Edward C. Dempsey

Smooth Muscle Cells Isolated From Discrete Compartments of the Mature Vascular Media Exhibit Unique Phenotypes and Distinct Growth Capabilities

Heterogeneity of smooth muscle cell (SMC) phenotype and function is rapidly emerging as an important concept. We have recently described that phenotypically distinct SMC subpopulations in bovine pulmonary arteries exhibit unique proliferative and matrix-producing responses to hypoxic pulmonary hypertension. To provide better understanding of the molecular mechanisms contributing to this phenomenon, experimental studies will require a reliable in vitro model. The purpose of the present study was first to determine if distinct SMC subpopulations, similar to those observed in vivo, could be selectively isolated from the mature arterial media, and then to evaluate whether select SMC subpopulations would exhibit heightened responses to growth-promoting stimuli and hypoxia. We were able to reproducibly isolate at least four phenotypically unique cell subpopulations from the inner, middle, and outer compartments of the arterial media. Differences in cell phenotype were demonstrated by morphological appearance and differential expression of muscle-specific proteins. The isolated cell subpopulations exhibited markedly different growth capabilities. Two SMC subpopulations grew slowly in 10% serum and were quiescent in plasma-based medium. The other two cell subpopulations, exhibiting nonmuscle characteristics, grew rapidly in 10% serum and proliferated in plasma-based medium and in response to hypoxia. Certain colonies of the nonmuscle-like cell subpopulations were found to grow autonomously under serum-deprived conditions and to secrete mitogenic factors. Our data, demonstrating that phenotypically distinct cells with enhanced growth potential exist within the normal arterial media, support the idea that these unique cells could contribute selectively to the pathogenesis of vascular disease.

Smooth Muscle Cell Heterogeneity in Pulmonary and Systemic Vessels Importance in Vascular Disease

Experimental evidence is rapidly accumulating which demonstrates that the arterial media in both pulmonary and systemic vessels is not composed of a phenotypically homogeneous population of smooth muscle cells (SMCs) but rather of heterogeneous subpopulations of cells with unique developmental lineages. In vivo and in vitro observations strongly suggest that marked differences in the phenotype, growth, and matrix-producing capabilities of phenotypically distinct SMC subpopulations exist and that these differences are intrinsic to the cell type. These data also suggest that differential proliferative and matrix-producing capabilities of distinct SMC subpopulations govern, at least in part, the pattern of abnormal cell proliferation and matrix protein synthesis observed in the pathogenesis of vascular disease. Within the pulmonary circulation, the observation that the isolated medial SMC subpopulations exhibit differential proliferative responses to hypoxic exposure is important, since this in vitro cell-model system can now be used to better understand the mechanisms that regulate increased responsiveness of specific medial cell subpopulations to low oxygen concentrations. Our data also support the idea that protein kinase C is likely to be one important determinant of differential cell growth responses to hypoxia. The data also suggest differential involvement of specific arterial SMC subpopulations in the elastogenic responses of the vessel wall to injury. We believe that a better understanding of the mechanisms contributing to the unique behavior of specific arterial cell subpopulations will provide important future directions for therapies aimed at preventing abnormal cell replication and matrix protein synthesis in vascular disease.

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 withdrawal, 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 to 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(β)‐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 17β‐estradiol was partially (62%) reproduced with 17‐alpha(α)‐estradiol, an isomer which does not bind the estrogen receptor. This suggested that 17β‐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 μM dihydrosphingosine and phorbol‐ester‐induced downregulation) were applied prior to 17α‐ or β‐estradiol and blocked the enhanced responses to PDGF. The synergistic effect of 17β‐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.

Neprilysin Null Mice Develop Exaggerated Pulmonary Vascular Remodeling in Response to Chronic Hypoxia

Neprilysin is a transmembrane metalloendopeptidase that degrades neuropeptides that are important for both growth and contraction. In addition to promoting carcinogenesis, decreased levels of neprilysin increases inflammation and neuroendocrine cell hyperplasia, which may predispose to vascular remodeling. Early pharmacological studies showed a decrease in chronic hypoxic pulmonary hypertension with neprilysin inhibition. We used a genetic approach to test the alternate hypothesis that neprilysin depletion increases chronic hypoxic pulmonary hypertension. Loss of neprilysin had no effect on baseline airway or alveolar wall architecture, vessel density, cardiac function, hematocrit, or other relevant peptidases. Only lung neuroendocrine cell hyperplasia and a subtle neuropeptide imbalance were found. After chronic hypoxia, neprilysin-null mice exhibited exaggerated pulmonary hypertension and striking increases in muscularization of distal vessels. Subtle thickening of proximal media/adventitia not typically seen in mice was also detected. In contrast, adaptive right ventricular hypertrophy was less than anticipated. Hypoxic wild-type pulmonary vessels displayed close temporal and spatial relationships between decreased neprilysin and increased cell growth. Smooth muscle cells from neprilysin-null pulmonary arteries had increased proliferation compared with controls, which was decreased by neprilysin replacement. These data suggest that neprilysin may be protective against chronic hypoxic pulmonary hypertension in the lung, at least in part by attenuating the growth of smooth muscle cells. Lung-targeted strategies to increase neprilysin levels could have therapeutic benefits in the treatment of this disorder.

