Maya Simionescu

Vascular endothelium in atherosclerosis

Their strategic location between blood and tissue and their constitutive properties allow endothelial cells (EC) to monitor the transport of plasma molecules, by employing bidirectional receptor-mediated and receptor-independent transcytosis and endocytosis, and to regulate vascular tone, cellular cholesterol and lipid homeostasis. These cells are also involved in signal transduction, immunity, inflammation and haemostasis. Cardiovascular risk factors, such as hyperlipaemia/dyslipidaemia trigger the molecular machinery of EC to respond to insults by modulation of their constitutive functions followed by dysfunction and ultimately by injury and apoptosis. The gradual activation of EC consists initially in the modulation of two constitutive functions: (1) permeability, i.e. increased transcytosis of lipoproteins, and (2) biosynthetic activity, i.e. enhanced synthesis of the basement membrane and extracellular matrix. The increased transcytosis and the reduced efflux of β-lipoproteins (βLp) lead to their retention within the endothelial hyperplasic basal lamina as modified lipoproteins (MLp) and to their subsequent alteration (oxidation, glycation, enzymatic modifications). MLp generate chemoattractant and inflammatory molecules, triggering EC dysfunction (appearance of new adhesion molecules, secretion of chemokines, cytokines), characterised by monocyte recruitment, adhesion, diapedesis and residence within the subendothelium. In time, EC in the athero-prone areas alter their net negative surface charge, losing their non-thrombogenic ability, become loaded with lipid droplets and turn into foam cells. Prolonged and/or repeated exposure to cardiovascular risk factors can ultimately exhaust the protective effect of the endogenous anti-inflammatory system within EC. As a consequence, EC may progress to senescence, lose their integrity and detach into the circulation.

Structural basis of permeability in sequential segments of the microvasculature of the diaphragm: II. Pathways followed by microperoxidase across the endothelium

Abstract Bipolar microvascular fields of mouse diaphragm were used to investigate the existence of characteristic modulations in the structural basis of permeability along successive segments of the microcirculation. Using hemeundecapeptide (microperoxidase; MW, ∼1900; mol diam, ∼20 A) as a probe molecule, the observations indicate that the timing and pathways followed by this tracer across the endothelium were different in each microvascular segment. The first permeated by these molecules were the pericytic venules at the level of their endothelial junctions. Among the latter, ∼25 to 30% are normally open to a space of ∼30 to 60 A. The vesicular transport is particularly extensive in capillaries and pericytic venules, and the transition from phase I to phase III is faster in the venular segments of capillaries, probably due to their high frequency in transendothelial channels formed by single vesicles. In the arteriolar and middle segments of capillaries, the H11P passed via vesicular transport and transendothelial channels. Endothelial junctions in arterioles and along the entire length of capillaries appeared to be impermeable to molecules of ≥20 A in diameter.

Transcriptional regulation of NADPH oxidase isoforms, Nox1 and Nox4, by nuclear factor-κB in human aortic smooth muscle cells

Inflammation-induced changes in the activity and expression of NADPH oxidases (Nox) play a key role in atherogenesis. The molecular mechanisms of Nox regulation are scantily elucidated. Since nuclear factor-kappaB (NF-kappaB) controls the expression of many genes associated to inflammation-related diseases, in this study we have investigated the role of NF-kappaB signaling in the regulation of Nox1 and Nox4 transcription in human aortic smooth muscle cells (SMCs). Cultured cells were exposed to tumor necrosis factor-alpha (TNFalpha), a potent inducer of both Nox and NF-kappaB, up to 24h. Lucigenin-enhanced chemiluminescence and dichlorofluorescein assays, real-time polymerase chain reaction, and Western blot analysis showed that inhibition of NF-kappaB pathway reduced significantly the TNFalpha-dependent up-regulation of Nox-derived reactive oxygen species production, Nox1 and Nox4 expression. In silico analysis indicated the existence of typical NF-kappaB elements in the promoters of Nox1 and Nox4. Transient overexpression of p65/NF-kappaB significantly increased the promoter activities of both isoforms. Physical interaction of p65/NF-kappaB proteins with the predicted sites was demonstrated by chromatin immunoprecipitation assay. These findings demonstrate that NF-kappaB is an essential regulator of Nox1- and Nox4-containing NADPH oxidase in SMCs. Elucidation of the complex relationships between NF-kappaB and Nox enzymes may lead to a novel pharmacological strategy to reduce both inflammation and oxidative stress in atherosclerosis and its associated complications.

