Comparison of the Latin America Regulation Landscape and International Reference Health Authorities to Hasten Drug Registration and Clinical Research Applications.

INTRODUCTION: Promptly providing new drugs to fulfill unmet medical needs requires changes in drug development and registration processes. Health Authorities (HAs) considered as reference due to their experience and acknowledgement (Food and Drug Administration [FDA] among others) already consider innovative clinical trial (CT) designs and flexible approval procedures, but Latin America (LATAM) regulations are still far. A comparison was performed to identify gaps. MATERIALS AND METHODS: CT requirements for drug Marketing Authorization Application (MAA) and CT approval regulations were compared between LATAM and reference HAs (FDA/European Medicines Agency [EMA]/Health-Canada/Swissmedic/Therapeutic Goods Administration [TGA]/Pharmaceuticals and Medical Devices Agency [PMDA]), as of August 2022. Procedure included reference HAs regulations review, item selection, identification in LATAM regulations, and International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines (ICH-E6[R2]/ICH-E8[R1]) implementation revision. RESULTS: For MAA, specific application requirements or ICH guideline M4(R4) on common technical document (CTD) adoption are generally stated, and phase-I/III performance is mandatory (explicitly/implicitly). Faster patient access procedures are infrequent: Priority-drug programs, conditional authorizations, or expedited procedures are scarce or non-existent. Regulatory reliance procedures are adopted through different pathways. Regarding CT approval, innovative/complex CT designs are not prohibited but usually omitted. Some countries implemented adapted CT conducting during the COVID-19 pandemic. Early scientific advice meetings (HA-sponsor) are occasionally considered. Most countries are not formally ICH-joined. CONCLUSIONS: LATAM regulations must adapt to new regulatory standards (FDA/EMA/ICH) through implementation of frequent updates, reliance/expedited procedures, early HA-sponsor interactions, innovative/complex CTs, mandatory phase-III reaching elimination, and decentralized elements for CT conducting.

iTRAQ proteomic analysis of extracellular matrix remodeling in aortic valve disease.

Degenerative aortic stenosis (AS) is the most common worldwide cause of valve replacement. The aortic valve is a thin, complex, layered connective tissue with compartmentalized extracellular matrix (ECM) produced by specialized cell types, which directs blood flow in one direction through the heart. There is evidence suggesting remodeling of such ECM during aortic stenosis development. Thus, a better characterization of the role of ECM proteins in this disease would increase our understanding of the underlying molecular mechanisms. Aortic valve samples were collected from 18 patients which underwent aortic valve replacement (50% males, mean age of 74 years) and 18 normal control valves were obtained from necropsies (40% males, mean age of 69 years). The proteome of the samples was analyzed by 2D-LC MS/MS iTRAQ methodology. The results showed an altered expression of 13 ECM proteins of which 3 (biglycan, periostin, prolargin) were validated by Western blotting and/or SRM analyses. These findings are substantiated by our previous results demonstrating differential ECM protein expression. The present study has demonstrated a differential ECM protein pattern in individuals with AS, therefore supporting previous evidence of a dynamic ECM remodeling in human aortic valves during AS development.

Proteomic characterization of human coronary thrombus in patients with ST-segment elevation acute myocardial infarction.

