Linda A. Gleaves

Increased atherosclerosis in LDL receptor–null mice lacking ACAT1 in macrophages

During atherogenesis, circulating macrophages migrate into the subendothelial space, internalize cholesterol-rich lipoproteins, and become foam cells by progressively accumulating cholesterol esters. The inhibition of macrophage acyl coenzyme A:cholesterol acyltransferase (ACAT), which catalyzes the formation of cholesterol esters, has been proposed as a strategy to reduce foam cell formation and to treat atherosclerosis. We show here, however, that hypercholesterolemic LDL receptor-deficient (LDLR(-/-)) mice reconstituted with ACAT1-deficient macrophages unexpectedly develop larger atherosclerotic lesions than control LDLR(-/-) mice. The ACAT1-deficient lesions have reduced macrophage immunostaining and more free cholesterol than control lesions. Our findings suggest that selective inhibition of ACAT1 in lesion macrophages in the setting of hyperlipidemia can lead to the accumulation of free cholesterol in the artery wall, and that this promotes, rather than inhibits, lesion development.

Macrophage lipoprotein lipase promotes foam cell formation and atherosclerosis in vivo

Expression of lipoprotein lipase (LPL) by the macrophage has been proposed to promote foam cell formation and atherosclerosis, primarily on the basis of in vitro studies. LPL-deficient mice might provide a model for testing the role of LPL secretion by the macrophage in an in vivo system. Unfortunately, homozygous deficiency of LPL in the mouse is lethal shortly after birth. Because the fetal liver is the major site of hematopoiesis in the developing fetus, transplantation of C57BL/6 mice with LPL-/- fetal liver cells (FLCs) was used to investigate the physiologic role of macrophage LPL expression in vivo. Thirty-four female C57BL/6 mice were lethally irradiated and reconstituted with FLCs from day 14 LPL+/+, LPL+/-, and LPL-/- donors. No significant differences were detected in plasma levels of post-heparin LPL activity or in serum cholesterol or triglyceride levels between the 3 groups on either a chow diet or an atherogenic diet. After 19 weeks on the atherogenic diet, aortae were collected for quantitative analysis of the extent of aortic atherosclerosis. LPL expression was detected by immunocytochemistry and in situ hybridization in macrophages of aortic atherosclerotic lesions of LPL+/+-->C57BL/6 and LPL+/--->C57BL/6 mice, but not in LPL-/--->C57BL/6 mice, whereas myocardial cells expressed LPL in all groups. The mean aortic lesion area was reduced by 55% in LPL-/--->C57BL/6 mice compared with LPL+/+-->C57BL/6 mice and by 45% compared with LPL+/--->C57BL/6 mice, respectively. These data demonstrate in vivo that LPL expression by macrophages in the artery wall promotes foam cell formation and atherosclerosis. off

Reduced Atherosclerotic Lesions in Mice Deficient for Total or Macrophage-Specific Expression of Scavenger Receptor-A

Abstract—The absence of the scavenger receptor A (SR-A)-I/II has produced variable effects on atherosclerosis in different murine models. Therefore, we examined whether SR-AI/II deficiency affected atherogenesis in C57BL/6 mice, an inbred strain known to be susceptible to diet-induced atherosclerotic lesion formation, and whether the deletion of macrophage SR-AI/II expression would modulate lesion growth in C57BL/6 mice and LDL receptor (LDLR)−/− mice. SR-AI/II–deficient (SR-AI/II−/−) female and male mice on the C57BL/6 background were challenged with a butterfat diet for 30 weeks. No differences were detected in plasma lipids between SR-AI/II−/− and SR-AI/II+/+ mice, whereas both female and male SR-AI/II−/− mice had a tremendous reduction (81% to 86%) in lesion area of the proximal aorta compared with SR-AI/II+/+ mice. Next, to analyze the effect of macrophage-specific SR-AI/II deficiency in atherogenesis, female C57BL/6 mice were lethally irradiated, transplanted with SR-AI/II−/− or SR-AI/II+/+ fetal liver cells, and challenged with the butterfat diet for 16 weeks. In a separate experiment, male LDLR−/− mice were reconstituted with SR-AI/II−/− or SR-AI/II+/+ fetal liver cells and challenged with a Western diet for 10 weeks. No significant differences in plasma lipids and lipoprotein profiles were noted between the control and experimental groups in either experiment. SR-AI/II−/−→C57BL/6 mice, however, had a 60% reduction in lesion area of the proximal aorta compared with SR-AI/II+/+→C57BL/6 mice. A similar level of reduction (60%) in lesion area was noted in the proximal aorta and the entire aorta en face of SR-AI/II−/−→LDLR−/− mice compared with SR-AI/II+/+→LDLR−/− mice. These results demonstrate in vivo that SR-AI/II expression has no impact on plasma lipid levels and that macrophage SR-AI/II contributes significantly to atherosclerotic lesion formation.

