Fluorescence versus white-light bronchoscopy for detection of preneoplastic lesions: a randomized study.

BACKGROUND: There are no currently approved methods for the screening and early detection of lung cancer. We compared the ability of conventional white-light bronchoscopy (WLB) and laser-induced fluorescence endoscopy (LIFE) to detect preneoplastic lung lesions in a randomized trial in which both the order of the procedures and the bronchoscopists were randomly assigned. METHODS: The study included high-risk subjects enrolled because of a cigarette smoking history of at least 30 pack-years, an air-flow obstruction, and either an abnormal sputum cytology (n = 48) or a previous or suspected lung cancer (n = 7). LIFE and WLB were performed on all patients. Biopsy specimens were assessed for histologic abnormalities, including the presence of angiogenic squamous dysplasia. All statistical tests were two-sided. RESULTS: A total of 391 biopsy specimens were taken from the 55 patients. Thirty-two patients (58%; 95% confidence interval [CI] = 44% to 71%) had at least one biopsy with moderate or severe dysplasia, and 19 (59%; 95% CI = 41% to 76%) of these patients could be diagnosed based solely on the results of LIFE. LIFE was statistically significantly more sensitive than WLB for detecting moderate dysplasia or worse (68.8% versus 21.9%, respectively) (difference = 46.9%; 95% CI = 25% to 68%; P< .001). The relative sensitivities (WLB = 1.0) were 3.1 (95% CI = 1.6 to 6.3) for LIFE and 3.7 (95% CI = 1.9 to 7.3) for LIFE and WLB combined. LIFE was less specific than WLB (69.6% versus 78.3%, respectively; P = .45), but the difference was not statistically significant. The relative specificities (WLB = 1.0) were 0.9 for LIFE (95% CI = 0.6 to 1.3) and 0.6 (95% CI = 0.4 to 1.0) for LIFE and WLB combined. The results were similar regardless of the order of the procedures or the order of the bronchoscopists. Also, LIFE was better at identifying angiogenic squamous dysplasia lesions than WLB (detection ratio [DR], which indicates the relative likelihood of getting a positive result in a sample with dysplasia compared with one without, for LIFE = 1.39 [95% CI = 1.17 to 1.65] versus DR for WLB = 0.67 [95% CI = 0.38 to 1.21]). CONCLUSION: LIFE was more sensitive than WLB in detecting preneoplastic bronchial changes in high-risk subjects. The prognostic implication of this finding is not yet clear.

Angiogenic squamous dysplasia in bronchi of individuals at high risk for lung cancer.

Lung carcinogenesis is assumed to be a multistep process, but detailed understanding of the sequential morphological and molecular changes preceding invasive lung cancer remains elusive. To better understand early lung carcinogenesis, we initiated a program of fluorescence bronchoscopy in smokers at high risk for lung cancer. In the bronchial biopsies from these subjects, we observed a unique lesion consisting of capillary blood vessels closely juxtaposed to and projecting into metaplastic or dysplastic squamous bronchial epithelium, angiogenic squamous dysplasia (ASD). Serial sections of the capillary projections confirmed that they represent intramucosal capillary loops. Microvessel density in ASD was elevated in comparison to normal mucosa (P = 0.0003) but not in comparison to other forms of hyperplasia or dysplasia. ASD thus represents a qualitatively distinct form of angiogenesis in which there is architectural rearrangement of the capillary microvasculature. Genetic analysis of surface epithelium in a random subset of lesions revealed loss of heterozygosity at chromosome 3p in 53% of ASD lesions. No confirmed p53 mutations were identified. Compared with normal epithelium, proliferative activity was markedly elevated in ASD lesions. ASD occurred in 54 of 158 (34%) high-risk smokers without carcinoma and in 6 of 10 patients with squamous carcinoma who underwent fluorescence bronchoscopy. One early-stage invasive carcinoma was noteworthy for the occurrence of ASD juxtaposed to invasive tumor. Seventy-seven (59%) of the ASD lesions were detected by abnormal fluorescence alone. Twenty bronchial sites (11 patients) were rebiopsied 1 year after the initial diagnosis. At nine (45%) of these sites, the lesion was found to persist. The lesion was not present in biopsies from 16 normal nonsmoker control subjects. The presence of this lesion in high-risk smokers suggests that aberrant patterns of microvascularization may occur at an early stage of bronchial carcinogenesis.

