Leni Moldovan - Academia.edu (original) (raw)

Papers by Leni Moldovan

The presence and supposed roles of reactive oxygen species (ROS) were reported in literature in a... more The presence and supposed roles of reactive
oxygen species (ROS) were reported in literature in a
myriad of instances. However, the breadth and depth of
their involvement in cellular physiology and pathology, as
well as their relationship to the redox environment can
only be guessed from specialized reports. Whatever their
circumstances of formation or consequences, ROS seem
to be conspicuous components of intracellular milieu. We
sought to verify this assertion, by collecting the available
evidence derived from the most recent publications in the
biomedical field. Unlike other reviews with similar objectives,
we centered our analysis on the subcellular
compartments, namely on organelles, grouped according
to their major functions. Thus, plasma membrane is a
major source of ROS through NAD(P)H oxidases located
on either side. Enzymes of the same class displaying low
activity, as well as their components, are also present free
in cytoplasm, regulating the actin cytoskeleton and cell
motility. Mitochondria can be a major source of ROS,
mainly in processes leading to apoptosis. The protein
synthetic pathway (endoplasmic reticulum and Golgi apparatus),
including the nucleus, as well as protein turnover,
are all exquisitely sensitive to ROS-related redox
conditions. The same applies to the degradation pathways
represented by lysosomes and peroxisomes. Therefore,
ROS cannot be perceived anymore as a mere harmful
consequence of external factors, or byproducts of altered
cellular metabolism. This may explain why the indiscriminate
use of anti-oxidants did not produce the expected
“beneficial” results in many medical applications
attempted so far, underlying the need for a deeper apprehension
of the biological roles of ROS, particularly in
the context of the higher cellular order of organelles.

Biochemical and Biophysical Research Communications, 2000

The small GTP-binding protein family including Rac proteins represents a paradigm for signaling m... more The small GTP-binding protein family including Rac proteins represents a paradigm for signaling molecules shared by animal and plants. In mammalian cells, Rac induces the activation of NADPH oxidase leading to superoxide production. In plants, evidence suggests that resistance to pathogens depends on superoxide that is generated via NADPH oxidase-like enzymes. We have identified four closely related Rho/ Rac genes from Zea mays that exhibit a high degree of homology to the human Rac. We hypothesized that these plant Rac proteins could function as their mammalian counterpart and activate an enzymatic complex that leads to superoxide production. Here, we show that like human Rac1, activated Zea mays Rac genes can induce superoxide production, when expressed in a mammalian system: NIH 3T3 cells. Our results suggest that in plants, Rac proteins can function as activators of oxidative burst and indicate the remarkable functional and structural conservation of Rho/Rac proteins between plant and animal kingdoms during evolution.

We studied the association between the production of reactive oxygen species, actin organization,... more We studied the association between the production of reactive oxygen species, actin organization, and cellular motility. We have used an endothelial cell monolayer-wounding assay to demonstrate that the cells at the margin of the wound thus created produced significantly more free radicals than did cells in distant rows. The rate of incorporation of actin monomers into filaments was fastest at the wound margin, where heightened production of free radicals was detected. We have tested the effect of decreasing reactive oxygen species production on the migration of endothelial cells and on actin polymerization. The NADPH inhibitor diphenylene iodonium and the superoxide dismutase mimetic manganese (III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP) virtually abolished cytochalasin D-inhibitable actin monomer incorporation at the fast-growing barbed ends of filaments. Moreover, endothelial cell migration within the wound was significantly retarded in the presence of both diphenylene iodonium and MnTMPyP. We conclude that migration of endothelial cells in response to loss of confluence includes the intracellular production of reactive oxygen species, which contribute to the actin cytoskeleton reorganization required for the migratory behavior of endothelial cells. (Circ Res. 2000;86:549-557.)

Cold Spring Harbor Symposia on Quantitative Biology, 2002

Reactive oxygen species (ROS) are acknowledged generally to be multi-faceted regulators of cellul... more Reactive oxygen species (ROS) are acknowledged generally to be multi-faceted regulators of cellular functions that trigger various pathological states when present chronically or transiently at non-physiologically high levels. Here we focus on the physiological involvement of ROS in cellular motility, with special emphasis on endothelial cells (EC). An important source of ROS within EC is the nonphagocytic NAD(P)H oxidase, and the small GTPase Rac1 plays a central role in activating this complex. Rac1 is one of the three Rho-family molecules (Rac, Rho and Cdc42) involved in the control of the actin cytoskeleton in response to various signals. In this review we examine the evidence linking ROS production, Rac1 activation and actin organization to EC motility, considering mechanisms for direct interaction of ROS and actin and the effects of ROS on proteins that regulate the actin cytoskeleton. The accumulated evidence suggests that ROS are important regulators of the actin cytoskeletal dynamics and cellular motility, and more in-depth studies are needed to understand the underlying mechanisms.

