Ultrastructural and Molecular Analysis of Vascular Smooth Muscle Cells During the Switch from a Physiological to a Pathological Phenotype

Abstract: Background/Objectives: Under physiological conditions, vascular smooth muscle cells (VSMCs) are in a quiescent contractile state, but under pathological conditions, such as atherosclerosis, they change their phenotype to synthetic, characterized by increased proliferation, migration, and production of an extracellular matrix. Furthermore, VSMCs can undergo calcification, switching to an osteoblast-like phenotype, contributing to plaque instability. Methods: In this study, we analyzed the phenotypic changes in VSMCs during the transition from a physiological to a pathological state, a key process in the progression of atherosclerosis, using confocal and transmission electron microscopy, real-time PCR, and intracellular calcium quantification. Results: Confocal and transmission electron microscopy revealed a prominent remodeling of the actin cytoskeleton, increasing autophagic vacuoles in synthetic VSMCs and the deposition of calcium microcrystals in calcified cells. Immunofluorescence analysis revealed differential expression of α-SMA (contractile marker) and galectin-3 (synthetic marker), confirming the phenotypic changes. Real-time PCR further validated these changes, showing upregulation of RUNX-2, a marker of osteogenic transition, in calcified VSMCs. Conclusions: This study highlights the dynamic plasticity of VSMCs and their role in atherosclerosis progression. Understanding the characteristics of these phenotypic transitions can help develop targeted therapies to mitigate vascular calcification and plaque instability, potentially countering cardiovascular disease

Plastic changes in the spinal cord in motor neuron disease.

In the present paper, we analyze the cell number within lamina X at the end stage of disease in a G93A mouse model of ALS; the effects induced by lithium; the stem-cell like phenotype of lamina X cells during ALS; the differentiation of these cells towards either a glial or neuronal phenotype. In summary we found that G93A mouse model of ALS produces an increase in lamina X cells which is further augmented by lithium administration. In the absence of lithium these nestin positive stem-like cells preferentially differentiate into glia (GFAP positive), while in the presence of lithium these cells differentiate towards a neuronlike phenotype (III-tubulin, NeuN, and calbindin-D28K positive). These effects of lithium are observed concomitantly with attenuation in disease progression and are reminiscent of neurogenetic effects induced by lithiumin the subependymal ventricular zone of the hippocampus

Proteomics Profiling of Neuron-Derived Small Extracellular Vesicles From Human Plasma: Enabling Single-Subject Analysis

Small extracellular vesicles have been intensively studied as a source of biomarkers in neurodegenerative disorders. The possibility to isolate neuron-derived small extracellular vesicles (NDsEV) from blood represents a potential window into brain pathological processes. To date, the absence of sensitive NDsEV isolation and full proteome characterization methods has meant their protein content has been underexplored, particularly for individual patients. Here, we report a rapid method based on an immunoplate covalently coated with mouse monoclonal anti-L1CAM antibody for the isolation and the proteome characterization of plasma-NDsEV from individual Parkinson’s disease (PD) patients. We isolated round-shaped vesicles with morphological characteristics consistent with exosomes. On average, 349 ± 38 protein groups were identified by liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis, 20 of which are annotated in the Human Protein Atlas as being highly expressed in the brain, and 213 were shared with a reference NDsEV dataset obtained from cultured human neurons. Moreover, this approach enabled the identification of 23 proteins belonging to the Parkinson disease KEGG pathway, as well as proteins previously reported as PD circulating biomarkers

Lamina X of the spinal cord in motor neuron disease

A number of plastic events were described in the spinal cord in the course of amyotrophic lateral sclerosis (ALS). These consist of various morphological effects, involving neurons, glia, and inflammatory cells, as well. Among plastic changes, an increase in neuronal progenitor cells (NPC) occurs within ependymal cells layer of lamina X. This stem cell-like activity is known to be weak in baseline conditions but it is known to increase significantly during spinal cord disorders, when it preferentially generates glial cells, due to the strong gliogenic effect of the spinal cord “milieu”. In the present work, we used immunohistochemistry and electron microscopy to analyze cell number within lamina X at the end stage of disease in the G93A mouse model of ALS in baseline conditions and following chronic lithium administration. These cells were identified by using GFAP, bIII-tubulin, NeuN, and calbindin- D28K immunostaining. In the absence of lithium we observed an increase of lamina X cells in ALS mice with a glial phenotype, while in G93A mice treated with lithium these cells differentiate towards neuronal-like phenotype. These effects of lithium are concomitant with slowed disease progression and are reminiscent of the neurogenetic effects described in the sub-ependymal ventricular zone of the hippocampus. The present data confirm the scarce NPC activity in the intact spinal cord which is enhanced by disease conditions; in the presence of chronic lithium, such increased NPCs differentiate towards a neuron-like rather than a glial phenotype

