Srikanth Karnati

Compromised peroxisomes in idiopathic pulmonary fibrosis, a vicious cycle inducing a higher fibrotic response via TGF-β signaling

Significance This study enhances the knowledge on the molecular pathogenesis of idiopathic pulmonary fibrosis (IPF). To date, there is no information available on the role of peroxisomes in lung fibrosis. In our study we demonstrate that peroxisomal biogenesis and metabolism is compromised in tissue samples as well as in fibroblasts of IPF patients and in bleomycin-induced fibrosis mouse model. Moreover, RNAi-mediated knockdown of peroxisomal biogenesis leads to a profibrotic response in control and IPF fibroblasts suggesting that the reduction of peroxisomal function in IPF would contribute to the profibrotic phenotype of this devastating disease. Our work opens a new field of research in the area of lung fibrosis and might lead to novel treatment strategies against IPF by modulating the peroxisomal compartment. Idiopathic pulmonary fibrosis (IPF) is a devastating disease, and its pathogenic mechanisms remain incompletely understood. Peroxisomes are known to be important in ROS and proinflammatory lipid degradation, and their deficiency induces liver fibrosis. However, altered peroxisome functions in IPF pathogenesis have never been investigated. By comparing peroxisome-related protein and gene expression in lung tissue and isolated lung fibroblasts between human control and IPF patients, we found that IPF lungs exhibited a significant down-regulation of peroxisomal biogenesis and metabolism (e.g., PEX13p and acyl-CoA oxidase 1). Moreover, in vivo the bleomycin-induced down-regulation of peroxisomes was abrogated in transforming growth factor beta (TGF-β) receptor II knockout mice indicating a role for TGF-β signaling in the regulation of peroxisomes. Furthermore, in vitro treatment of IPF fibroblasts with the profibrotic factors TGF-β1 or tumor necrosis factor alpha (TNF-α) was found to down-regulate peroxisomes via the AP-1 signaling pathway. Therefore, the molecular mechanisms by which reduced peroxisomal functions contribute to enhanced fibrosis were further studied. Direct down-regulation of PEX13 by RNAi induced the activation of Smad-dependent TGF-β signaling accompanied by increased ROS production and resulted in the release of cytokines (e.g., IL-6, TGF-β) and excessive production of collagen I and III. In contrast, treatment of fibroblasts with ciprofibrate or WY14643, PPAR-α activators, led to peroxisome proliferation and reduced the TGF-β–induced myofibroblast differentiation and collagen protein in IPF cells. Taken together, our findings suggest that compromised peroxisome activity might play an important role in the molecular pathogenesis of IPF and fibrosis progression, possibly by exacerbating pulmonary inflammation and intensifying the fibrotic response in the patients.

Cre-Mediated Stress Affects Sirtuin Expression Levels, Peroxisome Biogenesis and Metabolism, Antioxidant and Proinflammatory Signaling Pathways

Cre-mediated excision of loxP sites is widely used in mice to manipulate gene function in a tissue-specific manner. To analyze phenotypic alterations related to Cre-expression, we have used AMH-Cre-transgenic mice as a model system. Different Cre expression levels were obtained by investigation of C57BL/6J wild type as well as heterozygous and homozygous AMH-Cre-mice. Our results indicate that Cre-expression itself in Sertoli cells already has led to oxidative stress and lipid peroxidation (4-HNE lysine adducts), inducing PPARα/γ, peroxisome proliferation and alterations of peroxisome biogenesis (PEX5, PEX13 and PEX14) as well as metabolic proteins (ABCD1, ABCD3, MFP1, thiolase B, catalase). In addition to the strong catalase increase, a NRF2- and FOXO3-mediated antioxidative response (HMOX1 of the endoplasmic reticulum and mitochondrial SOD2) and a NF-κB activation were noted. TGFβ1 and proinflammatory cytokines like IL1, IL6 and TNFα were upregulated and stress-related signaling pathways were induced. Sertoli cell mRNA-microarray analysis revealed an increase of TNFR2-signaling components. 53BP1 recruitment and expression levels for DNA repair genes as well as for p53 were elevated and the ones for related sirtuin deacetylases affected (SIRT 1, 3-7) in Sertoli cells. Under chronic Cre-mediated DNA damage conditions a strong downregulation of Sirt1 was observed, suggesting that the decrease of this important coordinator between DNA repair and metabolic signaling might induce the repression release of major transcription factors regulating metabolic and cytokine-mediated stress pathways. Indeed, caspase-3 was activated and increased germ cell apoptosis was observed, suggesting paracrine effects. In conclusion, the observed wide stress-induced effects and metabolic alterations suggest that it is essential to use the correct control animals (Cre/Wt) with matched Cre expression levels to differentiate between Cre-mediated and specific gene-knock out-mediated effects.