High frequency of K-ras codon 12 mutations in bronchoalveolar lavage fluid of patients at high risk for second primary lung cancer.

A high frequency of K-ras mutations may indicate preneoplastic changes in the bronchial epithelium as a result of genotoxic injury. With the use of sensitive detection techniques, we report a higher prevalence of K-ras mutations in bronchoalveolar lavage than has been reported previously for lung cancer. A PCR/ligase chain reaction technique was used to determine K-ras codon 12 mutations in a group of 52 bronchoalveolar lavage specimens from patients at risk of a second lung cancer. Of the specimens examined, 84% contained at least one mutation in K-ras codon 12, corroborated by an allele-specific hybridization method. These results suggest that point mutations in K-ras codon 12 are widespread in the bronchial epithelium. Based on these preliminary findings, further evaluation of this efficient sensitive assay to monitor K-ras status should be conducted in larger clinical cohorts where clinical outcomes will ultimately be available. Such a trial will define the utility of K-ras codon 12 mutation status as a marker of lung cancer.

Peptide amidating activity in human bronchoalveolar lavage fluid.

Monitoring respiratory epithelial biology may reveal individuals with incipient lung cancer. The expression of neuroendocrine (NE) markers in pulmonary epithelium is thought to be central to lung development, repair of injury and may contribute to carcinogenesis. In this study, we evaluate several candidate NE markers to determine the feasibility of prospective analysis of clinical specimens. The potential NE markers include the enzyme L-DOPA decarboxylase (DDC), the neuropeptide gastrin releasing peptide (GRP), and peptidyl-glycine alpha-amidating monooxygenase (PAM), the bifunctional enzyme responsible for the final bioactivation step of many neuropeptides. A comparison of PAM activity and DDC levels in 30 lung cancer cell lines indicated that peptide amidating activity may be an indicator of NE status. Bronchoalveolar lavage (BAL) fluid from subjects at risk of developing second primary lung cancer and from volunteers was obtained. The activity of the first PAM enzyme, peptidylglycine alpha-hydroxylating monooxygenase (PHM), ranged from not detectable to 507 pmol/h/mg protein in 57 specimens. The second PAM enzyme, peptidylamidoglycolate lyase (PAL), ranged from not detectable to 414 pmol/h/mg protein in 56 specimens. Using cluster analysis by the average linkage method, a group of enzyme values with PHM greater than 230 pmol/h/mg protein was determined. Long-term follow-up of these patients for new second primary lung cancers may help to determine the potential predictive value of PAM detected in the BAL fluid.

Hypoxia-induced pulmonary vascular remodeling: contribution of the adventitial fibroblasts.

