Association of Women Leaders with Women Program Director and Trainee Representation Across US Academic Internal Medicine.

BACKGROUND: Women are underrepresented within internal medicine (IM). Whether women leaders attract women trainees is not well explored. OBJECTIVE: To characterize leader and trainee gender across US academic IM and to investigate the association of leader gender with trainee gender. DESIGN: Cross-sectional study. PARTICIPANTS: Leaders (chairs, chiefs, program directors (PDs)) in 2018 and trainees (residents, fellows) in 2012-2016 at medical school-affiliated IM and seven IM fellowship programs. EXPOSURE: Leadership (chair/chief and program director; and, for resident analyses, fellow) gender. MAIN MEASURES: Our primary outcome was percent women trainees (IM residents and, separately, subspecialty fellows). We used standard statistics to describe leadership and trainee gender. We created separate multivariable linear regressions to evaluate associations of leader gender and percent women fellows with percent women IM residents. We then created separate multivariable multilevel models (site as a random effect) to evaluate associations of leader gender with percent women subspecialty fellows. KEY RESULTS: Our cohort consisted of 940 programs. Women were 13.4% of IM chairs and <25% of chiefs in each fellowship subspecialty (cardiology: 2.6%; gastroenterology: 6.6%; pulmonary and critical care: 10.7%; nephrology: 14.4%; endocrinology: 20.6%; hematology-oncology: 23.2%; infectious diseases: 24.3%). IM PDs were 39.7% women; fellowship PDs ranged from nearly 25% (cardiology and gastroenterology) to nearly 50% (endocrinology and infectious disease) women. Having more women fellows (but not chairs or PDs) was associated with having more women residents (0.3% (95% CI: 0.2-0.5%) increase per 1% fellow increase, p<0.001); this association remained after adjustment (0.3% (0.1%, 0.4%), p=0.001). In unadjusted analyses, having a woman PD (increase of 7.7% (4.7%, 10.6%), p<0.001) or chief (increase of 8.9% (4.6%, 13.1%), p<0.001) was associated with an increase in women fellows; after adjustment, these associations were lost. CONCLUSIONS: Women held a minority of leadership positions in academic IM. Having women leaders was not independently associated with having more women trainees.

Intravenous stem cell dose and changes in quantitative lung fibrosis and DLCO in the AETHER trial: a pilot study.

OBJECTIVE: Our purpose was to compare quantitative CT-derived changes in lung fibrosis with pulmonary function, including DLCO, in human subjects with idiopathic pulmonary fibrosis who received an injection of one of two different intravenous doses of human bone-marrow-derived mesenchymal stem cells. PATIENTS AND METHODS: Two three-subject cohorts from the AETHER trial (Allogeneic Human Cells in subjects with Idiopathic Pulmonary Fibrosis via Intravenous Delivery) underwent high-resolution CT and clinical testing at baseline, 24 weeks, and 48 weeks after injection. Cohort 1 received 2x107 stem cells, and cohort 2 received 1x108 stem cells. CT scans were quantitatively analyzed for lung fibrosis using 510K cleared validated software. The percent predicted DLCO and other pulmonary function studies were obtained. RESULTS: The cohorts were well matched in lung fibrosis at baseline as assessed by CT scan and lung function. The mean QLF in cohort 1 increased from 13.1% at baseline to 17.1% at 48 weeks, while mean QLF in cohort 2 increased from 15.4% at baseline to 16.5% at 48 weeks. The subjects in cohort 2 progressed more slowly in whole lung fibrosis by a mean of 2.87% compared with cohort 1 (p=0.001 with adjustment of baseline covariates) during the baseline to the 48-week interval. The baseline DLCO was lower in cohort 2 than in cohort 1 (p<0.0001). Over 48 weeks of the study, cohort 2 subjects demonstrated a mean DLCO decline of only 2% compared with a decline of 17% in cohort 1 subjects (p=0.02). CONCLUSIONS: In this pilot study, the subjects receiving 1x108 stem cells demonstrated slower progression in quantitative lung fibrosis and a smaller decrease in DLCO than subjects receiving 2x107 stem cells.

The hedgehog processing pathway is required for NSCLC growth and survival.