Peroxisomes in mouse and human lung: their involvement in pulmonary lipid metabolism.

Only sparse information is available from the literature on the peroxisomal compartment and its enzyme composition in mouse and human lungs. Therefore, in the present investigation we have characterized peroxisomes in different cell types of adult mouse (C57BL/6J) and human lungs in a comprehensive study using a variety of light-, fluorescence- and electron microscopic as well as biochemical techniques and by the use of various peroxisomal marker proteins (Pex13p, Pex14p, ABCD3, β-oxidation enzymes and catalase). In contrast to previous reports, we have found that peroxisomes are present in all cell types in human and mouse lungs. However, they differ significantly and in a cell-type-specific manner in their structure, numerical abundance and enzyme composition. Whereas catalase showed significant differences between distinct cell types, Pex14p proved to be the marker of choice for labeling all lung peroxisomes. In alveolar type II cells and alveolar macrophages peroxisomes contained significant amounts of the lipid transporter ABCD3 and β-oxidation enzymes, suggesting their involvement in the modification and recycling of surfactant lipids and in the control of lipid mediators and ligands for nuclear receptors of the PPAR family. Possible connections between ROS and lipid metabolism of lung peroxisomes are discussed.

Peroxisomes in airway epithelia and future prospects of these organelles for pulmonary cell biology

Peroxisomes are intimately involved in the metabolism of reactive oxygen species, in the synthesis of ether lipids and of polyunsaturated fatty acids as well as in the β-oxidation of bioactive and toxic lipid derivatives. Therefore, the metabolic pathways of this organelle might play an important role in pulmonary biology by protection of inner pulmonary surface epithelia against oxidative stress, induced by the high oxygen levels in the air and/or by regulation of the lipid homeostasis in pulmonary epithelia and the pulmonary surfactant film. In this article, original results on the distribution of peroxisomal marker proteins, involved in the biogenesis, ROS- and lipid-metabolism of this organelle in the bronchiolar epithelium and the alveolar region of the adult human lung in comparison to newborn and adult murine lungs are presented. In addition, we investigated the expression of the PEX11β-mRNA, encoding a protein involved in peroxisomal division. Our study revealed significant differences in the abundance and distribution of peroxisomal proteins in distinct cell types of the lung and different developmental stages and led to the discovery of species-specific differences in the peroxisomal compartment in pulmonary epithelia between mouse and man. Finally, the structure and general biology of pulmonary airways—with special emphasis on Clara cells—are reviewed and discussed in relation to peroxisomal metabolism and proliferation. Future prospects of peroxisomes and Pex11 proteins for pulmonary cell biology are highlighted.

