Effects of Deletion of Macrophage ABCA7 on Lipid Metabolism and the Development of Atherosclerosis in the Presence and Absence of ABCA1

ABCA7, a close relative of ABCA1 which facilitates cholesterol efflux to lipid-poor apoproteins, has been implicated in macrophage lipid efflux and clearance of apoptotic cells in in vitro studies. In the current study, we investigated the in vivo effects of macrophage ABCA7 deficiency on lipid metabolism and atherosclerosis. Chimeras with dysfunctional ABCA7 in macrophages and other blood cells were generated by transplantation of bone marrow from ABCA7 knockout (KO) mice into irradiated low-density lipoprotein receptor (LDLr) KO mice. Unexpectedly, macrophage ABCA7 deficiency did not significantly affect atherosclerosis susceptibility of LDLr KO mice after 10 weeks Western-type diet feeding. However, ABCA7 deficiency was associated with 2-fold (p<0.05) higher macrophage ABCA1 mRNA expression levels. Combined disruption of ABCA1 and ABCA7 in bone-marrow-derived cells increased atherosclerotic lesion development (1.5-fold (p>0.05) as compared to wild type transplanted mice. However, single deletion of ABCA1 had a similar effect (1.8-fold, p<0.05). Macrophage foam cell accumulation in the peritoneal cavity was reduced in ABCA1/ABCA7 dKO transplanted animals as compared to single ABCA1 KO transplanted mice, which was associated with increased ABCG1 expression. Interestingly, spleens of ABCA1/ABCA7 double KO transplanted mice were significantly larger as compared to the other 3 groups and showed massive macrophage lipid accumulation, a reduction in CD3+ T-cells, and increased expression of key regulators of erythropoiesis. In conclusion, deletion of ABCA7 in bone marrow-derived cells does not affect atherogenesis in the arterial wall neither in the absence or presence of ABCA1. Interestingly, combined deletion of bone marrow ABCA1 and ABCA7 causes severe splenomegaly associated with cellular lipid accumulation, a reduction in splenic CD3+ T cells, and induced markers of erythropoeisis. Our data indicate that ABCA7 may play a role in T cell proliferation and erythropoeisis in spleen

Marked alveolar apoptosis/proliferation imbalance in end-stage emphysema

BACKGROUND: Apoptosis has recently been proposed to contribute to the pathogenesis of emphysema. METHODS: In order to establish if cell fate plays a role even in end-stage disease we studied 16 lungs (9 smoking-associated and 7 α1antitrypsin (AAT)-deficiency emphysema) from patients who had undergone lung transplantations. Six unused donor lungs served as controls. Apoptosis was evaluated by TUNEL analysis, single-stranded DNA laddering, electron microscopy and cell proliferation by an immunohistochemical method (MIB1). The role of the transforming growth factor (TGF)-β1 pathway was also investigated and correlated with epithelial cell turnover and with the severity of inflammatory cell infiltrate. RESULTS: The apoptotic index (AI) was significantly higher in emphysematous lungs compared to the control group (p ≤ 0.01), particularly if only lungs with AAT-deficiency emphysema were considered (p ≤ 0.01 vs p = 0.09). The proliferation index was similar in patients and controls (1.9 ± 2.2 vs 1.7 ± 1.1). An increased number of T lymphocytes was observed in AAT-deficiency lungs than smoking-related cases (p ≤ 0.05). TGF-β1 expression in the alveolar wall was higher in patients with smoking-associated emphysema than in cases with AAT-deficiency emphysema (p ≤ 0.05). A positive correlation between TGF-βRII and AI was observed only in the control group (p ≤ 0.005, r(2 )= 0.8). A negative correlation was found between the TGF-β pathway (particularly TGF-βRII) and T lymphocytes infiltrate in smoking-related cases (p ≤ 0.05, r(2 )= 0.99) CONCLUSION: Our findings suggest that apoptosis of alveolar epithelial cells plays an important role even in end-stage emphysema particularly in AAT-deficiency disease. The TGFβ-1 pathway does not seem to directly influence epithelial turnover in end-stage disease. Inflammatory cytokine different from TGF-β1 may differently orchestrate cell fate in AAT and smoking-related emphysema types

Bcl-2 protein family: Implications in vascular apoptosis and atherosclerosis

Apoptosis has been recognized as a central component in the pathogenesis of atherosclerosis, in addition to the other human pathologies such as cancer and diabetes. The pathophysiology of atherosclerosis is complex, involving both apoptosis and proliferation at different phases of its progression. Oxidative modification of lipids and inflammation differentially regulate the apoptotic and proliferative responses of vascular cells during progression of the atherosclerotic lesion. Bcl-2 proteins act as the major regulators of extrinsic and intrinsic apoptosis signalling pathways and more recently it has become evident that they mediate the apoptotic response of vascular cells in response to oxidation and inflammation either in a provocative or an inhibitory mode of action. Here we address Bcl-2 proteins as major therapeutic targets for the treatment of atherosclerosis and underscore the need for the novel preventive and therapeutic interventions against atherosclerosis, which should be designed in the light of molecular mechanisms regulating apoptosis of vascular cells in atherosclerotic lesions

Endometrial apoptosis and neutrophil infiltration during menstruation exhibits spatial and temporal dynamics that are recapitulated in a mouse model.