Considerable interest has been generated from the results of recent clinical trials using smoothened (SMO) antagonists to inhibit the growth of hedgehog (HH) signaling-dependent tumors. This interest is tempered by the discovery of SMO mutations mediating resistance, underscoring the rationale for developing therapeutic strategies that interrupt HH signaling at levels distinct from those inhibiting SMO function. Here, we demonstrate that HH-dependent non-small cell lung carcinoma (NSCLC) growth is sensitive to blockade of the HH pathway upstream of SMO, at the level of HH ligand processing. Individually, the use of different lentivirally delivered shRNA constructs targeting two functionally distinct HH-processing proteins, skinny hedgehog (SKN) or dispatched-1 (DISP-1), in NSCLC cell lines produced similar decreases in cell proliferation and increased cell death. Further, providing either an exogenous source of processed HH or a SMO agonist reverses these effects. The attenuation of HH processing, by knocking down either of these gene products, also abrogated tumor growth in mouse xenografts. Finally, we extended these findings to primary clinical specimens, showing that SKN is frequently overexpressed in NSCLC and that higher DISP-1 expression is associated with an unfavorable clinical outcome. Our results show a critical role for HH processing in HH-dependent tumors, identifies two potential druggable targets in the HH pathway, and suggest that similar therapeutic strategies could be explored to treat patients harboring HH ligand-dependent cancers.

ET-1 induces mitogenesis in ovine airway smooth muscle cells via ETA and ETB receptors.

The proliferation of airway smooth muscle cells is a characteristic feature of asthma. Endothelin (ET)-1, a member of a family of three isopeptides (ET-1, ET-2, and ET-3), functions as a spasmogen and mitogen for airway smooth muscle cells. Two types of ET receptors have been identified in mammalian species (ETA and ETB). Because the respective roles of ETA and ETB receptors in ET-1-induced mitogenesis are not known, we determined the effect of two selective ETA and ETB antagonists (BQ-610 and BQ-788) on ET-1-induced mitogenesis of cultured ovine airway smooth muscle cells. Both BQ-610 and BQ-788 inhibited ET-1-induced mitogenesis in a concentration-dependent manner, with BQ-788 exhibiting more potent antagonism [half-maximal inhibitory concentration (IC50) = 3.5 nM, slope of 0.49] compared with BQ-610 (IC50 = 20 nM, slope of 0.27). The combined ETA-ETB antagonist, bosentan, also inhibited ET-1-induced mitogenesis (IC50 = 20 nM, slope of 0.60). The effects of BQ-788 and bosentan appear to be mediated via the same receptor (ETB), as their slopes are comparable. These observations suggest that both receptor subtypes are utilized in ET-1-induced proliferation of ovine airway smooth muscle. ET receptor expression may be important in the increase in airway smooth muscle mass seen in the airways of patients with bronchial asthma.

Endothelial and epithelial sources of endothelin-1 in sheep bronchi.

Airway smooth muscle tone and growth is regulated by endothelin-1 (ET-1), but the sources of ET-1 in the airway wall have not been clearly defined. We therefore wished to estimate the relative contributions of the epithelium and endothelium to ET-1 production in the bronchi (mean ID 1.7-4.9 mm) of mature normal sheep. Morphometric assessment of bronchial cross-sections revealed a number of epithelial cells three times greater than endothelial cells by direct cell count. In contrast, the overall cell surface density was five to six times greater, and the airway smooth muscle-centered cell surface density was three to four times greater for endothelial cells than for epithelial cells. The expression of preproendothelin-1 mRNA was detected in cultured aortic endothelial cells (as a substitute for bronchial endothelial cells) but not in cultured bronchial epithelial cells, and the former secreted seven times more immunoreactive ET-1 than the latter. These findings show that topographically the endothelium is better positioned for the regulation of ovine bronchial smooth muscle than the epithelium. Furthermore, the findings suggest greater constitutive ET-1 secretion by cultured endothelium than by epithelium.

Characterization of endothelin receptor subtypes on airway smooth muscle cells.

