Doina Popov

Nicolae Simionescu Institute of Cellular Biology and Pathology, Bucureşti, Bucureşti, Romania

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Publications (41)105.09 Total impact

  • Doina Popov
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    ABSTRACT: Abstract The interaction between antioxidant glutathione and the free thiol in susceptible cysteine residues of proteins leads to reversible protein S-glutathionylation. This reaction ensures cellular homeostasis control (as a common redox-dependent post-translational modification associated with signal transduction) and intervenes in oxidative stress-related cardiovascular pathology (as initiated by redox imbalance). The purpose of this review is to evaluate the recent knowledge on protein S-glutathionylation in terms of chemistry, broad cellular intervention, specific quantification, and potential for therapeutic exploitation. The data bases searched were Medline and PubMed, from 2009 to 2014 (term: glutathionylation). Protein S-glutathionylation ensures protection of protein thiols against irreversible over-oxidation, operates as a biological redox switch in both cell survival (influencing kinases and protein phosphatases pathways) and cell death (by potentiation of apoptosis), and cross-talks with phosphorylation and with S-nitrosylation. Collectively, protein S-glutathionylation appears as a valuable biomarker for oxidative stress, with potential for translation into novel therapeutic strategies.
    Archives of Physiology and Biochemistry 08/2014; DOI:10.3109/13813455.2014.944544
  • Doina Popov
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    ABSTRACT: Abstract Hydrogen sulphide (H2S) is the most recently discovered gasotransmitter. It is endogenously generated in mammalian vascular cells and attracts substantial interest by its function as physiological relevant signalling mediator, and by its dysfunction in metabolic diseases like obesity, type 2 diabetes and their associated complications. The purpose of this review is to highlight the novel findings on vascular H2S homeostasis, pathology-associated dysregulation, cell signalling, and therapeutic potential. The data bases searched were Medline and PubMed, from 2008 to 2012 (terms: hydrogen sulphide, sulfhydration). The new reports definitely assess the vasculoprotectant role of H2S in health, and its reduced biosynthesis/systemic levels in obesity, diabetes, atherosclerosis and hypertension. One of the mechanisms of H2S signalling discussed here is S-sulfhydration of catalytic cysteine residue of PTP1B, a negative regulator of insulin and leptin signalling. Finally, the review critically evaluates the compounds able to regulate vascular H2S bioavailability, and with potential in therapeutic exploitation.
    Archives of Physiology and Biochemistry 06/2013; DOI:10.3109/13813455.2013.803578
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    ABSTRACT: Circulating microparticles (MPs) and endothelial progenitor cells (EPCs) correlate with endothelial dysfunction and contribute to the pathogenesis of atherosclerosis. In this context, we explored whether the angiotensin II type I receptor antagonist, irbesartan, exerts a pharmacological control in the atherosclerotic process by the improvement of EPC mobilization and inhibitory effects on MP release and VEGF and SDF-1α levels in the hypertensive-hypercholesterolemic (HH) hamster model. The HH hamsters were treated with irbesartan (50mg/kg b.w/day administered by gavage) for 4 month (HHI). We analyzed MP/EPC infiltration in vascular wall before and after irbesartan administration as well as the endothelial function and expression of VEGF/SDF-1α in plasma and tissue and of molecular pathways activated by them. The results showed that treatment with irbesartan significantly increased EPC infiltration and decreased MP infiltration. The mechanisms underlying this response include the reduction/increase of a number of specific membrane receptors exposed by MPs (TF, P-Selectin, E-Selectin, PSGL-1, Rantes), respectively by EPCs (β2-Integrins, α4β1-integrin), the augmentation of endothelium-mediated vasodilation and the reduction of protein expression of VEGF/SDF-1α followed by: 1) the diminishment of pro-inflammatory endothelial cytokines: VEGFR1, VEGFR2, CXCR4, Tie2, PIGF with role in EPC homing to sites of damaged endothelium; and 2) the increase of protein expression of COX-2, PGI2 synthase molecules with role in the improvement of arterial wall vasodilatation. In conclusion, the study underlines that irbesartan administration therapeutically improves/reduces EPC, respectively MP mobilization and this action may be of salutary relevance contributing to its beneficial cardiovascular effects.