Abstract Menstruation is characterised by synchronous shedding and restoration of tissue integrity. An in vivo model of menstruation is required to investigate mechanisms responsible for regulation of menstrual physiology and to investigate common pathologies such as heavy menstrual bleeding (HMB). We hypothesised that our mouse model of simulated menstruation would recapitulate the spatial and temporal changes in the inflammatory microenvironment of human menses. Three regulatory events were investigated: cell death (apoptosis), neutrophil influx and cytokine/chemokine expression. Well-characterised endometrial tissues from women were compared with uteri from a mouse model (tissue recovered 0, 4, 8, 24 and 48 h after removal of a progesterone-secreting pellet). Immunohistochemistry for cleaved caspase-3 (CC3) revealed significantly increased staining in human endometrium from late secretory and menstrual phases. In mice, CC3 was significantly increased at 8 and 24 h post-progesterone-withdrawal. Elastase+ human neutrophils were maximal during menstruation; Ly6G+ mouse neutrophils were maximal at 24 h. Human endometrial and mouse uterine cytokine/chemokine mRNA concentrations were significantly increased during menstrual phase and 24 h post-progesterone-withdrawal respectively. Data from dated human samples revealed time-dependent changes in endometrial apoptosis preceding neutrophil influx and cytokine/chemokine induction during active menstruation. These dynamic changes were recapitulated in the mouse model of menstruation, validating its use in menstrual research

Toll-Like Receptor-2 Mediates Diet and/or Pathogen Associated Atherosclerosis: Proteomic Findings

BACKGROUND. Accumulating evidence implicates a fundamental link between the immune system and atherosclerosis. Toll-like receptors are principal sensors of the innate immune system. Here we report an assessment of the role of the TLR2 pathway in atherosclerosis associated with a high-fat diet and/or bacteria in ApoE+/- mice. METHODS AND RESULTS. To explore the role of TLR2 in inflammation- and infection-associated atherosclerosis, 10 week-old ApoE+/--TLR2+/+, ApoE+/--TLR2+/- and ApoE+/--TLR2-/- mice were fed either a high fat diet or a regular chow diet. All mice were inoculated intravenously, once per week for 24 consecutive weeks, with 50 μl live Porphyromonas gingivalis (P.g) (107 CFU) or vehicle (normal saline). Animals were euthanized 24 weeks after the first inoculation. ApoE+/--TLR2+/+ mice showed a significant increase in atheromatous lesions in proximal aorta and aortic tree compared to ApoE+/--TLR2+/- and ApoE+/--TLR2-/- mice for all diet conditions. They also displayed profound changes in plaque composition, as evidenced by increased macrophage infiltration and apoptosis, increased lipid content, and decreased smooth muscle cell mass, all reflecting an unstable plaque phenotype. SAA levels from ApoE+/--TLR2+/+ mice were significantly higher than from ApoE+/--TLR2+/- and ApoE+/--TLR2-/- mice. Serum cytokine analysis revealed increased levels of pro-inflammatory cytokines in ApoE+/--TLR2+/+ mice compared to ApoE+/--TLR2+/- and TLR2-/- mice, irrespective of diet or bacterial challenge. ApoE+/--TLR2+/+ mice injected weekly for 24 weeks with FSL-1 (a TLR2 agonist) also demonstrated significant increases in atherosclerotic lesions, SAA and serum cytokine levels compared to ApoE+/--TLR2-/- mice under same treatment condition. Finally, mass-spectrometry (MALDI-TOF-MS) of aortic samples analyzed by 2-dimentional gel electrophoresis differential display, identified 6 proteins upregulated greater than 2-fold in ApoE+/--TLR2+/+ mice fed the high fat diet and inoculated with P.g compared to any other group. CONCLUSION. Genetic deficiency of TLR2 reduces diet- and/or pathogen-associated atherosclerosis in ApoE+/- mice, along with differences in plaque composition suggesting greater structural stability while TLR-2 ligand-specific activation triggers atherosclerosis. The present data offers new insights into the pathophysiological pathways involved in atherosclerosis and paves the way for new pharmacological interventions aimed at reducing atherosclerosis.National Heart, Lung, and Blood Institute (R01 HL076801

Alteration of proliferation and apoptotic markers in normal and premalignant tissue associated with prostate cancer

