Prothrombin Knockdown Protects Podocytes and Reduces Proteinuria in Glomerular Disease.

Chronic kidney disease (CKD) is a leading cause of death, and its progression is driven by glomerular podocyte injury and loss, manifesting as proteinuria. Proteinuria includes urinary loss of coagulation zymogens, cofactors, and inhibitors. Importantly, both CKD and proteinuria significantly increase the risk of thromboembolic disease. Prior studies demonstrated that anticoagulants reduced proteinuria in rats and that thrombin injured cultured podocytes. Herein we aimed to directly determine the influence of circulating prothrombin on glomerular pathobiology. We hypothesized that (pro)thrombin drives podocytopathy, podocytopenia, and proteinuria. Glomerular proteinuria was induced with puromycin aminonucleoside (PAN) in Wistar rats. Circulating prothrombin was either knocked down using a rat-specific antisense oligonucleotide or elevated by serial intravenous infusions of prothrombin protein, which are previously established methods to model hypo- (LoPT) and hyper-prothrombinemia (HiPT), respectively. After 10 days (peak proteinuria in this model) plasma prothrombin levels were determined, kidneys were examined for (pro)thrombin co-localization to podocytes, histology, and electron microscopy. Podocytopathy and podocytopenia were determined and proteinuria, and plasma albumin were measured. LoPT significantly reduced prothrombin colocalization to podocytes, podocytopathy, and proteinuria with improved plasma albumin. In contrast, HiPT significantly increased podocytopathy and proteinuria. Podocytopenia was significantly reduced in LoPT vs. HiPT rats. In summary, prothrombin knockdown ameliorated PAN-induced glomerular disease whereas hyper-prothrombinemia exacerbated disease. Thus, (pro)thrombin antagonism may be a viable strategy to simultaneously provide thromboprophylaxis and prevent podocytopathy-mediated CKD progression.

Optimizing extracellular vesicles' isolation from chronic lymphocytic leukemia patient plasma and cell line supernatant.

In chronic lymphocytic leukemia (CLL) and very likely all cancer types, extracellular vesicles (EVs) are a common mechanism by which intercellular messages are communicated between normal, diseased, and transformed cells. Studies of EVs in CLL and other cancers have great variability and often lack reproducibility. For CLL patient plasma and cell lines, we sought to characterize current approaches used in isolating EV products and understand whether cell culture-conditioned media or complex biological fluids confound results. Utilizing nanoparticle tracking analysis, protein quantification, and electron microscopy, we show that ultracentrifugation with an OptiPrep cushion can effectively minimize contaminants from starting materials including plasma and conditioned media of CLL cell lines grown in EV-depleted complete RPMI media but not grown in the serum-free media AIM V commonly used in CLL experimental work. Moreover, we confirm the benefit of including 25 mM trehalose in PBS during EV isolation steps to reduce EV aggregation, to preserve function for downstream applications and characterization. Furthermore, we report the highest particles/µg EVs were obtained from our CLL cell lines utilizing the CELLine bioreactor flask. Finally, we optimized a proliferation assay that offers a functional evaluation of our EVs with minimal sample requirements.

A Morphometric Analysis of Platelet Dense Granules of Patients with Unexplained Bleeding: A New Entity of Delta-Microgranular Storage Pool Deficiency.

One thousand and eighty patients, having prolonged bleeding times, frequent epistaxis, menorrhagia or easy bruising or other bleeding manifestations, and excluding those with von Willebrand's disease, were evaluated for platelet dense granule deficiency. The mean diameter of platelet dense granules was determined for all patients using image analysis. Four hundred and ninety-nine had "classic" dense (delta) granule storage pool deficiency (δ-SPD). Five hundred and eighty-one individuals (53.8%) were found to have a normal mean number of dense granules, but for some of these patients, the dense granules were smaller than for the controls. Of the patients having a normal number of dense granules, 165 (28.4%) were found to have significantly smaller granules than the platelets obtained from the control subjects. Their average granule diameter was 123.35 ± 0.86 nm, that is more than three standard deviations below the mean of the control data. Total δ-granule storage pool volumes (TDGV)/platelet were calculated using these measurements. Individuals with δ-SPD had half the number of granules (2.25 ± 0.04 DG/PL) and storage pool volume (3.88 ± 1.06 × 106 nm3) when compared to our control data (4.64 ± 0.11 DG/PL; 10.79 × 106 nm3 ± 0.42). Individuals having a bleeding history but a normal average of small dense granules had a calculated storage pool volume statistically different than controls and essentially the same storage pool volume as patients with δ-SPD. We have identified a sub-classification of δ-SPD that we have defined as micro-granular storage pool deficiency (δ-MGSPD).

