell proliferation by approximately 45%. Collectively, these observations provide new evidence that the tachykinin SP induces ASM cell proliferation, and that this action is mediated by transmembrane signaling coupled to selective activation of the NK1 receptor.

The insulin-like growth factor (IGF) axis is involved in regulating proliferation in a variety of cell types, including airway smooth muscle. Because airway hyperplasia is a characteristic feature of asthma and other lung diseases, we examined the interaction of the potent proinflammatory eicosanoid leukotriene D4 (LTD4) with the IGF axis in regulating airway smooth muscle cell mitogenesis. In cultured rabbit airway smooth muscle cells, IGF-I but not LTD4 was mitogenic at submaximal concentrations. The combination of the two agents exerted a significant synergistic effect on airway smooth muscle cell mitogenesis. Analysis of airway smooth muscle cell conditioned medium by Western ligand blotting demonstrated a marked LTD4-induced reduction in the levels of the predominant IGF binding protein IGFBP-2, which is elaborated into the conditioned medium. The latter effect on IGFBP-2 release was not associated with a reduction in IGFBP-2 mRNA levels; however, LTD4-treated airway smooth muscle conditioned medium demonstrated the presence of a lower molecular weight form of IGFBP-2 by cross-linking to IGFs and specific proteolysis of radiolabeled IGFBP-2. IGFBP-2 was also noted to be associated with airway smooth muscle cell membranes, where it was protected from LTD4-induced proteolysis. Finally, exogenous administration of IGFBP-2 was found to inhibit the promitogenic effect of IGF-I in a dose-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin-like growth factors (IGFs) mediate cell proliferation and differentiation and bind with high affinities and specificities to IGF receptors and IGF-binding proteins (IGFBPs). We examined the roles of these three groups of proteins in cultured rabbit airway smooth muscle (ASM) cells. Affinity cross-linking of IGF-I and IGF-II to membranes of ASM cells revealed type 1 and type 2 IGF receptors. Western ligand blot analysis of ASM cell-conditioned medium revealed the presence of a single IGFBP band that precipitated with an antibody specific to IGFBP-2. ASM cells secreted radioimmunoassayable IGF-II; however, no IGF-I was detected under the same conditions. Two molecular weight forms of IGF-II were produced by the ASM cells. Exposure of cells to 1,000 ng/ml of IGF-I stimulated them to proliferate to 230 +/- 9.7% of their respective controls. Exposure to 1,000 ng/ml of IGF-II was approximately 40% as effective as exposure to 1,000 ng/ml of IGF-I. Both IGF-I and IGF-II exhibited binding to the type 1 IGF receptor. In summary, IGFs are mitogens for cultured rabbit ASM cells, and their actions are most likely mediated through the type 1 IGF receptor. The ASM cells secrete IGF-II and IGFBP-2, and the latter could modulate the actions of the IGFs in these cells.

To evaluate the potential role of adrenergic receptors in regulating airway smooth muscle (ASM) cell proliferation, the mitogenic effects and mechanisms of action of selective alpha- and beta-adrenoceptor activation were investigated in cultured rabbit ASM cells. The alpha 1-adrenoceptor agonists, phenylephrine and methoxamine, elicited significant dose-dependent (10(-10)-10(-4) M) stimulation of ASM cell mitogenesis, with mean +/- SE peak increases in ASM cell count amounting to 17.0 +/- 3.5 and 44.0 +/- 6.8% above unstimulated (control) levels, respectively. Similarly, the alpha 2-adrenoceptor agonist clonidine (10(-8)-10(-4)) also induced ASM cell proliferation, with a 41.1 +/- 5.5% peak increase in cell count above control. The promitogenic responses to alpha-adrenoceptor activation were blocked by pertussis toxin (PT; 100 ng/ml), which ADP-ribosylates G protein negatively coupled to adenylate cyclase activation. In additional studies, we found that 1) treatment with agents inducing intracellular adenosine 3',5'-cyclic monophosphate (cAMP) accumulation including the beta-adrenergic agonist, isoproterenol, the cAMP analogue, 8-(4-chlorophenylthio)-cAMP and forskolin, all produced significant dose-dependent inhibition of serum-stimulated ASM cell growth; 2) alpha-adrenoceptor activation inhibited isoproterenol-induced cAMP accumulation; and 3) the anti-proliferative effects of isoproterenol and PT were additive. Collectively, the above findings provide new evidence that adrenergic receptors exert an opposing duality of action in regulating ASM cell proliferation, wherein receptors which are negatively coupled (i.e., alpha-adrenergic) and those which are positively coupled (i.e., beta-adrenergic) to activation of the adenylate cyclase/cAMP signal-transduction pathway are promitogenic and growth inhibitory to ASM cells, respectively.

