Distinct Ultrastructural Changes Research Articles

Abstract 1555: CDK2 inhibition & disruption of centrosome stoichiometry: a clinically tractable death program for aneuploid cancer cells

Abstract Genomic instability is a cancer hallmark. Aberrant centrosome numbers often occur in cancer and is associated with genomic instability. Mitosis with supernumerary centrosomes leads to abnormal chromosome segregation and aneuploidy in progeny cells. While many cells with abnormal chromosomes are eliminated through apoptosis, some gain a growth advantage and acquire resistance to antineoplastic agents. This aneuploidy is associated with aggressive tumor biology and unfavorable clinical outcomes. Cyclin-dependent kinase 2 (CDK2) plays a major role regulation of cell cycle progression, centrosome duplication and clustering. We previously reported that CDK2 inhibition prevented supernumerary centrosomes clustering, causing multipolar mitosis and anaphase catastrophe in lung and other aneuploid cancers. To elucidate engaged mechanisms focused ion beam scanning electron microscope (FIB-SEM) and immunofluorescent staining were each used to interrogate the ultrastructure of multipolar mitosis after CDK2 inhibition by CYC065-treatment (a CDK2/9 inhibitor). CYC065-treatment induced distinct ultrastructural changes in aneuploid lung cancer cells including formation of chromosome rings and multipolarity. As compared to vehicle controls, CYC065-treatment markedly extended the duration of chromosome rings in lung cancer cells and conferred aberrant mitosis in live cell imagining. Intriguingly, this treatment disrupted centrosome and centriole stoichiometry in cells with chromosome rings or multipolarity. These findings were confirmed using immunofluorescence assays of lung and other cancer cells having supernumerary centrosomes. To elucidate if CDK2 or CDK9 activity directly altered centriole stoichiometry, chromosome rings or multipolarity, immunofluorescent assays were performed in lung cancer cells that independently knocked down CDK2 or CDK9 using siRNAs relative to controls. Centriole stoichiometry abnormalities in cancer cells having chromosome rings or multipolarity were statistically significantly higher in CDK2 knock-down but not in CDK9 knock-down transfectants or controls. Engineered gain of CDK2 but not CDK9 expression rescued CYC065 mediated chromosome rings and multipolarity in aneuploid lung cancer cells. Taken together, this study uncovered that CDK2 inhibition disrupted centrosome stoichiometry, causing apoptotic death of aneuploid cancer cells. This is a clinically-tractable pharmacologic mechanism that conferring cancer cell death. Current work determines if similar mechanisms are engaged in vivo in syngeneic mouse lung cancer models undergoing CDK2 antagonism. Citation Format: Zibo Chen, Xi Liu, Masanori Kawakami, Xiuxia Liu, Allison Baker, Aayush Bhatawadekar, Liliya Tyutyunyk-Massey, Ethan Dmitrovsky, Kedar Narayan. CDK2 inhibition & disruption of centrosome stoichiometry: a clinically tractable death program for aneuploid cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1555.

Plastid diversity and chromoplast biogenesis in differently coloured carrots: role of the DcOR3Leu gene.

Distinct plastid types and ultrastructural changes are associated with differences in carotenoid pigment profiles in differently coloured carrots, and a variant of the OR gene, DcOR3Leu is vital for chromoplast biogenesis. Accumulation of different types and amounts of carotenoids in carrots impart different colours to their taproots. In this study, the carotenoid pigment profiles, morphology, and ultrastructure of plastids in 25 carrot varieties with orange, red, yellow, or white taproots were investigated by ultra-high performance liquid chromatography as well as light and transmission electron microscopy. α-/β-Carotene and lycopene were identified as colour-determining carotenoids in orange and red carrots, respectively. In contrast, lutein was identified as the colour-determining carotenoid in almost all tested yellow and white carrots. The latter contained only trace amounts of lutein as a unique detectable carotenoid. Striking differences in plastid types that coincided with distinct carotenoid profiles were observed among the differently coloured carrots. Microscopic analysis of the different carotenoid pigment-loaded plastids revealed abundant crystalloid chromoplasts in the orange and red carrots, whereas amyloplasts were dominant in most of the yellow and white carrots, except for the yellow carrot 'Yellow Stone', where yellow chromoplasts were observed. Plastoglobuli and crystal remnants, the carotenoid sequestering substructures, were identified in crystalloid chromoplasts. Crystal remnants were often associated with a characteristic undulated internal membrane in orange carrots or several undulated membranes in red carrots. No crystal remnants, but some plastoglobuli, were observed in the plastids of all tested yellow and white carrots. In addition, the presence of chromoplast in carrot taproots was found to be associated with DcOR3Leu, a natural variant of DcOR3, which was previously reported to be co-segregated with carotene content in carrots. Knocking out DcOR3Leu in the orange carrot 'Kurodagosun' depressed chromoplast biogenesis and led to the generation of yellow carrots. Our results support that DcOR3Leu is vital but insufficient for chromoplasts biogenesis in carrots, and add to the understanding of the formation of chromoplasts in carrots.