Journal of Cellular and Molecular Medicine, 2006

Objective: We have previously shown that monocytes/macrophages (MC/Mph) influence neovascularizat... more Objective: We have previously shown that monocytes/macrophages (MC/Mph) influence neovascularization by extracellular matrix degradation, and by direct incorporation into growing microvessels. To date, neither the phenotype of these cells, nor the stages of their capillary-like conversion were sufficiently characterized. Methods: We isolated mouse peritoneal Mph from transgenic mice expressing fluorescent proteins either ubiquitously, or specifically in the myelocytic lineage. These Mph were embedded in Matrigel which contained fluorescent protease substrates, exposed to an MCP-1 chemotactic gradient, and then examined by confocal microscopy after various intervals. Results: Within 3 hrs after gel embedding, we detected TIMP-1 and MMP-12 dependent proteolysis of the matrix surrounding Mph, mostly in the direction of high concentrations of MCP-1. After 2 days, Mph developed intracellular vacuoles containing degradation product. At 5 days these vacuoles were enlarged and/or fused to generate trans-cellular lumens in approximately 10% of cells or more (depending on animal's genetic background). At this stage, Mph became tubular, and occasionally organized in three-dimensional structures resembling branched microvessels. Conclusion: Isolated mouse peritoneal Mph penetrate Matrigel and form tunnels via a metalloprotease-driven proteolysis and phagocytosis. Following a morphological adjustment driven by occurrence, enlargement and/or fusion process of intracellular vacuoles, similar to that described in bona fide endothelium, a subpopulation of these cells end up by lining a capillary-like lumen in vitro. Thus we show that adult Mph, not only the more primitive ‘endothelial progenitors’, have functional properties until now considered defining of the endothelial phenotype.

Blood Coagulation & Fibrinolysis, 1994

Qualitative ultrastructural autoradiography was used to study the binding of the vascular anticoa... more Qualitative ultrastructural autoradiography was used to study the binding of the vascular anticoagulant alpha (annexin V) to normal and atherosclerotic (AS) rabbit aortic intima. Recombinant annexin V was labelled with 125I by the Iodogen method. Rabbits were fed a hypercholesterolemic (HC) diet up to 10 months. After laparotomy and exsanguination, the aortae were perfused with dilutions of 125I-annexin V (I-AV) for 10-15 min, either on ice or at 22 degrees C and then perfusion-fixed with aldehydes. Fragments of the labelled aortae were used for en face contact autoradiography, followed by Sudan Black staining of intimal lipid. Specimens were also included in Epon and sectioned for light- and electron-microscopic autoradiography. The binding of I-AV was increased on the AS aortae as compared with the normal ones, with an apparent preference for the lesioned areas. Microscopically, I-AV was found at the luminal front of aortic intima, on endothelial cells (EC), on macrophage foam cells, and on their disrupted remnants. The presence of the AV binding sites (reportedly known to interact with high affinity with phosphatidylserine) in the rabbit AS aortic intima, together with other known procoagulant conditions, may contribute to the initiation of coagulation events into the lesioned vascular wall, and may offer a rationale for the use of annexin V as an anticoagulant drug.

Antioxidants & Redox Signaling, 1999

The small GTPase rac1 controls actin redistribution to membrane ruffles in fibroblasts and other ... more The small GTPase rac1 controls actin redistribution to membrane ruffles in fibroblasts and other cell types, as well as the activation of the NADPH oxidase in phagocytes. We explored the possibility that these two processes could be related. We used a replication-deficient adenoviral vector to overexpress the constitutively active form of rac1, racV12, in human and mouse aortic endothelial cells. We show here that, in addition to membrane ruffle formation, racV12 induced an increase in the total amount of F-actin within endothelial cells. Concurrently, racV12-overexpressing cells produced significantly higher amounts of free radicals, as detected by the fluorescent probe 5-(and-6)-chloromethyl-2',7'-dichloro-dihydrofluorescein diacetate, than cells infected with a control virus encoding the bacterial beta-galactosidase (Ad-betaGal). To assess the specific role of superoxide in racV12-induced actin reorganization, we co-expressed the human enzyme Cu,Zn-superoxide dismutase (SOD), by means of another adenoviral vector construct. Overexpressed SOD reduced the concentration of superoxide detected in Ad-racV12-transfected cells and reversed the effects of Ad-racV12 on the content of filamentous actin. MnTMPyP, an SOD mimetic, as well as the antioxidant N-acetyl cysteine, had similar effects, in that they reduced not only the free radicals production, but also ruffle formation and the concentration of F-actin within racV12-overexpressing endothelial cells. Our data support the hypothesis that superoxide is one of the important mediators acting downstream of rac1 on the pathway of actin cytoskeleton remodeling in endothelial cells.