Thromboxane (Tx)A2 has been reported to play an important role in modulating airway contractility under various conditions associated with airways inflammation. To identify its potential role in contributing to airway smooth muscle (ASM) hyperplasia, a characteristic feature of asthmatic airways, the mitogenic effect and mechanism of action of TxA2 were investigated in cultured rabbit ASM cells. The stable TxA2 mimetics, carbocyclic TxA2 (CTA2) and U-46619, elicited dose-dependent (10(-12) to 10(-6) M) increases in ASM cell number and induced acute augmentation of intracellular inositol 1,4,5-trisphosphate accumulation. The latter action was blocked by neomycin, a phospholipase C inhibitor; however, neomycin had no effect on the promitogenic action of the TxA2 mimetics. In contrast, TxA2-induced ASM cell proliferation was inhibited by inhibitors of phospholipase A2 and 5-lipoxygenase, as well as blockade of the leukotriene (LT)D4 receptor. Moreover, in complementary studies, we found that exogenous administration of LTD4 (10(-14) to 10(-6) M) potently induced ASM cell proliferation and that the TxA2 mimetics evoked the enhanced release of endogenous leukotrienes from the cultured ASM cells. Taken together, these observations provide new evidence that 1) TxA2 stimulates ASM cell proliferation; 2) the promitogenic effect of TxA2 is associated with activation of phospholipase A2; and 3) the latter mediates ASM cell proliferation via the release and autocrine mitogenic action of leukotrienes. The findings support a potential role for TxA2 in contributing to the characteristic increase in ASM cell mass obtained in asthma and other chronic airway diseases.

Increased expression of the potent vasoconstrictor and bronchoactive peptide, endothelin-1 (ET-1), has recently been demonstrated in airway epithelial and endothelial cells of asthmatic patients. To identify its potential role in contributing to airway smooth muscle (ASM) hyperplasia, a characteristic feature of asthmatic airways, the mitogenic action of ET-1 was investigated in cultured rabbit ASM cells. ET-1 elicited significant dose-dependent (10(-12)-10(-6) M) increases in ASM cell number, with a mean potency (i.e., -log mean effective dose) of action of 9.82-log M. ET-1 also acutely stimulated intracellular inositol 1,4,5-trisphosphate accumulation. The latter response was blocked by phospholipase C inhibition with neomycin; however, neomycin had no effect on the promitogenic action of ET-1. By contrast, the ASM cell proliferative response to ET-1 was independently inhibited by pertussis toxin, inhibitors of phospholipase A2, cyclooxygenase, and thromboxane A2 (TxA2) synthesis, as well as blockade of the TxA2 receptor. Moreover, in complementary studies, we found that administration of the stable TxA2 mimetics, carbocyclic TxA2 (CTA2) and U-46619, induced ASM cell proliferation and that ET-1 evoked the release of endogenous TxA2 from the ASM cells. Collectively, these observations provide new evidence that 1) ET-1 is a potent mitogen of ASM cells, 2) the promitogenic effect of ET-1 is associated with activation of a pertussis toxin-sensitive G protein coupled to stimulation of phospholipase A2, and 3) the latter mediates ASM cell proliferation via the release and autocrine mitogenic action of TxA2. The findings support a potential role for ET-1 in mediating the characteristic hyperplasia of ASM in asthma.