    European journal of pharmacology 04/2013; DOI:10.1016/j.ejphar.2013.04.004 · 2.68 Impact Factor
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    ABSTRACT: Aim The purpose of this project was to evaluate the influence of circulating endothelial progenitor cells (EPCs) and platelet microparticles (PMPs) on blood platelet function in experimental hypertension associated with hypercholesterolemia. Methods Golden Syrian hamsters were divided in six groups: (i) control, C; (ii) hypertensive-hypercholesterolemic, HH; (iii) ‘prevention’, HHin-EPCs, HH animals fed a HH diet and treated with EPCs; (iv) ‘regression’, HHfin-EPCs, HH treated with EPCs after HH feeding; (v) HH treated with PMPs, HH-PMPs, and (vi) HH treated with EPCs and PMPs, HH-EPCs-PMPs. Results Compared to HH group, the platelets from HHin-EPCs and HHfin-EPCs groups showed a reduction of: (i) activation, reflected by decreased integrin 3β, FAK, PI3K, src protein expression; (ii) secreted molecules as: SDF-1, MCP-1, RANTES, VEGF, PF4, PDGF and (iii) expression of pro-inflammatory molecules as: SDF-1, MCP-1, RANTES, IL-6, IL-1β; TFPI secretion was increased. Compared to HH group, platelets of HH-PMPs group showed increased activation, molecules release and proteins expression. Compared to HH-PMPs group the combination EPCs with PMPs treatment induced a decrease of all investigated platelet molecules, however not comparable with that recorded when EPC individual treatment was applied. Conclusion EPCs have the ability to reduce platelet activation and to modulate their pro-inflammatory and anti-thrombogenic properties in hypertension associated with hypercholesterolemia. Although, PMPs have several beneficial effects in combination with EPCs, these did not improve the EPC effects. These findings reveal a new biological role of circulating EPCs in platelet function regulation, and may contribute to understand their cross talk, and the mechanisms of atherosclerosis.
    PLoS ONE 01/2013; 8(1):e52058. DOI:10.1371/journal.pone.0052058 · 3.53 Impact Factor
  • Doina Popov
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    ABSTRACT: Growing evidence links the stress at the endoplasmic reticulum (ER) to pathologies such as diabetes mellitus, obesity, liver, heart, renal and neurodegenerative diseases, endothelial dysfunction, atherosclerosis, and cancer. Therefore, identification of molecular pathways beyond ER stress and their appropriate modulation might alleviate the stress, and direct toward novel tools to fight this disturbance. An interesting resident of the ER membrane is protein tyrosine phosphatase 1B (PTP1B), an enzyme that negatively regulates insulin and leptin signaling, contributing to insulin and leptin resistance. Recently, new functions of PTP1B have been established linked to ER stress response. This review evaluates the novel data on ER stressors, discusses the mechanisms beyond PTP1B function in the ER stress response, and emphasizes the potential therapeutic exploitation of PTP1B to relieve ER stress.
    Biochemical and Biophysical Research Communications 05/2012; 422(4):535-8. DOI:10.1016/j.bbrc.2012.05.048 · 2.28 Impact Factor
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    ABSTRACT: Cardiovascular diseases are one of the leading causes of morbidity and mortality in industrialized countries, and although many processes play a role in the development of vascular disease, thrombosis is the primary event that precipitates stroke and acute coronary syndromes. The blood platelets are of significant importance in medicine. These cells are involved in many physiological processes, particularly haemostasis through their ability to aggregate and form clots in response to activation. In addition, these dynamic cells display activities that extend beyond thrombosis, including an important role in initiating and sustaining vascular inflammation. The expansion of knowledge from basic and clinical research has highlighted the critical position of platelets in several inflammatory diseases such as arthritis and atherosclerosis. Platelets are emerging as important mediators of inflammation and provide important signals to mediate phenotype of other blood and vascular cells. The important role of platelets in arterial thrombosis and the onset of acute myocardial infarction after atherosclerotic plaque rupture make inhibition of platelet aggregation a critical step in preventing thrombotic events associated with stroke, heart attack, and peripheral arterial thrombosis. However, the use of platelet inhibitors for thrombosis prevention must seek a delicate balance between inhibiting platelet activation and an associated increased bleeding risk. The aim of this review is to up-date the knowledge on platelets physiology and dysfunction in pathologies, such as diabetes mellitus, hypercholesterolemia, and hypertension, emphasizing the link between platelets and the inflammation-related atherosclerosis. The review evaluates the opportunities offered by the novel platelet inhibitors to efficiently alleviate the thrombotic events.