Collagen fibril abnormalities in human and mice abdominal aortic aneurysm.

Vascular diseases like abdominal aortic aneurysms (AAA) are characterized by a drastic remodeling of the vessel wall, accompanied with changes in the elastin and collagen content. At the macromolecular level, the elastin fibers in AAA have been reported to undergo significant structural alterations. While the undulations (waviness) of the collagen fibers is also reduced in AAA, very little is understood about changes in the collagen fibril at the sub-fiber level in AAA as well as in other vascular pathologies. In this study we investigated structural changes in collagen fibrils in human AAA tissue extracted at the time of vascular surgery and in aorta extracted from angiotensin II (AngII) infused ApoE-/- mouse model of AAA. Collagen fibril structure was examined using transmission electron microscopy and atomic force microscopy. Images were analyzed to ascertain length and depth of D-periodicity, fibril diameter and fibril curvature. Abnormal collagen fibrils with compromised D-periodic banding were observed in the excised human tissue and in remodeled regions of AAA in AngII infused mice. These abnormal fibrils were characterized by statistically significant reduction in depths of D-periods and an increased curvature of collagen fibrils. These features were more pronounced in human AAA as compared to murine samples. Thoracic aorta from Ang II-infused mice, abdominal aorta from saline-infused mice, and abdominal aorta from non-AAA human controls did not contain abnormal collagen fibrils. The structural alterations in abnormal collagen fibrils appear similar to those reported for collagen fibrils subjected to mechanical overload or chronic inflammation in other tissues. Detection of abnormal collagen could be utilized to better understand the functional properties of the underlying extracellular matrix in vascular as well as other pathologies. STATEMENT OF SIGNIFICANCE: Several vascular diseases including abdominal aortic aneurysm (AAA) are characterized by extensive remodeling in the vessel wall. Although structural alterations in elastin fibers are well characterized in vascular diseases, very little is known about the collagen fibril structure in these diseases. We report here a comprehensive ultrastructural evaluation of the collagen fibrils in AAA, using high-resolution microscopy techniques like transmission electron microscopy (TEM) and atomic force microscopy (AFM). We elucidate how abnormal collagen fibrils with compromised D-periodicity and increased fibril curvature are present in the vascular tissue in both clinical AAA as well as in murine models. We discuss how these abnormal collagen fibrils are likely a consequence of mechanical overload accompanying AAA and could impact the functional properties of the underlying tissue.

Modulation of biomimetic mineralization of collagen by soluble ectodomain of discoidin domain receptor 2.

Collagen fibrils serve as the major template for mineral deposits in both biologically derived and engineered tissues. In recent years certain non-collagenous proteins have been elucidated as important players in differentially modulating intra vs. extra-fibrillar mineralization of collagen. We and others have previously shown that the expression of the collagen receptor, discoidin domain receptor 2 (DDR2) positively correlates with matrix mineralization. The objective of this study was to examine if the ectodomain (ECD) of DDR2 modulates intra versus extra-fibrillar mineralization of collagen independent of cell-signaling. For this purpose, a decellularized collagenous substrate, namely glutaraldehyde fixed porcine pericardium (GFPP) was subjected to biomimetic mineralization protocols. GFPP was incubated in modified simulated body fluid (mSBF) or polymer-induced liquid precursor (PILP) solutions in the presence of recombinant DDR2 ECD (DDR2-Fc) to mediate extra or intra-fibrillar mineralization of collagen. Thermogravimetric analysis revealed that DDR2-Fc increased mineral content in GFPP calcified in mSBF while no significant differences were observed in PILP mediated mineralization. Electron microscopy approaches were used to evaluate the quality and quantity mineral deposits. An increase in the matrix to mineral ratio, frequency of particles and size of mineral deposits was observed in the presence of DDR2-Fc in mSBF. Von Kossa staining and immunohistochemistry analysis of adjacent sections indicated that DDR2-Fc bound to both the matrix and mineral phase of GFPP. Further, DDR2-Fc was found to bind to hydroxyapatite (HAP) particles and enhance the nucleation of mineral deposits in mSBF solutions independent of collagen. Taken together, our results elucidate DDR2 ECD as a novel player in the modulation of extra-fibrillar mineralization of collagen.

