Margherita Aglianò

Lymphatics at the crossroads of angiogenesis and lymphangiogenesis

The lymphatic system is implicated in interstitial fluid balance regulation, immune cell trafficking, oedema and cancer metastasis. However, the sequence of events that initiate and coordinate lymphatic vessel development (lymphangiogenesis) remains obscure. In effect, the understanding of physiological regulation of lymphatic vasculature has been overshadowed by the greater emphasis focused on angiogenesis, and delayed by a lack of specific markers, thereby limiting this field to no more than a descriptive characterization. Recently, new insights into lymphangiogenesis research have been due to the discovery of lymphatic‐specific markers and growth factors of vascular endothelial growth factor (VEGF) family, such as VEGF‐C and VEGF‐D. Studies using transgenic mice overexpressing VEGF‐C and VEGF‐D have demonstrated a crucial role for these factors in tumour lymphangiogenesis. Knowledge of lymphatic development has now been redefined at the molecular level, providing an interesting target for innovative therapies. This review highlights the recent insights and advances into the field of lymphatic vascular research, outlining the most important aspects of the embryo development, structure, specific markers and methods applied for studying lymphangiogenesis. Finally, molecular mechanisms involved in the regulation of lymphangiogenesis are described.

The structure of superficial lymphatics in the human thigh: precollectors

Little is known about the morphology of precollectors, the lymphatic vessels connecting the absorbing and the collecting vessels, which are regarded as the initial drainage routes of lymph. The aim of this study was to describe the structural features of human precollectors.

Colchicine myopathy and neuromyopathy: two cases with different characteristics.

Colchicine, a long established anti-inflammatory agent now used in several rheumatologic conditions, acts by inhibiting microtubular polymerization, as it binds equimolarly to tubulin molecules. Cytoskeletal microtubules are crucial in processes of cell viability, such as mitosis and intracellular vesicle motility.Gastrointestinal side effects are quite common and often minor in nature or duration, whereas neuromuscular toxicity is rare. We report 2 cases of colchicine myopathy in the context of very different clinical settings.

Antiangiogenic VEGF Isoform in Inflammatory Myopathies

Objective. To investigate expression of vascular endothelial growth factor (VEGF) antiangiogenic isoform A-165b on human muscle in idiopathic inflammatory myopathies (IIM) and to compare distribution of angiogenic/antiangiogenic VEGFs, as isoforms shifts are described in other autoimmune disorders. Subjects and Methods. We analyzed VEGF-A165b and VEGF-A by western blot and immunohistochemistry on skeletal muscle biopsies from 21 patients affected with IIM (polymyositis, dermatomyositis, and inclusion body myositis) and 6 control muscle samples. TGF-β, a prominent VEGF inductor, was analogously evaluated. Intergroup differences of western blot bands density were statistically examined. Endomysial vascularization, inflammatory score, and muscle regeneration, as pathological parameters of IIM, were quantitatively determined and their levels were confronted with VEGF expression. Results. VEGF-A165b was significantly upregulated in IIM, as well as TGF-β. VEGF-A was diffusely expressed on unaffected myofibers, whereas regenerating/atrophic myofibres strongly reacted for both VEGF-A isoforms. Most inflammatory cells and endomysial vessels expressed both isoforms. VEGF-A165b levels were in positive correlation to inflammatory score, endomysial vascularization, and TGF-β. Conclusions. Our findings indicate skeletal muscle expression of antiangiogenic VEGF-A165b and preferential upregulation in IIM, suggesting that modulation of VEGF-A isoforms may occur in myositides.

Eosinophilia-associated muscle disorders: an immunohistological study with tissue localisation of major basic protein in distinct clinicopathological forms