    Thrombosis Research 02/2012; 129(2):116-26. DOI:10.1016/j.thromres.2011.09.026 · 2.43 Impact Factor
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    ABSTRACT: Diabetes mellitus is one of the most common metabolic diseases in the world and the vascular dysfunction represents a challenging clinical problem. In diabetes, endothelial cells (ECs), lining the inner wall of blood vessels, do not function properly and contribute to impaired vascular function. Circulating endothelial progenitor cells (EPCs), the precursor of mature EC, actively participate in endothelial repair, by moving to the vascular injury site to form mature EC and new blood vessels. Knowing that the therapeutic interventions can improve only a part of EC dysfunction in diabetes, this review addresses recent findings on the use of EPCs for cell therapy. The strategies proposed in review are based on in vivo and in vitro studies and, thus, their physiological relevance is confirmed. EPC therapy shows great promise for the prevention and cure of diabetes-induced vascular dysfunction.
    European journal of pharmacology 08/2011; 669(1-3):1-6. DOI:10.1016/j.ejphar.2011.07.035 · 2.68 Impact Factor
  • Doina Popov
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    ABSTRACT: Resistance to the hormones insulin and leptin are hallmarks in common for type 2 diabetes mellitus and obesity. Both conditions are associated with increased activity and expression of protein tyrosine phosphatase (PTP)1B. Therefore, inhibition of PTP1B activity or down-regulation of its expression should ameliorate insulin and leptin resistance, and may hold therapeutic utility in type 2 diabetes mellitus and obesity control. This background has motivated the fervent search for PTP1B inhibitors, carried out in the recent years. The purpose of this review is to provide the most recent advances in understanding the structural details of PTP1B molecule relevant to the interactions with inhibitors, and the progress towards compounds with enhanced membrane permeability, affinity, specificity, and potency on intracellular PTP1B; several inhibitors of benefit in type 2 diabetes mellitus and obesity control are presented and discussed.
    Biochemical and Biophysical Research Communications 06/2011; 410(3):377-81. DOI:10.1016/j.bbrc.2011.06.009 · 2.28 Impact Factor
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    ABSTRACT: The arterial endothelial dysfunction in aging and diabetes remains a clinical problem. We questioned the effect of the low-molecular-weight heparin, enoxaparin, on arterioles contractility in aging and in aging associated with diabetes, and investigated the involvement of the mitogen-activated protein (MAP) kinases pathway in the enoxaparin-mediated effect. The experiments were performed on the isolated resistance arteries of young (4 months old), aged (16 months old), and aged-diabetic hamsters (16 months old and 5 months since streptozotocin injection). The techniques used were myography, molecular biology, and immunoblotting. The results showed that 60 μg/ml enoxaparin has favorable effects on the arteriole reactivity in aged and aged-diabetic conditions, reducing the contractile response to 10-10 mol/l noradrenaline. The diminishment of contractility is exerted via MAP kinase pathway, and involves reduction of c-fos gene expression and of transcription factor AP-1 protein expression. These results suggest that enoxaparin preserves the arterial endothelial function in a mechanism independent of its anticoagulant activity. Understanding the signal transduction mechanisms involved in the altered contractility of vascular wall could provide useful information on the development of specific MAP kinase inhibitors with therapeutic benefits and reduced side effects.
    Blood coagulation & fibrinolysis: an international journal in haemostasis and thrombosis 03/2011; 22(4):310-6. DOI:10.1097/MBC.0b013e328345123d · 1.38 Impact Factor
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    ABSTRACT: Chronic venous insufficiency (CVI) is a common medical problem that may result in significant morbidity and mortality. Platelets are key players in haemostasis and thrombosis, but their role in the development of venous thrombosis is more controversial. The purpose of this study was to investigate platelet properties in CVI and their interaction with the venular endothelium. Human peripheral venules were explanted during leg surgery of patients with CVI and of healthy subjects (C); concurrently, the platelets were isolated from blood samples collected. The techniques used were: fluorescence and electron microscopy and Western-blotting. Compared with the C group, the platelets of patients with CVI are activated, as demonstrated by: (i) cellular modifications, such as alteration of the discoidal shape by the development of extended cytoplasmic filopodia and changes of the cells normal ultrastructure, (ii) biochemical modifications, such as the enhanced protein levels of FAK, p85 PI3K, Akt and src, accounting for activation of alphaIIbbeta3 outside-in signaling, and (iii) apparent higher adhesion to the venular endothelium. We demonstrate in addition, that CVI is accompanied by severe modifications of the ultrastructure of the cells within the venular wall. In CVI, platelets circulate in an activated state and may contribute to the altered dysfunctional response of the venous wall and to the development of this pathology.
