Canine glioblastoma-derived extracellular vesicles as precise carriers for glioblastoma imaging: Targeting across the blood-brain barrier.

The treatment of glioblastoma (GBM) faces significant challenges due to the difficulty of delivering drugs through the blood-brain barrier (BBB). Extracellular vesicles (EVs) have emerged as potential carriers for targeted drug delivery to brain tumors. However, their use and distribution in the presence of an intact BBB and their ability to target GBM tissue are still under investigation. This study explored the use of EVs for GBM targeting across the BBB. Canine plasma EVs from healthy dogs and dogs with glioma were isolated, characterized, and loaded with diagnostic agents. Biodistribution studies were conducted in healthy murine models and a novel intranasal model that preserved BBB integrity while initiating early-stage GBM growth. This model assessed EVs' potential for delivering the contrast agent gadoteric acid to intracranial tumors. Imaging techniques, such as bioluminescence and MRI, confirmed EVs' targeting and delivery capabilities thus revealing a selective accumulation of canine glioma-derived EVs in brain tissue under physiological conditions. In the model of brain tumor, MRI experiments demonstrated the ability of EVs to accumulate gadoteric acid within GBM to enhance contrast of the tumoral mass, even when BBB integrity is maintained. This study underscores the potential of EVs derived from glioma for the targeted delivery of drugs to glioblastoma. EVs from dogs with glioma showed capacity to traverse the BBB and selectively accumulate within the brain tumor. Overall, this research represents a foundation for the application of autologous EVs to precision glioblastoma treatment, addressing the challenge of BBB penetration and targeting specificity in brain cancer therapy.

Primary HIV infection features colonic damage and neutrophil inflammation yet containment of microbial translocation.

INTRODUCTION: Impairment of the gastrointestinal barrier leads to microbial translocation and peripheral immune activation, which are linked to disease progression. Data in the setting of primary HIV/SIV infection suggest that gut barrier damage is one of the first events of the pathogenic cascade, preceding mucosal immune dysfunction and microbial translocation. We assessed gut structure and immunity as well as microbial translocation in acutely and chronically-infected, combination antiretroviral therapy (cART)-naive individuals. METHODS: Fifteen people with primary HIV infection (P-HIV) and 13 with chronic HIV infection (C-HIV) c-ART-naive participants were cross-sectionally studied. Gut biopsies were analysed in terms of gut reservoirs (total, integrated and unintegrated HIV DNA); tight junction proteins (E-cadherin, Zonula Occludens-1), CD4 + expression, neutrophil myeloperoxidase (histochemical staining); collagen deposition (Masson staining). Flow cytometry was used to assess γδ T-cell frequency (CD3 + panγδ+Vδ1+/Vδ2+). In plasma, we measured microbial translocation (LPS, sCD14, EndoCAb) and gut barrier function (I-FABP) markers (ELISA). RESULTS: P-HIV displayed significantly higher tissue HIV DNA, yet neutrophil infiltration and collagen deposition in the gut were similar in the two groups. In contrast, microbial translocation markers were significantly lower in P-HIV compared with C-HIV. A trend to higher mucosal E-cadherin, and gut γδ T-cells was also observed in P-HIV. CONCLUSION: Early HIV infection features higher HIV DNA in the gut, yet comparable mucosal alterations to those observed in chronic infection. In contrast, microbial translocation is contained in primary HIV infection, likely because of a partial preservation of E-cadherin and mucosal immune subsets, namely γδ T-cells.

The Potential of JAG Ligands as Therapeutic Targets and Predictive Biomarkers in Multiple Myeloma.

