Astrocytoma and glioblastoma IDH1-wildtype cells colonize tumor vessels and deploy vascular mimicry.

Gliomas are the most prevalent type of malignant brain tumors with a very dismal prognosis. Angiogenesis in glioma has recently gotten more attention and its molecular aspects have been published; however, these were not complemented with ultrastructural evidence. Our ultrastructural examination of glioma vessels reveals several unique and critical features related to their mechanisms of progression and metastasis strategy. The detailed ultrastructural survey of 18 isocitrate dehydrogenase-wildtype (IDH1-wt) glioblastomas and 12 isocitrate dehydrogenase-mutant (IDH1-mt) High-grade gliomas indicated that tumor vessels of both types had undergone deformities such as the thickening of the vessel wall (VW) and proliferation of the basement membrane, contour distortions, abnormal and discontinuous basal lamina, tumor cells' invasion and colonization of VW, disappearance of endothelial cells (ECs), pericytes, and smooth muscle cells, as well as the formation of a continuous ring of tumor cells attached to the luminal side of VW in numerous cases. The latter feature is a clear sign of vascular mimicry (VM) that was previously suggested in gliomas but never shown by TEM. Additionally, the vascular invasion was carried out by a large number of tumor cells and was accompanied by the accumulation of tumor lipids in the vessels' lumina and VWs; these two features are distinct for gliomas and may alter the course of the clinical presentation and overall prognosis. This raises the issue of how to specifically target tumor cells involved in vascular invasion in order to optimize prognosis and overcome these mechanisms employed by the tumor cells.

Comparative survey of mitochondrial ultrastructure in IDH1-mutant astrocytoma and IDH1-wildtype glioblastoma (GBM).

Gliomas are the most common malignant brain tumors with poor prognosis. The WHO's classification recognizes isocitrate dehydrogenase 1 (IDH1) mutant astrocytoma and IDH1-wildtype glioblastoma (GBM). The IDH1 mutation confers a survival advantage over the wildtype. There are several explanations for the metabolic advantage of the IDH1 mutation, some involve mitochondrial implications. Since an ultrastructural comparison of both tumor genotypes is still lacking, we surveyed the ultrastructural effects of the IDH1 mutation on the mitochondria of the IDH1-mutant astrocytoma (n = 15) and IDH1-wildtype glioblastoma (n = 15) tumors. Our results show that both IDH1 genotypes have degenerate and uncoupled mitochondria; this has not been reported before. The presence of ample lipid inclusions and lipid droplets in the cytoplasm of both genotypes support our conclusion of dysfunctional uncoupled mitochondria. Thus, the IDH1 mutation may have no ultrastructural consequences on the mitochondria, and the aberrant mitochondria in both genotypes may be the result of other unknown mutations. The status of the mitochondria in these genotypes portends a clinical challenge since tumor cells with uncoupled mitochondria are more primitive, aggressive, and considerably treatment resistant.

Astrocytoma and IDH1-Wildtype Glioblastoma (GBM) Cells Colonize Tumor Vessels and Deploy Vascular Mimicry.

Gliomas are the most prevalent type of malignant brain tumors with a very dismal prognosis. Angiogenesis in glioma has recently gotten more attention and its molecular aspects have been published; however, these were not complemented with ultrastructural evidence. Our ultrastructural examination of glioma vessels reveals several unique and critical features related to their mechanisms of progression and metastasis strategy. The detailed ultrastructural survey of 18 IDH1 -wildtype glioblastomas (GBM) and 12 IDH1 -mutant High-grade gliomas indicated that tumor vessels of both types had undergone deformities such as the thickening of the vessel wall (VW) and proliferation of the basement membrane, contour distortions, abnormal and discontinuous basal lamina, tumor cells' invasion and colonization of VW, disappearance of endothelial cells (ECs), pericytes, and smooth muscle cells, as well as the formation of a continuous ring of tumor cells attached to the luminal side of VW in numerous cases. The latter feature is a clear sign of vascular mimicry (VM) that was previously suggested in gliomas but never shown by TEM. Additionally, the vascular invasion was carried out by a large number of tumor cells and was accompanied by the accumulation of tumor lipids in the vessels' lumina and VWs; these two features are distinct for gliomas and may alter the course of the clinical presentation and overall prognosis. This raises the issue of how to specifically target tumor cells involved in vascular invasion in order to optimize prognosis and overcome these mechanisms employed by the tumor cells.