Dose-Dependent Degeneration of Leydig Cells Following Kisspeptin-10 Administration: An Ultrastructural Study.

The discovery of kisspeptin signaling as a key regulator of gonadotropin- releasing hormone (GnRH) secretion from the hypothalamus enhanced our understanding of the neuroendocrine regulation of mammalian reproduction. Effects of central and peripheral administration of kisspeptin on plasma gonadotropins, testosterone, and spermatogenesis are studied in detail. The present study was conducted to check the ultrastructure of Leydig cells in prepubertal male rats in response to the administration of a range of kisspeptin doses. We administered a range of kisspeptin-10 doses (1 μg, 1 ηg, and 10 ρg) intraperitoneally to prepubertal male Sprague-Dawley rats (PND 35) twice daily after every 12 hours. Control rats were injected with physiological saline in parallel. At the end of the treatment, plasma concentrations of testosterone were measured by competitive binding radioimmunoassay, and small pieces of rat testicular tissue were processed for electron microscopy to examine the ultrastructure of Leydig cells. Plasma testosterone concentration was reduced significantly at 1ηg (P<0.05) and 1μg (P<0.01) doses as compared to control. Distinct ultrastructural changes categorized as dilatation of cytoplasmic organelles, irregularly shaped nuclei with nuclear membrane invaginations, reduced nuclear sizes, degeneration, and vacuolation were observed in the kisspeptin-10 treated Leydig cells as compared to control. Quantification of the data showed reduced Leydig cell indices and hyperplasia of the interstitial cells. It is concluded that chronic intermittent administration of kisspeptin-10 has a dose-dependent degenerative effect on the plasma testosterone levels and Leydig cells ultrastructure in prepubertal male rats.

Morphologic, cytochemical, and ultrastructural features of gray eosinophils in nine cats.

Gray eosinophils, resembling those in sighthound dog breeds, have not previously been reported in cats. We aimed to provide a morphologic, cytochemical, and ultrastructural description of gray eosinophils in cats. Blood films examined as part of routine hematology profiles in cats from May 2015 to July 2018 were evaluated for the presence of gray eosinophils. When identified with modified Wright stain, cells were morphologically assessed and additionally stained with Diff-Quik, ALP, Luna, and Luxol fast blue stains and compared with feline controls. Two cases were prepared for transmission electron microscopy (TEM) and compared with a feline control. Gray eosinophils were identified in 9 of 2641 cats during the study period. Compared with typical feline eosinophils, these cells contained abundant round granules instead of the characteristic rod-shaped specific granules. These granules lacked the characteristic intense pink/red staining with Romanowsky stains and did not stain with ALP, Luna, or Luxol fast blue stains. On TEM, the classical electron-dense core of these granules was replaced by a core with fragmented or amorphous internal material. Typical eosinophils were not identified in any cat in which gray eosinophils were identified. The distinct morphologic, cytochemical, and ultrastructural changes in gray feline eosinophils might be associated with a reduction or lack of major basic protein (MBP) in specific granule cores. Similar to canine gray eosinophils, accurate recognition of these cells is essential to prevent their misclassification as toxic neutrophils.

Stress-induced changes in the ultrastructure of the photosynthetic apparatus of green microalgae.