Vascular repair in response to injury or stress (often referred to as remodeling) is a common complication of many cardiovascular abnormalities including pulmonary hypertension, systemic hypertension, atherosclerosis, vein graft remodeling and restenosis following balloon dilatation of the coronary artery. It is not surprising that repair and remodeling occurs frequently in the vasculature in that exposure of blood, vessels to either excessive hemodynamic stress (e.g. hypertension), noxious blood borne agents (e.g. atherogenic lipids), locally released cytokines, or unusual environmental conditions (e.g. hypoxia), requires readily available mechanisms to counteract these adverse stimuli and to preserve structure and function of the vessel wall. The responses, which were presumably evolutionarily developed to repair an injured tissue, often escape self-limiting control and can result, in the case of blood vessels, in lumen narrowing and obstruction to blood flow. Each cell type (i. e. endothelial cells, smooth muscle cells, and fibroblasts) in the vascular wall plays a specific role in the response to injury. However, while the roles of the endothelial cells and smooth muscle cells (SMC) in vascular remodeling have been extensively studied, relatively little attention has been given to the adventitial fibroblasts. Perhaps this is because the fibroblast is a relatively ill-defined cell which, at least compared to the SMC, exhibits few specific cellular markers. Importantly though, it has been well demonstrated that fibroblasts possess the capacity to express several functions such as migration, rapid proliferation, synthesis of connective tissue components, contraction and cytokine production in response to activation or stimulation. The myriad of responses exhibited by the fibroblasts, especially in response to stimulation, suggest that these cells could play a pivotal role in the repair of injury. This fact has been well documented in the setting of wound healing where a hypoxic environment has been demonstrated to be critical in the cellular responses. As such it is not surprising that fibroblasts may play an important role in the vascular response to hypoxia and/or injury. This paper is intended to provide a brief review of the changes that occur in the adventitial fibroblasts in response to vascular stress (especially hypoxia) and the role the activated fibroblasts might play in hypoxia-mediated pulmonary vascular disease.

Hypoxia induces cell-specific changes in gene expression in vascular wall cells: implications for pulmonary hypertension.

Mammals respond to reduced oxygen concentrations (hypoxia) in many different ways at the systemic, local, cellular and molecular levels. Within the pulmonary circulation, exposure to chronic hypoxia has been demonstrated to illicit increases in pulmonary artery pressure as well as dramatic structural changes in both large and small vessels. It has become increasingly clear that the response to hypoxia in vivo is differentially regulated at the level of specific cell types within the vessel wall. For instance, in large pulmonary blood vessels there is now convincing evidence to suggest that the medial layer is made up of many different subpopulations of smooth muscle cells. In response to hypoxia there are remarkable differences in the proliferative and matrix producing responses of these cells to the hypoxic environment. Some cell populations proliferate and increase matrix protein synthesis, while in other cell populations no apparent change in the proliferative or differentiation state of the cell takes place. In more peripheral vessels, the predominant proliferative changes in response to hypoxia in the pulmonary circulation occur in the adventitial layer rather than in the medial layer. Here again, specific increases in proliferation and matrix protein synthesis take place. Accumulating evidence suggests that the unique responses exhibited by specific cell types of hypoxia in vivo can be modeled in vitro. We have isolated, in culture, specific medial cell populations which demonstrate significant increases in proliferation in response to hypoxia, and others which exhibit no change or, in fact, a decrease in proliferation under hypoxic conditions. We have also isolated and cloned several unique populations of adventitial fibroblasts. There is good evidence that only certain fibroblast populations are capable of responding to hypoxia with an increase in proliferation. We have begun to elucidate the signaling pathways which are activated in those cell populations that exhibit proliferative responses to hypoxia. We show that hypoxia, in the absence of serum or mitogens, specifically activates select members of the protein kinase C isozyme family, as well as members of the mitogen-activated protein kinase (MAPK) family of proteins. This selective activation appears to take place in response to hypoxia only in those cells exhibiting a proliferative response, and antagonists of this pathway inhibit the response. Thus, there appear to be cells within each organ that demonstrate unique responses to hypoxia. A better understanding of why these cells exist and how they specifically transduce hypoxia-mediated signals will lead to a better understanding of how the changes in the pulmonary circulation take place under conditions of chronic hypoxia.

Neonatal lung side population cells demonstrate endothelial potential and are altered in response to hyperoxia-induced lung simplification.