JAK/STAT Signaling Pathway Regulates Nox1 and Nox4-Based NADPH Oxidase in Human Aortic Smooth Muscle Cells

Objective—Oxidative stress mediated by Nox1- and Nox4-based NADPH oxidase (Nox) plays a key role in vascular diseases. The molecular mechanisms involved in the regulation of Nox are not entirely elucidated. Because JAK/STAT regulates many genes linked to inflammation, cell proliferation, and differentiation, we questioned whether this pathway is involved in the regulation of Nox1 and Nox4 in human aortic smooth muscle cells (SMCs). Methods and Results—Cultured SMCs were exposed to interferon γ (IFNγ) for 24 hours. Using lucigenin-enhanced chemiluminescence and dihydroethidium assays, real-time polymerase chain reaction, and Western blot analysis, we found that JAK/STAT inhibitors significantly diminished the IFNγ-dependent upregulation of Nox activity, Nox1 and Nox4 expression. In silico analysis revealed the presence of highly conserved GAS elements within human Nox1, Nox4, p22phox, p47phox, and p67phox promoters. Transient overexpression of STAT1/STAT3 augmented the promoter activities of each subunit. JAK/STAT blockade reduced the Nox subunits transcription. Chromatin immunoprecipitation demonstrated the physical interaction of STAT1/STAT3 proteins with the predicted GAS elements from Nox1 and Nox4 promoters. Conclusions—JAK/STAT is a key regulator of Nox1 and Nox4 in human vascular SMCs. Inhibition of JAK/STAT pathway and the consequent Nox-dependent oxidative stress may be an efficient therapeutic strategy to reduce atherogenesis.

Designing of ‘intelligent’ liposomes for efficient delivery of drugs

The liposome‐ vesicles made by a double phospholipidic layers which may encapsulate aqueous solutions‐ have been introduced as drug delivery vehicles due to their structural flexibility in size, composition and bilayer fluidity as well as their ability to incorporate a large variety of both hydrophilic and hydrophobic compounds. With time the liposome formulations have been perfected so as to serve certain purposes and this lead to the design of “intelligent” liposomes which can stand specifically induced modifications of the bilayers or can be surfaced with different ligands that guide them to the specific target sites. We present here a brief overview of the current strategies in the design of liposomes as drug delivery carriers and the medical applications of liposomes in humans.

Factors secreted by mesenchymal stem cells and endothelial progenitor cells have complementary effects on angiogenesis in vitro.

Stem cell-based therapy for myocardial regeneration has reported several functional improvements that are attributed mostly to the paracrine effects stimulating angiogenesis and cell survival. This study was conducted to comparatively evaluate the potential of factors secreted by mesenchymal stem cells (MSCs) in normoxic and hypoxic conditions to promote tissue repair by sustaining endothelial cell (EC) adhesion and proliferation and conferring protection against apoptosis. To this aim, a conditioned medium (CM) was generated from MSCs after 24-h incubation in a serum-free normal or hypoxic environment. MSCs exhibited resistance to hypoxia, which induced increased secretion of vascular endothelial growth factor (VEGF) and decreased levels of other cytokines, including stromal-derived factor-1 (SDF). The CM derived from normal (nMSC-CM) and hypoxic cells (hypMSC-CM) induced similar protective effects on H9c2 cells in hypoxia. Minor differences were noticed in the potential of normal versus hypoxic CM to promote angiogenesis, which were likely connected to SDFα and VEGF levels: the nMSC-CM was more effective in stimulating EC migration, whereas the hypMSC-CM had an enhanced effect on EC adhesion. However, the factors secreted by MSCs in normoxic or hypoxic conditions supported adhesion, but not proliferation, of ECs in vitro, as revealed by impedance-based dynamic assessments. Surprisingly, factors secreted by other stem/progenitor cells, such as endothelial progenitor cells (EPCs), had complementary effects to the MSC-CM. Thus, the EPC-CM, in either a normal or hypoxic environment, supported EC proliferation, but did not sustain EC adhesion. Combined use of the MSC-CM and EPC-CM promoted both EC adhesion and proliferation, suggesting that the local angiogenesis at the site of ischemic injury might be better stimulated by simultaneous releasing of factors secreted by multiple stem/progenitor cell populations.