In this study, the expression of extracellular matrix genes by vascular cells from human iliac blood vessels was characterized on the mRNA steady-state level by slot blot and Northern transfer analyses, as well as by in situ hybridization. Endothelial cells were isolated from adult human iliac arteries and veins, as well as from umbilical veins; smooth muscle cells were isolated from adult human iliac arteries and inferior vena cava. The results show that confluent umbilical vein endothelial cells expressed the genes that encode types I, III, IV and VI collagens, as well as fibronectin and laminin. In contrast, the iliac endothelial cells expressed the genes for types IV and V collagens, fibronectin and laminin; mRNA transcripts for types I, III and VI collagens were not detectable. The smooth muscle cells from iliac arteries or inferior vena cava displayed gene expression for types I, III, IV, V and VI collagens, fibronectin and laminin. The results indicate major differences in gene expression for the various types of collagens by human iliac endothelial and smooth muscle cells. Furthermore, the fetal-derived umbilical endothelial cells displayed differential collagen gene expression from that of adult iliac endothelial cells.

High-level expression of human tissue-type plasminogen activator was accomplished in endothelial cells by a novel approach to dihydrofolate reductase (DHFR) coamplification in DHFR+ cells. A tripartite mammalian expression vector coding for DHFR, neomycin phosphotransferase, and the t-PA gene was introduced into bovine endothelial cells by transfection and selection for G418 resistance. Upon methotrexate selection of these transformants, we obtained endothelial cells that had amplified the plasmid-encoded DHFR and t-PA genes. As a result, cell lines were isolated that efficiently produced t-PA (greater than 4 pg/cell.day). This t-PA was purified and compared with recombinant t-PA produced in Chinese hamster ovary cells. These two t-PA samples differed in carbohydrate composition, and amounts of 530 and 527 amino acid forms but had similar in vitro activity.

Bovine fetal aortic endothelial cells cultured in serum-containing medium accumulate angiotensin I-converting enzyme (ACE) activity and also release it into the culture medium. Following subcultivation of a confluent culture using trypsin-EDTA, cellular ACE activity falls 50% within 8 h, but no ACE activity is detected in the medium, suggesting intracellular loss of the enzyme activity. ACE activity reappears in both the cell lysate and culture medium after the culture becomes confluent. The rate of accumulation of ACE activity released into the medium is always greater than that for cellular activity. For example, 21 days following subcultivation 80-85% of the total culture activity is detected in the medium. Both cellular and medium-associated ACE decrease proportionately as the culture progresses through its in vitro lifespan.

Angiotensin I-converting enzyme (ACE) activity was measured in lysates of cloned and uncloned cultures of bovine fetal aortic endothelial cells. The expression of ACE activity in these cells was complex, and influenced by subcultivation, cell density, serum, cumulative population doublings, and clonal heterogeneity. The ACE specific activity at any point in the in vitro lifespan was determined, at least in part, by interaction of these culture variables. After subcultivation to subconfluent densities, cellular ACE specific activity decreased markedly and did not reach detectable levels until cells attained confluent densities. The use of different suppliers' lots of serum in the growth medium resulted in different cellular ACE specific activities. The ACE specific activity decreased as cultures were serially subcultivated, but remained detectable throughout the lifespan, suggesting a linkage between the proliferative history of an endothelial cell and its remaining capacity to express ACE. Increased ACE activity was observed when cells at the end of their lifespan were cultured at high densities. Cloned strains behaved similarly to the uncloned parent culture, except that they exhibited a wide range of ACE specific activities.

We have utilized clonal strains of bovine fetal aortic endothelial cells to study cellular senescence in a differentiated cell type of physiological significance. Serial subcultivation of nine endothelial clones derived from three fetal calf aortas revealed proliferative life-spans in vitro of 53--125 population doublings (PDs), compared with 60 and 143 PDs for two lines of bovine fetal lung cells and 85 and 147 PDs for two lines of bovine vascular smooth muscle cells. Serial growth curves showed marked reductions associated with endothelial cellular senescence both in cellular growth rate and culture plateau density. Studies of the 24-hour [3H]-thymidine labeling index versus percentage of proliferative life-span completed indicated that clonal endothelial cultures contained a large proportion (greater than 90%) of rapidly cycling cells until about 75% of the lifespans were completed. Senescent endothelial cells showed evidence of large increases in cell area, cell volume, and protein content. In those clones examined, one specialized endothelial function, Factor VIII antigen expression, was retained qualitatively throughout the life-spans.