The NOTCH ligands JAG1 and JAG2 have been correlated in vitro with multiple myeloma (MM) cell proliferation, drug resistance, self-renewal and a pathological crosstalk with the tumor microenvironment resulting in angiogenesis and osteoclastogenesis. These findings suggest that a therapeutic approach targeting JAG ligands might be helpful for the care of MM patients and lead us to explore the role of JAG1 and JAG2 in a MM in vivo model and primary patient samples. JAG1 and JAG2 protein expression represents a common feature in MM cell lines; therefore, we assessed their function through JAG1/2 conditional silencing in a MM xenograft model. We observed that JAG1 and JAG2 showed potential as therapeutic targets in MM, as their silencing resulted in a reduction in the tumor burden. Moreover, JAG1 and JAG2 protein expression in MM patients was positively correlated with the presence of MM cells in patients' bone marrow biopsies. Finally, taking advantage of the Multiple Myeloma Research Foundation (MMRF) CoMMpass global dataset, we showed that JAG2 gene expression level was a predictive biomarker associated with patients' overall survival and progression-free survival, independently from other main molecular or clinical features. Overall, these results strengthened the rationale for the development of a JAG1/2-tailored approach and the use of JAG2 as a predictive biomarker in MM.

Thyroid cancer harboring PTEN and TP53 mutations: A peculiar molecular and clinical case report.

To date, the molecular mechanisms that underline aggressiveness and resistance to tyrosine kinase inhibitors in some thyroid carcinomas (TCs) are not known yet. We report the case of a young patient with a metastatic poorly differentiated (PDTC) and follicular thyroid carcinoma (FTC) refractory to conventional therapies and to Sorafenib. The patient, despite an initial partial response, died of progressive disease 21 months after diagnosis. The genetic analysis performed on the primary tumor and on lymph nodes and distant metastases allowed to identify a frameshift mutation (p.P248Tfs*5) in the PTEN gene, never described in TC. This mutation was present in the primary tumor and, with a lower allelic frequency, in metastases diagnosed after treatment with Sorafenib. Mutations in TP53 (p.C135Y and c.920-2A>G previously detected in anaplastic carcinomas and p.M133R never found in TC) were also detected in the primary tissue together with a mono-allelic expression of the p.C135Y mutant at RNA level. At metastatic sites level, we found only the TP53 splicing mutation c.920-2A>G. The presence of defects in mismatch repair (MMR) proteins and genomic instability was also evaluated. The primary tumor showed a partial expression of MMR proteins together with a strong genomic instability. In conclusion, we demonstrated that the rare combination of somatic PTEN and TP53 mutations in a patient with a metastatic FTC, together with the presence of tumor heterogeneity and genomic instability, might be associated with a high tumor aggressiveness and resistance to treatments.

Evidence of SARS-CoV-2 in nasal brushings and olfactory mucosa biopsies of COVID-19 patients.

The aim of the present study is to detect the presence of SARS-CoV-2 of patients affected by COVID-19 in olfactory mucosa (OM), sampled with nasal brushing (NB) and biopsy, and to assess whether a non-invasive procedure, such as NB, might be used as a large-scale procedure for demonstrating SARS-CoV-2 presence in olfactory neuroepithelium. Nasal brushings obtained from all the COVID-19 patients resulted positive to SARS-CoV-2 immunocytochemistry while controls were negative. Double immunofluorescence showed that SARS-CoV-2 positive cells included supporting cells as well as olfactory neurons and basal cells. OM biopsies showed an uneven distribution of SARS-CoV-2 positivity along the olfactory neuroepithelium, while OM from controls were negative. SARS-CoV-2 was distinctively found in sustentacular cells, olfactory neurons, and basal cells, supporting what was observed in NB. Ultrastructural analysis of OM biopsies showed SARS-CoV-2 viral particles in the cytoplasm of sustentacular cells. This study shows the presence of SARS-CoV-2 at the level of the olfactory neuroepithelium in patients affected by COVID-19. For the first time, we used NB as a rapid non-invasive tool for assessing a potential neuroinvasion by SARS-CoV-2 infection.

MICA/B-targeted antibody promotes NK cell-driven tumor immunity in patients with intrahepatic cholangiocarcinoma.