Lung side population (SP) cells are resident lung precursor cells with both epithelial and mesenchymal potential that are believed to play a role in normal lung development and repair. Neonatal hyperoxic exposure impairs lung development leading to a long-term decrease in gas exchange surfaces. The hypothesis that lung SP cells are altered during impaired lung development has not been studied. To address this issue, we characterized the endothelial potential of neonatal lung SP and subsets of lung SP from neonatal mice following hyperoxic exposure during room air recovery. Lung SP cells were isolated and sorted on the basis of their capacity to efflux Hoechst 33342. The lung SP was further sorted based on expression of Flk-1 and CD45. In vitro, both CD45(pos)/Flk-1(pos) and CD45(neg)/Flk-1(pos) bind isolectin B4 and incorporate LDL and form networks in matrigel, indicating that these populations have endothelial cell characteristics. Hyperoxic exposure of neonatal mice resulted in subtle changes in vascular and alveolar density on P13, which persisted with room air recovery to P41. During room air recovery, a decrease in lung SP cells was detected in the hyperoxic-exposed group on postnatal day 13 followed by an increase on day 41. Within this group, the lung SP subpopulation of cells expressing CD45 increased on day 21, 41, and 55. Here, we show that lung SP cells demonstrate endothelial potential and that the population distribution changes in number as well as composition following hyperoxic exposure. The hyperoxia-induced changes in lung SP cells may limit their ability to effectively contribute to tissue morphogenesis during room air recovery.

Mice deficient in galectin-1 exhibit attenuated physiological responses to chronic hypoxia-induced pulmonary hypertension.

Pulmonary hypertension (PH) is characterized by sustained vasoconstriction, with subsequent extracellular matrix (ECM) production and smooth muscle cell (SMC) proliferation. Changes in the ECM can modulate vasoreactivity and SMC contraction. Galectin-1 (Gal-1) is a hypoxia-inducible beta-galactoside-binding lectin produced by vascular, interstitial, epithelial, and immune cells. Gal-1 regulates SMC differentiation, proliferation, and apoptosis via interactions with the ECM, as well as immune system function, and, therefore, likely plays a role in the pathogenesis of PH. We investigated the effects of Gal-1 during hypoxic PH by quantifying 1) Gal-1 expression in response to hypoxia in vitro and in vivo and 2) the effect of Gal-1 gene deletion on the magnitude of the PH response to chronic hypoxia in vivo. By constructing and screening a subtractive library, we found that acute hypoxia increases expression of Gal-1 mRNA in isolated pulmonary mesenchymal cells. In wild-type (WT) mice, Gal-1 immunoreactivity increased after 6 wk of hypoxia. Increased expression of Gal-1 protein was confirmed by quantitative Western analysis. Gal-1 knockout (Gal-1(-/-)) mice showed a decreased PH response, as measured by right ventricular pressure and the ratio of right ventricular to left ventricular + septum wet weight compared with their WT counterparts. However, the number and degree of muscularized vessels increased similarly in WT and Gal-1(-/-) mice. In response to chronic hypoxia, the decrease in factor 8-positive microvessel density was similar in both groups. Vasoreactivity of WT and Gal-1(-/-) mice was tested in vivo and with use of isolated perfused lungs exposed to acute hypoxia. Acute hypoxia caused a significant increase in RV pressure in wild-type and Gal-1(-/-) mice; however, the response of the Gal-1(-/-) mice was greater. These results suggest that Gal-1 influences the contractile response to hypoxia and subsequent remodeling during hypoxia-induced PH, which influences disease progression.

Divergent contractile and structural responses of the murine PKC-epsilon null pulmonary circulation to chronic hypoxia.