The biogenesis protein PEX14 is an optimal marker for the identification and localization of peroxisomes in different cell types, tissues, and species in morphological studies

Abstract Catalase and ABCD3 are frequently used as markers for the localization of peroxisomes in morphological experiments. Their abundance, however, is highly dependent on metabolic demands, reducing the validity of analyses of peroxisomal abundance and distribution based solely on these proteins. We therefore attempted to find a protein which can be used as an optimal marker for peroxisomes in a variety of species, tissues, cell types and also experimental designs, independently of peroxisomal metabolism. We found that the biogenesis protein peroxin 14 (PEX14) is present in comparable amounts in the membranes of every peroxisome and is optimally suited for immunoblotting, immunohistochemistry, immunofluorescence, and immunoelectron microscopy. Using antibodies against PEX14, we could visualize peroxisomes with almost undetectable catalase content in various mammalian tissue sections (submandibular and adrenal gland, kidney, testis, ovary, brain, and pancreas from mouse, cat, baboon, and human) and cell cultures (primary cells and cell lines). Peroxisome labeling with catalase often showed a similar tissue distribution to the mitochondrial enzyme mitochondrial superoxide dismutase (both responsible for the degradation of reactive oxygen species), whereas ABCD3 exhibited a distinct labeling only in cells involved in lipid metabolism. We increased the sensitivity of our methods by using QuantumDots™, which have higher emission yields compared to classic fluorochromes and are unsusceptible to photobleaching, thereby allowing more exact quantification without artificial mistakes due to heterogeneity of individual peroxisomes. We conclude that PEX14 is indeed the best marker for labeling of peroxisomes in a variety of tissues and cell types in a consistent fashion for comparative morphometry.

Observations on the microvasculature of bone defects filled with biodegradable nanoparticulate hydroxyapatite

The microvascularization of metaphyseal bone defects filled with nanoparticulate, biodegradable hydroxyapatite biomaterial with and without platelet factors enrichment was investigated in a minipig model. Results from morphological analysis and PECAM-1 immunohistochemistry showed the formation of new blood vessels into the bone defects by sprouting and intussusception of pre-existing ones. However, no significant differences were observed in the microvascularization of the different biomaterials applied (pure versus platelet factors-enriched hydroxyapatite), concerning the number of vessels and their morphological structure at day 20 after operation. The appearance of VEGFR-2 positive endothelial progenitor cells in the connective tissue between hydroxyapatite particles was also found to be independent from platelet factors enrichment of the hydroxyapatite bone substitute. In both groups formation of lymphatic vessels was detected with a podoplanin antibody. No differences were noted between HA/PLF- and HA/PLF+ implants with respect to the podoplanin expression level, the staining pattern or number of lymphatic vessels. In conclusion, the present study demonstrates different mechanisms of blood and lymphatic vessel formation in hydroxyapatite implants in minipigs.

Mammalian SOD2 is exclusively located in mitochondria and not present in peroxisomes.

Superoxide dismutases (SODs) are metalloenzymes that belong to the essential antioxidant enzyme systems of virtually all oxygen-respiring organisms. SODs catalyze the dismutation of highly reactive superoxide radicals into hydrogen peroxide and molecular oxygen. For the subcellular localization of the manganese superoxide dismutase (SOD2) in eukaryotic cells, a dual mitochondrial localization and peroxisomal localization were proposed in the literature. However, our own observation from immunofluorescence preparations of human and mouse tissues suggested that SOD2 serves as an excellent marker protein for mitochondria but never co-localized with peroxisomes. To clarify whether our observations were correct, we have carefully reinvestigated the subcellular localization of SOD2 using sensitive double-immunofluorescence methods on frozen and paraffin sections as well as in cell culture preparations. In addition, ultrastructural analyses were performed with post-embedding immunoelectron microscopy on LR White sections as well as labeling of ultrathin cryosections with various immunogold techniques. In all morphological experiments, the SOD2 localization was compared to one of the catalase, a typical marker protein for peroxisomes, solely localized in these organelles. Moreover, biochemical subcellular fractions of mouse liver was used to isolate enriched organelles and highly purified peroxisomal fractions for Western blot analyses of the exact subcellular distributions of SOD2 and catalase. All results with the various methodologies, tissues, and cell types used revealed that catalase and SOD2 were always confined to distinct and separate subcellular compartments. SOD2 was unequivocally in mitochondria, but never present in peroxisomes. Furthermore, our results are supported by accumulating database information on organelle proteomes that also indicate that SOD2 is a pure mitochondrial protein.