The major histocompatibility complex-class I chain related proteins A and B (MICA/B) is upregulated because of cellular stress and MICA/B shedding by cancer cells causes escape from NKG2D recognition favoring the emergence of cancers. Cholangiocarcinoma (CCA) is a relatively rare, though increasingly prevalent, primary liver cancer characterized by a late clinical presentation and a dismal prognosis. We explored the NKG2D-MICA/B axis in NK cells from 41 patients with intrahepatic cholangiocarcinoma (iCCA). The MICA/B-specific 7C6 mAb was used for ex vivo antibody-dependent cytotoxicity (ADCC) experiments using circulating, non tumor liver- and tumor-infiltrating NK cells against the HuCCT-1 cell line and patient-derived primary iCCA cells as targets. MICA/B were more expressed in iCCA than in non-tumoral tissue, MICA transcription being higher in moderately-differentiated compared with poorly-differentiated cancer. Serum MICA was elevated in iCCA patients in line with higher expression of ADAM10 and ADAM17 that are responsible for proteolytic release of MICA/B from tumor. Addition of 7C6 significantly boosted peripheral, liver- and tumor-infiltrating-NK cell degranulation and IFNγ production toward MICA/B-expressing established cell lines and autologous iCCA patient target cells. Our data show that anti-MICA/B drives NK cell anti-tumor activity, and provide preclinical evidence in support of 7C6 as a potential immunotherapeutic tool for iCCA.

Therapeutic Effect of an Antibody-Derived Peptide in a Galleria mellonella Model of Systemic Candidiasis.

The synthetic peptide T11F (TCRVDHRGLTF), with sequence identical to a fragment of the constant region of human IgM, and most of its alanine-substituted derivatives proved to possess a significant candidacidal activity in vitro. In this study, the therapeutic efficacy of T11F, D5A, the derivative most active in vitro, and F11A, characterized by a different conformation, was investigated in Galleria mellonella larvae infected with Candida albicans. A single injection of F11A and D5A derivatives, in contrast with T11F, led to a significant increase in survival of larvae injected with a lethal inoculum of C. albicans cells, in comparison with infected animals treated with saline. Peptide modulation of host immunity upon C. albicans infection was determined by hemocyte analysis and larval histology, highlighting a different immune stimulation by the studied peptides. F11A, particularly, was the most active in eliciting nodule formation, melanization and fat body activation, leading to a better control of yeast infection. Overall, the obtained data suggest a double role for F11A, able to simultaneously target the fungus and the host immune system, resulting in a more efficient pathogen clearance.

Hofbauer Cells and COVID-19 in Pregnancy.

CONTEXT.­: SARS-CoV-2 can undergo maternal-fetal transmission, heightening interest in the placental pathology findings from this infection. Transplacental SARS-CoV-2 transmission is typically accompanied by chronic histiocytic intervillositis together with necrosis and positivity of syncytiotrophoblast for SARS-CoV-2. Hofbauer cells are placental macrophages that have been involved in viral diseases, including HIV and Zika virus, but their involvement in SARS-CoV-2 is unknown. OBJECTIVE.­: To determine whether SARS-CoV-2 can extend beyond the syncytiotrophoblast to enter Hofbauer cells, endothelium, and other villous stromal cells in infected placentas of liveborn and stillborn infants. DESIGN.­: Case-based retrospective analysis by 29 perinatal and molecular pathology specialists of placental findings from a preselected cohort of 22 SARS-CoV-2-infected placentas delivered to pregnant women testing positive for SARS-CoV-2 from 7 countries. Molecular pathology methods were used to investigate viral involvement of Hofbauer cells, villous capillary endothelium, syncytiotrophoblast, and other fetal-derived cells. RESULTS.­: Chronic histiocytic intervillositis and trophoblast necrosis were present in all 22 placentas (100%). SARS-CoV-2 was identified in Hofbauer cells from 4 of 22 placentas (18.2%). Villous capillary endothelial staining was positive in 2 of 22 cases (9.1%), both of which also had viral positivity in Hofbauer cells. Syncytiotrophoblast staining occurred in 21 of 22 placentas (95.5%). Hofbauer cell hyperplasia was present in 3 of 22 placentas (13.6%). In the 7 cases having documented transplacental infection of the fetus, 2 (28.6%) occurred in placentas with Hofbauer cell staining positive for SARS-CoV-2. CONCLUSIONS.­: SARS-CoV-2 can extend beyond the trophoblast into the villous stroma, involving Hofbauer cells and capillary endothelial cells, in a small number of infected placentas. Most cases of SARS-CoV-2 transplacental fetal infection occur without Hofbauer cell involvement.