In photosynthetic organisms including unicellular algae, acclimation to and damage by environmental stresses are readily apparent at the level of the photosynthetic apparatus. Phenotypic manifestations of the stress responses include rapid and dramatic reduction of photosynthetic activity and pigment content aimed at mitigating the risk of photooxidative damage. Although the physiological and molecular mechanisms of these events are well known, the ultrastructural picture of the stress responses is often elusive and frequently controversial. We analyzed an extensive set of transmission electron microscopy images of the microalgal cells obtained across species of Chlorophyta and in a wide range of growth conditions. The results of the analysis allowed us to pinpoint distinct ultrastructural changes typical of normal functioning and emergency reduction of the chloroplast membrane system under high light exposure and/or mineral nutrient starvation. We demonstrate the patterns of the stress-related ultrastructural changes including peculiar thylakoid rearrangements and autophagy-like processes and provide an outlook on their significance for implementation of the stress responses.

A new iron(III) complex-containing sulfadiazine inhibits the proliferation and induces cystogenesis of Toxoplasma gondii.

We have previously shown that metallocomplexes can control the growth of Toxoplasma gondii, the agent that causes toxoplasmosis. In order to develop new metallodrugs to treat this disease, we investigated the influence of the coordination of sulfadiazine (SDZ), a drug used to treat toxoplasmosis, on the biological activity of the iron(III) complex [Fe(HBPClNOL)Cl2]·H2O, 1, (H2BPClNOL=N-(2-hydroxybenzyl)-N-(2-pyridylmethyl)(3-chloro)(2-hydroxy)-propylamine). The new complex [(Cl)(SDZ)Fe(III)(μ-BPClNOL)2Fe(III)(SDZ)(Cl)]·2H2O, 2, which was obtained by the reaction between complex 1 and SDZ, was characterized using a range of physico-chemical techniques. The cytotoxic effect of the complexes and the ability of T. gondii to infect LLC-MK2 cells were assessed. It was found that both complexes reduced the growth of T. gondii while also causing low cytotoxicity in the host cells. After 48h of treatment, complex 2 reduced the parasite's ability to proliferate by about 50% with an IC50 of 1.66μmol/L. Meanwhile, complex 1 or SDZ alone caused a 40% reduction in proliferation, and SDZ displayed an IC50 of 5.3μmol/L. In addition, complex 2 treatment induced distinct morphological and ultrastructural changes in the parasites and triggered the formation of cyst-like forms. These results show that the coordination of SDZ to the iron(III) complex is a good strategy for increasing the anti-toxoplasma activity of these compounds.

Effects of mechanical trauma to the human tympanic membrane: an experimental study using transmission electron microscopy

Hypothesis: To examine the cellular events following induced superficial lesions of the human tympanic membrane (TM). Such information could lead to enhanced appreciation of repair mechanisms and novel strategies to restore TM perforations.Background: Persistent perforation of the TM in chronic otitis media is a major global health problem and frequently necessitates surgical intervention. Most TM perforations heal spontaneously and swiftly, but sometimes healing fails. The underlying mechanisms and the reason for incomplete repair are often elusive, although some mechanisms have been proposed.Methods: Here, five healthy adult human TMs were sampled during vestibular schwannoma surgery. Three days before harvesting, three TMs were superficially lesioned, including the epithelial and sub-epithelial layers, using a needle and two TMs served as controls. Light and transmission electron microscopy were performed.Results: Surrounding lesion showed distinct ultrastructural changes. This included a keratinocyte frontier with electron-dense cells with abundant ribosomes and nuclei metamorphosis. Beneath, were activated fibroblasts and invaded/transformed free cells and signs of increased transcellular activity of adjacent blood vessels.Conclusions: The study describes dynamic morphological events of a human lesioned TM. The human model may be used for further investigations and understanding of TM healing mechanisms.

Electron microscopic features of nasal mucosa treated with topical and circumostial injection of mitomycin C: implications in dacryocystorhinostomy.