Acute myocardial infarction with ST-segment elevation (STEMI) initiates with intraluminal thrombosis and results in total occlusion of the coronary artery. To date, characterization of the coronary thrombus proteome in STEMI patients has not been yet accomplished. Therefore, we aimed to perform an in-depth proteomic characterization of the human coronary thrombus by means of three different approaches: 2-DE followed by mass spectrometry (MALDI MS/MS), 1-DE combined either with liquid chromatography coupled to mass spectrometry in a MALDI TOF/TOF (LC-MALDI-MS/MS), or in a LTQ-Orbitrap (LC-ESI-MS/MS). This approach allowed us to identify a total of 708 proteins in the thrombus. Expression in coronary thrombi (n=20) of 14 proteins was verified, and the expression of fibrin and 6 cell markers (platelets, monocytes, neutrophils, eosinophils, T-cells and B-cells) quantified by selected reaction monitoring (SRM). A positive correlation of 5 proteins (fermitin homolog 3, thrombospondin-1, myosin-9, beta parvin and ras-related protein Rap-1b) with CD41 was found, pointing out the potential activation of a focal adhesion pathway within thrombus platelets. DIDO1 protein was found to correlate negatively with thrombus fibrin, and was found up-regulated in the plasma of these STEMI patients, which may constitute a starting point for further analyses in the search for biomarkers of thrombosis. BIOLOGICAL SIGNIFICANCE: The proteomic characterization of the human coronary thrombus may contribute to a better understanding of the mechanisms involved in acute coronary syndrome, and thus pave the road for the identification of new therapeutic targets that may help addressing this and other thrombotic diseases. A novel methodology to characterize thrombus composition and expression of a sub-group of proteins is hereby described, which allowed linking protein expression with cellular and ECM matrix composition of the thrombus. Five proteins (fermitin homolog 3, thrombospondin-1, myosin-9, beta parvin and ras-related protein Rap-1b) co-express within the human coronary thrombus with CD41, pointing out the potential activation of a focal adhesion pathway within thrombus platelets during thrombus formation. Besides, the protein death-inducer obliterator 1, found to be expressed within the human coronary thrombus, has been proved to increase in the plasma of STEMI patients, which constitutes an important starting point for further analyses in the search for biomarkers of thrombosis.

Identification of a circulating microvesicle protein network involved in ST-elevation myocardial infarction.

Membrane microvesicles (MVs) are released from activated cells, most notably platelets, into the circulation. They represent an important mode of intercellular communication, and their number is increased in patients with acute coronary syndromes. We present here a differential proteomic analysis of plasma MVs from ST-elevation myocardial infarction (STEMI) patients and stable coronary artery disease (SCAD) controls. The objective was the identification of MVs biomarkers/drug targets that could be relevant for the pathogenesis of the acute event. Proteome analysis was based on 2D-DIGE, and mass spectrometry. Validations were by western blotting in an independent cohort of patients and healthy individuals. A systems biology approach was used to predict protein-protein interactions and their relation with disease. Following gel image analysis, we detected 117 protein features that varied between STEMI and SCAD groups (fold change cut-off ≥2; p<0.01). From those, 102 were successfully identified, corresponding to 25 open-reading frames (ORFs). Most of the proteins identified are involved in inflammatory response and cardiovascular disease, with 11 ORFs related to infarction. Among others, we report an up-regulation of α2-macroglobulin isoforms, fibrinogen, and viperin in MVs from STEMI patients. Interestingly, several of the proteins identified are involved in thrombogenesis (e.g. α2-macroglobulin, and fibrinogen). In conclusion, we provide a unique panel of proteins that vary between plasma MVs from STEMI and SCAD patients and that might constitute a promising source of biomarkers/drug targets for myocardial infarction.

The plasma proteomic signature as a strategic tool for early diagnosis of acute coronary syndrome.

BACKGROUND: Acute coronary syndrome is the major cause of death in developed countries. Despite its high prevalence, there is still a strong need for new biomarkers which permit faster and more accurate diagnostics and new therapeutic drugs. The basis for this challenge lay in improving our understanding of the whole atherosclerotic process from atherogenesis to atherothrombosis. In this study, we conducted two different proteomic analyses of peripheral blood plasma from non-ST elevation acute coronary syndrome and ST elevation acute coronary syndrome patients vs healthy controls. RESULTS: Two-dimensional Fluorescence Difference in Gel Electrophoresis and mass spectrometry permitted the identification of 31 proteins with statistical differences (p < 0.05) between experimental groups. Additionally, validation by Western blot and Selected Reaction Monitoring permitted us to confirm the identification of a different and characteristic plasma proteomic signature for NSTEACS and STEACS patients. CONCLUSIONS: We purpose the severity of hypoxia as the cornerstone for explaining the differences observed between both groups.

Differential protein expression analysis of degenerative aortic stenosis by iTRAQ labeling.

Degenerative aortic stenosis is the most common worldwide cause of valve replacement. While it shares certain risk factors with coronary artery disease, it is not delayed or reversed by reducing exposure to risk factors (e.g., therapies that lower lipids). Therefore, it is necessary to better understand its pathophysiology for preventive measures to be taken. In order to identify proteins that are involved in AS, we designed a simple method for protein extraction, digestion, labeling, identification, and differential protein expression analysis of both normal and stenotic aortic valve to identify biomarkers of this pathology, which may facilitate early diagnosis and treatment.

Inside human aortic stenosis: a proteomic analysis of plasma.