Brainstem neuropathology in two cases of COVID-19: SARS-CoV-2 trafficking between brain and lung.

INTRODUCTION: SARS-CoV-2 might spread through the nervous system, reaching respiratory centers in the brainstem. Because we recently reported neurophysiological brainstem reflex abnormalities in COVID-19 patients, we here neuropathologically assessed structural brainstem damage in two COVID-19 patients. MATERIALS AND METHODS: We assessed neuropathological features in two patients who died of COVID-19 and in two COVID-19 negative patients as controls. Neuronal damage and corpora amylacea (CA) numbers /mm2 were histopathologically assessed. Other features studied were the immunohistochemical expression of the SARS-CoV-2 nucleoprotein (NP) and the Iba-1 antigen for glial activation. RESULTS: Autopsies showed normal gross brainstem anatomy. Histopathological examination demonstrated increased neuronal and CA damage in Covid-19 patients' medulla oblongata. Immunohistochemistry disclosed SARS-CoV-2 NP in brainstem neurons and glial cells, and in cranial nerves. Glial elements also exhibited a widespread increase in Iba-1 expression. Sars-Co-V2 was immunohistochemically detected in the vagus nerve fibers. DISCUSSION: Neuropathologic evidence showing SARS-CoV-2 in the brainstem and medullary damage in the area of respiratory centers strongly suggests that the pathophysiology of COVID-19-related respiratory failure includes a neurogenic component. Sars-Co-V2 detection in the vagus nerve, argues for viral trafficking between brainstem and lung.

Endothelial-Mesenchymal Transition in COVID-19 lung lesions.

Sars-Cov-2 infection is still a healthcare emergency and acute respiratory distress failure with Diffuse Alveolar Damage (DAD) features is the main causes of patients' death. Pathogenic mechanisms of the disease are not clear yet, but new insights are necessary to improve therapeutic management, to prevent fatal irreversible multi-organ damage and to adequately follow up those patients who survive. Here we investigated, by histochemistry and immunohistochemistry, a wide number of mapped lung specimens taken from whole body autopsies of 7 patients dead of COVID-19 disease. Our data confirm morphological data of other authors, and enlarge recent reports of the literature suggesting that Endothelial-Mesenchymal Transition might be central to COVID-19 lung fibrosing lesions. Furthermore, based upon recent acquisition of new roles in immunity and vascular pathology of the CD31 molecule, we hypothesize that this molecule might be important in the development and treatment of COVID-19 pulmonary lesions. These preliminary findings need further investigations to shed light on the complexity of Sars-Cov-2 disease.

Evidence of SARS-CoV-2 Transcriptional Activity in Cardiomyocytes of COVID-19 Patients without Clinical Signs of Cardiac Involvement.

AIMS: A considerable proportion of patients affected by coronavirus respiratory disease (COVID-19) develop cardiac injury. The viral impact in cardiomyocytes deserves, however, further investigations, especially in asymptomatic patients. METHODS: We investigated for SARS-CoV-2 presence and activity in heart tissues of six consecutive COVID-19 patients deceased from respiratory failure showing no signs of cardiac involvement and with no history of heart disease. Cardiac autopsy samples were collected within 2 h after death, and then analysed by digital PCR, Western blot, immunohistochemistry, immunofluorescence, RNAScope, and transmission electron microscopy assays. RESULTS: The presence of SARS-CoV-2 into cardiomyocytes was invariably detected in all assays. A variable pattern of cardiomyocyte injury was observed, spanning from absence of cell death and subcellular alterations hallmarks, to intracellular oedema and sarcomere ruptures. In addition, we found active viral transcription in cardiomyocytes, by detecting both sense and antisense SARS-CoV-2 spike RNA. CONCLUSIONS: In this autopsy analysis of patients with no clinical signs of cardiac involvement, the presence of SARS-CoV-2 in cardiomyocytes has been detected, determining variable patterns of intracellular damage. These findings suggest the need for cardiologic surveillance in surviving COVID-19 patients not displaying a cardiac phenotype.

The molecular and gene/miRNA expression profiles of radioiodine resistant papillary thyroid cancer.