Repetitive intratracheal bleomycin models several features of idiopathic pulmonary fibrosis

Single-dose intratracheal bleomycin has been instrumental for understanding fibrotic lung remodeling, but fails to recapitulate several features of idiopathic pulmonary fibrosis (IPF). Since IPF is thought to result from recurrent alveolar injury, we aimed to develop a repetitive bleomycin model that results in lung fibrosis with key characteristics of human disease, including alveolar epithelial cell (AEC) hyperplasia. Wild-type and cell fate reporter mice expressing β-galactosidase in cells of lung epithelial lineage were given intratracheal bleomycin after intubation, and lungs were harvested 2 wk after a single or eighth biweekly dose. Lungs were evaluated for fibrosis and collagen content. Bronchoalveolar lavage (BAL) was performed for cell counts. TUNEL staining and immunohistochemistry were performed for pro-surfactant protein C (pro-SP-C), Clara cell 10 (CC-10), β-galactosidase, S100A4, and α-smooth muscle actin. Lungs from repetitive bleomycin mice had marked fibrosis with prominent AEC hyperplasia, similar to usual interstitial pneumonia (UIP). Compared with single dosing, repetitive bleomycin mice had greater fibrosis by scoring, morphometry, and collagen content; increased TUNEL+ AECs; and reduced inflammatory cells in BAL. Sixty-four percent of pro-SP-C+ cells in areas of fibrosis expressed CC-10 in the repetitive model, suggesting expansion of a bronchoalveolar stem cell-like population. In reporter mice, 50% of S100A4+ lung fibroblasts were derived from epithelial mesenchymal transition compared with 33% in the single-dose model. With repetitive bleomycin, fibrotic remodeling persisted 10 wk after the eighth dose. Repetitive intratracheal bleomycin results in marked lung fibrosis with prominent AEC hyperplasia, features reminiscent of UIP.

Retroviral Gene Therapy in ApoE-Deficient Mice ApoE Expression in the Artery Wall Reduces Early Foam Cell Lesion Formation

BACKGROUND Apolipoprotein E (apoE) has long been known to play an important role in the clearance of plasma lipoproteins. More recently, a direct role for apoE in delaying atherogenesis has been proposed. Macrophage production of apoE in the artery wall has been demonstrated to provide protection against atherosclerotic lesion development independently from its role in lipoprotein clearance. However, whether macrophage apoE can affect lesion growth at all stages of atherogenesis remains to be established. METHODS AND RESULTS To evaluate the role of macrophage apoE in different stages of atherogenesis, as well as to establish a novel gene therapy approach to atherosclerotic vascular disease, we used an apoE-expressing retrovirus to transduce apoE-deficient (-/-) bone marrow for transplantation into apoE(-/-) recipient mice. Three weeks after bone marrow transplantation, apoE was expressed from arterial macrophages and was detectable in plasma associated with lipoproteins at 0.5% to 1% of normal levels but did not affect plasma cholesterol levels. We used 2 groups of recipient mice: younger mice with lesions consisting primarily of foam cells and older mice with more advanced lesions. When either the mouse or human apoE transgenes were expressed in mice from 5 to 13 weeks of age, there was a significant reduction in lesion area, whereas no effects were detected in mice that expressed apoE from 10 to 26 weeks of age. CONCLUSIONS We demonstrate that arterial macrophage apoE secretion can delay atherogenesis if expressed during foam cell formation but is not beneficial during the later stages of atherogenesis. These data also provide evidence that apoE transgene expression from arterial macrophages may have therapeutic applications.