BACKGROUND: Molecular markers identifying alterations in proliferation and apoptotic pathways could be particularly important in characterizing high-risk normal or pre-neoplastic tissue. We evaluated the following markers: Ki67, Minichromosome Maintenance Protein-2 (Mcm-2), activated caspase-3 (a-casp3) and Bcl-2 to determine if they showed differential expression across progressive degrees of intraepithelial neoplasia and cancer in the prostate. To identify field effects, we also evaluated whether high-risk expression patterns in normal tissue were more common in prostates containing cancer compared to those without cancer (supernormal), and in histologically normal glands adjacent to a cancer focus as opposed to equivalent glands that were more distant. METHODS: The aforementioned markers were studied in 13 radical prostatectomy (RP) and 6 cystoprostatectomy (CP) specimens. Tissue compartments representing normal, low grade prostatic intraepithelial neoplasia (LGPIN), high grade prostatic intraepithelial neoplasia (HGPIN), as well as different grades of cancer were mapped on H&E slides and adjacent sections were analyzed using immunohistochemistry. Normal glands within 1 mm distance of a tumor focus and glands beyond 5 mm were considered "near" and "far", respectively. Randomly selected nuclei and 40 × fields were scored by a single observer; basal and luminal epithelial layers were scored separately. RESULTS: Both Ki-67 and Mcm-2 showed an upward trend from normal tissue through HGPIN and cancer with a shift in proliferation from basal to luminal compartment. Activated caspase-3 showed a significant decrease in HGPIN and cancer compartments. Supernormal glands had significantly lower proliferation indices and higher a-casp3 expression compared to normal glands. "Near" normal glands had higher Mcm-2 indices compared to "far" glands; however, they also had higher a-casp3 expression. Bcl-2, which varied minimally in normal tissue, did not show any trend across compartments or evidence for field effects. CONCLUSION: These results demonstrate that proliferation and apoptosis are altered not only in preneoplastic lesions but also in apparently normal looking epithelium associated with cancer. Luminal cell expression of Mcm-2 appears to be particularly promising as a marker of high-risk normal epithelium. The role of apoptotic markers such as activated caspase-3 is more complex, and might depend on the proliferation status of the tissue in question

Human antimicrobial peptide LL-37 is present in atherosclerotic plaques and induces death of vascular smooth muscle cells: a laboratory study

BACKGROUND: Death of smooth muscle cells in the atherosclerotic plaques makes the plaques more prone to rupture, which can initiate an acute ischemic event. The development of atherosclerosis includes the migration of immune cells e.g. monocytes/macrophages and T lymphocytes into the lesions. Immune cells can release antimicrobial peptides. One of these, human cathelicidin antimicrobial peptide hCAP-18, is cleaved by proteinase 3 generating a 4.5 kDa C-terminal fragment named LL-37, which has been shown to be cytotoxic. The aim of the study was to explore a potential role of LL-37 in the pathophysiology of atherosclerosis. METHODS: We investigated the presence of LL-37 in human atherosclerotic lesions obtained at autopsy using immunohistochemistry. The direct effects of LL-37 on cultured vascular smooth muscle cells and isolated neutrophil granulocytes were investigated with morphological, biochemical and flow cytometry analysis. RESULTS: The neointima of atherosclerotic plaques was found to contain LL-37-like immunoreactivity, mainly in macrophages. In cultured smooth muscle cells, LL-37 at 30 μg/ml caused cell shrinkage, membrane blebbing, nuclear condensation, DNA fragmentation and an increase in caspase-3 activity as studied by microscopy, ELISA and enzyme activity assay, respectively. Flow cytometry demonstrated that LL-37 in a subset of the cells caused a small but rapidly developing increase in membrane permeability to propidium iodide, followed by a gradual development of FITC-annexin V binding. Another cell population stained heavily with both propidium iodide and FITC-annexin V. Neutrophil granulocytes were resistant to these effects of LL-37. CONCLUSION: This study shows that LL-37 is present in atherosclerotic lesions and that it induces death of vascular smooth muscle cells. In a subset of cells, the changes indicate the development of apoptosis triggered by an initial mild perturbation of plasma membrane integrity. The findings suggest a role for LL-37 as a mediator of immune cell-induced death of vascular smooth muscle cells in atherosclerosis

Improved Control of Tuberculosis and Activation of Macrophages in Mice Lacking Protein Kinase R

Host factors that microbial pathogens exploit for their propagation are potential targets for therapeuic countermeasures. No host enzyme has been identified whose genetic absence benefits the intact mammalian host in vivo during infection with Mycobacterium tuberculosis (Mtb), the leading cause of death from bacterial infection. Here, we report that the dsRNA-dependent protein kinase (PKR) is such an enzyme. PKR-deficient mice contained fewer viable Mtb and showed less pulmonary pathology than wild type mice. We identified two potential mechanisms for the protective effect of PKR deficiency: increased apoptosis of macrophages in response to Mtb and enhanced activation of macrophages in response to IFN-gamma. The restraining effect of PKR on macrophage activation was explained by its mediation of a previously unrecognized ability of IFN-gamma to induce low levels of the macrophage deactivating factor interleukin 10 (IL10). These observations suggest that PKR inhibitors may prove useful as an adjunctive treatment for tuberculosis