Valvular aortic stenosis (AS) produces a slowly progressive obstruction in left ventricular outflow track. For this reason, aortic valve replacement is warranted when the valvular stenosis is hemodinamically significant, becoming the most common worldwide cause of aortic valve surgery. Recent epidemiologic studies have revealed an association between degenerative AS and cardiovascular risk factors for atherosclerosis, althought reducing the exposure to such factors and statin therapies both fail to delay or reverse the pathology. Hence, a deeper understanding of the pathophysiology of this disease is required to identify appropriate preventive measures. A proteomic analysis of plasma will permit to know and identify the changes in protein expression induced by AS in this tissue. Using two-dimensional difference gel electrophoresis (2D-DIGE) followed by mass spectrometry (MS), we compared the crude (not pre-fractioned) and pre-fractioned plasma from AS patients and control subjects. We sought to identify plasma proteins whose expression is modified in AS. In addition we investigated if crude plasma presented some alterations in the more abundant proteins since to date, has never been studied before. We also further investigated the link between this disease and atherosclerosis with a view to identifying new potential markers and therapeutic targets.

Aportaciones de la proteómica al laboratorio clínico / The role of proteomics in the clinical laboratory

La irrupción de la proteómica en la última década ha abierto nuevas perspectivas en la investigación clínica, principalmente en la búsqueda de nuevos biomarcadores de diagnóstico, pronóstico, recuperación y respuesta a un tratamiento farmacológico. En su aplicación clínica, la proteómica está llamada a revolucionar la definición actual de enfermedad, al desarrollar un nuevo concepto que englobe el conjunto de cambios fisiológicos y patológicos derivados de una enfermedad y estableciendo perfiles proteicos que permitan realizar un diagnóstico más preciso. En este artículo se recogen, mediante una descripción detallada, las principales aportaciones que la proteómica ofrece a la práctica clínica (AU)

The irruption of proteomics in the last decade has opened new insights in clinical research, mainly in the search for new biomarkers of diagnosis, prognosis and recovery, as well as the response to pharmacological treatments. In its clinical application, proteomics is beginning to revolutionise the current definition of disease, developing a new concept that includes both the physiological changes and pathological origins of a disease, and establishing proteomic profiles that allow the clinician to make a more precise diagnosis. In this article presents a detailed description of the main contributions proteomics offers to clinical practice (AU)

Development of an optimal protocol for the proteomic analysis of stenotic and healthy aortic valves.

INTRODUCTION AND OBJECTIVES: For many years, degenerative aortic stenosis was thought to be a passive process secondary to calcium deposition in aortic valves. Although its etiology remains unknown, several authors have pointed out that degenerative aortic stenosis is associated with the same risk factors as coronary artery disease. Furthermore, histological similarities have been found between aortic valve stenosis and atherosclerotic plaque, giving rise to the hypothesis that degenerative aortic stenosis is an inflammatory process similar to atherosclerosis. Nevertheless, some data do not fit with this hypothesis and, consequently, greater understanding of the condition is needed. The main aim of this study was to develop a practical protocol for extracting protein for use in proteomic analysis from both stenotic and healthy aortic valves. METHODS: The study was carried out using a number of different proteomic methods: two-dimensional electrophoresis, mass spectrometry and additional techniques. RESULTS: We developed a simple and reproducible methodology in the laboratory for carrying out the proteomic analysis of human aortic valves and for identifying their component proteins. CONCLUSIONS: We developed a simple and reproducible method for extracting protein that can be used with mass spectrometry and that makes it possible to carry out large-scale proteomic analysis of stenotic aortic valves. Furthermore, the methodology will significantly increase our understanding of the valve proteome.

Obtención de un protocolo óptimo para el análisis proteómico de válvulas aórticas humanas sanas y estenóticas / Development of an optimal protocol for the proteomic analysis of stenotic and healthy aortic valves