To evaluate the ultrastructural effects of topical and circumostial injection of mitomycin C (COS-MMC) on nasal mucosa and compare them with the controls. The study also aimed at classifying the subcellular effects in detail. The nasal mucosa of 6 patients were subjected to 0.02% of mitomycin C for 3 minutes (3 patients) and 0.02% COS-MMC (3 patients) as per standard protocol, during endoscopic dacryocystorhinostomy. Normal nasal mucosa from untreated areas (2 each from topical and COS-MMC groups) were taken as controls after harvesting the treated areas. Full thickness tissues (5 mm × 5 mm) were collected for transmission electron microscopy, and ultrastructural effects were evaluated. Both topical and COS-MMC showed significant and distinct ultrastructural changes involving the epithelial, glandular, vascular, and fibrocollagenous tissues compared with the controls. There were profound changes within fibroblasts with intracellular edema, pleomorphic and vesicular mitochondria, dilated smooth and rough endoplasmic reticulum, and chromatin condensation. In addition, COS-MMC samples showed subepithelial hypocellularity with limited disorganization of structure. The changes in both the MMC groups were restricted to treated areas only. Both topical and COS-MMC show profound changes in nasal mucosa with more marked changes in COS-MMC group. These changes being limited in nature may help in enhancing the success of dacryocystorhinostomy by preventing cicatricial changes of the ostium, especially in high-risk cases such as revision and post-traumatic dacryocystorhinostomy.

Staphylococcus aureus biofilm susceptibility to small and potent β2,2-amino acid derivatives

Small antimicrobial β2,2-amino acid derivatives (Mw < 500 Da) are reported to display high antibacterial activity against suspended Gram-positive strains combined with low hemolytic activity. In the present study, the anti-biofilm activity of six β2,2-amino acid derivatives (A1–A6) against Staphylococcus aureus (ATCC 25923) was investigated. The derivatives displayed IC50 values between 5.4 and 42.8 μM for inhibition of biofilm formation, and concentrations between 22.4 and 38.4 μM had substantial effects on preformed biofilms. The lead derivative A2 showed high killing capacity (log R), and it caused distinct ultrastructural changes in the biofilms as shown by electron and atomic force microscopy. The anti-biofilm properties of A2 was preserved under high salinity conditions. Extended screening showed also high activity of A2 against Escherichia coli (XL1 Blue) biofilms. These advantageous features together with high activity against preformed biofilms make β2,2-amino acid derivatives a promising class of compounds for further development of anti-biofilm agents.

An ultrastructural analysis of platelets, erythrocytes, white blood cells, and fibrin network in systemic lupus erythematosus

The study suggests that patients with systemic lupus erythematosus (SLE) present with distinct inflammatory ultrastructural changes such as platelets blebbing, generation of platelet-derived microparticles, spontaneous formation of massive fibrin network and fusion of the erythrocytes membranes. Lupoid platelets actively interact with other inflammatory cells, particularly with white blood cells (WBCs), and the massive fibrin network facilitates such an interaction. It is possible that the concerted actions of platelets, erythrocytes and WBC, caught in the inflammatory fibrin network, predispose to pro-thrombotic states in patients with SLE.

Microvillous Inclusion Disease—An Ultrastructural Diagnosis: With a Review of the Literature

Microvillous inclusion disease is a rare disorder of infancy associated with protracted diarrhea. This malady reveals distinct ultrastructural changes. The surface enterocytes of the duodenum show vesicles lined with microvilli and the surface microvilli are poorly formed. The authors present one case of microvillous inclusion disease with a review of the literature.

Prion Protein with an Insertional Mutation Accumulates on Axonal and Dendritic Plasmalemma and Is Associated with Distinctive Ultrastructural Changes

Prion diseases are fatal neurological diseases characterized by central nervous system deposition of abnormal forms of a membrane glycoprotein designated PrP (prion protein). Tg(PG14) transgenic mice express PrP that harbor a nine-octapeptide insertional mutation homologous to one described in a familial prion disease of humans. Tg(PG14) mice spontaneously develop a fatal neurological illness accompanied by massive apoptosis of cerebellar granule neurons and accumulation of an aggregated and weakly protease-resistant form of PrP that is not infectious. Previous light microscopic analyses of these mice left open questions regarding the subcellular distribution of the mutant protein and the nature of the neuropathological lesions produced. To address these questions, we undertook an immunogold electron microscopic study of Tg(PG14) mice. We found that mutant PrP is localized primarily on the plasma membrane of dendrites and unmyelinated axons in the hippocampus and cerebellum, with little labeling of either neuronal cell bodies or intracellular organelles. PrP deposits were shown to be associated with degenerative changes in dendritic structure. We also describe for the first time marked pathology in myelinated axons, and alterations in the axon/oligodendrocyte interface. Taken together, our results suggest cellular mechanisms by which mutant PrPs produce pathology. In addition, they highlight distinctions between familial and infectious prion disorders at the ultrastructural level that correlate with differences in cellular trafficking of the disease-associated PrP forms.