Lethal H1N1 influenza A virus infection alters the murine alveolar type II cell surfactant lipidome.

Alveolar type II (ATII) epithelial cells are the primary site of influenza virus replication in the distal lung. Development of acute respiratory distress syndrome in influenza-infected mice correlates with significant alterations in ATII cell function. However, the impact of infection on ATII cell surfactant lipid metabolism has not been explored. C57BL/6 mice were inoculated intranasally with influenza A/WSN/33 (H1N1) virus (10,000 plaque-forming units/mouse) or mock-infected with virus diluent. ATII cells were isolated by a standard lung digestion protocol at 2 and 6 days postinfection. Levels of 77 surfactant lipid-related compounds of known identity in each ATII cell sample were measured by ultra-high-performance liquid chromatography-mass spectrometry. In other mice, bronchoalveolar lavage fluid was collected to measure lipid and protein content using commercial assay kits. Relative to mock-infected animals, ATII cells from influenza-infected mice contained reduced levels of major surfactant phospholipids (phosphatidylcholine, phosphatidylglycerol, and phosphatidylethanolamine) but increased levels of minor phospholipids (phosphatidylserine, phosphatidylinositol, and sphingomyelin), cholesterol, and diacylglycerol. These changes were accompanied by reductions in cytidine 5'-diphosphocholine and 5'-diphosphoethanolamine (liponucleotide precursors for ATII cell phosphatidylcholine and phosphatidylethanolamine synthesis, respectively). ATII cell lamellar bodies were ultrastructurally abnormal after infection. Changes in ATII cell phospholipids were reflected in the composition of bronchoalveolar lavage fluid, which contained reduced amounts of phosphatidylcholine and phosphatidylglycerol but increased amounts of sphingomyelin, cholesterol, and protein. Influenza infection significantly alters ATII cell surfactant lipid metabolism, which may contribute to surfactant dysfunction and development of acute respiratory distress syndrome in influenza-infected mice.

Collagen Fibril Ultrastructure in Mice Lacking Discoidin Domain Receptor 1.

The quantity and quality of collagen fibrils in the extracellular matrix (ECM) have a pivotal role in dictating biological processes. Several collagen-binding proteins (CBPs) are known to modulate collagen deposition and fibril diameter. However, limited studies exist on alterations in the fibril ultrastructure by CBPs. In this study, we elucidate how the collagen receptor, discoidin domain receptor 1 (DDR1) regulates the collagen content and ultrastructure in the adventitia of DDR1 knock-out (KO) mice. DDR1 KO mice exhibit increased collagen deposition as observed using Masson's trichrome. Collagen ultrastructure was evaluated in situ using transmission electron microscopy, scanning electron microscopy, and atomic force microscopy. Although the mean fibril diameter was not significantly different, DDR1 KO mice had a higher percentage of fibrils with larger diameter compared with their wild-type littermates. No significant differences were observed in the length of D-periods. In addition, collagen fibrils from DDR1 KO mice exhibited a small, but statistically significant, increase in the depth of the fibril D-periods. Consistent with these observations, a reduction in the depth of D-periods was observed in collagen fibrils reconstituted with recombinant DDR1-Fc. Our results elucidate how DDR1 modulates collagen fibril ultrastructure in vivo, which may have important consequences in the functional role(s) of the underlying ECM.

Adrenal gland inclusions in patients treated with aldosterone antagonists (Spironolactone/Eplerenone): incidence, morphology, and ultrastructural findings.