Aims: (a) To evaluate tissue eosinophil density, location of eosinophil cytotoxic products, histopathological muscle changes and inflammatory cell types in different eosinophilia-associated myopathies that are clinicopathologically heterogeneous. (b) To determine the immunohistological range of tissue eosinophil density in non-eosinophilic inflammatory myopathies. Methods: Muscle biopsy specimens from seven patients with blood and/or tissue eosinophilia and clinicolaboratory myopathic signs (five chronic course myopathies, one subacute onset fasciitis/myositis, one acute myositis), and from 18 non-eosinophilic inflammatory myopathies, underwent routine staining, inflammatory infiltrate immunophenotyping, immunostaining for eosinophil major basic protein (MBP) and transmission electron microscopy examination. Eosinophil and total inflammatory cell counts were statistically analysed. Results: Histological examination showed occasional or no infiltrating eosinophils in all cases. MBP staining showed that tissue eosinophil density and percentages in eosinophilia-associated myopathies were significantly higher than in idiopathic myositides. Extracellular MBP diffusion, the hallmark of eosinophil cytotoxicity, was recurrent on sarcolemma and endothelium. Electron microscopy showed eosinophils close to sarcolemma, abundant mast cells, and capillary endothelial swelling. Immunostaining detected a higher mean eosinophil density in idiopathic myositides than previously assessed histologically. Conclusions: MBP immunohistology on skeletal muscle, previously performed only for acute eosinophilic polymyositis, suggests that eosinophil-mediated injury of muscle cells may occur in a wider spectrum of less aggressive eosinophilia-associated myopathies than previously thought. As conventional histology is likely to underestimate this leucocyte subset, MBP staining may be a useful tool in the analysis of tissue infiltration of eosinophils as a possible treatment target.

Prophylactic role of acetyl-l-carnitine on knee lesions and associated pain in a rat model of osteoarthritis

AIMS in the present study, our aim was to validate in vivo the prophylactic role of acetyl-l-carnitine (ALC) using an established knee osteoarthritis (OA) animal model which mimics the pathological changes of OA in humans, targeting cartilage and causing chondrocyte death. MAIN METHODS animal model was obtained by an intra-articular injection of monosodium iodoacetate (MIA) into rat femorotibial joint space. Pain was measured in animals submitted to MIA model by paw pressure and compression behavioral tests in the presence or absence of ALC. KEY FINDINGS morphological analysis of knee-joint from MIA and ALC co-treated rats showed that the total pathological score attributed to histological findings was dramatically lower in rats treated with MIA in the presence of ALC. OA chondrocyte overexpression of pathogenic collagenase matrix-metallopeptidase-13 (MMP13) could be decreased in knee-cartilage from MIA/ALC rats; whereas type II collagen (COL2) expression level could be partially increased to control value. ALC twice daily treatment was able to attenuate pain in OA rat knee as revealed by mechanical behavioral tests. SIGNIFICANCE in our experiments, pain that is usually associated with OA, was correlated with the severity of histopathological findings. Our findings show that there is a place for ALC as chondroprotective agents in cartilage degradation and strongly support the prophylactic and therapeutic potentials of ALC in knee-OA patients.

Megadoses of ascorbate as a new chemotherapeutic approach in uveal melanoma: a preliminary in vitro investigation

Background: Despite the more recent advances, there is still no effective systemic therapy for Uveal Melanoma (UM). However, a better understanding of the role of oxidative stress in cancer, has more recently led to a completely new approach to the systemic therapy of cancer, and modulators of the oxidative balance, such as sodium ascorbate (ASC) or arsenic trioxide (ATO), have already entered advanced phases of preclinical and clinical development. Since high doses of ASC have already demonstrated a strong cytotoxic effect on different human cancer cell lines, we have undertaken the present investigation in order to test the sensitivity of OCM1 and C918 uveal melanoma (UM) cell lines to high doses of ASC, in vitro, as compared to ATO, a pro-oxidant drug which has already undergone extensive in vitro and pre-clinical investigation in UM. Methods: Both OCM1 and C918 UM cell lines have been exposed to increasing doses of either ASC or ATO, to build a dose-response curve around the peak plasma concentrations reached by both chemicals. The assessment of cell count and viability was performed with flow cytometry. Results: Both OCM1 and C918 UM cell lines are highly sensitive to ASC in the range of millimolar (mM) concentrations which can be usually reached by the intravenous injection of high doses of the compound. ATO at the dosages used in this study, never reached the LC50. When the exposure to ASC was reduced to two hours, it still had significant effects on survival of both OCM1 and C918 UM cells. Conclusions: This report shows that ASC is highly cytotoxic for both OCM1 and C918 UM cells, when used in high, pro-oxidant doses. To our knowledge, this is the first report showing that UM cells can be efficiently killed, in vitro, with high doses of ASC.