Morphological analysis of genetic modulation of PINK1 on mitochondrial alterations, autophagy and cell death

Mutations in the PTEN-induced putative kinase1 (PINK1) represent the second most common cause of autosomal recessive Parkinson’s disease. The PINK1 protein has a mitochondrial localization and interacts with a variety of proteins, including the pro-autophagy protein beclin1 and the ubiquitin-ligase parkin. In particular, PINK1 is able to recruit parkin to the surface of dysfunctional mitochondria, to promote the ubiquitination of several mitochondrial proteins and the subsequent activation of the mitophagy cascade. Aim of this study was to use a dopaminergic cell model and transmission electron microscopy to characterize whether the modulation of PINK1 expression: (i) modifies the number and morphology of mitochondria and of autophagy organelles (autophagosomes); (ii) alters the recruitment of beclin1, parkin and ubiquitin to the mitochondria; (iii) affects cell survival. We used PC12 cells transfected either with the empty vector (pcDNA), or vectors expressing wild type PINK1 (PINK1wt), a pathogenic mutant (PINK1W437X), shRNA against rat PINK1 (shPINK1) or scramble (shSCR). Samples were analyzed both in baseline conditions and following methamphetamine (METH) treatment to provide a neurotoxic, autophagy-dependent stimulation. We showed that, especially upon METH exposure, the modulation of PINK1 levels dramatically affected the morphology and clearance of mitochondria. In fact, the number of abnormal mitochondria was reduced in PINK1wt, while it was significantly increased upon shPINK1 and also, to a lesser extent, in PINK1W437X cells. In keeping with this, mitochondrial ubiquitin clusters and mitochondrial levels of parkin and beclin1 were increased in PINK1wt cells while they were reduced both in PINK1 silenced and PINK1W437X cells. Interestingly, the number of autophagic vacuoles was unaffected by PINK1 modulation in baseline conditions, and was significantly reduced only in cells lacking functional PINK1 and upon METH exposure. All these effects were significantly associated with a modulation of apoptotic cell death. Our data provide robust sub-cellular evidence that PINK1 counteracts neurodegeneration by simultaneously recruiting beclin1, parkin and ubiquitin and thus enhancing the clearance of damaged mitochondria. In the absence of functional PINK1 and upon autophagy stress, we observed a failure of the autophagy system at large, with marked accumulation of dysfunctional mitochondria and dramatic increase in apoptotic cell death

Rapamycin promotes differentiation increasing βIII-tubulin, NeuN, and NeuroD while suppressing nestin expression in glioblastoma cells

Glioblastoma cells feature mammalian target of rapamycin (mTOR) up-regulation which relates to a variety of effects such as: lower survival, higher infiltration, high stemness and radio- and chemo-resistance. Recently, it was demonstrated that mTOR may produce a gene shift leading to altered protein expression. Therefore, in the present study we administered different doses of the mTOR inhibitor rapamycin to explore whether the transcription of specific genes are modified. By using a variety of methods we demonstrate that rapamycin stimulates gene transcription related to neuronal differentiation while inhibiting stemness related genes such as nestin. In these experimental conditions, cell phenotype shifts towards a pyramidal neuron-like shape owing long branches. Rapamycin suppressed cell migration when exposed to fetal bovine serum (FBS) while increasing the cell adhesion protein phospho-FAK (pFAK). The present study improves our awareness of basic mechanisms which relate mTOR activity to the biology of glioblastoma cells. These findings apply to a variety of effects which can be induced by mTOR regulation in the brain. In fact, the ability to promote neuronal differentiation might be viewed as a novel therapeutic pathway to approach neuronal regeneration

Rapamycin induces neuronal differentiation and decreases prion-like proteins in human glioblastoma cell cultures