An Ultrastructural Perspective on Cell Death.

In the field of cell death, there is still a wide gap between the molecular models and their ultrastructural phenotypes. Because only very few published works included electron microscopy (EM) images, many ultrastructural features have not yet been incorporated into the descriptions of death modes. Some of the EM features that appear in dying cells have not been incorporated in describing death modes. It includes the accumulation of lipid droplets and glycogen, the appearance of extranuclear chromatin in the cytoplasm, and the various ways mitochondria become damaged. We argue that electron microscopy should be routinely included in these studies because it exposes some new features that molecular studies do not. It has successfully recognized new modes of cell death, such as entosis, methuosis, and paraptosis. Elucidating the precise sequence of events in death modes could be the cornerstone for offering the proper therapy of many diseases by slowing down or stopping the progression of degeneration. This review presents our own experience applying ultrastructural interpretations to death modes and explaining their biochemical implications. We complement the molecular and biochemical data and point out missing features that should be considered and studied.

Gain-of-function mutations in ALPK1 cause an NF-κB-mediated autoinflammatory disease: functional assessment, clinical phenotyping and disease course of patients with ROSAH syndrome.

OBJECTIVES: To test the hypothesis that ROSAH (retinal dystrophy, optic nerve oedema, splenomegaly, anhidrosis and headache) syndrome, caused by dominant mutation in ALPK1, is an autoinflammatory disease. METHODS: This cohort study systematically evaluated 27 patients with ROSAH syndrome for inflammatory features and investigated the effect of ALPK1 mutations on immune signalling. Clinical, immunologic and radiographical examinations were performed, and 10 patients were empirically initiated on anticytokine therapy and monitored. Exome sequencing was used to identify a new pathogenic variant. Cytokine profiling, transcriptomics, immunoblotting and knock-in mice were used to assess the impact of ALPK1 mutations on protein function and immune signalling. RESULTS: The majority of the cohort carried the p.Thr237Met mutation but we also identified a new ROSAH-associated mutation, p.Tyr254Cys.Nearly all patients exhibited at least one feature consistent with inflammation including recurrent fever, headaches with meningeal enhancement and premature basal ganglia/brainstem mineralisation on MRI, deforming arthritis and AA amyloidosis. However, there was significant phenotypic variation, even within families and some adults lacked functional visual deficits. While anti-TNF and anti-IL-1 therapies suppressed systemic inflammation and improved quality of life, anti-IL-6 (tocilizumab) was the only anticytokine therapy that improved intraocular inflammation (two of two patients).Patients' primary samples and in vitro assays with mutated ALPK1 constructs showed immune activation with increased NF-κB signalling, STAT1 phosphorylation and interferon gene expression signature. Knock-in mice with the Alpk1 T237M mutation exhibited subclinical inflammation.Clinical features not conventionally attributed to inflammation were also common in the cohort and included short dental roots, enamel defects and decreased salivary flow. CONCLUSION: ROSAH syndrome is an autoinflammatory disease caused by gain-of-function mutations in ALPK1 and some features of disease are amenable to immunomodulatory therapy.

Retinal and choroidal capillaries contribution to age-related macular degeneration (AMD) phenotypes in murine models of the disease.

Mouse models of age-related macular degeneration (AMD) such as Ccl2-/- and Ccl2-/-/Cx3cr1-/- have not yet been fully characterized ultrastructurally. Although we have previously shown extranuclear DNA (enDNA) leakage into the cytoplasm and damaged mitochondria in the retinal pigment epithelium (RPE) of these AMD mouse models, little is known about the state of their vascular capillaries of the retina and choroid. Our ultrastructural survey shows that the aberrations were not restricted to the RPE cells, but also extended to the vasculature of the retina and choroid. Their endothelial aberrations included cytoplasmic degeneration, pyknotic DNA, hypertrophic nuclei, and loss of fenestration in addition to duplication of basement membrane and loss of density in Bruch's membrane. Moreover, the state of the vasculature in the mutant mice models suggests that the capillaries could also be active contributors to the pathological findings seen in AMD. The goal of this study is to gain insights into the early events of AMD that may lead to a better understanding of AMD's pathogenesis, improve our preventative measures, and formulate designed therapeutic regimens that are tailored to target the initial pathological events.Abbreviations: AMD: age-related macular degeneration; BM: Bruch's membrane; DPC: degenerate pericyte; EN: endothelial nucleus; enDNA: extranuclear DNA; GCL: ganglion cell layer; HEN: hypertrophic endothelial nucleus; IPL: inner plexiform layer; NFL: nerve fiber layer; OPL: outer plexiform layer; RBC: red blood cell; RPE: retinal pigment epithelium; SNPs: Single nucleotide polymorphisms.