BACKGROUND: Papillary thyroid cancer (PTC) is the most frequent endocrine tumor. Radioiodine (RAI) treatment is highly effective in these tumors, but up to 60% of metastatic cases become RAI-refractory. Scanty data are available on either the molecular pattern of radioiodine refractory papillary thyroid cancers (PTC) or the mechanisms responsible for RAI resistance. METHODS: We analyzed the molecular profile and gene/miRNA expression in primary PTCs, synchronous and RAI-refractory lymph node metastases (LNMs) in correlation to RAI avidity or refractoriness. We classified patients as RAI+/D+ (RAI uptake/disease persistence), RAI-/D+ (absent RAI uptake/disease persistence), and RAI+/D- (RAI uptake/disease remission), and analyzed the molecular and gene/miRNA profiles, and the expression of thyroid differentiation (TD) related genes. RESULTS: A different molecular profile according to the RAI class was observed: BRAFV600E cases were more frequent in RAI-/D+ (P = 0.032), and fusion genes in RAI+/D+ cases. RAI+/D- patients were less frequently pTERT mutations positive, and more frequently wild type for the tested mutations/fusions. Expression profiles clearly distinguished PTC from normal thyroid. On the other hand, in refractory cases (RAI+/D+ and RAI-/D+) no distinctive PTC expression patterns were associated with either tissue type, or RAI uptake, but with the driving lesion and BRAF-/RAS-like subtype. Primary tumors and RAI-refractory LNMs with BRAFV600E mutation display transcriptome similarity suggesting that RAI minimally affects the expression profiles of RAI-refractory metastases. Molecular profiles associated with the expression of TPO, SLC26A4 and TD genes, that were found more downregulated in BRAFV600E than in gene fusions tumors. CONCLUSIONS: The present data indicate a different molecular profile in RAI-avid and RAI-refractory metastatic PTCs. Moreover, BRAFV600E tumors displayed reduced differentiation and intrinsic RAI refractoriness, while PTCs with fusion oncogenes are RAI-avid but persistent, suggesting different oncogene-driven mechanisms leading to RAI refractoriness.

Jagged Ligands Enhance the Pro-Angiogenic Activity of Multiple Myeloma Cells.

Multiple myeloma (MM) is an incurable plasma cell malignancy arising primarily within the bone marrow (BM). During MM progression, different modifications occur in the tumor cells and BM microenvironment, including the angiogenic shift characterized by the increased capability of endothelial cells to organize a network, migrate and express angiogenic factors, including vascular endothelial growth factor (VEGF). Here, we studied the functional outcome of the dysregulation of Notch ligands, Jagged1 and Jagged2, occurring during disease progression, on the angiogenic potential of MM cells and BM stromal cells (BMSCs). Jagged1-2 expression was modulated by RNA interference or soluble peptide administration, and the effects on the MM cell lines' ability to induce human pulmonary artery cells (HPAECs) angiogenesis or to indirectly increase the BMSC angiogenic potential was analyzed in vitro; in vivo validation was performed on a zebrafish model and MM patients' BM biopsies. Overall, our results indicate that the MM-derived Jagged ligands (1) increase the tumor cell angiogenic potential by directly triggering Notch activation in the HPAECs or stimulating the release of angiogenic factors, i.e., VEGF; and (2) stimulate the BMSCs to promote angiogenesis through VEGF secretion. The observed pro-angiogenic effect of Notch activation in the BM during MM progression provides further evidence of the potential of a therapy targeting the Jagged ligands.

Correction to: Immunohistochemical evaluation of microsatellite instability in resected colorectal liver metastases: a preliminary experience.

The original version of this article unfortunately contained a mistake. All the authors first name and second name has been inadvertently interchanged. Now the authors name are corrected.

CD10 and CD34 as markers in vascular malformations with PIK3CA and TEK mutations.