Loss of PKC-epsilon limits the magnitude of acute hypoxic pulmonary vasoconstriction (HPV) in the mouse. Therefore, we hypothesized that loss of PKC-epsilon would decrease the contractile and/or structural response of the murine pulmonary circulation to chronic hypoxia (Hx). However, the pattern of lung vascular responses to chronic Hx may or may not be predicted by the acute HPV response. Adult PKC-epsilon wild-type (PKC-epsilon(+/+)), heterozygous null, and homozygous null (PKC-epsilon(-/-)) mice were exposed to normoxia or Hx for 5 wk. PKC-epsilon(-/-) mice actually had a greater increase in right ventricular (RV) systolic pressure, RV mass, and hematocrit in response to chronic Hx than PKC-epsilon(+/+) mice. In contrast to the augmented PA pressure and RV hypertrophy, pulmonary vascular remodeling was increased less than expected (i.e., equal to PKC-epsilon(+/+) mice) in both the proximal and distal PKC-epsilon(-/-) pulmonary vasculature. The contribution of increased vascular tone to this pulmonary hypertension (PHTN) was assessed by measuring the acute vasodilator response to nitric oxide (NO). Acute inhalation of NO reversed the increased PA pressure in hypoxic PKC-epsilon(-/-) mice, implying that the exaggerated PHTN may be due to a relative deficiency in nitric oxide synthase (NOS). Despite the higher PA pressure, chronic Hx stimulated less of an increase in lung endothelial (e) and inducible (i) NOS expression in PKC-epsilon(-/-) than PKC-epsilon(+/+) mice. In contrast, expression of nNOS in PKC-epsilon(+/+) mice decreased in response to chronic Hx, while lung levels in PKC-epsilon(-/-) mice remained unchanged. In summary, loss of PKC-epsilon results in increased vascular tone, but not pulmonary vascular remodeling in response to chronic Hx. Blunting of Hx-induced eNOS and iNOS expression may contribute to the increased vascular tone. PKC-epsilon appears to be an important signaling intermediate in the hypoxic regulation of each NOS isoform.

Exocrine pancreatic insufficiency syndrome in CBA/J mice. II. Biochemical studies.

Premature activation of proteolytic zymogens (trypsinogen, chymotrypsinogen) as an early step in the pathogenesis of exocrine pancreatic insufficency (EPI) syndrome in CBA/J mice was investigated in electrophoresed pancreatic homogenates. Polyacrylamide gels containing extracts from control pancreas required prior activation of trypsinogen and chymotrypsinogen (with exogenously added enterokinase and trypsin, respectively) to produce activity staining with specific synthetic substrates. On the contrary, bands of activity staining in gels containing homogenates from mice with EPI syndrome could be readily detected without trypsin or enterokinase preincubation. Subcellular fractionation of control and diseased pancreas revealed that the premature intracellular proteolysis was confined to the zymogren granule fraction, which, even in very moderately affected pancreases (10 to 30% acinar cell autolysis), was very labile in vitro. These proteolytic events reflect the biochemical consequences of zymogen granule destabilization that were observed at the ultrastructural level.

Enhanced growth of fetal and neonatal pulmonary artery adventitial fibroblasts is dependent on protein kinase C.

The earliest and most striking proliferative changes in the neonatal pulmonary arterial wall occur in the adventitia where the fibroblast resides. The protein kinase C (PKC) pathway is developmentally regulated and important in vascular cell growth. We tested the hypothesis that developmental differences in growth of pulmonary artery adventitial fibroblasts would be detectable in vitro and dependent on PKC. Fibroblasts were isolated from bovine fetal, neonatal, and adult pulmonary arteries. Growth was measured by [3H]thymidine incorporation and cell counts. Under serum-stimulated conditions, fetal and neonatal pulmonary artery fibroblasts grew faster and reached higher plateau densities than adult cells. Increased growth of fetal cells in vitro was dependent on time of harvest during fetal life (early > late). Under quiescent conditions, fetal and neonatal fibroblasts had increased DNA synthesis compared with adult cells in response to the PKC agonist phorbol 12-myristate 13-acetate. To test whether the developmental differences in fibroblast growth were dependent on PKC, three different inhibitor strategies were used (dihydrosphingosine, phorbol-ester-induced downregulation, and heparin). Fetal and neonatal fibroblasts were more susceptible than adult cells to each antagonist strategy. Finally, we measured whole cellular PKC catalytic activity and found it correlated with growth and susceptibility to PKC inhibition (i.e., fetal PKC activity > neonatal > adult). We conclude that PKC-dependent developmental differences in growth of pulmonary artery fibroblasts are detectable in vitro and that the enhanced growth capacity of fetal and neonatal cells may contribute to the dramatic adventitial thickening seen in vivo after hypoxic exposure in the neonatal calf.