Genetic Deficiency of Plasminogen Activator Inhibitor-1 Promotes Cardiac Fibrosis in Aged Mice Involvement of Constitutive Transforming Growth Factor-β Signaling and Endothelial-to-Mesenchymal Transition

Background— Elevated levels of plasminogen activator inhibitor-1 (PAI-1), a potent inhibitor of urokinase plasminogen activator and tissue plasminogen activator, are implicated in the pathogenesis of tissue fibrosis. Paradoxically, lack of PAI-1 in the heart is associated with the development of cardiac fibrosis in aged mice. However, the molecular basis of cardiac fibrosis in aged PAI-1-deficient mice is unknown. Here, we investigated the molecular and cellular bases of myocardial fibrosis. Methods and Results— Histological evaluation of myocardial tissues derived from aged PAI-1-deficient mice revealed myocardial fibrosis resulting from excessive accumulation of collagen. Immunohistochemical characterization revealed that the levels of matrix metalloproteinase-2, matrix metalloproteinase-9, and transforming growth factor-&bgr;1/2 and the number of Mac3-positive and fibroblast specific protein-1-positive cells were significantly elevated in aged PAI-1-deficient myocardial tissues compared with controls. Zymographic analysis revealed that matrix metalloproteinase-2 enzymatic activity was elevated in PAI-1-deficient mouse cardiac endothelial cells. Real-time quantitative polymerase chain reaction analyses of RNA from myocardial tissues revealed the upregulation of profibrotic markers in aged PAI-1-deficient mice. The numbers of phosphorylated Smad2–, phosphorylated Smad3–, and phosphorylated ERK1/2 MAPK-, but not pAkt/PKB-, positive cells were significantly increased in PAI-1-deficient myocardial tissues. Western blot and immunocytochemical analysis revealed that PAI-1-deficient mouse cardiac endothelial cells were more susceptible to endothelial-to-mesenchymal transition in response to transforming growth factor-&bgr;2. Conclusions— These results indicate that spontaneous activation of both Smad and non-Smad transforming growth factor-&bgr; signaling may contribute to profibrotic responses in aged PAI-1-deficient mice hearts and establish a possible link between endothelial-to-mesenchymal transition and cardiac fibrosis in PAI-1-deficient mice.

A Critical Role for Macrophages in Promotion of Urethane-Induced Lung Carcinogenesis

Macrophages have established roles in tumor growth and metastasis, but information about their role in lung tumor promotion is limited. To assess the role of macrophages in lung tumorigenesis, we developed a method of minimally invasive, long-term macrophage depletion by repetitive intratracheal instillation of liposomal clodronate. Compared with controls treated with repetitive doses of PBS-containing liposomes, long-term macrophage depletion resulted in a marked reduction in tumor number and size at 4 mo after a single i.p. injection of the carcinogen urethane. After urethane treatment, lung macrophages developed increased M1 macrophage marker expression during the first 2–3 wk, followed by increased M2 marker expression by week 6. Using a strategy to reduce alveolar macrophages during tumor initiation and early promotion stages (weeks 1–2) or during late promotion and progression stages (weeks 4–16), we found significantly fewer and smaller lung tumors in both groups compared with controls. Late-stage macrophage depletion reduced VEGF expression and impaired vascular growth in tumors. In contrast, early-stage depletion of alveolar macrophages impaired urethane-induced NF-κB activation in the lungs and reduced the development of premalignant atypical adenomatous hyperplasia lesions at 6 wk after urethane injection. Together, these studies elucidate an important role for macrophages in lung tumor promotion and indicate that these cells have distinct roles during different stages of lung carcinogenesis.