Inherited Myopathies and Muscular Dystrophies

The inherited myopathies and muscular dystrophies are a diverse group of muscle diseases presenting with common complaints and physical signs: weakness, motor delay, and respiratory and bulbar dysfunction. The myopathies are caused by genetic defects in the contractile apparatus of muscle, and defined by distinctive histochemical or ultrastructural changes on muscle biopsy. The muscular dystrophies, in contrast, are diseases of muscle membrane or supporting proteins, which are generally characterized by pathological evidence of ongoing muscle degeneration and regeneration. Diagnosis of these disorders is contingent on a targeted history and examination, biochemical and neurophysiological assessment, muscle biopsy, and genetic testing. Treatment is focused on symptomatic management and rehabilitation, and monitoring for disease complications.

Cardiotoxicity Profile of Trastuzumab

Trastuzumab is a monoclonal antibody that targets the human epidermal growth factor receptor tyrosine kinase HER2/ErbB2. This agent has shown a highly significant antitumour effect for patients with HER2-positive breast cancer, and is now considered part of the standard regimens for the treatment of this disease in both the metastatic and adjuvant setting. Cardiotoxicity has been associated with trastuzumab, and this issue has now been studied and documented in a number of adjuvant trials for which data have now been released. Cardiotoxicity has been shown to be potentiated when the agent is used concurrently or sequentially with an anthracycline, and this has limited the use of trastuzumab in some patients. Determining the overall impact of trastuzumab is further complicated by the administration of other cardiotoxic agents such as the taxanes and cyclophosphamide as well as by pre-existing cardiac disease. The incidence of severe congestive heart failure (New York Heart Association class III or IV) was 0-3.9% in the trastuzumab arms versus 0-1.3% in the control arms in the five major randomized adjuvant trials. Only one cardiac death was related to trastuzumab whereas two cardiac deaths occurred in the control arms. Ejection fraction decline of >or=10% or 15% was reported in 3-34% of trastuzumab recipients in these trials. Patients affected by trastuzumab-related cardiotoxicity do not exhibit the cellular death and distinctive ultrastructural myocardial changes seen on electron microscopy with anthracycline-induced cardiotoxicity. The cardiotoxicity of trastuzumab also differs from traditional chemotherapy-induced cardiotoxicity in that it appears to be at least partially reversible, not related to the cumulative dose, and re-challenge is generally tolerated. There remain a number of uncertainties regarding the diagnosis and management of trastuzumab-related cardiotoxicity. While no formal guidelines or consensus statements exist at present regarding cardiac monitoring during use of trastuzumab, proposed recommendations include a careful assessment of ejection fraction prior to initiating trastuzumab, avoidance of concurrent administration of trastuzumab with anthracyclines, and regular monitoring of symptoms and cardiac function during and for several years after therapy. Increased vigilance is appropriate for higher risk patients.

Perturbation of the vesicle cycle in reticulospinal synapses of lamprey after presynaptic microinjections of GTPγS

The synaptic vesicle cycle sustains neurotransmission and keeps exo- and endocytosis in synapses in dynamic equilibrium. GTP-binding proteins function as key regulators of this cycle. The large GTPase dynamin is implicated in the fission of clathrin-coated vesicles from presynaptic membrane during endocytosis. The present study addresses the effect of the nonhydrolysable GTP analog GTPγS on assembly of the dynamin fission complex in situ. Intraaxonal microinjections of GTPγS induced the following distinct ultrastructural changes in the synapses: the number of synaptic vesicles in a cluster decreased while the number of the docked vesicles at the active zone increased; at the same time, the clathrin-coated intermediates also increased in number, indicating the inhibition of synaptic vesicle recycling. Unusual clathrin-coated intermediates were found. At low concentrations of GTPγS, they were presented by long tubules wreathed with a dynamin helix (spiral) and topped with a clathrin-coated vesicle. At high concentrations of GTPγS the tubular structures were much shorter and branched, with each branch topped with a clathrin-coated vesicle. The spiral pitch and the tubule diameter were significantly reduced as the concentration of GTPγS built up (23.1 ± 0.4 and 26.6 ± 0.4 nm, respectively, at low and 19.0 ± 0.5 and 23.3 ± 0.4 nm at high concentration of GTPγS, p < 0.001). We suggest that these ultrastructural changes reflect different steps in dynamin-mediated fission of clathrin-coated vesicles and propose a model for this process. The model implies that at first, GTP hydrolysis leads to a fast elongation of the helix due to a straightening of its dynamin dimmers. This entails an increase both in a pitch and a diameter of the dynamin helix. The shift in diameter disrupts local hydrophobic interactions between the inner and the outer lipid layers of the membrane at the sites of dynamin binding. Concurrent stretching of the helix and the clathrin-coated vesicle’s neck disintegrates the neck membrane and results finally in a release of the clathrincoated vesicle.