Introducción y objetivos. Durante años, la estenosisaórtica (EA) degenerativa ha sido considerada como un proceso pasivo secundario al depósito de calcio en la válvula aórtica. Aunque no se conoce su etiología, diversos autores han señalado que comparte los mismos factores de riesgo que la enfermedad arterial coronaria. Además, se han encontrado similitudes histológicas en la válvula aórtica estenótica y la placa de ateroma, lo que ha llevado a la hipótesis de que la EA degenerativa es un proceso inflamatorio similar a la aterosclerosis. No obstante, existen algunos datos discordantes con esta teoría, lo que hace necesario profundizar en el conocimiento de esta patología. El principal objetivo de este trabajo es la puesta a punto de un protocolo de extracción proteínica eficaz paraválvulas estenóticas aórticas y válvulas aórticas sanas, compatible con la realización de estudios proteómicos. Métodos. Para el desarrollo del objetivo planteado, sehan utilizado diferentes abordajes proteómicos: electroforesis bidimensional, espectrometría de masas y técnicas complementarias. Resultados. Hemos puesto a punto una metodología, sencilla, reproducible y desarrollada en el laboratorio, que permite el análisis proteómico de la válvula aórtica humana, así como la identificación de las proteínas quelas componen. Conclusiones. Obtención de un método de extracción de proteínas sencillo, reproducible y compatible con la espectrometría de masas, que permite el análisis a gran escala del proteoma de válvulas con estenosis aórtica. Además, esta metodología aumentará de forma significativa nuestro conocimiento del proteoma valvular (AU)

Introduction and objectives. For many years, degenerative aortic stenosis was thought to be a passive process secondary to calcium deposition in aortic valves. Although its etiology remains unknown, several authors have pointed out that degenerative aortic stenosis is associated with the same risk factors as coronary artery disease. Furthermore, histological similarities have been found between aortic valve stenosis and atherosclerotic plaque, giving rise to the hypothesis that degenerative aortic stenosis is an inflammatory process similar to atherosclerosis. Nevertheless, some data do not fit with this hypothesis and, consequently, greater understanding of the condition is needed. The main aim of this study was to develop a practical protocol for extracting protein for use in proteomic analysis from both stenotic and healthy aortic valves. Methods. The study was carried out using a number of different proteomic methods: two-dimensional electrophoresis, mass spectrometry and additional techniques. Results. We developed a simple and reproducible methodology in the laboratory for carrying out the proteomic analysis of human aortic valves and for identifying their component proteins. Conclusions. We developed a simple and reproducible method for extracting protein that can be used with mass spectrometry and that makes it possible to carry outlarge-scale proteomic analysis of stenotic aortic valves. Furthermore, the methodology will significantly increase our understanding of the valve proteome (AU)

Novel mutation (H402R) in the S1 domain of KCNH2-encoded gene associated with long QT syndrome in a Spanish family.

Long-QT syndrome is a congenital cardiac disease resulting in ventricular arrhythmias and sudden death. Genetic mutations in two protein ion-channel genes, KCNQ1 and KCNH2. The mutations position in these genes provides additional information about the evaluation of the risk-stratification. In a Spanish family in whom previous repetitive syncope episodes, sudden death and pathological prolongation of the QT interval were documented, a novel heterozygous mutation in the KCNH2 gene (A1218>G) was identified. This mutation loading to amino acid substitution H420R in the S1 transmembrane domain of KCNH2. The new A1218>G mutation in the KCNH2 gene detected in this Spanish family causes arrhythmia manifestation in the carriers.

Relationship between vitamin D binding protein and aspirin resistance in coronary ischemic patients: a proteomic study.

Our aim was to analyze the plasma proteome in aspirin (acetylsalicylic acid [ASA])-sensitive and ASA-resistant coronary ischemic patients. Plasma from 19 ASA-sensitive and 19 ASA-resistant patients was analyzed. For the proteomic study, two-dimensional electrophoresis was performed. The expression of one isotype of the fibrinogen gamma chain and three isotypes of haptoglobin was increased in ASA-resistant patients. Three vitamin D binding protein isotypes were increased in ASA-resistant patients. In vitro incubation of vitamin D binding protein (DBP) with blood from healthy volunteers reduced the inhibitory effect of ASA on thromboxane A2 production. DBP may be a new regulator of the inhibitory effect of ASA on platelets.

Case report of a Spanish patient with arrhythmogenic right ventricular cardiomyopathy and palmoplantar keratoderma without plakoglobin and desmoplakin gene modifications.