A Direct Role for the Macrophage Low Density Lipoprotein Receptor in Atherosclerotic Lesion Formation

To evaluate the contribution of the macrophage low density lipoprotein receptor (LDLR) to atherosclerotic lesion formation, we performed bone marrow transplantation studies in different mouse strains. First, LDLR(−/−) mice were transplanted with either LDLR(+/+) marrow or LDLR(−/−) marrow and were challenged with an atherogenic Western type diet. The diet caused severe hypercholesterolemia of a similar degree in the two groups, and no differences in the aortic lesion area were detected. Thus, macrophage LDLR expression does not influence foam cell lesion formation in the setting of extreme LDL accumulation. To determine whether macrophage LDLR expression affects foam cell formation under conditions of moderate, non-LDL hyperlipidemia, we transplanted C57BL/6 mice with either LDLR(−/−) marrow (experimental group) or LDLR(+/+) marrow (controls). Cholesterol levels were not significantly different between the two groups at baseline or after 6 weeks on a butterfat diet, but were 40% higher in the experimental mice after 13 weeks, mostly due to accumulation of β-very low density lipoprotein (β-VLDL). Despite the increase in cholesterol levels, mice receiving LDLR(−/−) marrow developed 63% smaller lesions than controls, demonstrating that macrophage LDLR affects the rate of foam cell formation when the atherogenic stimulus is β-VLDL. We conclude that the macrophage LDLR is responsible for a significant portion of lipid accumulation in foam cells under conditions of dietary stress.

Bronchoscopic Cryobiopsy for the Diagnosis of Diffuse Parenchymal Lung Disease

Background Although in some cases clinical and radiographic features may be sufficient to establish a diagnosis of diffuse parenchymal lung disease (DPLD), surgical lung biopsy is frequently required. Recently a new technique for bronchoscopic lung biopsy has been developed using flexible cryo-probes. In this study we describe our clinical experience using bronchoscopic cryobiopsy for diagnosis of diffuse lung disease. Methods A retrospective study of subjects who had undergone bronchoscopic cryobiopsy for evaluation of DPLD at an academic tertiary care center from January 1, 2012 through January 15, 2013 was performed. The procedure was performed using a flexible bronchoscope to acquire biopsies of lung parenchyma. H&E stained biopsies were reviewed by an expert lung pathologist. Results Twenty-five eligible subjects were identified. With a mean area of 64.2 mm2, cryobiopsies were larger than that typically encountered with traditional transbronchial forceps biopsy. In 19 of the 25 subjects, a specific diagnosis was obtained. In one additional subject, biopsies demonstrating normal parenchyma were felt sufficient to exclude diffuse lung disease as a cause of dyspnea. The overall diagnostic yield of bronchoscopic cryobiopsy was 80% (20/25). The most frequent diagnosis was usual interstitial pneumonia (UIP) (n = 7). Three of the 25 subjects ultimately required surgical lung biopsy. There were no significant complications. Conclusion In patients with suspected diffuse parenchymal lung disease, bronchoscopic cryobiopsy is a promising and minimally invasive approach to obtain lung tissue with high diagnostic yield.

Antiproliferative Agents Alter Vascular Plasminogen Activator Inhibitor-1 Expression. A Potential Prothrombotic Mechanism of Drug-Eluting Stents

Objectives—Drug eluting stents (DES) reduce the incidence of restenosis after coronary angioplasty. Enthusiasm has been tempered by a possible increased risk of in-stent thrombosis. We examined the effects of paclitaxel and rapamycin on the endothelial transcriptome to identify alterations in gene expression associated with thrombosis. Methods and Results—Gene expression profiling was performed on human coronary artery endothelial cells treated with rapamycin or paclitaxel. Plasminogen activator inhibitor-1 (PAI-1) was the most consistently induced transcript in rapamycin-treated human coronary artery endothelial cells. RT-PCR and ELISA were performed to confirm positive findings. Transgenic mice engineered to express enhanced green fluorescent protein under control of the human PAI-1 promoter were also treated. Rapamycin and paclitaxel treated endothelial cells produced dose-dependent increases in PAI-1. There was a variable effect on endothelial tissue-type plasminogen activator (t-PA) expression. Enhanced expression of PAI-1 and enhanced green fluorescent protein were detected in coronary arteries, the aorta, and kidney of the mice. Conclusion—Antiproliferative agents stimulate the expression of prothrombotic genes. PAI-1 expression may also play a role in the prevention of restenosis through an antimigratory mechanism. The effects of antiproliferatives on vascular gene expression deserve further scrutiny in view of the increasing utilization of drug-eluting stents.