Stem Cells and Development, 2004

Linear arrays of cells, or cell columns, have been observed in the extracellular matrix prior to ... more Linear arrays of cells, or cell columns, have been observed in the extracellular matrix prior to neovascularization, but their nature and significance remains elusive. Based on the emerging evidence implicating a role for monocytes and macrophages (MC/MPH) in vasculogenesis, we hypothesized that MC/MPH also can form linear or branched columns, facilitating the co-migration and the spatial arrangement of other cell types. To test this hypothesis, we studied the distribution of MC/MPH effected by chemotactic migration in novel in vitro and in vivo models of development. We induced transversal and lateral migration of THP-1 monocytoid cells in Matrigel in vitro. The effect of this process on co-localization of other micro-objects was assessed using erythrocytes and micron-sized plastic beads. In vivo, we analyzed MC/MPH infiltration in subcutaneously implanted Matrigel plugs containing angiogenic factors and across a microporous filter comprising the wall of a chamber filled with Matrigel, also placed subcutaneously in mice. In vitro, we found that migrating THP-1 cells induced the lasting degradation of Matrigel and produced cell columns, a process amplified by monocyte chemoattractant protein-1 (MCP-1). We also report the co-localization of erythrocytes with THP-1 cells in cell columns. Endothelium-free tunnels containing MC/MPH, neutrophils, or erythrocytes were also observed in the Matrigel-filled chambers. In free subcutaneous Matrigel plugs, we found MC/MPH-based columns harboring isolated Tie-2 ؉ cells (a marker of endothelial progenitor phenotype), as well as fibroblasts, dendritic cells, and adypocytes. Many of these cell columns displayed conspicuous branching. Our data demonstrate formation of branched MC/MPH cell columns in vitro and in vivo, a previously unrecognized pattern of penetration of extracellular matrices by inflammatory cells. Thus, monocytes and macrophages influence the distribution of neovessels as well as their branching points. These cells are the "architects of development," assisting organogenesis, tumorigenesis, and wound healing by patterning the tissular space. 665

Cytometry Part A, 2005

BackgroundEvaluation of chemotaxis assays traditionally relies on cumbersome and at times inaccur... more BackgroundEvaluation of chemotaxis assays traditionally relies on cumbersome and at times inaccurate visual counting. Moreover, many physiologic parameters that could be evaluated in conjunction with chemotactic migration, aside from morphologic changes, usually are not assessed due to the lack of a simultaneous method of analysis. We tested the suitability of laser scanning cytometry (LSC) as a convenient platform for counting migrated cells and for concurrent analysis of some features associated with their physiologic status.Evaluation of chemotaxis assays traditionally relies on cumbersome and at times inaccurate visual counting. Moreover, many physiologic parameters that could be evaluated in conjunction with chemotactic migration, aside from morphologic changes, usually are not assessed due to the lack of a simultaneous method of analysis. We tested the suitability of laser scanning cytometry (LSC) as a convenient platform for counting migrated cells and for concurrent analysis of some features associated with their physiologic status.MethodsWe induced migration of THP-1 monocytes across Nuclepore filters with monocyte chemotactic protein-1 or vascular endothelial growth factor, alone or in combination. Filters were collected, and cells were fixed on filters and stained with the nuclear stain propidium iodide. Chemotactic indices were obtained by counting representative microscopic fields and by scanning the filters in LSC mode.We induced migration of THP-1 monocytes across Nuclepore filters with monocyte chemotactic protein-1 or vascular endothelial growth factor, alone or in combination. Filters were collected, and cells were fixed on filters and stained with the nuclear stain propidium iodide. Chemotactic indices were obtained by counting representative microscopic fields and by scanning the filters in LSC mode.ResultsWe found an excellent correlation between direct counting and LSC. In addition, the software tools embodied in the LSC instrument allowed the observation of changes in nuclear compactness (increase in propidium iodide brightness) and morphology (increase in nuclear area and perimeter) that occurred in transmigrated cells. Monocyte chemotactic protein-1 and vascular endothelial growth factor acted as additive stimuli on these parameters.We found an excellent correlation between direct counting and LSC. In addition, the software tools embodied in the LSC instrument allowed the observation of changes in nuclear compactness (increase in propidium iodide brightness) and morphology (increase in nuclear area and perimeter) that occurred in transmigrated cells. Monocyte chemotactic protein-1 and vascular endothelial growth factor acted as additive stimuli on these parameters.ConclusionsLSC analysis of cells undergoing chemotaxis provides a reliable and comprehensive assessment of the numbers and distribution of migrated cells and some of their nuclear parameters. The method can be easily extended to include the assessment of coincident molecular changes in cells due to chemotactic stimulation. © 2005 Wiley-Liss, Inc.LSC analysis of cells undergoing chemotaxis provides a reliable and comprehensive assessment of the numbers and distribution of migrated cells and some of their nuclear parameters. The method can be easily extended to include the assessment of coincident molecular changes in cells due to chemotactic stimulation. © 2005 Wiley-Liss, Inc.