Minimal Efficacy of Nitisinone Treatment in a Novel Mouse Model of Oculocutaneous Albinism, Type 3.

Purpose: Oral nitisinone has been shown to increase fur and ocular pigmentation in a mouse model of oculocutaneous albinism (OCA) due to hypomorphic mutations in tyrosinase (TYR), OCA1B. This study determines if nitisinone can improve ocular and/or fur pigmentation in a mouse model of OCA type 3 (OCA3), caused by mutation of the tyrosinase-related protein 1 (Tyrp1) gene. Methods: Mice homozygous for a null allele in the Tyrp1 gene (C57BL/6J-Tyrp1 b-J/J) were treated with 8 mg/kg nitisinone or vehicle every other day by oral gavage. Changes in fur and ocular melanin pigmentation were monitored. Mature ocular melanosome number and size were quantified in pigmented ocular structures by electron microscopy. Results: C57BL/6J-Tyrp1 b-J/J mice carry a novel c.403T>A; 404delG mutation in Tyrp1, predicted to result in premature truncation of the TYRP1 protein. Nitisinone treatment resulted in an approximately 7-fold increase in plasma tyrosine concentrations without overt toxicity. After 1 month of treatment, no change in the color of fur or pigmented ocular structures was observed. The distribution of melanosome cross-sectional area was unchanged in ocular tissues. There was no significant difference in the number of pigmented melanosomes in the RPE/choroid of nitisinone-treated and control groups. However, there was a significant difference in the number of pigmented melanosomes in the iris. Conclusions: Treatment of a mouse model of OCA3 with oral nitisinone did not have a favorable clinical effect on melanin production and minimally affected the number of pigmented melanosomes in the iris stroma. As such, treatment of OCA3 patients with nitisinone is unlikely to be therapeutic.

CaMK4 compromises podocyte function in autoimmune and nonautoimmune kidney disease.

Podocyte malfunction occurs in autoimmune and nonautoimmune kidney disease. Calcium signaling is essential for podocyte injury, but the role of Ca2+/calmodulin-dependent kinase (CaMK) signaling in podocytes has not been fully explored. We report that podocytes from patients with lupus nephritis and focal segmental glomerulosclerosis and lupus-prone and lipopolysaccharide- or adriamycin-treated mice display increased expression of CaMK IV (CaMK4), but not CaMK2. Mechanistically, CaMK4 modulated podocyte motility by altering the expression of the GTPases Rac1 and RhoA and suppressed the expression of nephrin, synaptopodin, and actin fibers in podocytes. In addition, it phosphorylated the scaffold protein 14-3-3ß, which resulted in the release and degradation of synaptopodin. Targeted delivery of a CaMK4 inhibitor to podocytes preserved their ultrastructure, averted immune complex deposition and crescent formation, and suppressed proteinuria in lupus-prone mice and proteinuria in mice exposed to lipopolysaccharide-induced podocyte injury by preserving nephrin/synaptopodin expression. In animals exposed to adriamycin, podocyte-specific delivery of a CaMK4 inhibitor prevented and reversed podocyte injury and renal disease. We conclude that CaMK4 is pivotal in immune and nonimmune podocyte injury and that its targeted cell-specific inhibition preserves podocyte structure and function and should have therapeutic value in lupus nephritis and podocytopathies, including focal segmental glomerulosclerosis.

Mitochondrial autophagosomes as a mechanism of drug resistance in breast carcinoma.