Glioblastoma (GB, grade IV astrocytoma) is highly proliferating, infiltrating, and relapsing. So far, no therapy cures the disease and on average lethality occurs within 2 years from diagnosis. Despite the intimate molecular mechanisms of GB remain unknown, the mammalian Target Of Rapamycin (mTOR) is constantly upregulated within GB and in other astrocytomas. Consistently, mTOR was proposed as a target to cure GB. In fact, the mTOR inhibitor rapamycin was tested in order to prevent the growth of GB in human primary cultures and in mouse xenograft. Cytopathology of GB is highly heterogeneous for the co-existence of various cell types and the occurrence progressive steps in cell differentiation. Remarkably, the non differentiated stem-like cells represent primary GB precursors which initiate the tumor growth. Altered transmission of pathological proteins was recently claimed to underly the growth of GB and its infiltration. These proteins are controlled by the mTOR pathway and spread from cell to cell suggesting a prion-like mechanism in GB. Therefore, in the present study, using the human GB cell line U87MG, we evaluated the effect of a wide range of rapamycin concentrations (between 1 nM to 1 x 103 nM) at 24 h on: cell survival; cell morphology and electrophysiology; cell differentiation and migration; expression of PrP and other prion-like proteins. We found that effects of rapamycin on U87MG are dose-dependent. While high doses (100 nM up to 1000 nM) reduce cell viability, lower rapamycin doses (1nm up to 10 nM) produce cell differentiation consisting of morphological and immunohistochemical changes. Rapamycin promotes a neuronal shape with process elongation and increased cell size. This associates with the loss of the staminal marker nestin while the early neuronal marker betaIII-tubulin increases. At 24 h rapamycin inhibits U87MG cell migration dose-dependently. The neuronal morphology associates with altered intracellular calcium flux but not neuronal electrophysiological properties. Finally, expression of prionoids which we found to be high in baseline conditions was suppressed by rapamycin. This is consistent with our ultrastructural findings which substantiate the dramatic effects induced by mTOR inhibition. Our results suggests pathological cell communication in GB while providing cellular evidence supporting the use of rapamycin as well rapalogs as effective drugs to treat malignant astrocytoma. This work was supported by a research grant from PRIN 2010/2011

Genetic modulation of PINK1 differentially affects mitophagy compared with autophagy disclosing common mechanisms of genetic and environmental parkinsonism

The second most frequent cause of autosomal recessive Parkinson’s disease is represented by mutations in the PTEN-induced putative kinase1 (PINK1). The PINK1 protein mainly localizes to mitochondria which are considered the target organelles mainly affected in Parkinson’s disease. In fact, parkinsonism-inducing neurotoxins such as rotenone, MPTP and methamphetamine all damage mitochondria. Therefore, the ability to counteract mitochondrial toxicity and promoting mitochondrial renewal by mithophagy and mitochondrial biogenesis is critical to cure Parkinsonism. For instance the autophagy-dependent removal of altered mitochondria known as mitophagy is supposed to be key in conteracting mitochondrial toxicity. Interestingly mitochondrial PINK1 is known to interact with autophagy proteins such as beclin1 and the ubiquitin-ligase parkin. Therefore, in the present study we evaluated whether such an interaction produced downstream effects leading to autophagy activation. This was evaluated through the simultaneous analysis of co-localization of parkin and beclin1 with the autophagy initiator ubiquitin. These phenomena were analyzed both at mitochondrial level and throughout the cytosol by analyzing autophagy-like vacuoles and LC3-II positive structures. Interestingly, despite increased mitophagy PINK1 overexpression did not produce a general activation of the autophagy pathway. It is likely that such a selective fashion of autophagy activation only limited to mitochondrial removal could explain the relevance of PINK1 for Parkinson’s disease but not for other neurodegenerative, autophagy-related disorders. The present data were obtained through several experimental settings featuring PINK1 overexpression, mutation, deletion and silencing of the gene. The effects were analyzed in baseline conditions but were supplemented by experiments in the presence of methamphetamine used here both as a mitochondrial neurotoxin and an autophagy-dependent Parkinsonism inducing compound. Data revealed that PINK1 was critical for mitochondria and cell viability already in baseline conditions though such an effect was magnified upon methamphetamine exposure. The present findings while explaining the molecular interactions which are likely to induce PINK1-dependent genetic Parkinsonism, provide a further evidence on the critical role of genetic and environmental alterations in the genesis of Parkinson’s disease

Schwann cell hamartoma: case report

<p>Abstract</p> <p>Background</p> <p>Colorectal polyps of mesenchymal origin represent a small percentage of gastrointestinal (GI) lesions. Nevertheless, they are encountered with increasing frequency since the widespread adoption of colonoscopy screening.</p> <p>Case presentation</p> <p>We report a case of a small colonic polyp that presented as intramucosal diffuse spindle cell proliferation with a benign cytological appearance, strong and diffuse immunoreactivity for S-100 protein, and pure Schwann cell phenotype. Careful morphological, immunohistochemical and clinical evaluation emphasize the differences from other stromal colonic lesions and distinguish it from schwannoma, a circumscribed benign nerve sheath tumor that rarely arises in the GI tract.</p> <p>Conclusion</p> <p>As recently proposed, this lesion was finally described as mucosal Schwann cell hamartoma.</p