Journal of Cellular and Molecular Medicine, 2005

The tyrosine kinase Tie2/Tek (the receptor for angiopoietins) is considered one of the most relia... more The tyrosine kinase Tie2/Tek (the receptor for angiopoietins) is considered one of the most reliable markers of the endothelial phenotype, across organisms, organs, and developmental stages. However, endothelium is intrinsically heterogeneous in origin, composition and function, presenting an arteriolar/venular asymmetry. In this regard, the expression of Tie2 along the vascular tree, although thought to be homogenous, has not been systematically investigated. Therefore we questioned whether the activity of Tie2 promoter is uniform in the microvascular endothelium. To this end, we analyzed in situ the expression of the markers β-galactosidase [LacZ(Tie2)] and green fluorescent protein (GFP) [GFP(Tie2)], placed under the Tie2 promoter in transgenic mice, in whole mount tissue samples, which allow the simultaneous evaluation of its relative distribution in various microvascular compartments. In the mesenteries of LacZ(Tie2) and GFP(Tie2) mice, we found that the activity of Tie2 promoter is asymmetrically distributed, being much stronger in arteries and arterioles than on the venular side of the vascular tree. This observation was replicated in the diaphragm of LacZ(Tie2) mice. The capillaries presented a mosaic pattern of Tie2 promoter activity. Stimulation of angiogenesis either by wounding, or by intraperitoneal injection of Vascular Endothelial Growth Factor (VEGF), revealed that the arteriolar/venular asymmetry is established at endothelial cellular level early during new capillary formation, even before the starting of the microvasular blood flow. In conclusion, a strong Tie2 promoter activity qualifies as a novel marker of the arteriolar phenotype in microvascular endothelium.

Histochemistry and Cell Biology, 2004

The presence and supposed roles of reactive oxygen species (ROS) were reported in literature in a... more The presence and supposed roles of reactive oxygen species (ROS) were reported in literature in a myriad of instances. However, the breadth and depth of their involvement in cellular physiology and pathology, as well as their relationship to the redox environment can only be guessed from specialized reports. Whatever their circumstances of formation or consequences, ROS seem to be conspicuous components of intracellular milieu. We sought to verify this assertion, by collecting the available evidence derived from the most recent publications in the biomedical field. Unlike other reviews with similar objectives, we centered our analysis on the subcellular compartments, namely on organelles, grouped according to their major functions. Thus, plasma membrane is a major source of ROS through NAD(P)H oxidases located on either side. Enzymes of the same class displaying low activity, as well as their components, are also present free in cytoplasm, regulating the actin cytoskeleton and cell motility. Mitochondria can be a major source of ROS, mainly in processes leading to apoptosis. The protein synthetic pathway (endoplasmic reticulum and Golgi apparatus), including the nucleus, as well as protein turnover, are all exquisitely sensitive to ROS-related redox conditions. The same applies to the degradation pathways represented by lysosomes and peroxisomes. Therefore, ROS cannot be perceived anymore as a mere harmful consequence of external factors, or byproducts of altered cellular metabolism. This may explain why the indiscriminate use of anti-oxidants did not produce the expected “beneficial” results in many medical applications attempted so far, underlying the need for a deeper apprehension of the biological roles of ROS, particularly in the context of the higher cellular order of organelles.