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.

Pregnancy stimulation of DNA synthesis and uterine blood flow in the guinea pig.

Pregnancy stimulates DNA synthesis in uterine artery smooth muscle cells. Unknown is whether DNA synthesis increases in all layers of the vessel wall in uterine or nonuterine vessels, the distribution and time course of the proliferative response in relation to the rise in uterine blood flow, and the extent to which a pregnancy-induced rise in DNA synthesis can be mimicked by chronic estradiol treatment. To measure DNA synthesis, we implanted bromodeoxyuridine (BrdU, 400 mg) s.c. for 14-d periods in three nonpregnant, nine pregnant, three vehicle, and five estradiol (2.5 mg/14 d)-treated guinea pigs. Uterine blood flow was measured in four nonpregnant and 18 pregnant animals using radiolabeled microspheres. Pregnancy stimulated DNA synthesis in the adventitia, media, and intima of the uterine artery, radial artery (the vessels deriving from the main uterine artery and entering the uterine wall), and uterine vein but not in the aorta or mesenteric artery. Maximal uterine artery medial area and labeling indices in all layers of the uterine artery, uterine vein, and the radial artery adventitia were attained by mid-pregnancy(d 28-42 of the guinea pigs 63-day gestation), whereas DNA synthesis increased progressively until term in the radial artery media and intima. The greatest rise in uterine artery blood flow (y) occurred after peak proliferation in the uterine artery and in concert with radial artery medial and intimal proliferation (y = 1.99·100.023x where x is day postconception). 17β-Estradiol treatment for 14 d in ovariectomized guinea pigs increased DNA synthesis in the radial artery adventitia and tended (p = 0.08) to increase labeling indices in the media of all vessels examined but did not fully reproduce the effects of pregnancy. We concluded that pregnancy-related, possibly hormonal stimuli prompted growth in all layers of the uterine artery wall by mid-pregnancy and served to initiate a rise in uterine blood flow. The resultant increase in flow and shear stress likely stimulated DNA synthesis in the radial artery which helped sustain the rise in flow near term.

Alveolar Hypoxia Promotes Murine Lung Tumor Growth through a VEGFR-2/EGFR-Dependent Mechanism

Patients with chronic obstructive pulmonary disease (COPD) are at an increased risk for the development of lung cancer, the mechanisms for which are incompletely understood. We hypothesized that the hypoxic pulmonary microenvironment present in COPD would augment lung carcinogenesis. Mice were subjected to chemical carcinogenesis protocols and placed in either hypoxia or normoxia. Mice exposed to chronic hypoxia developed tumors with increased volume compared with normoxic controls. Both lungs and tumors from hypoxic mice showed a preferential stabilization of HIF-2α and increased expression of VEGF-A, FGF2, and their receptors as well as other survival, proliferation, and angiogenic signaling pathways regulated by HIF-2α. We showed that tumors arising in hypoxic animals have increased sensitivity to VEGFR-2/EGFR inhibition, as chemoprevention with vandetanib showed markedly increased activity in hypoxic mice. These studies showed that lung tumors arising in a hypoxic microenvironment express increased growth, angiogenic, and survival signaling that could contribute to the increased lung cancer risk in COPD. Furthermore, the differential sensitivity of tumors arising in hypoxia to VEGFR-2/EGFR inhibition suggests that the altered signaling present in tumors arising in hypoxic lung might be therapeutically exploited in patients with underlying COPD. Cancer Prev Res; 5(8); 1061–71. ©2012 AACR.

Impact of customized videotape education on quality of life in patients with chronic obstructive pulmonary disease.

PURPOSE To compare the impact of a library of pulmonary rehabilitation videotapes versus an older videotape and usual care on quality of life and ability to perform activities of daily living in persons with chronic obstructive pulmonary disease. METHODS Two hundred fourteen patients diagnosed with chronic obstructive pulmonary disease, emphysema, or chronic bronchitis were recruited and randomized to receive customized videotapes, standard videotapes, or usual care. Outcome measures included the Fatigue Impact Scale, Seattle Obstructive Lung Disease Questionnaire, and the SF-36(R) Health Survey. RESULTS Differences in coping skills and emotional functioning on the Seattle Obstructive Lung Disease Questionnaire were found among the 174 subjects who completed the study. The customized videotape group improved by 8.6 and 4.8 points, respectively, whereas the score of the other groups decreased by less than 1 point for the coping skills, and the scores of the standard video and the control groups decreased by 3.0 and 2.1 points, respectively, for emotional functioning (P < .05, all comparisons). The scores using the Fatigue Impact Scale also improved for the customized videotape group, whereas the scores of the others remained unchanged. Videotape users demonstrated better conversion to and retention of exercise habits, with over 80% of customized videotape subjects who reported exercise habits at baseline continuing the habits as compared with 40% in the usual care group. Sedentary subjects at baseline were more likely to begin and maintain exercise if randomized to videotapes. CONCLUSIONS These findings demonstrate increased quality of life, lower fatigue, and better compliance with a prescribed exercise regimen among subjects using the customized videotapes. There was a significant improvement in emotional functioning and coping skills among customized videotape subjects.