    Clinical laboratory 01/2011; 57(7-8):527-34. · 1.08 Impact Factor
  • Doina Popov
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    ABSTRACT: Hyperglycemia, the hallmark of Diabetes mellitus, is a major risk factor for endothelial dysfunction and vascular complication. In recent years, significant achievements have been made in understanding endothelial cell dysfunction triggered by high glucose concentration. The purpose of this review is to discuss the results of these recent developments. First, the remarkable plasticity of vascular endothelial cell in response to the high glucose insult is emphasized. This is evident through the switch in the cell’s normal quiescent profile into new phenotypes, endowed with biosynthetic, inflammatory, adhesive, proliferative, migratory, pro-atherogenic, and pro-coagulant properties, frequently overlapping each other. Then, we underline the imbalanced expression and activity of transcription and signaling pathways, and the intense metabolic activity that accompanies the change in endothelial cell phenotype. As an adaptation to the high glucose-induced biochemical modification, a severe alteration of cell structure is produced. The review concludes with the clinical outcomes of the subject, emphasizing the high glucose-associated endothelial cell dysfunctional molecules of potential for targeting, and for reducing the impact of hyperglycemia on vascular endothelium. Such interventions may lead to a more efficient therapy for the benefit of those diabetic patients who are at increased cardiovascular risk.
    International Journal of Diabetes Mellitus 12/2010; 2(3):189-195. DOI:10.1016/j.ijdm.2010.09.002
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    ABSTRACT: The aim of the present study was to examine the effects of obesity alone and obesity associated with Type 2 diabetes on the structure, vascular reactivity and response to insulin of isolated human subcutaneous fat arterioles; these effects were correlated with the expression of insulin signalling proteins. Periumbilical subcutaneous adipose tissue was explanted during surgery, small arterioles (internal diameter 220 ± 40 μm) were dissected out and investigated by electron microscopy, myography and immunoblotting. Compared with the subcutaneous arterioles of lean subjects, obesity activated the endothelium, enhanced the accumulation of collagen within vascular wall and increased the sensitivity of adrenergic response; obesity also diminished eNOS (endothelial NO synthase) protein expression, NO production, and endothelium-dependent and insulin-induced vasodilatation, as well as the protein expression of both IRS (insulin receptor substrates)-1 and IRS-2 and of the downstream molecules in the insulin signalling pathway, such as PI3K (phosphoinositide 3-kinase), phospho-Akt and Akt. When obesity was associated with Type 2 diabetes, these changes were significantly augmented. In conclusion, obesity alone or obesity associated with Type 2 diabetes alters human periumbilical adipose tissue arterioles in terms of structure, function and biochemsitry, including diminished eNOS expression and reduced levels of IRS-1, IRS-2, PI3K and Akt in the insulin signalling pathway.
    Clinical Science 10/2010; 120(10):463-72. DOI:10.1042/CS20100355 · 5.63 Impact Factor
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    ABSTRACT: This article provides an overview of the current knowledge on intraplatelet oxidative/nitrative stress, an abnormality associated with platelet activation and hyper-reactivity. The first issue discussed is related to induction of platelet endogenous stress by the molecules present within the circulating (extracellular) milieu that bathes these cells. The second issue concerns the intraplatelet oxidative/nitrative stress associated with specific pathologies or clinical procedures and action of particular molecules and platelet agonists as well as of the specialized intraplatelet milieu and its redox system; the biomarkers of endogenous oxidative/nitrative stress are also briefly outlined. Next, the association between intraplatelet oxidative/nitrative stress and the risk factors of the metabolic syndrome is presented. Then, the most recent strategies aimed at the control/regulation of platelet endogenous oxidative/nitrative stress, such as exploitation of circulating extracellular reactive oxygen species scavengers, manipulation of platelet molecules, and the use of antioxidants, are discussed. Finally, the results of studies on platelet-dependent redox mechanisms, which deserve immediate attention for potential clinical exploitation, are illustrated.