AIMS: Vascular malformations (vMs) encompass a wide range of diseases often associated with somatic or, more rarely, germinal genetic mutations. A mutation in the PIK3Ca/mTOR pathway is more often involved in various vMs. CD10 and CD34 are cellular markers that may play a role in mesenchymal differentiation and proliferation. The aim of our study was to find a possible link between the immunohistochemical expression of CD10 and CD34 in vMs and their relationship with mutations in the PIK3CA/mTOR signaling pathway. METHODS AND RESULTS: Our study on 58 samples of vMs showed that in endothelial cells, CD10 was significantly expressed in PIK3CA-mutated samples compared with samples without any mutation (p < 0.05), especially and even more consistently when compared with samples with mutation in other pathways (p < 0.0001). Conversely, in the same PIK3CA-mutated samples, CD34 expression in endothelial cells was significantly reduced compared with samples either without any mutation or mutations in other pathways (p < 0.05 and p < 0.0005). Compared with samples with mutations in other pathways, a significant overexpression of endothelial CD10 was also found in samples with TEK/TIE2 mutation, a gene linked to the PIK3CA/mTOR pathway (p < 0.01). However, CD34 expression was not altered. In samples with PIK3CA mutation, the CD10 expression was significantly increased in the stroma compared with samples with TEK/TIE2 gene or other gene mutations (p < 0.05). CONCLUSION: Therefore, the CD10 and CD34 immunohistochemical profile could suggest/support the presence of mutations in the PIK3CA/mTOR pathway in samples of vMs.

EPIRETINAL FIBROSIS REMOVAL IN AN ARGUS II-IMPLANTED EYE: Histological Characteristics and Functional Results.

PURPOSE: 1) To investigate morphologic and histochemical characteristics of an epiretinal fibrosis removed in an Argus II-implanted eye; 2) to evaluate the Argus II function before and after the fibrosis removal, and 3) to compare morphologic and functional data. METHODS: Fibrosis, which developed between the Argus II prosthesis and the retina two years after implant, was surgically removed. Its morphologic and histochemical characteristics were evaluated both in light and transmission electron microscopy, with special stains and immunohistochemistry. The Argus II function was evaluated during the follow-up before surgical removal and 1 month later. RESULTS: Fibrosis was successfully removed. It was composed of a fibrotic tissue with spindle cells arranged in nodular aggregates with a symmetric distribution, mixed with an inflammatory infiltrate. Extra- and intracellular, irregular, small iron particles were found and confirmed ultrastructural characterization with degenerative cellular changes. The repositioned Argus II restored, and its function was partially nearly to normal values 1 month after surgery. CONCLUSION: Fibrosis can develop between the Argus II and the retina with increasing reduced function. Morphologic characteristics of the removed fibrosis suggested a pathogenesis based on an inflammatory process involved in a foreign body reaction with progressing connective tissue deposition leading to sclerosis. Adequate clinical follow-up is critical to successful removal of the fibrosis with reactivation of the Argus II function.

Candida isolates causing candidemia show different degrees of virulence in Galleria mellonella.

We aim to assess intra- and interspecies differences in the virulence of Candida spp. strains causing candidemia using the invertebrate Galleria mellonella model. We studied 739 Candida spp. isolates (C. albicans [n = 373], C. parapsilosis [n = 203], C. glabrata [n = 92], C. tropicalis [n = 53], and C. krusei [n = 18]) collected from patients with candidemia admitted to Gregorio Marañon Hospital (Madrid, Spain). Species-specific infecting inocula (yeast cells/larva) were adjusted (5 × 105 [C. albicans, and C. tropicalis], 2 × 106-5 × 106 [C. parapsilosis, C. glabrata, and C. krusei]) and used to infect 10 larvae per isolate; percentage of survival and median survival per isolate were calculated. According to the interquartile range of the median survival, isolates with a median survival under P25 were classified as of high-virulence and isolates with a median survival over P75 as of low virulence. The median survival of larvae infected with different species was variable: C. albicans (n = 2 days, IQR <1-3 days), C. tropicalis (n = 2 days, IQR 1.5-4 days), C. parapsilosis (n = 2 days, IQR 2-3.5 days), C. glabrata (n = 3 days, IQR 2-3 days), and C. krusei (n = 7 days, 6.5->8 days) (P < .001). Differences in virulence among species were validated by histological examination (day +1 post-infection) in the larvae infected by the isolates of each virulence category and species. Virulence-related gene expression in C. albicans isolates did not reach statistical significance. We report species-specific virulence patterns of Candida spp. and show that isolates within a given species have different degrees of virulence in the animal model.