Apoptotic process induced by oxaliplatin in rat hippocampus causes memory impairment

Aspects of memory involved in cognitive mechanisms were investigated in rat after oxaliplatin (OX) chemotherapy using animal behavioural assessment of passive avoidance and social learning paradigms, which are both hippocampus‐sensitive. Rodents, previously subjected to 2‐week OX treatment, showed passive avoidance and social learning impairment and apoptotic processes in the hippocampus. Apoptosis rate significantly increased in cultured hippocampal cells exposed to OX at increasing doses, and this effect was dose‐dependent. Ex vivo experiments showed that cell damage and apoptosis were blocked in the hippocampus from OX rats cotreated with copper sulphate (CS) which precludes OX transport inside the cell. In vivo, passive avoidance and social learning impairment could not be observed in OX rats co‐administered with CS. Thus, a site of action of OX treatment on memory impairment appears to be located at the hippocampus. These findings strongly support that cellular damage induced by OX in rodent hippocampus underlies the weakening of some memory functions.

Histopathological findings in systemic sclerosis-related myopathy: fibrosis and microangiopathy with lack of cellular inflammation

Objectives: The objective of this study was to identify specific histopathological features of skeletal muscle involvement in systemic sclerosis (SSc) patients. Methods: A total of 35 out of 112 SSc-patients (32%, including 81% female and 68% diffuse scleroderma) presenting clinical, biological and electromyographic (EMG) features of muscle weakness, were included. Patients underwent vastus lateralis biopsy, assessed for individual pathologic features including fibrosis [type I collagen (Coll-I), transforming growth factor β (TGF-β)], microangiopathy [cluster of differentiation 31 (CD31), pro-angiogenic vascular endothelial growth factor A (VEGF-A), anti-angiogenic VEGF-A165b], immune/ inflammatory response [CD4, CD8, CD20, human leucocyte antigens ABC (HLA-ABC)], and membranolytic attack complex (MAC). SSc biopsies were compared with biopsies of (n = 35) idiopathic inflammatory myopathies (IIMs) and to (n = 35) noninflammatory myopathies (NIMs). Ultrastructural abnormalities of SSc myopathy were also analyzed by transmission electron microscopy (TEM). Results: Fibrosis in SSc myopathy (81%) is higher compared with IIM (32%, p < 0.05) and with NIM (18%, p < 0.05). Vascular involvement is dominant in SSc muscle (92%), and in IIM (78%) compared with NIM (21%, p < 0.05). In particular, CD31 shows loss of endomysial vessels in SSc myopathy compared with IIM (p < 0.05) and with NIM (p < 0.01). VEGF-A is downregulated in SSc myopathy compared with IIM (p < 0.05) and NIM (p < 0.05). Conversely, VEGF-A165b is upregulated in SSc myopathy. The SSc immune/inflammatory response suggested humoral process with majority (85%) HLA-ABC fibral neoexpression and complement deposits on endomysial capillaries MAC, compared with IIM (p < 0.05), characterized by CD4+/CD8+/B-cell infiltrate, and NIM (p < 0.05). TEM analysis showed SSc vascular alterations consisting of thickening and lamination of basement membrane and endothelial cell ‘swelling’ coupled to endomysial/perimysial fibrosis. Conclusions: Fibrosis, microangiopathy and humoral immunity are predominant in SSc myopathy, even if it is difficult to identify specific histopathological hallmarks of muscle involvement in SSc, since they could be present also in other (IIM/NIM) myopathies.

Geometric complexity identifies platelet activation in familial hypercholesterolemic patients

Familial hypercholesterolemia (FH), a genetic disease, is associated with a severe incidence of athero‐thrombotic events, related, also, to platelet hyperreactivity. A plethora of methods have been proposed to identify those activated circulating platelets, none of these has proved really effective. We need efficient methods to identify the circulating platelet status in order to follow the patients after therapeutic procedures. We propose the use of computerized fractal analysis for an objective characterization of the complexity of circulating platelet shapes observed by means of transmission electron microscopy in order to characterize the in vivo hyperactivated platelets of familial hypercholesterolemic patients, distinguishing them from the in vivo resting platelets of healthy individuals. Platelet boundaries were extracted by means of automatically image analysis. Geometric complexity (fractal dimension, D) by box counting was automatically calculated. The platelet boundary observed by electron microscopy is fractal, the shape of the circulating platelets is more complex in FH (n = 6) than healthy subjects (n = 5, P < 0.01), with 100% correct classification in selected individuals. In vitro activated platelets from healthy subjects show an analogous increase of D. The observed high D in the platelet boundary in FH originates from the in vivo platelet activation. Computerized fractal analysis of platelet shape observed by transmission electron microscopy can provide accurate, quantitative data to study platelet activation in familial hypercholesterolemia and after administration of drugs or other therapeutic procedures. Microsc. Res. Tech. 78:519–522, 2015.