    Trends in cardiovascular medicine 10/2010; 20(7):232-8. DOI:10.1016/j.tcm.2011.11.007 · 2.07 Impact Factor
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    ABSTRACT: Protein activation by phosphorylation reactions (exerted by various kinases) is a universal mechanism of signal transduction that controls the amplitude of signaling response. In physiological conditions, this mechanism is fine tuned by dephosphorylation reactions (exerted by phosphatases), that control the rate and the duration of signaling response. In this study we questioned on the in vivo effect of circulating high glucose concentration on the balance between phosphorylating kinases and dephosphorylating protein tyrosine phosphatase 1B operating in the aorta, an issue still uncovered. Experiments were conducted on Type 1 diabetic golden Syrian hamsters, at 4 weeks after streptozotocin injection (control: normal hamsters); animals were severely diabetic (plasma glucose concentration: 44020 mg/dl), the pancreatic β cells showed progressive destruction and apoptosis, and the thoracic aorta displayed dysfunctional endothelium and smooth muscle cells. In comparison with normal animals, hyperglycaemia produced the following effects on aortic signaling proteins expression: (i) did not significantly alter the protein level of insulin receptor substrate 2 (IRS2, that mediates effects of insulin, insulin-like growth factor 1, and other cytokines by acting as a molecular adaptor between diverse receptor tyrosine kinases and downstream effectors), (ii) up-regulates expression of Protein Tyrosine Phosphatase-1B (PTP-1B, that interacts with and dephosphorylates the activated insulin receptor), and (iii) down-regulates the levels of Phosphoinositide 3-kinase (PI3K, that interacts with the insulin receptor substrate and regulates intracellular glucose uptake through a series of phosphorylation events), and AKT (a member of the serine/threonine-specific protein kinase family). The imbalance in hyperglycaemia -induced phosphorylation /dephosphorylation events are likely to intervene in diabetes-associated aortic wall dysfunction.
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    ABSTRACT: We aimed to investigate whether polymorphisms LEP G-2548A and LEPR Q223R in the human leptin (LEP), and leptin receptor (LEPR) genes are associated with obesity and metabolic traits in a sample of Romanian population. Two hundred and two subjects divided in obese (body mass index, BMI30 kg/m(2)), and non-obese were included in this study. The polymorphisms were genotyped using polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) analysis. The results showed no significant differences in LEP and LEPR genotype and allele frequencies between obese and non-obese subjects. Logistic regression analysis showed that LEP -2548GG genotype presented an increased risk of obesity (p=0.013, OR=1.003, 95% CI=1.000-1.007), after adjusting for age and gender. The association analysis with metabolic syndrome quantitative traits showed that homozygous for LEP -2548G allele had significantly higher leptin levels (17.2+/-6.6 ng/ml vs. 13.2+/-4.9 ng/ml, p=0.011), and carriers of R allele had higher levels of triglycerides (p=0.017) and glucose (p=0.040), and enhanced systolic (p=0.015) and diastolic blood pressure (p=0.026), after adjustment for age, gender, and BMI. These results indicate that LEP G-2548A and LEPR Q223R SNPs may not be considered as genetic risk factors for obesity in a sample of Romanian population. However, LEP -2548GG genotype appear to be important in regulating leptin levels, whereas the LEPR 223R allele might predispose healthy subjects to develop metabolic disturbances.
    Biochemical and Biophysical Research Communications 11/2009; 391(1):282-6. DOI:10.1016/j.bbrc.2009.11.050 · 2.28 Impact Factor
  • Doina Popov
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    ABSTRACT: Protein Tyrosine Phosphatases (PTPs) are important contributors to vascular cells normal function, by balancing signaling proteins activation exerted by phosphorylating kinases. Type 2 diabetes related insults, such as hyperglycemia, oxidative stress, and insulin resistance disturb the phosphorylation/dephosphorylation equilibrium towards an abnormal augmented phosphorylation of signaling proteins associated with changes in PTPs expression, enzymatic activity and interaction with cellular substrates. We briefly review here: (i) the new findings on receptor and non-receptor PTPs and their role in vascular cells, (ii) several data on oxidation and phosphorylation of these molecules in endothelial and smooth muscle cells, (iii) vascular PTPs intrinsic activity and dysregulation under the insults of diabetic milieu, and (iv) the potential use of PTPs and their inhibitors as therapeutic targets in Type 2 diabetes-associated vascular dysfunction.