We have previously described the process by which mitochondria donate their membranes for the formation of autophagosomes, and in this study we show that the same process could be involved in drug sequestration and exocytosis resulting in multidrug-resistant cancerous cells. We examine the implications of mitochondrial vesicle formation of mitoautophagosomes (MAPS) in response to the cytotoxic drug MKT-077, which targets mortalin, in a drug-resistant breast carcinoma cell line overexpressing P-glycoprotein (P-gp). The breast cancer cell line MCF-7Adr is derived from MCF-7, but differs from its ancestral line in tolerance of MKT-077-induced mitochondrial toxicity. Our ultrastructural observations suggest that autophagy in the MCF-7Adr cells entails regional sequestration of MKT077 in multilamellar LC3-labeled MAPS, which then separate from their mitochondria, and fuse with or engulf each other. MAPS appeared to be migrating through the cytoplasm and fusing with the plasma membrane, thus carrying out exocytotic secretion. This mechanism, which seems ineffective in the ancestral cell line, provides a resistance mechanism for MKT-077 by enhancing the efflux process of the cells. After 8 hr of MKT-077 exposure, a fraction of the resistant cells appeared viable and contained larger number of smaller sized mitochondria. Mitoautophagosomes, therefore, provide a potentially novel model for multidrug resistance in cancerous cells and may contribute to the P-gp efflux process.

Aberrant tRNA processing causes an autoinflammatory syndrome responsive to TNF inhibitors.

OBJECTIVES: To characterise the clinical features, immune manifestations and molecular mechanisms in a recently described autoinflammatory disease caused by mutations in TRNT1, a tRNA processing enzyme, and to explore the use of cytokine inhibitors in suppressing the inflammatory phenotype. METHODS: We studied nine patients with biallelic mutations in TRNT1 and the syndrome of congenital sideroblastic anaemia with immunodeficiency, fevers and developmental delay (SIFD). Genetic studies included whole exome sequencing (WES) and candidate gene screening. Patients' primary cells were used for deep RNA and tRNA sequencing, cytokine profiling, immunophenotyping, immunoblotting and electron microscopy (EM). RESULTS: We identified eight mutations in these nine patients, three of which have not been previously associated with SIFD. Three patients died in early childhood. Inflammatory cytokines, mainly interleukin (IL)-6, interferon gamma (IFN-γ) and IFN-induced cytokines were elevated in the serum, whereas tumour necrosis factor (TNF) and IL-1ß were present in tissue biopsies of patients with active inflammatory disease. Deep tRNA sequencing of patients' fibroblasts showed significant deficiency of mature cytosolic tRNAs. EM of bone marrow and skin biopsy samples revealed striking abnormalities across all cell types and a mix of necrotic and normal-appearing cells. By immunoprecipitation, we found evidence for dysregulation in protein clearance pathways. In 4/4 patients, treatment with a TNF inhibitor suppressed inflammation, reduced the need for blood transfusions and improved growth. CONCLUSIONS: Mutations of TRNT1 lead to a severe and often fatal syndrome, linking protein homeostasis and autoinflammation. Molecular diagnosis in early life will be crucial for initiating anti-TNF therapy, which might prevent some of the severe disease consequences.

Loss of endothelial planar cell polarity and cellular clearance mechanisms in age-related macular degeneration.

Apoptosis, autophagosomes, and lysosomes are lacking in the retinal pigment epithelium (RPE) of age-related macular degeneration (AMD) eyes. Necrosis, not apoptosis, appeared to be the prominent type of cell death in RPE, which led to the accumulation of cell debris within and on both sides of Bruch's membrane. The endothelium of the choriocapillaris had an altered planar cell polarity which encompassed the disappearance of fenestrations, the thickening of cytoplasm, and anterior nuclear dislocation. There were no significant differences in RPE and choroidal aberrations between macular and temporal regions. Loss of endothelial polarity could be at the crux of AMD initiation and progression.

Adverse effects of antiretroviral therapy on liver hepatocytes and endothelium in HIV patients: An ultrastructural perspective.

Human immunodeficiency virus and antiretroviral therapy (ART) together can be far more detrimental to liver cells than either of the two unaided. However, ultrastructural aspects of the synergistic effects of HIV and ART have been understudied. In a patient cohort receiving ART, this study characterizes ultrastructurally sinusoidal degeneration, hepatocytic aberrations, mitochondrial dysfunction, accumulation of bulky lipid droplets (steatosis), and occlusion of sinusoidal lumina. Mitochondrial dysfunction causes the accumulation of acetyl-CoA which leads to insulin upregulation and resistance, lipid synthesis, and steatosis. Lipid droplets deposited in the sinusoids could be the source of the blood's lipid profile alterations in HIV patients on ART.