Decreased Neprilysin and Pulmonary Vascular Remodeling in Chronic Obstructive Pulmonary Disease

RATIONALE Studies with genetically engineered mice showed that decreased expression of the transmembrane peptidase neprilysin (NEP) increases susceptibility to hypoxic pulmonary vascular remodeling and hypertension; in hypoxic wild-type mice, expression is decreased early in distal pulmonary arteries, where prominent vascular remodeling occurs. Therefore, in humans with smoke- and hypoxia-induced vascular remodeling, as in chronic obstructive pulmonary disease (COPD), pulmonary activity/expression of NEP may likewise be decreased. OBJECTIVES To test whether NEP activity and expression are reduced in COPD lungs and pulmonary arterial smooth muscle cells (SMCs) exposed to cigarette smoke extract or hypoxia and begin to investigate mechanisms involved. METHODS Control and advanced COPD lung lysates (n = 13-14) were analyzed for NEP activity and protein and mRNA expression. As a control, dipeptidyl peptidase IV activity was analyzed. Lung sections were assessed for vascular remodeling and oxidant damage. Human pulmonary arterial SMCs were exposed to cigarette smoke extract, hypoxia, or H₂O₂, and incubated with antioxidants or lysosomal/proteasomal inhibitors. MEASUREMENTS AND MAIN RESULTS COPD lungs demonstrated areas of vascular rarification, distal muscularization, and variable intimal and prominent medial/adventitial thickening. NEP activity was reduced by 76%; NEP protein expression was decreased in alveolar walls and distal vessels; mRNA expression was also decreased. In SMCs exposed to cigarette smoke extract, hypoxia, and H₂O₂, NEP activity and expression were also reduced. Reactive oxygen species inactivated NEP activity; NEP protein degradation appeared to be substantially induced. CONCLUSIONS Mechanisms responsible for reduced NEP activity and protein expression include oxidative reactions and protein degradation. Maintaining or increasing lung NEP may protect against pulmonary vascular remodeling in response to chronic smoke and hypoxia.

A Randomized Phase II Chemoprevention Trial of 13-CIS Retinoic Acid with Or without α Tocopherol or Observation in Subjects at High Risk for Lung Cancer

No chemoprevention strategies have been proven effective for lung cancer. We evaluated the effect of 13-cis retinoic acid (13-cis RA), with or without α tocopherol, as a lung cancer chemoprevention agent in a phase II randomized controlled clinical trial of adult subjects at high risk for lung cancer as defined by the presence of sputum atypia, history of smoking, and airflow obstruction, or a prior surgically cured nonsmall cell lung cancer (disease free, >3 years). Subjects were randomly assigned to receive either 13-cis RA, 13-cis RA plus α tocopherol (13-cis RA/α toco) or observation for 12 months. Outcome measures are derived from histologic evaluation of bronchial biopsy specimens obtained by bronchoscopy at baseline and follow-up. The primary outcome measure is treatment “failure” defined as histologic progression (any increase in the maximum histologic score) or failure to return for follow-up bronchoscopy. Seventy-five subjects were randomized (27/22/26 to obervations/13-cis RA/13-cis RA/α toco); 59 completed the trial; 55 had both baseline and follow-up bronchoscopy. The risk of treatment failure was 55.6% (15 of 27) and 50% (24 of 48) in the observation and combined (13 cis RA plus 13 cis RA/α toco) treatment arms, respectively (odds ratio adjusted for baseline histology, 0.97; 95% confidence interval, 0.36-2.66; P = 0.95). Among subjects with complete histology data, maximum histology score in the observation arm increased by 0.37 units and by 0.03 units in the treated arms (difference adjusted for baseline, −0.18; 95% confidence interval, −1.16 to 0.81; P = 0.72). Similar (nonsignificant) results were observed for treatment effects on endobronchial proliferation as assessed by Ki-67 immunolabeling. Twelve-month treatment with 13-cis RA produced nonsignificant changes in bronchial histology, consistent with results in other trials. Agents advancing to phase III randomized trials should produce greater histologic changes. The addition of α tocopherol did not affect toxicity.