BACKGROUND: Spironolactone is often used to treat hypertension caused by hyperaldosteronism, and as a result, can form concentrically laminated electron dense spironolactone body inclusions within the adrenal gland. Spironolactone bodies have not been investigated in a contemporary cohort or in patients treated with the more recently approved aldosterone antagonist, eplerenone. METHODS: Spironolactone bodies were retrospectively investigated in patients treated for hyperaldosteronism (n=15) from 2012-2013 that underwent a subsequent adrenalectomy. RESULTS: Inclusions were identified in 33% of patients treated with aldosterone antagonists, far less than previously reported. Remarkably, 50% of patients treated with spironolactone had inclusions while no patients using eplerenone alone had inclusions. Two patients treated with spironolactone had bodies present longer than the duration described in prior studies. Inclusions unexpectedly persisted in 1 patient despite increased duration of discontinued pharmacological treatment. A spectrum of histologic and ultrastructural findings were encountered within an adrenal cortical adenoma from a patient treated with both spironolactone and eplerenone. Ultrastructural examination revealed laminated electron dense bodies with the appearance of classic spironolactone inclusions as well as electron dense bodies without laminations and laminated bodies without electron dense cores. CONCLUSIONS: Our incidence rate of spironolactone bodies was much lower than previously reported, with no inclusions seen in patients treated solely with the newer aldosterone antagonist, eplerenone. Pathologists should be aware of these infrequently encountered inclusions, particularly as the clinical history of hyperaldosteronism and pharmacologic treatment may not be provided. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/4597918761268031.

Ultrastructural findings in adrenal cortical adenomas clinically mimicking pheochromocytoma: a comparison with other adrenal tumors and tissue preparation techniques.

Adrenal cortical tumors clinically mimicking pheochromocytomas are extremely rare, with 14 cases in the literature. The authors describe 2 patients with adrenal cortical adenoma (ACA) and catecholamine elevations. The impact of tissue preparation methods on electron microscopy (EM) images was assessed in ACA mimicking pheochromocytoma, pheochromocytoma, and ACA lacking pheochromocytoma-like symptoms. Ten adrenal cortical tumors were examined using EM after a variety of tissue preparation techniques, including fixation with glutaraldehyde, formalin for varying lengths of time followed by glutaraldehyde, and/or formalin followed by paraffin embedding. Electron micrographs were assessed for image quality and the presence of dense secretory granules and eccentric, norepinephrine (NE)-type granules. Images created from tissue fixed in glutaraldehyde and/or formalin and embedded in resin were of good quality, while those derived from paraffin-embedded specimens were poor with disrupted cellular architecture. When pheochromocytoma was fixed in glutaraldehyde for 24 h or in formalin for 8 days, eccentric granules were identified. These granules were absent when tissue was fixed in formalin for 20 days or was obtained from a paraffin block. ACA without pheochromocytoma-like symptoms and ACA mimicking pheochromocytoma both had noneccentric dense-core granules on EM regardless of tissue preparation, and eccentric NE-type granules were absent. ACA is a rare cause of pheochromocytoma-like symptoms. These tumors lack eccentric, NE-type dense-core granules present in pheochromocytoma. Glutaraldehyde alone or formalin fixation followed by glutaraldehyde produces electron micrographs that may aid in the diagnosis of adrenal cortical tumors, whereas formalin-fixed, paraffin-embedded tissue results in images that are inadequate.

Sterols with antileishmanial activity isolated from the roots of Pentalinon andrieuxii.

A new cholesterol derivative, pentalinonsterol (cholest-4,20,24-trien-3-one, 1), and a new polyoxygenated pregnane sterol glycoside, pentalinonside (2), together with 18 known compounds, including 14 sterols (3-16), three coumarins (17-19), and a triterpene (20), were isolated from a n-hexane partition of a methanol extract of the roots of the Mexican medicinal plant Pentalinon andrieuxii. Structure elucidation of compounds 1 and 2 was accomplished by spectroscopic data interpretation. All isolates were evaluated in vitro for their antileishmanial activity. Among these compounds, 6,7-dihydroneridienone (15) was found to be the most potent principle against promastigotes of Leishmania mexicana (L. mexicana). The cholesterol analogue, pentalinonsterol (1), together with two known sterols, 24-methylcholest-4,24(28)-dien-3-one (3) and neridienone (16), also exhibited significant leishmanicidal activity in this same bioassay. Compounds 1, 3, 15, 16, cholest-4-en-3-one (4), and cholest-5,20,24-trien-3ß-ol (7), showed strong antileishmanial activity against amastigotes of L. mexicana, and 4 was found to be the most potent agent with an IC(50) value of 0.03µM. All the isolates were also evaluated for their cytotoxicity in non-infected bone marrow-derived macrophages, but none of these compounds was found active towards this cell line. The intracellular parasites treated with compounds 1, 3, 4, 15, and 16 were further studied by electron microscopy; morphological abnormalities and destruction of the amastigotes were observed, as a result of treatment with these compounds.