Pregnancy-stimulated growth of vascular smooth muscle cells: importance of protein kinase C-dependent synergy between estrogen and platelet-derived growth factor.

Dramatic smooth muscle cell (SMC) growth occurs in the uterine artery during pregnancy. The potential for pregnancy-associated growth may also exist at other vascular sites. We tested the hypothesis that increased growth of uterine artery SMC isolated from pregnant (vs. nonpregnant) guinea pigs would be detectable in culture, that pregnancy-associated phenotypic changes would also be found in nonuterine vascular cells (aortic SMC), and that the enhanced growth would be dependent on estrogen, peptide growth factors like platelet-derived growth factor (PDGF), and protein kinase C (PKC). Growth responses were measured by [3H]-thymidine incorporation and cell counts. Uterine artery SMC from pregnant guinea pigs grew to a higher plateau density with serum stimulation, had increased spontaneous DNA synthesis and persistent growth following serum with-drawal, and were more responsive to 3-30 ng/ml PDGF-BB than nonpregnant cells. Aortic SMC from pregnant animals also grew to a higher plateau density and had enhanced responsiveness of PDGF-BB. This increased response to PDGF-BB by pregnant uterine artery and aortic SMC (40-233% increase over nonpregnant PDGF result) was reproduced in nonpregnant cells by pretreatment for 1-24 h with 17-beta(beta)-estradiol (30-100 nM). Neither the pregnancy-induced difference nor the estradiol pretreatment was associated with increased PDGF-BB binding activity. The synergistic effect of 17 beta-estradiol was partially (62%) reproduced with 17-alpha(alpha)-estradiol, an isomer which does not bind the estrogen receptor. This suggested that 17 beta-estradiol modulates the PDGF-BB response by both estrogen-receptor- and nonreceptor-mediated mechanisms. To test if the estrogen effects were dependent on PKC, two different antagonist strategies (3 microM dihydrosphingosine and phorbol-ester-induced downregulation) were applied prior to 17 alpha- or beta-estradiol and blocked the enhanced responses to PDGF. The synergistic effect of 17 beta-estradiol on PDGF was then reproduced by 1 h pretreatment with the cell-permeable PKC activator, 10 nM PMA. We conclude that pregnancy stimulates increased growth of uterine and aortic SMC in vitro which is dependent on estrogen, PDGF, and PKC and may be important in vascular remodeling during pregnancy.

Enhanced growth capacity of neonatal pulmonary artery smooth muscle cells in vitro: dependence on cell size, time from birth, insulin-like growth factor I, and auto-activation of protein kinase C.

Based on the unique susceptibility of the neonatal pulmonary circulation to hypoxia-induced structural alteration in vivo, we hypothesized that pulmonary artery (PA) smooth muscle cells (SMC) from the neonate would demonstrate enhanced growth capacity in vitro compared to adult cells. To test this hypothesis, matched neonatal and adult bovine SMC were tested for differences in size, serum-stimulated proliferation, susceptibility to senescence, resistance to serum withdrawal, autocrine growth capacity, and responsiveness to a locally important growth factor (insulin-like growth factor I; IGF-I) and an activator of protein kinase C (PKC) (phorbol 12-myristate 13-acetate; PMA). Neonatal PA SMC were smaller, grew faster, reached a higher plateau density, and were less susceptible to senescence. They were more resistant to serum withdrawal, had spontaneous autocrine growth capacity, and were more responsive to IGF-I, PMA, and the combination. Acquisition of increased growth factor responsiveness occurred between d5 and d14 after birth. Increased neonatal growth to IGF-I was associated with reduced IGF-I binding activity, implicating a post-receptor mechanism in enhanced responsiveness. Increased membrane-bound PKC catalytic activity was found in serum-deprived neonatal SMC. This basal increase was equal to that stimulated by 1 nM PMA in adult SMC, a pretreatment that caused these cells to become as responsive to IGF-I as untreated neonatal ones. We conclude that neonatal bovine PA SMC have marked enhancement of growth capacity in vitro, the acquisition of which is dependent on time from birth and is associated with auto-activation of PKC, These increased growth properties detected in vitro may contribute to the striking hyperplasia of neonatal PA SMC found in vivo following hypoxic exposure.

