Antonio Pavan

Shedding of the Matrix Metalloproteinases MMP-2, MMP-9, and MT1-MMP as Membrane Vesicle-Associated Components by Endothelial Cells

Production of matrix-degrading proteases, particularly matrix metalloproteinases (MMPs), by endothelial cells is a critical event during angiogenesis, the process of vessel neoformation that occurs in normal and pathological conditions. MMPs are known to be highly regulated at the level of synthesis and activation, however, little is known about the regulation of MMP secretion by endothelial cells. We found that cultured human umbilical vein endothelial cells shed vesicles (300 to 600 nm) originating from localized areas of the cell plasma membrane, as revealed by ultrastructural analysis. Normal and reverse zymography, Western blot, and immunogold analyses of the vesicles showed two gelatinases, MMP-2 and MMP-9, in both the active and proenzyme forms, the MT1-MMP proenzyme located on the external side of the vesicle membrane and the two inhibitors TIMP-1 and TIMP-2. Serum and the angiogenic factors, fibroblast growth factor-2 and vascular endothelial growth factor, stimulated the shedding of MMPs as vesicle components. Shedding the vesicle was rapid, as it was already completed after 4 hours. Addition of shed vesicles to human umbilical vein endothelial cells resulted in autocrine stimulation of invasion through a layer of reconstituted basement membrane (Matrigel) and cord formation on Matrigel. We conclude that endothelial cells shed MMP-containing vesicles and this may be a mechanism for regulating focalized proteolytic activity vital to invasive and morphogenic events during angiogenesis.

Platelet-Rich Plasma Versus Focused Shock Waves in the Treatment of Jumper’s Knee in Athletes

Background: Tendinopathies represent a serious challenge for orthopaedic surgeons involved in treatment of athletes. Purpose: To compare the effectiveness and safety of platelet-rich plasma (PRP) injections and focused extracorporeal shock wave therapy (ESWT) in athletes with jumper’s knee. Study Design: Randomized controlled trial; Level of evidence, 1. Methods: Forty-six consecutive athletes with jumper’s knee were selected for this study and randomized into 2 treatment groups: 2 autologous PRP injections over 2 weeks under ultrasound guidance (PRP group; n = 23), and 3 sessions of focused extracorporeal shock wave therapy (2.400 impulses at 0.17-0.25 mJ/mm2 per session) (ESWT group; n = 23). The outcome measures were Victorian Institute of Sports Assessment–Patella (VISA-P) questionnaire, pain visual analog scale (VAS), and modified Blazina scale. A reviewer who was blinded as to the group allocation of participants performed outcome assessments before treatment and at 2, 6, and 12 months after treatment. Nonparametric tests were used for within-group (Friedman/Wilcoxon test) and between-group (Kruskal-Wallis/Fisher test) testing, and the significance level was set at .05. Results: The 2 groups were homogeneous in terms of age, sex, level of sports participation, and pretreatment clinical status. Patients in both groups showed statistically significant improvement of symptoms at all follow-up assessments. The VISA-P, VAS, and modified Blazina scale scores showed no significant differences between groups at 2-month follow-up (P = .635, .360, and .339, respectively). The PRP group showed significantly better improvement than the ESWT group in VISA-P, VAS scores at 6- and 12-month follow-up, and modified Blazina scale score at 12-month follow-up (P < .05 for all). Conclusion: Therapeutic injections of PRP lead to better midterm clinical results compared with focused ESWT in the treatment of jumper’s knee in athletes.

Impairment of Caveolae Formation and T-System Disorganization in Human Muscular Dystrophy with Caveolin-3 Deficiency

Caveolin-3, a muscle specific caveolin-related protein, is the principal structural protein of caveolar membranes. We have recently identified an autosomal dominant form of limb girdle muscular dystrophy (LGMD-1C) that is due to caveolin-3 deficiency and caveolin-3 gene mutations. Here, we studied by electron microscopy, including freeze-fracture and lanthanum staining, the distribution of caveolae and the organization of the T-tubule system in caveolin-3 deficient human muscle fibers. We found a severe impairment of caveolae formation at the muscle cell surface, demonstrating that caveolin-3 is essential for the formation and organization of caveolae in muscle fibers. In addition, we also detected a striking disorganization of the T-system openings at the sub-sarcolemmal level in LGMD-1C muscle fibers. These observations provide new perspectives in our understanding of the role of caveolin-3 in muscle and of the pathogenesis of muscle weakness in caveolin-3 deficient muscle.

Identification of an optimal concentration of platelet gel for promoting angiogenesis in human endothelial cells.

BACKGROUND: Numerous studies have supported the use of topical blood components to improve wound healing and tissue regeneration. Platelet gel (PG), a hemocomponent obtained from mix of activated platelets (PLTs) and cryoprecipitate, is currently being used clinically in an attempt to improve tissue healing. The present study sought to define the most effective PG concentration to promote angiogenesis in vitro.

Monosialoganglioside GM3 induces CD4 internalization in human peripheral blood T lymphocytes

Gangliosides modulate the expression of CD4 molecules on the cell surface of T lymphocytes. We report here that treatment of human peripheral blood lymphocytes with exogenous monosialoganglioside GM3 induces a rapid down‐modulation of the CD4 molecules on the plasma membrane of CD4+ T lymphocytes, as assessed by cytofluorimetric analysis and quantitative immunoelectron microscopy. The CD4 down‐modulation was gang Hoside‐dose dependent and was already evident after 5 min of treatment, reaching the maximum after 20 min. The expression of other surface antigens was not affected by GM3 treatment. The immunoelectron microscopic analysis showed that, following GM3 addition, gold labelled CD4 molecules were rapidly redistributed on the cell surface, clustered and internalized via endocytic pits and vesicles. These results indicate that CD4 down‐modulation induced by GM3 occurs through an endocytic mechanism. A persistent low level of CD4 expression on the cell surface up to 24 h after GM3 treatment, compared with a stable expression of either CD4 in untreated cells and CD3 in GM3‐treated cells, suggests intracellular degradation of the internalized CD4 molecules.