Acute epididymitis induces alterations in sperm protein composition

OBJECTIVE To use a proteomic approach to evaluate possible postinflammatory alterations in the protein composition of motile sperm in patients 3 months after acute epididymitis. DESIGN Prospective case-control study. SETTING University medical school research laboratory. PATIENT(S) Eight patients 3 months after acute unilateral epididymitis and 10 healthy controls. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Proteome analysis of sperm samples collected by swim-up from control and acute epididymitis patients analyzed by two-dimensional gel electrophoresis and subsequent protein identification by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry; immunofluorescence staining for mitochondrial ATP synthase subunit β (ATP5B), α-tubulin (TUBA1A), and tubulin-β2c (TUBB4B) for validation purposes. RESULT(S) Proteome analysis identified 35 proteins in sperm from epididymitis patients that were down-regulated, irrespective of subcellular localization and biologic function. Furthermore, immunofluorescence microscopy confirmed ATP5B, TUBA1A, and TUBB4B were less abundantly expressed in epididymitis samples compared with controls. CONCLUSION(S) Despite normal semen parameters observed by conventional semen analysis in patients after epididymitis, significant changes to sperm protein composition were observed. These changes may be implicated as additional factors contributing to subfertility/infertility in men after episodes of epididymitis.

PEX6 is Expressed in Photoreceptor Cilia and Mutated in Deafblindness with Enamel Dysplasia and Microcephaly

Deafblindness is part of several genetic disorders. We investigated a consanguineous Egyptian family with two siblings affected by congenital hearing loss and retinal degeneration, initially diagnosed as Usher syndrome type 1. At teenage, severe enamel dysplasia, developmental delay, and microcephaly became apparent. Genome‐wide homozygosity mapping and whole‐exome sequencing detected a homozygous missense mutation, c.1238G>T (p.Gly413Val), affecting a highly conserved residue of peroxisomal biogenesis factor 6, PEX6. Biochemical profiling of the siblings revealed abnormal and borderline plasma phytanic acid concentration, and cerebral imaging revealed white matter disease in both. We show that Pex6 localizes to the apical extensions of secretory ameloblasts and differentiated odontoblasts at early stages of dentin synthesis in mice, and to cilia of retinal photoreceptor cells. We propose PEX6, and possibly other peroxisomal genes, as candidate for the rare cooccurrence of deafblindness and enamel dysplasia. Our study for the first time links peroxisome biogenesis disorders to retinal ciliopathies.

BDNF and its TrkB receptor in human fracture healing.

Fracture healing is a physiological process of repair which proceeds in stages, each characterized by a different predominant tissue in the fracture gap. Matrix reorganization is regulated by cytokines and growth factors. Neurotrophins and their receptors might be of importance to osteoblasts and endothelial cells during fracture healing. The aim of this study was to examine the presence of brain-derived neurotrophic factor (BDNF) and its tropomyosin-related kinase B receptor (TrkB) during human fracture healing. BDNF and TrkB were investigated in samples from human fracture gaps and cultured cells using RT-PCR, Western blot, and immunohistochemistry. Endothelial cells and osteoblastic cell lines demonstrated a cytoplasmic staining pattern of BDNF and TrkB in vitro. At the mRNA level, BDNF and TrkB were expressed in the initial and osteoid formation phase of human fracture healing. In the granulation tissue of fracture gap, both proteins--BDNF and TrkB--are concentrated in endothelial and osteoblastic cells at the margins of woven bone suggesting their involvement in the formation of new vessels. There was no evidence of BDNF or TrkB during fracture healing in chondrocytes of human enchondral tissue. Furthermore, BDNF is absent in mature bone. Taken together, BDNF and TrkB are involved in vessel formation and osteogenic processes during human fracture healing. The detection of BDNF and its TrkB receptor during various stages of the bone formation process in human fracture gap tissue were shown for the first time. The current study reveals that both proteins are up-regulated in human osteoblasts and endothelial cells in fracture healing.