A Systems Biology Interpretation of Array Comparative Genomic Hybridization (aCGH) Data through Phylogenetics.

Array Comparative Genomic Hybridization (aCGH) is a rapid screening technique to detect gene deletions and duplications, providing an overview of chromosomal aberrations throughout the entire genome of a tumor, without the need for cell culturing. However, the heterogeneity of aCGH data obfuscates existing methods of data analysis. Analysis of aCGH data from a systems biology perspective or in the context of total aberrations is largely absent in the published literature. We present here a novel alternative to the functional analysis of aCGH data using the phylogenetic paradigm that is well-suited to high dimensional datasets of heterogeneous nature, but has not been widely adapted to aCGH data. Maximum parsimony phylogenetic analysis sorts out genetic data through the simplest presentation of the data on a cladogram, a graphical evolutionary tree, thus providing a powerful and efficient method for aCGH data analysis. For example, the cladogram models the multiphasic changes in the cancer genome and identifies shared early mutations in the disease progression, providing a simple yet powerful means of aCGH data interpretation. As such, applying maximum parsimony phylogenetic analysis to aCGH results allows for the differentiation between drivers and passenger genes aberrations in cancer specimens. In addition to offering a novel methodology to analyze aCGH results, we present here a crucial software suite that we wrote to carry out the analysis. In a broader context, we wish to underscore that phylogenetic analysis of aCGH data is a non-parametric method that circumvents the pitfalls and frustrations of standard analytical techniques that rely on parametric statistics. Organizing the data in a cladogram as explained in this research article provides insights into the disease common aberrations, as well as the disease subtypes and their shared aberrations (the synapomorphies) of each subtype. Hence, we report the method and make the software suite publicly and freely available at http://software.phylomcs.com so that researchers can test alternative and innovative approaches to the analysis of aCGH data.

NLRP3 Upregulation in Retinal Pigment Epithelium in Age-Related Macular Degeneration.

Inflammation and oxidative stress are involved in age-related macular degeneration (AMD) and possibly associated with an activation of neuronal apoptosis inhibitor protein/class II transcription activator of the Major Histocompatibility Complex (MHC)/heterokaryon incompatibility/telomerase-associated protein 1, leucine-rich repeat or nucleotide-binding domain, leucine-rich repeat-containing family, and pyrin domain-containing 3 (NLRP3) inflammasome. In the present study, we used a translational approach to address this hypothesis. In patients with AMD, we observed increased mRNA levels of NLRP3, pro-interleukin-1 beta (IL-1ß) and pro-IL-18 in AMD lesions of the retinal pigment epithelium (RPE) and photoreceptor. In vitro, a similar increase was evoked by oxidative stress or lipopolysaccharide (LPS) stimulation in the adult retinal pigment epithelium (ARPE-19) cell line, and the increase was reduced in siRNA transfected cells to knockdown NLRP3. Ultrastructural studies of ARPE-19 cells showed a swelling of the cytoplasm, mitochondrial damage, and occurrence of autophagosome-like structures. NLRP3 positive dots were detected within autophagosome-like structures or in the extracellular space. Next, we used a mouse model of AMD, Ccl2/Cx3cr1 double knockout on rd8 background (DKO rd8) to ascertain the in vivo relevance. Ultrastructural studies of the RPE of these mice showed damaged mitochondria, autophagosome-like structures, and cytoplasmic vacuoles, which are reminiscent of the pathology seen in stressed ARPE-19 cells. The data suggest that the NLRP3 inflammasome may contribute in AMD pathogenesis.

Carney triad, SDH-deficient tumors, and Sdhb+/- mice share abnormal mitochondria.