Endothelin-1 (ET-1) has constrictor and mitogenic effects on airway smooth muscle strips and cultured cells, respectively. This study addresses the type of the ET receptor subtype(s) present on ovine airway smooth muscle cells and the possibility of autocrine effects. The expression of the preproendothelin-1 gene was demonstrated by Northern analysis, and the medium obtained from these cells contained immunoreactive-ET-1. Competitive binding experiments between [125I]ET-1 and ET-1, ET-3, and two ET-receptor subtype selective-ligands, BQ-123 (ETA) and sarafotoxin S6c (ETB), yielded IC50 values of 1.1 +/- 0.1, 227 +/- 13, 12 +/- 1, and 194 +/- 21 nM, respectively. ET-3 also gave a limited number of higher affinity sites. In the presence of BQ-123 (1 microM), the binding of [125I]ET-1 was decreased by 80-85%, and the IC50 values with ET-1, ET-3, and S6c were 2.0 +/- 0.4, 3.6 +/- 0.6, and 1.1 +/- 0.9 nM, respectively. In similar experiments with 0.1 microM sarafotoxin S6c, the respective IC50 values for ET-1 and ET-3 were 2.4 +/- 0.4 and 300 +/- 20 nM. These results demonstrate that about 85 +/- 5% of ET-1 binding to airway smooth muscle cells is to ETA receptors and that these cells produce ET-1 in vitro.

A transformed murine Leydig cell line expresses the ETA receptor subtype.

We recently demonstrated that transformed murine Leydig cells (MA-10) responded to endothelin-1 (ET-1) via increased steroidogenesis. This study addresses the endothelin receptor subtype present on this cell line and whether or not the cells produce ET-1. The expression of the preproendothelin-1 (PPET-1) gene was investigated by Northern blot analysis, and PPET-1 mRNA was found to be < 0.2% of that present in pulmonary endothelial cells. The medium from MA-10 cells, maintained under serum-free conditions, was analyzed by radio-immunoassay to determine immunoreactive-ET-1 production and ET-1 levels were found to be below the sensitivity of the assay (< 10 pg/ml). The data from competitive binding experiments with [125I]ET-1 and unlabeled ET-1, ET-3 and receptor subtype selective ligands yielded a single class of high affinity binding sites with ETA receptor subtype characteristics. The results of this study demonstrate that MA-10 cells possess the ETA receptor subtype but do not produce significant quantities of ET-1 under basal conditions.

Endothelin-1 promotes mitogenesis in airway smooth muscle cells.

Endothelin exists as three isoforms (ET-1, ET-2, and ET-3) and exhibits vasoconstricting, bronchoconstricting, and growth-promoting properties in vascular smooth muscle. In the airways, ET-1 immunoreactivity and mRNA have been detected and localized to the epithelium, smooth muscle, and endothelium in different species, including humans. It has been suggested that ET-1 may have a role in the airway smooth muscle hyperplasia and hypertrophy seen in patients with bronchial asthma. We studied ovine airway smooth muscle cells (SMC) in vitro and showed saturable binding of [125I]ET-1 with a dissociation constant (Kd) of 0.4 nM and high affinity binding sites (Bmax) for ET-1 (104 fmol/10(6) cells). This binding was functional as ET-1 promoted mitogenesis of these muscle cells as measured by increased cell number in the absence of serum. Twenty-four hours after exposing the cells to graded doses of ET-1 from 1 pM to 1 microM, cell number increased significantly over control in a dose-dependent manner. ET-1 also enhanced the transient expression of c-fos mRNA by 2.5-fold over control, with maximal expression occurring at 30 min. These observations provide evidence that: (1) airway SMC possess high affinity binding sites for ET-1, and (2) ET-1 is mitogenic for airway SMC as determined by increased cell number and amplification of c-fos mRNA expression. ET-1 may have a fundamental role in influencing the growth of smooth muscle in the airways.

Endothelin-1 promotes steroidogenesis and stimulates protooncogene expression in transformed murine Leydig cells.

The effects of endothelin-1 (ET-1), a potent vasoconstrictor and mitogen to various cell types, on proliferation and differentiated functions of the murine Leydig tumor cell line MA-10 were investigated. Kinetic binding experiments at room temperature showed that [125I] ET-1 bound to MA-10 cells and reached equilibrium in 2 h. The data from competitive binding experiments yielded an apparent single class of high affinity binding sites characterized by a Kd and maximum binding capacity of 1 nM and 59 fmol/10(6) cells, respectively. For steroidogenic assays, cells were incubated with ET-1 (1 pM to 1 microM) and with epidermal growth factor (10 ng/ml) for 4 h at 37 C, and the progesterone levels in the medium were measured by RIA. Like epidermal growth factor, ET-1 caused about a 6-fold increase in progesterone production. ET-1 also enhanced the transient expression of the protooncogenes c-jun and c-myc by 3- and 2-fold, respectively. For proliferation studies, ET-1 (1 pM to 1 microM) was added to quiescent MA-10 cells for 24 h, and cell counts were performed; no increase in cell number was observed. The results of this study demonstrate that MA-10 cells possess high affinity binding sites for ET-1 and that ET-1 stimulates progesterone production and protooncogene expression, but not mitosis in this cell line.