Ultrastructural changes and location of β-1,3-glucanase in resistant and susceptible cotton callus cells in response to treatment with toxin of Verticillium dahliae and salicylic acid

Calli from two cotton cultivars susceptible and resistant to Verticillium wilt, were treated with a crude toxin of Verticillium dahliae (VD-toxin) plus salicylic acid (SA). Cells treated with VD-toxin showed distinct ultrastructural changes. Cells from the susceptible cultivar displayed damage to plasma membrane and cytoplasm. The deleterious effect on cells of the resistant cultivar, with an accumulation of electron-dense precipitate in the vacuoles, was less noticeable. Exogenous SA protected callus cells from VD-toxin. We also report the localization of β-1,3-glucanase in callus cells with immunofluorescence labeling. Stronger fluorescence was observed in the extra-cellular space in resistant than in susceptible cotton; strongest in resistant cotton after 5 days of treatment with VD-toxin plus SA. The findings reported here indicate an important role of exogenous salicylic acid in the induction of resistance to VD-toxin in cotton. Coupled with an increase in β-1,3-glucanase, cellular integrity is maintained and damage to cell wall and plasma membrane is avoided.

Histopathologic analysis of hamster hepatocytes submitted to experimental infection with Leishmania donovani.

Hepatocytes from different vertebrates are used increasingly as models of environmentally driven cell structure plasticity and for the investigation of ultrastructural pathological patterns induced by cell injury. The present study was carried out to assess the morphological changes in hamster hepatocytes subjected to chronic infection by amastigote forms of Leishmania donovani. Liver fragments were processed for routine light and transmission electron microscopy. For cytochemical visualization of peroxisomes, liver slices were incubated in alkaline 3,3'-diaminobenzidine (DAB) medium at pH 10.0. The results showed that the presence of Leishmania donovani induced distinct ultrastructural changes in the liver acinus (zone 2). The significant pathological changes in hepatocytes consisted of disruption of the endomembrane system and alterations of both the peroxisomal compartment and the distribution of hepatic glycogen. Particularly, hepatic peroxisomes exhibited different shapes and sizes, with modifications of the peroxisomal matrix, including absence of the catalase reaction. These observations suggest an adaptive response of hepatocytes, with cytological reorganization after parasitic infection. The presence of DAB-negative peroxisomes could be morphological evidence of a metabolic disturbance of this organelle. The parasitic infection, through deregulation of the cytokene network, is probably responsible for those structural alterations, since similar changes have been observed in vivo and in vitro.

Ultrastructural Localization of Collagen IV, Fibronectin, and Laminin in the Trabecular Meshwork of Normal and Glaucomatous Eyes