    Biochemical and Biophysical Research Communications 09/2009; 389(1):1-4. DOI:10.1016/j.bbrc.2009.08.110 · 2.28 Impact Factor
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    ABSTRACT: Hyperglycemia stimulates a plethora of intracellular signaling pathways within the cells of the vascular wall resulting in dysfunction-associated pathologies. Most of the studies reported so far explored the effect of rather short-time exposure of smooth muscle cells to high glucose concentrations. To mimic situation in Type 2 diabetes in which vascular wall is constantly exposed to circulating hyperglycemia, we report here the long-term (7days) effect of high glucose concentration on human media artery smooth muscle cells. This consists in up-regulation of PTP1B protein expression, down-regulation of basal Akt phosphorylation, and elevation of basal ERK1/2 activation. Acute stimulation of cells in high glucose with insulin down-regulated PTP1B expression, slightly decreased ERK1/2 activity, and activated Akt, whereas oxidative stress up-regulated Akt and ERK1/2 phosphorylation. In conclusion, long-term high glucose and acute oxidative stress and insulin stimulation imbalance the expression of activated kinases Akt and ERK1/2 and of dephosphorylating PTP1B in the insulin signaling pathway.
    Biochemical and Biophysical Research Communications 09/2009; 388(1):51-5. DOI:10.1016/j.bbrc.2009.07.141 · 2.28 Impact Factor
  • Maya Simionescu, Doina Popov, Anca Sima
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    ABSTRACT: 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 initial 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.
    Cell and Tissue Research 11/2008; 335(1):27-40. DOI:10.1007/s00441-008-0688-3 · 3.33 Impact Factor
  • Doina Popov, Elena Constantinescu
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    ABSTRACT: Given the important role of smooth muscle cells in arterial wall dysfunction in diabetes, as well as in diabetes associated with accelerated atherosclerosis, we provide a brief review of the recent achievements in identification of signalling molecules underlying their altered cellular responses, and examine the consequences of these pathological insults on smooth muscle cells properties. The original results emerging from the Golden Syrian hamster model (rendered diabetic or simultaneously hyperlipidaemic-diabetic) and from human aortic smooth muscle cells cultured in 25 mM glucose (to mimic diabetic condition) or sera of obese type 2 diabetic patients (to mimic the metabolic syndrome condition) are presented in this context. We conclude this review with several open issues disclosed by the most recent literature that deserve essential attention for targeting the translational medicine.
    Archives of Physiology and Biochemistry 05/2008; 114(2):150-60. DOI:10.1080/13813450802033990
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    ABSTRACT: In normal ageing, as well as in diabetes mellitus, blood platelets are exposed to increasing amounts of reactive oxygen species. Because occurrence of carbonyl groups is the first step in oxidative damage of proteins, we questioned the formation of carbonylated proteins (i) in vitro: platelets exposed to oxidant hydrogen peroxide (H2O2) and agonist thrombin (in the presence/absence of antioxidants), and (ii) in vivo: during ageing progression, and in type 2 diabetes. Platelets were isolated from the blood of experimental animals (rats and hamsters) and humans (healthy, young and elderly), as well as from type 2 diabetics (matures and elderly). 2,4-Dinitrophenyl derivatization of the amino acid side chains was used to quantify protein carbonyls (spectrophotometry) and to immunodetect carbonylated protein bands (Western blotting). In animal models and humans, H2O2 produced dose-dependent increases in carbonylation of platelet proteins (vs. basal condition). Thrombin activation stimulated protein carbonyl formation in a process quenched by antioxidant catalase, suggesting that carbonylation was induced by the oxidative stress generated by activated platelets. Progression of ageing caused increased carbonylation of platelet proteins (vs. young age); enriched in carbonylated proteins, platelets of elderly subjects were less sensitive to H2O2. Type 2 diabetes additionally enhanced carbonylation of human platelet proteins (vs. the levels at young and elderly healthy subjects). In all experiments, protein carbonyl concentrations were correlated with changes in intensity of 2,4-dinitrophenyl-hydrazine-reactive protein bands on immunoblots. The results suggest that exogenous oxidative stress, thrombin activation, progression of ageing and type 2 diabetes lead to protein carbonyls formation in platelets, and this modification can be attenuated by antioxidant enzymes.
    Clinical Chemistry and Laboratory Medicine 02/2008; 46(4):528-36. DOI:10.1515/CCLM.2008.104 · 2.96 Impact Factor