Carney triad (CTr) describes the association of paragangliomas (PGL), pulmonary chondromas, and gastrointestinal (GI) stromal tumors (GISTs) with a variety of other lesions, including pheochromocytomas and adrenocortical tumors. The gene(s) that cause CTr remain(s) unknown. PGL and GISTs may be caused by loss-of-function mutations in succinate dehydrogenase (SDH) (a condition known as Carney-Stratakis syndrome (CSS)). Mitochondrial structure and function are abnormal in tissues that carry SDH defects, but they have not been studied in CTr. For the present study, we examined mitochondrial structure in human tumors and GI tissue (GIT) of mice with SDH deficiency. Tissues from 16 CTr tumors (n=12), those with isolated GIST (n=1), and those with CSS caused by SDHC (n=1) and SDHD (n=2) mutations were studied by electron microscopy (EM). Samples of GIT from mice with a heterozygous deletion in Sdhb (Sdhb(+) (/-), n=4) were also studied by EM. CTr patients presented with mostly epithelioid GISTs that were characterized by plump cells containing a centrally located, round nucleus and prominent nucleoli; these changes were almost identical to those seen in the GISTs of patients with SDH. In tumor cells from patients, regardless of diagnosis or tumor type, cytoplasm contained an increased number of mitochondria with a 'hypoxic' phenotype: mitochondria were devoid of cristae, exhibited structural abnormalities, and were of variable size. Occasionally, mitochondria were small and round; rarely, they were thin and elongated with tubular cristae. Many mitochondria exhibited amorphous fluffy material with membranous whorls or cystic structures. A similar mitochondrial hypoxic phenotype was seen in Sdhb(+) (/-) mice. We concluded that tissues from SDH-deficient tumors, those from mouse GIT, and those from CTr tumors shared identical abnormalities in mitochondrial structure and other features. Thus, the still-elusive CTr defect(s) is(are) likely to affect mitochondrial function, just like germline SDH-deficiency does.

CD47-dependent immunomodulatory and angiogenic activities of extracellular vesicles produced by T cells.

Intercellular communication is critical for integrating complex signals in multicellular eukaryotes. Vascular endothelial cells and T lymphocytes closely interact during the recirculation and trans-endothelial migration of T cells. In addition to direct cell-cell contact, we show that T cell derived extracellular vesicles can interact with endothelial cells and modulate their cellular functions. Thrombospondin-1 and its receptor CD47 are expressed on exosomes/ectosomes derived from T cells, and these extracellular vesicles are internalized and modulate signaling in both T cells and endothelial cells. Extracellular vesicles released from cells expressing or lacking CD47 differentially regulate activation of T cells induced by engaging the T cell receptor. Similarly, T cell-derived extracellular vesicles modulate endothelial cell responses to vascular endothelial growth factor and tube formation in a CD47-dependent manner. Uptake of T cell derived extracellular vesicles by recipient endothelial cells globally alters gene expression in a CD47-dependent manner. CD47 also regulates the mRNA content of extracellular vesicles in a manner consistent with some of the resulting alterations in target endothelial cell gene expression. Therefore, the thrombospondin-1 receptor CD47 directly or indirectly regulates intercellular communication mediated by the transfer of extracellular vesicles between vascular cells.

Implications of DNA leakage in eyes of mutant mice.

Extranuclear DNA (enDNA) is not well studied ultrastructurally in the retinal pigment epithelium (RPE). We analyzed the retina and vastus medialis muscle of four mouse strains that are related to focal retinal degeneration by transmission electron microscopy (TEM) and EM immunolabeling. Evaluation of enDNA would imply the involvements of enDNA is either limited to the affected tissue or generalized in the whole body. Ultrastructural analysis and EM immunolabeling revealed that enDNA was present in the RPE cells but not in the muscle. These data suggest that enDNA could be unique to unhealthy RPE and a potential biomarker for cellular abnormality.

Upregulation of hypoxia-inducible factors and autophagy in von Hippel-Lindau-associated retinal hemangioblastoma.

PURPOSE: To describe pathological and molecular changes of three patients with clinically severe von Hippel-Lindau (VHL)-associated retinal hemangioblastoma (RH) with rapid progression. METHODS: Medical records, ocular histopathology, and transmission electron microscopy from three cases of VHL-associated RHs at the National Eye Institute were retrospectively reviewed. One eye of each patient was enucleated. Hypoxia-inducible factor (HIF) 1α and HIF2α expressions were identified by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunohistochemistry. RESULTS: All three cases had rapidly growing RHs that were resistant to multiple conventional therapies and two (patients 1 and 2) were also resistant to multiple intravitreal anti-vascular endothelial growth factor (VEGF) treatments. Macroscopically, all the enucleated eyes had multiple RHs, serous retinal detachment, severe retinal disorganization and focal hemorrhages. Histopathology showed typical RHs composed of vacuolated foamy VHL cells and capillary networks. Retinal gliosis and hemorrhages were also presented. Additionally, T lymphocytes and macrophages infiltrated in the tumors of two patients resistant to anti-VEGF therapy. Immunohistochemistry, and qRT-PCR found upregulation of HIF1α in the retinal lesions of all eyes. Importantly, upregulation of HIF2α was exclusively detected in the two cases with inflammatory infiltration and resistance to anti-VEGF therapy. Ultrastructural images showed autophagy, lipid droplets, glycogen aggregations, and cytoplasmic degeneration in many VHL cells. CONCLUSIONS: Based on the histopathological and molecular pathological findings, autophagy, inflammation, and/or upregulation of HIF2α could potentially contribute to the aggressive course of RHs, resulting in the resistance to multiple anti-VEGF and radiation therapies in these patients.