Purpose: To determine whether differences in the ultrastructural characteristics or composition of the basement membranes of the trabecular lamellae and Schlemm’s canal exist in normal eyes and eyes with primary open-angle glaucoma (POAG). Basement membranes play key roles in the attachment of the overlying trabecular cells and Schlemm’s canal cells. Methods: Electron microscopy used in conjunction with immunogold labeling was used to examine the ultrastructure of the basement membranes in the trabecular meshwork and to determine the presence of collagen IV, laminin, and fibronectin in 6 normal eyes and 6 eyes with POAG. To determine which cells in the meshwork synthesized these molecules in situ hybridization was studied in an additional 8 normal eyes. Results: No distinctive ultrastructural changes were found in the basement membranes of glaucomatous eyes, whether early or advanced disease, when compared with normal eyes. Label for all three proteins was present in the basement membranes of the trabecular lamellae, Schlemm’s canal, and in scattered patches within the juxtacanalicular tissue. Laminin and fibronectin were most abundant in the periphery of the sheath material surrounding the elastic tendons in the juxtacanalicular tissue. In contrast to previously published light microscopic studies, no increase in fibronectin was found in glaucoma. Regions of the basement membrane of the canal underlying giant vacuoles were similar to regions without giant vacuoles in both appearance and labeling. In situ hybridization revealed that mRNA for all three proteins was present in most trabecular cells throughout the meshwork; no regional differences in cellular labeling within were observed. Conclusion: The ultrastructural characteristics and immunogold labeling of basement membranes were similar in normal and glaucomatous eyes; no additional structures were labeled in POAG eyes that were not also labeled in normal eyes. Label of the patches of amorphous fibrogranular material within the juxtacanalicular tissue suggests it is basement membrane in origin, while the sheath material which is known to accumulate in POAG was not heavily labeled and does not appear to be basement membrane in origin.

Evidence of foam cell and cholesterol crystal formation in macrophages incubated with oxidized LDL by fluorescence and electron microscopy.

Macrophage-derived foam cells are a prominent component of developing atherosclerotic lesions. We describe an in vitro model of foam cell formation which mimics some aspects of the evolution of foam cells in mature atherosclerotic lesions. Thioglycollate-elicited mouse peritoneal macrophages were incubated with copper-oxidized LDL (ox-LDL) for periods up to 168 hr. Identifiable foam cells were present after incubation with ox-LDL at 24, 72, and 168 hr. Control cells incubated without ox-LDL did not form foam cells. Fluorescence microscopy after staining with Nile red exhibited progressive accumulation of lipids, and transmission electron microscopy (TEM) showed distinct ultrastructural changes over time. Macrophages at 24 hr had a few non-membrane-bound lipid droplets but were otherwise identical to control cells. These lipid droplets fluoresced yellow-gold after Nile red staining. After 72 hr of incubation with ox-LDL, in addition to increased numbers of non-membrane-bound lipid inclusions, macrophages contained membrane-bound multilamellar lipoid structures. These multilamellar structures corresponded to areas of reddish-orange fluorescence after Nile red staining. In macrophages incubated with ox-LDL for 168 hr, the amount of cellular lipid was further increased and cholesterol crystal profiles were apparent within some multilamellar lipoid structures. Biochemical analysis showed that the total cholesterol content steadily increased over 168 hr. The increase in total cholesterol was accompanied by a dramatic increase in free cholesterol between 72 and 168 hr. These results demonstrate that long-term incubation of macrophages with ox-LDL increased lipid deposition in cultured cells and that, under the conditions studied, cholesterol crystals formed in macrophage foam cells. Moreover, this system allows investigation of the evolution of foam cells showing some characteristics of those found in atherosclerotic lesions.

Progression of foam-cell formation in vitro in elicited macrophages after incubation with ox-LDL

Thioglycollate elicited mouse peritoneal macrophages incubated with copper-oxidized LDL(ox-LDL) developed foam cell morphology as observed by Nile red fluorescent staining for neutral lipids and by electron microscopic observations. Total cellular cholesterol content rose steadily over 168 hrs and esterified vs free cholesterol ratios also increased.Cultured macrophages were incubated with ox-LDL, fixed, and stained with Nile red or processed for electron microscopy. Macrophages stained with Nile red exhibited progressive accumulation of lipid over 24, 72 and 168 hrs (Fig. 1-3). The percentage of cells with lipid inclusions and the size and number of inclusions per cell increased progressively over 168 hrs. Control cells incubated without ox-LDL did not have staining indicative of foam cell formation.Transmission electron microscopy of cultured cells at 24, 72, and 168 hrs showed distinct ultrastructural changes (Fig. 4-6). Macrophages at 24 hrs possessed non-membrane bound lipid droplets. Following 72 hrs, lysosomes containing multilamellar lipoid structures were also present. Cells at 168 hrs had characteristics similar to those at 24 and 72 hrs with the addition of cholesterol crystal profiles.