Microvascular and macrovascular endothelial cells produce different constrictor substances.

The media from cultured microvascular and macrovascular endothelial cells (conditioned media, CM) were collected and tested for constrictor activity in sheep coronary artery rings and tracheal smooth muscle strips in vitro (isometric force), expressed as percentage of contraction produced by 80 mM KCl. Both microvascular (micro) and macrovascular (macro) CM caused a sustained slow-onset contraction (P less than 0.05) of the coronary artery rings by 71 +/- 10% (micro; n = 7) and 67 +/- 8% (macro; n = 6) and tracheal smooth muscle strips by 33 +/- 14% (micro; n = 6) and 34 +/- 6% (macro; n = 11); the calcium antagonist gallopamil (10(-7) M) attenuated these effects by 25-55%. Unconditioned medium and medium conditioned by cultured tracheal smooth muscle cells had no constrictor activity on coronary artery rings or tracheal smooth muscle strips. Synthetic endothelin (ET-1) also produced contraction of coronary artery rings and tracheal smooth muscle strips. The mean levels of ET-1 measured by radioimmunoassay were 1,200 pg/ml in the macro CM and 33 pg/ml in the micro CM. Depleting macro CM of ET-1 by affinity columns constructed with protein A agarose and anti-ET-1 antibody removed the contractile activity for coronary artery rings and tracheal smooth muscle strips. Thus ET-1 did not appear to be the contractile substance in the micro CM. Preliminary characterization of the contractile substance in micro CM revealed that it was heat stable, had a molecular weight of less than 10,000, was inactivated by trypsin, and retained its activity after two cycles of freeze-thawing.(ABSTRACT TRUNCATED AT 250 WORDS)

Endothelin-1 from pulmonary artery and microvessels acts on vascular and airway smooth muscle.

The conditioned medium (CM) from microvascular and macrovascular endothelial cell (EC) cultures was tested for constrictor activity. Sheep coronary artery rings, under 1 g tension, and sheep tracheal smooth muscle strips, under 2 g tension, were hung in organ baths containing Krebs-Henseleit solution (39 degrees C) and were equilibrated with 95% O2 and 5% CO2. Isometric force was measured in response to 80 mM KCl and constrictor responses to 100% CM were expressed as a percentage of maximum KCl response. Serum-free CM from confluent microvascular endothelial cells caused a sustained, slow-onset contraction of the coronary rings similar to that obtained with CM from macrovascular ECs. Indomethacin (5 microM) added to either the microvascular or macrovascular CM enhanced the constrictor responses by 1.6- and 1.8-fold, respectively, and the constrictor effects of both media were reduced by the calcium antagonist gallopamil (10 nM). CM from macrovascular EC caused a sustained contraction of tracheal strips similar to microvascular CM. In both cases, constriction was preceded by a brief relaxation as noted in vascular smooth muscle. Unconditioned medium had no constrictor activity on either vascular or airway smooth muscle. Microvascular-derived constrictor activity was heat stable. There was a slight loss of activity in the heat-treated CM from the macrovascular cells compared with control CM. Constrictor effects on tracheal smooth muscle were partially reversed by 1 microM gallopamil.

Cultured endothelial cells derived from the human iliac arteries.

Cells derived from the endothelium of human iliac arteries were cultured in vivo. The cells were isolated, grown, and subcultured in HEPES buffered Medium 199 supplemented with 20% heat inactivated human whole blood serum, human alpha-thrombin, and commercial endothelial cell growth supplement derived from bovine brain. The cells were viable in culture for 8 to 10 passages at a split ratio of 1:3. After the 10th passage, the cells began to enlarge and their growth rate was reduced. No cultures were viable after the 12th passage. The cells were determined to be of endothelial origin by their morphology at confluence; their ultrastructural characteristics, including the presence of Weibel-Palade bodies; the production and release of factor VIII-related antigen; and by their maintenance of a surface that prevented platelet attachment. The cultured arterial endothelial cells released prostacyclin in response to challenge with thrombin and protamine sulfate but not in response to bradykinin or the platelet-derived growth factor. Although the cultures described in this report were derived from patients with varying degrees of atherosclerotic disease, there were no significant differences in morphological or physiological parameters among these cultures or in comparison with commonly studied cells derived from human umbilical veins.