Therapeutic Effect of Iron Citrate in Blocking Calcium Deposition in High Pi-Calcified VSMC: Role of Autophagy and Apoptosis.

In chronic kidney disease (CKD), the first cause of mortality is cardiovascular disease induced mainly by vascular calcification (VC). Recently, iron-based phosphate binders have been proposed in advanced CKD to treat hyperphosphatemia. We studied the effect of iron citrate (iron) on the progression of calcification in high-phosphate (Pi) calcified VSMC. Iron arrested further calcification when added on days 7-15 in the presence of high Pi (1.30 ± 0.03 vs 0.61 ± 0.02; OD/mg protein; day 15; Pi vs Pi + Fe, p < 0.01). We next investigated apoptosis and autophagy. Adding iron to high-Pi-treated VSMC, on days 7-11, decreased apoptotic cell number (17.3 ± 2.6 vs 11.6 ± 1.6; Annexin V; % positive cells; day 11; Pi vs Pi + Fe; p < 0.05). The result was confirmed thorough analysis of apoptotic nuclei both in VSMCs and aortic rings treated on days 7-15 (3.8 ± 0.2 vs 2.3 ± 0.3 and 4.0 ± 0.3 vs 2.2 ± 0.2; apoptotic nuclei; arbitrary score; day 15; Pi vs Pi + Fe; VSMCs and aortic rings; p < 0.05). Studying the prosurvival axis GAS6/AXL, we found that iron treatment on days 9-14 counteracted protein high-Pi-stimulated down-regulation and induced its de novo synthesis. Moreover, iron added on days 9-15 potentiated autophagy, as detected by an increased number of autophagosomes with damaged mitochondria and an increase in autophagic flux. Highlighting the effect of iron on apoptosis, we demonstrated its action in blocking the H2O2-induced increase in calcification added both before high Pi treatment and when the calcification was already exacerbated. In conclusion, we demonstrate that iron arrests further high Pi-induced calcium deposition through an anti-apoptotic action and the induction of autophagy on established calcified VSMC.

High-phosphate induced vascular calcification is reduced by iron citrate through inhibition of extracellular matrix osteo-chondrogenic shift in VSMCs.

BACKGROUND: High serum phosphate (Pi) levels strongly associate with cardiovascular morbidity and mortality in chronic kidney disease (CKD) patients with vascular calcification playing a major role in the pathogenesis of related cardiovascular disease. High-Pi challenged vascular smooth muscle cells (VSMCs) undergo simil-osteoblastic transformation and actively deposit calcium-phosphate crystals. Iron-based Pi-binders are used to treat hyperphosphatemia in CKD patients. METHODS: In this study, we investigated the direct effect of iron citrate on extracellular matrix (ECM) modification induced by high-Pi, following either prophylactic or therapeutic approach. RESULTS: Iron prophylactically prevents and therapeutically blocks high-Pi induced calcification. Masson's staining highlights the changes of muscular ECM that after high-Pi stimulation becomes fibrotic and which modifications are prevented or partially reverted by iron. Interestingly, iron preserves glycogen granules and either prevents or partially reverts the formation of non-glycogen granules induced by high-Pi. In parallel, iron addition is able to either prevent or block the high-Pi induced acid mucin deposition. Iron inhibited calcification also by preventing exosome osteo-chondrogenic shift by reducing phosphate load (0,61 ±â€¯0.04vs0,45 ±â€¯0.05, PivsPi + Fe, p < 0,05, nmol Pi/mg protein) and inducing miRNA 30c (0.62 ± 0.05vs3.07 ±â€¯0.62; PivsPi + Fe, p < 0.01, relative expression). Studying aortic rings, we found that iron significantly either prevents or reverts the high-Pi induced collagen deposition and the elastin decrease, preserving elastin structure (0.7 ± 0.1 vs 1.2 ± 0.1; Pi vs Pi + Fe, p < 0.05, elastin mRNA relative expression). CONCLUSIONS: Iron directly either prevents or partially reverts the high-Pi induced osteo-chondrocytic shift of ECM. The protection of muscular nature of VSMC ECM may be one of the mechanisms elucidating the anti-calcific effect of iron.