Platelet-derived growth factor (PDGF)-C inhibits neuroretinal apoptosis in a murine model of focal retinal degeneration.

Platelet-derived growth factor (PDGF)-C is a member of the PDGF family and is critical for neuronal survival in the central nervous system. We studied the possible survival and antiapoptotic effects of PDGF-C on focal retinal lesions in Ccl2(-/-)/Cx3cr1(-/-) on C57BL/6N [Crb1(rd8)] (DKO rd8) background mice, a model for progressive and focal retinal degeneration. We found no difference in transcript and protein expression of PDGF-C in the retina between DKO rd8 mice and wild type (WT, C57BL/6N). Recombinant PDGF-CC protein (500 ng/eye) was injected intravitreally into the right eye of DKO rd8 mice with phosphate-buffered saline as controls into the left eye. The retinal effects of PDGF-C were assessed by fundoscopy, ocular histopathology, A2E levels, apoptotic molecule analysis, and direct flat mount retinal vascular labeling. We found that the PDGF-CC-treated eyes showed slower progression or attenuation of the focal retinal lesions, lesser photoreceptor and retinal pigment epithelial degeneration resulting in better-preserved photoreceptor structure. Lower expression of apoptotic molecules was detected in the PDGF-CC-treated eyes than in controls. In addition, no retinal neovascularization was observed after PDGF-CC treatment. Our results demonstrate that PDGF-C potently ameliorates photoreceptor degeneration via the suppression of apoptotic pathways without inducing retinal angiogenesis. The protective effects of PDGF-C suggest a novel alternative approach for potential age-related retinal degeneration treatment.

An integrative CGH, MSI and candidate genes methylation analysis of colorectal tumors.

BACKGROUND: Different DNA aberrations processes can cause colorectal cancer (CRC). Herein, we conducted a comprehensive molecular characterization of 27 CRCs from Iranian patients. MATERIALS AND METHODS: Array CGH was performed. The MSI phenotype and the methylation status of 15 genes was established using MSP. The CGH data was compared to two established lists of 41 and 68 cancer genes, respectively, and to CGH data from African Americans. A maximum parsimony cladogram based on global aberrations was established. RESULTS: The number of aberrations seem to depend on the MSI status. MSI-H tumors displayed the lowest number of aberrations. MSP revealed that most markers were methylated, except RNF182 gene. P16 and MLH1 genes were primarily methylated in MSI-H tumors. Seven markers with moderate to high frequency of methylation (SYNE1, MMP2, CD109, EVL, RET, LGR and PTPRD) had very low levels of chromosomal aberrations. All chromosomes were targeted by aberrations with deletions more frequent than amplifications. The most amplified markers were CD248, ERCC6, ERGIC3, GNAS, MMP2, NF1, P2RX7, SFRS6, SLC29A1 and TBX22. Most deletions were noted for ADAM29, CHL1, CSMD3, FBXW7, GALNS, MMP2, NF1, PRKD1, SMAD4 and TP53. Aberrations targeting chromosome X were primarily amplifications in male patients and deletions in female patients. A finding similar to what we reported for African American CRC patients. CONCLUSION: This first comprehensive analysis of CRC Iranian tumors reveals a high MSI rate. The MSI tumors displayed the lowest level of chromosomal aberrations but high frequency of methylation. The MSI-L were predominantly targeted with chromosomal instability in a way similar to the MSS tumors. The global chromosomal aberration profiles showed many similarities with other populations but also differences that might allow a better understanding of CRC's clinico-pathological specifics in this population.