Size and stability of dipalmitoylphosphatidylcholine/cholesterol unilamellar vesicles are affected by interaction with proteins.

The effect of entrapping the enzyme ascorbate oxidase into dipalmitoylphosphatidylcholine/cholesterol vesicles, was studied by conventional transmission electron microscopy and freeze-fracture. The freeze-fracture technique has definitely demonstrated the unilamellar nature of empty and enzyme-loaded vesicles. Images of freeze-fractured and label-fractured liposomes also indicate that the observed reduction of vesicles volume could be related to the localization of ascorbate oxidase across the membrane. The membrane localization of ascorbate oxidase may explain the oxidation of externally added ascorbate by intact enzyme-loaded liposomes. Finally, the ageing of liposomes appears to be accelerated in the presence of proteins.

Regional distribution of Sindbis virus glycoproteins on the plasma membrane of infected baby hamster kidney cells

Sindbis virus-infected baby hamster kidney (BHK) cells were analysed in surface replicas or conventional thin sections after specific immunolabelling with antiviral glycoprotein antibodies in conjunction with colloidal gold-conjugated protein A. Newly synthesized viral glycoproteins were detected, beginning 1 1/2 h after infection, while the virus maturation started 3 h after infection. The glycoproteins appeared to be inserted on the plasma membrane in large spots located mainly in the central area of the cells: no clustering of the labelling was detected. Later, the glycoproteins appeared to arrange linearly in regions in the medial portion of the cells. No labelling was found in the peripheral area or on the cell edges. A drastic change in the surface labelling was detected following the commencement of virus maturation: gold particles were organized mostly in small clusters, each labelling a budding virus. Very few glycoproteins appeared not to be involved in budding figures. The maturation of the virus was clearly regionalized, but during this time it also involved the peripheral area and the cell edges; preferential budding in narrow cellular processes was often observed. It appeared thus that either isolated glycoproteins soon after infection, or clustered glycoproteins at later times, are strictly regionalized on the plasma membrane: however, the early post-infection distribution is clearly different from that seen later during virus maturation. Our experiments support the concept of discrete plasma membrane domains even in cells that do not display distinct specialization of their surface.

Molecular cytochemistry of CD3 and CD4 antigens in human lymphocytes as studied by label-fracture and by fracture-label

Label-fracture and fracture-label membrane immunocytochemistry are used to analyze the surface distribution, dynamics and partition on fracture of CD3 and CD4 antigens of human T lymphocytes. Redistribution of the antigens, induced by treatment at 37 degrees C with specific monoclonal antibodies, results in patching and capping of the labeling as observed in label-fractured specimens. Examination of platinum/carbon replicas of freeze-fractured plasma membranes of antibody-treated cells does not reveal recognizable domains of intramembrane particles. However, in cells where the aggregation of intramembrane particles is induced by incubation with glycerol, colloidal gold-labeled CD3 and CD4 molecules are seen confined to particulate domains of the membrane. Therefore, the lack of visible aggregation of intramembrane particles in patched or capped regions of the membrane implies that migration of CD3 and CD4 antigens with concentration in domains of the membrane is achieved contemporaneously with export of other non-capped integral membrane proteins from the same regions, in a process of diffusional equilibrium. Examination of fracture-labeled specimens shows that CD4 molecules partition on fracture with the inner protoplasmic face of the plasma membrane. This partition illustrates the transmembrane attitude of the antigen molecule and is a probable consequence of interaction of the protein with other components of the membrane or with the cytoskeleton.

Patching and capping of LFA-1 molecules on human lymphocytes.

SummaryThe distribution and dynamics of LFA-1 molecules over the surface of human lymphocytes were analysed using immunogold label-fracture and fracture-flip methods. Patching and capping were induced by incubation at 37°C with antibodies directed against the alpha and beta chains respectively of the heterodimeric LFA-1 molecule, and were followed by immunofluorescence. Treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) to link LFA-1 molecules to the cytoskeleton increased the percentage of capped cells, implying a faster and more efficient process of capping. At all times of clustering or upon phorbol ester treatment, the concentration of LFA-1 in patches and then in caps was not accompained by a parallel concentration of membrane particles on the freeze-fractured plasma membranes. Our results support the role of the cytoskeleton in regulating the capping phenomenon and in controlling the structural organization of the plasma membranes.

Nonrandom distribution of epidermal growth factor receptors on the plasma membrane of human A431 cells.

The localization of epidermal growth factor (EGF) receptors over the plasma membranes of human epidermoid carcinoma A431 cells was analyzed at the electron microscopic level using surface replica techniques and conventional thin sections, in combination with immunocytochemistry. Immunolabeling was performed using two distinct monoclonal antibodies directed against the extracellular portion of the receptor, followed by protein A-colloidal gold conjugates. Unexpectedly, with the first monoclonal antibody used, the distribution of the receptors in both unfixed and glutaraldehyde-fixed cells was clearly regionalized, showing a preferential localization of the immunolabeling at the cell periphery as well as over the areas rich in microvilli and in coated and uncoated pits. A similar pattern of distribution was observed also with the other monoclonal antibody, but only when the cells were fixed with glutaraldehyde before immunolabeling. Treatment with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate modifies this distribution, inducing a more disperse pattern. Our observations suggest that a minor group of EGF receptors, which may represent the high-affinity receptors, presents a regional distribution, similar to that described for typical recycling receptors.