Inhibition of collagen fibrillogenesis by cells expressing soluble extracellular domains of DDR1 and DDR2.

Collagen fiber assembly affects many physiological processes and is tightly controlled by collagen-binding proteins. However, to what extent membrane-bound versus cell-secreted collagen-binding proteins affect collagen fibrillogenesis is not well understood. In our previous studies, we had demonstrated that the membrane-anchored extracellular domain (ECD) of the collagen receptor discoidin domain receptor 2 (DDR2) inhibits fibrillogenesis of collagen endogenously secreted by the cells. These results led to a novel functional role of the DDR2 ECD. However, since soluble forms of DDR1 and DDR2 containing its ECD are known to naturally exist in the extracellular matrix, in this work we investigated if these soluble DDR ECDs may have a functional role in modulating collagen fibrillogenesis. For this purpose, we created mouse osteoblast cell lines stably secreting DDR1 or DDR2 ECD as soluble proteins. Transmission electron microscopy, fluorescence microscopy, and hydroxyproline assays were used to demonstrate that DDR ECD expression reduced the rate and quantity of collagen deposition and induced significant changes in fiber morphology and matrix mineralization. Collectively, our studies advance our understanding of DDR receptors as powerful regulators of collagen deposition in the ECM and elucidate their multifaceted role in ECM remodeling.

Regulation of collagen fibrillogenesis by cell-surface expression of kinase dead DDR2.

The assembly of collagen fibers, the major component of the extracellular matrix (ECM), governs a variety of physiological processes. Collagen fibrillogenesis is a tightly controlled process in which several factors, including collagen binding proteins, have a crucial role. Discoidin domain receptors (DDR1 and DDR2) are receptor tyrosine kinases that bind to and are phosphorylated upon collagen binding. The phosphorylation of DDRs is known to activate matrix metalloproteases, which in turn cleave the ECM. In our earlier studies, we established a novel mechanism of collagen regulation by DDRs; that is, the extracellular domain (ECD) of DDR2, when used as a purified, soluble protein, inhibits collagen fibrillogenesis in-vitro. To extend this novel observation, the current study investigates how the DDR2-ECD, when expressed as a membrane-anchored, cell-surface protein, affects collagen fibrillogenesis by cells. We generated a mouse osteoblast cell line that stably expresses a kinase-deficient form of DDR2, termed DDR2/-KD, on its cell surface. Transmission electron microscopy, fluorescence microscopy, and hydroxyproline assays demonstrated that the expression of DDR2/-KD reduced the rate and abundance of collagen deposition and induced significant morphological changes in the resulting fibers. Taken together, our observations extend the functional roles that DDR2 and possibly other membrane-anchored, collagen-binding proteins can play in the regulation of cell adhesion, migration, proliferation and in the remodeling of the extracellular matrix.

Ultrastructure as a diagnostic adjunct in the evaluation of lung allograft biopsies.

Humoral immunity contributes significantly to lung graft dysfunction. Recognizing a role of ultrastructural studies in the evaluation and diagnosis of chronic humoral allograft rejection in the kidney, the authors sought to explore its utility as a diagnostic adjunct in lung allograft biopsies. Ultrastructural studies were conducted on 44 biopsies from 26 lung transplant recipients. Endothelial cell activation and necrosis were seen in the setting of acute humoral allograft rejection. Septal chronic vasculopathic changes of thickening and lamellation of the basement membrane zone (BMZ) and BMZ collagen deposition were correlated with greater numbers of humoral allograft rejection episodes and with the development of chronic graft dysfunction/bronchiolitis obliterans syndrome. There was a positive correlation between the extent of septal fibrosis and certain chronic vasculopathic changes, namely collagen deposition in the BMZ and BMZ wrinkling. Patients with chronic graft dysfunction and multiple rejection episodes manifested low diffusion capacities (less than 50% predicted). The results indicate that ultrastructural analysis is useful in identification of septal fibrosis and chronic vasculopathy of the septal microvasculature, correlating with chronic graft dysfunction, encompassing not only fibrotic sequelae of the bronchial wall but also irreversible terminal lung parenchymal changes, the latter associated with repeated episodes of humoral rejection.