American Journal of Pathology, 2006

The potential of monocytes/macrophages (MC/Mph) to contribute to neovascularization has recently ... more The potential of monocytes/macrophages (MC/Mph) to contribute to neovascularization has recently become a topic of intense scrutiny. Here, we characterized the behavior of MC/Mph in cellular infiltrates, with emphasis on their spatial organization and localization in newly formed microvessels. To this end, we studied MC/Mph migration and assembly in basic fibroblast growth factor-supplemented Matrigel plugs placed in transgenic Tie2-␤-galactosidase mice for up to 4 weeks. In these plugs, along with Nile Red-positive adipocytes, we found MC/Mph distributed in cell cords, also containing various mature and progenitor tissue cells; and functional Tie2-positive or -negative microvessels embedded in bundles of fibrillar collagen surrounded by F4/80-positive MC/Mph. At earlier stages of infiltration, we found tubular destruction of the matrix (tunnels) and MC/Mph-lined capillary-like structures occasionally containing erythrocytes, indicating their propensity for endothelial trans-differentiation. We also analyzed in vitro the MCP-1-induced chemotactic migration of fluorescently labeled peritoneal MC/Mph incorporated in Matrigel-containing fluorescent protease substrates. Many of these MC/ Mph produced MMP-12-and TIMP-1-dependent tunnels coupled with acquisition of a lumen. In conclusion, long-term implantation of Matrigel plugs qualifies as a novel experimental model of tissue regeneration, in which neovascularization intimately couples with fibrosis and organogenesis and in which cells of MC/Mph phenotype play a key structural role.

Atherosclerosis

Objective: Peritoneal macrophages are used in many studies related to atherosclerosis. In situ, t... more Objective: Peritoneal macrophages are used in many studies related to atherosclerosis. In situ, they are non-adherent and upon culturing, they adhere and function as scavengers of modified lipoproteins and dead apoptotic cells. They also produce growth factors, suggesting that they may provide life-supporting function as well. In this study, we propose that macrophage adherence plays a major role in their function and propose a novel concept that non-adherent macrophages are poor scavengers and may delay the process of apoptosis by secretion of growth factors. Methods and results: We analyzed non-adherent and adherent macrophages for changes in receptor expression, growth factor production and function by microarrays, real-time PCR, and western blot analyses. Our results indicate that adherent macrophages have increased expression of scavenger receptors as compared to fresh peritoneal cells. While genes for many growth factors were expressed in both nonadherent and adherent macrophages, the milk fat globule-epidermal growth factor 8 protein (MFG-E8) that recognizes and takes up apoptotic cells was specifically enhanced in non-adherent cells. Furthermore, early apoptotic endothelial cells demonstrated signs of delayed apoptosis when incubated in the presence of peritoneal lavage fluid that was shown to contain MFG-E8. Functional arrays indicated that peritoneal non-adherent macrophages represent a class of macrophages, distinct from either blood monocytes or adherent cultured macrophages. Conclusions: These results suggest that the adherence status of macrophages may play a major role in their functions.

Biochemical and Biophysical Research Communications, 2000

Cold Spring Harbor Symposia on Quantitative Biology, 2002

Journal of Cellular and Molecular Medicine, 2006

Blood Coagulation & Fibrinolysis, 1994

Antioxidants & Redox Signaling, 1999

Stem Cells and Development, 2004

Cytometry Part A, 2005

Journal of Cellular and Molecular Medicine, 2005

Histochemistry and Cell Biology, 2004

The presence and supposed roles of reactive oxygen species (ROS) were reported in literature in a... more The presence and supposed roles of reactive oxygen species (ROS) were reported in literature in a myriad of instances. However, the breadth and depth of their involvement in cellular physiology and pathology, as well as their relationship to the redox environment can only be guessed from specialized reports. Whatever their circumstances of formation or consequences, ROS seem to be conspicuous components of intracellular milieu. We sought to verify this assertion, by collecting the available evidence derived from the most recent publications in the biomedical field. Unlike other reviews with similar objectives, we centered our analysis on the subcellular compartments, namely on organelles, grouped according to their major functions. Thus, plasma membrane is a major source of ROS through NAD(P)H oxidases located on either side. Enzymes of the same class displaying low activity, as well as their components, are also present free in cytoplasm, regulating the actin cytoskeleton and cell motility. Mitochondria can be a major source of ROS, mainly in processes leading to apoptosis. The protein synthetic pathway (endoplasmic reticulum and Golgi apparatus), including the nucleus, as well as protein turnover, are all exquisitely sensitive to ROS-related redox conditions. The same applies to the degradation pathways represented by lysosomes and peroxisomes. Therefore, ROS cannot be perceived anymore as a mere harmful consequence of external factors, or byproducts of altered cellular metabolism. This may explain why the indiscriminate use of anti-oxidants did not produce the expected “beneficial” results in many medical applications attempted so far, underlying the need for a deeper apprehension of the biological roles of ROS, particularly in the context of the higher cellular order of organelles.

American Journal of Pathology, 2006

Atherosclerosis