Protein kinase C activation allows pulmonary artery smooth muscle cells to proliferate to hypoxia.

Pulmonary artery (PA) smooth muscle cell (SMC) proliferation occurs with hypoxic pulmonary hypertension in vivo. However, proliferation of cultured PA SMC to hypoxia has not been demonstrated, and thus the mechanism by which these cells respond to hypoxia is unknown. Because protein kinase C (PKC) plays a role in intracellular transduction of proliferative signals, we asked whether PKC activation 1) causes proliferation of bovine PA SMC and 2) is important in PA SMC proliferative response to hypoxia. By measuring [3H]thymidine incorporation and cell counts, we found that quiescent PA SMC from four different cows proliferated with the PKC activator, phorbol 12-myristate 13-acetate (PMA), in a concentration-dependent manner. The proliferation was blocked with a PKC inhibitor, dihydrosphingosine, or by downregulating SMC PKC. We tested whether "priming" PA SMC by PKC activation was required for in vitro SMC proliferative response to hypoxia. Each SMC population was treated with PMA and then exposed for 24 h to 20, 10, 7, 3 or 0% O2. These cells proliferated with hypoxia reaching a peak response at 3% O2. The magnitude of the response to PMA and hypoxia was different for each cell population tested. No hypoxic proliferation occurred in control cells (no PMA). Dihydrosphingosine blocked the hypoxic response to the same extent that it inhibited the initial PMA conditioning stimulus. PKC-downregulated PA SMC did not proliferate to PMA or to subsequent hypoxia. The hypoxic response was not due to a reduction in O2 radical-mediated antiproliferative effect; rather, the PMA-primed cells seemed to "acquire" the ability to directly sense hypoxia and proliferate. In summary, PKC activation caused proliferation of PA SMC in vitro and allowed an additional proliferative response to hypoxia. Activation of PKC may be a requisite step for PA SMC to respond directly to hypoxia.

BQ123, an ETA receptor antagonist, inhibits endothelin-1-mediated proliferation of human pulmonary artery smooth muscle cells.

Endothelin (ET-1) has been shown to be co-mitogenic for vascular smooth muscle cells (SMC) from human systemic arteries. A more modest growth-promoting effect has also been described in SMC from the bovine and porcine pulmonary circulation. Whether ET-1 has mitogenic properties in the human pulmonary circulation, and which ET receptor subtype mediates this response, is unknown. We first examined the effects of ET-1, ET-3, and the selective ETB agonist, Sarafotoxin 6c, on human pulmonary artery SMC growth. Cells were harvested from normal lung transplant donors. Growth was assessed by change in cell number 3 days after stimulation of quiescent cells. ET-1 in the presence of 0.3% serum produced a dose-dependent increase (82 +/- 1.5%) in cell number (threshold, 10(-11) M; maximal, 10(-7) M). ET-3 also stimulated growth (36 +/- 3.8%) but was less potent than ET-1 (threshold, 10(-9) M; maximal, 10(-7) M). The ETB selective agonist Sarafotoxin 6c had no proliferative effect. The effects of BQ123, a selective ETA receptor antagonist, on ET-1-induced growth were then assessed. BQ123 inhibited (threshold, 1.5 x 10(-7) M; maximal, 1.5 x 10(-5) M) ET-1-induced growth but had no effect on proliferation stimulated by the non-ET receptor-mediated growth factors, platelet-derived growth factor BB and 5-hydroxytryptamine. These results suggest that ET-1 is a potent co-mitogen for human proximal pulmonary artery SMC and that this effect is transduced by selective activation of the ETA receptor.