AP-1–Dependent Transcriptional Regulation of NADPH Oxidase in Human Aortic Smooth Muscle Cells Role of p22phox Subunit

Objective—NADPH oxidase (NADPHox) is the major source of reactive oxygen species in vascular diseases; the mechanisms of enzyme activation are not completely elucidated. AP-1 controls the expression of many genes linked to vascular smooth muscle cells (SMCs) dysfunction. In this study we searched for the role of AP-1 in the regulation of NADPHox expression and function in human aortic SMCs exposed to proinflammatory conditions. Methods and Results—Cultured SMCs were exposed to either angiotensin II (Ang II) or tumor necrosis factor (TNF)-&agr;. The lucigenin-enhanced chemiluminescence assay and real-time polymerase chain reaction analysis revealed that AP-1 and mitogen-activated protein kinase inhibitors reduced both Ang II or TNF-&agr;-dependent upregulation of NADPHox activity and mRNA expression (NOX1, NOX4, p67phox, p47phox, p22phox). Inhibitors of AP-1 significantly diminished the Ang II or TNF-&agr;-stimulated p22phox promoter activity and protein level. Transient overexpression of c-Jun/c-Fos upregulated p22phox promoter activity. Transcription factor pull-down assay and chromatin immunoprecipitation demonstrated the physical interaction of c-Jun protein with predicted AP-1–binding sites in the p22phox gene promoter. Conclusions—In SMCs exposed to Ang II or TNF-&agr;, inhibition of AP-1–related pathways reduces NADPHox expression and the O2− production. The physical interaction of AP-1 with p22phox gene promoter facilitates NADPHox regulation.

Structural basis of permeability in sequential segments of the microvasculature of the diaphragm: I. Bipolar microvascular fields

Abstract A new method is described for the reliable identification of successive segments in the microvasculature of skeletal muscle. The procedure relies on the existence in the postero-lateral regions of the mouse diaphragm of bipolar microvascular fields (BMFs) in which the supplying arteriole enters and the draining venules leave the capillary bed from opposite ends. In such fields the different segments of the microvasculature were initially recognized at the light microscope level in whole mounts of the muscle; their identity was confirmed by successive examination under the light and the electron microscope. In these BMFs, the average capillary path length is ∼580 μm, the average inner diameter increases progressively from ∼3 μm for the arteriolar, to ∼3.5 μm for the venular segments of capillaries; from this level, there is a sharp increase to the inner diameters of the postcapillary venules (∼11.5 μm). The new procedure has the advantage of allowing precise sampling in the muscle microvasculature and can be used to study regional differences in the structure and function of the small vessels of the peripheral vasculature.

Endothelial transcytosis in health and disease

The visionaries predicted the existence of transcytosis in endothelial cells; the cell biologists deciphered its mechanisms and (in part) the molecules involved in the process; the cell pathologists unravelled the presence of defective transcytosis in some diseases. The optimistic perspective is that transcytosis, in general, and receptor-mediated transcytosis, in particular, will be greatly exploited in order to target drugs and genes to exclusive sites in and on endothelial cells (EC) or underlying cells. The current recognition that plasmalemmal vesicles (caveolae) are the vehicles involved in EC transcytosis has moved through various phases from intial considerations of caveolae as unmovable sessile non-functional plasmalemma invaginations to the present identification of a multitude of molecules and a crowd of functions associated with these ubiquitous structures of endothelial and epithelial cells. Further understanding of the molecular machinery that precisely guides caveolae through the cells so as to reach the target membrane (fission, docking, and fusion), to avoid lysosomes, or on the contrary, to reach the lysosomes, and discharge the cargo molecules will assist in the design of pathways that, by manipulating the physiological route of caveolae, will carry molecules of choice (drugs, genes) at controlled concentrations to precise destinations.

Endothelial cell dysfunctions

General: Endothelial Cell Response to Normal and Abnormal Stimuli (M. Simionescu). Endothelial Cell Regrowth (M.A. Reidy, V. Lindner). Endothelial Cell in Inflammation and Immunity: LeukocyteMediated Endothelial Injury (R.K. Winn et al.). Endothelial Cell Adhesive Interactions (E. Dejana et al.). Endothelial Cells in the Viral Infection: Response of Human Vascular Cells to Viral Infection (N.A. Kefalides). Endothelial Cells in Hypertension, Hyperlipidemia, and Atherosclerosis: Endothelial Dysfunction and Atherosclerosis (R. Ross). Endothelial Cells in Diabetes: Vascular Endothelium and Diabetes Mellitus (R.S. Bar). Endothelial Cells in Neoplasia and Metastasis: The Microvascular Phases of Metastasis (L. Weiss, F.W. Orr). Endothelial Cells in Other Disturbances Twentythree additional articles. Index