We report a case of a 43 year old man from Spain, who has been diagnosed with Naxos disease. It is a hereditary disorder characterized by palmoplantar keratoderma, woolly hair and cardiomyopathy, which has been associated with a mutation in plakoglobin encoding gene in chromosome 17q21. In the patient, the direct sequencing of the plakoglobin gene discarded TG deletion at 2157 characteristic of Naxos disease. Analysis of the reported desmoplakin mutations associated with Carvajal Syndrome, another ARVC disease, that it is also accompanied with a skin and hair disorder, also failed to reveal mutations in desmoplakin gene. These results suggest the existence of other causative genes and/or other putative sites in desmoplakin/plakoglobin encoding genes than those recently published.

Direct effect of F12511, a systemic inhibitor of Acyl-CoA cholesterol acyltransferase on bovine aortic endothelial cells.

F12511(S)-2',3',5'-trimethyl-4'-hydroxy-alpha-dodecylthio-alpha-phenylacetanilide (F12511) is a new Acyl-CoA cholesterol acyltransferase (ACAT) inhibitor that not only reduces the plasma cholesterol levels but also has anti-atherosclerotic actions in animals models. The study's aim was to analyze if F12511 may directly modify the ability of tumor necrosis factor--alpha (TNF-alpha)-incubated bovine aortic endothelial cells (BAEC) to express endothelial nitric oxide synthase (eNOS) protein and inflammatory-related proteins such as platelet endothelial cell adhesion molecule (PECAM) and CD40 ligand (CD40L). The addition of increasing concentrations of F12511 (10 to 10 mol/L) failed to modify the level of eNOS protein expressed in control BAEC. TNF-alpha (10 ng/mL) reduced the expression of eNOS protein. In TNF-alpha--incubated BAEC, F12511 protected eNOS expression in a concentration-dependent manner. TNF-alpha stimulated the expression of both CD40L and PECAM in cultured BAEC. F12511 (10 mol/L) failed to modify the expression of CD40L and PECAM in control and TNF-alpha-incubated BAEC. Reverse transcriptase polymerase chain reaction showed a marked expression of the ACAT-2 isoform and absent of expression of the ACAT-1 isoform in BAEC. The presence of ACAT-2 isoform in BAEC was further confirmed by Western blot. F12511 failed to modify the expression of the proinflammatory associated proteins PECAM and CD40L in the endothelium but protected eNOS expression in the endothelial cells exposed to inflammatory conditions.

Proteomic study of plasma from moderate hypercholesterolemic patients.

Proteomics is a technology to detect and identify several proteins and their isoforms in a single sample. We used proteomics to analyze modifications in the protein map of plasma after simvastatin treatment of moderate hypercholesterolemic patients. Plasma from hypercholesterolemic patients (n = 9) was compared before and after 12 weeks of simvastatin treatment (40 mg/day). Patients with similar cardiovascular risk factors were used as controls (CR group). By using two-dimensional electrophoresis and mass spectrometry, we identified the different protein isoforms. The plasma expression of three fibrinogen gamma chain isoforms (FGG) was enhanced, whereas the expression of two isoforms of the fibrinogen beta chain (FGB) was reduced in the hypercholesterolemic patients compared with the CR group. The expression of apolipoprotein A-IV and three haptoglobin isoforms was higher in hypercholesterolemic patients. Simvastatin treatment modified the plasma expression of FGG chain isoform 1, FGB chain isoforms 1 and 2, vitamin D binding protein isoform 3, apo A-IV, and haptoglobin isoform 2. The modification of FGG chain isoform 1 and FGB chain isoforms 1 and 2 was positively correlated with total plasma cholesterol level. Proteomic analysis of plasma may help to know more in depth the molecular mechanism modified by simvastatin treatment.

Proteomic approach to identify changes in protein expression modified by 17beta-oestradiol in bovine vascular smooth muscle cells.

The aim of the present study was to use proteomics to analyse modifications in the level of expression of different proteins in BVSMCs (bovine vascular smooth muscle cells) incubated in the absence and presence of 17beta-oestradiol. By using two-dimensional electrophoresis with a pH range of 4-7, we identified several areas on the gels in which the level of expression of proteins were different between control BVSMCs and cells incubated for 24 h with 17beta-oestradiol. Changes in several isoforms of alpha-enolase, HSP60 (heat-shock protein 60), vimentin and PDI (protein disulphide-isomerase) were observed in BVSMCs. The expression of alpha-enolase isoform 1 was enhanced after 17beta-oestradiol treatment. The expression of HSP60 isoform 3, vimentin isoforms 2 and 3 and caldesmon was reduced by 17beta-oestradiol. Finally, the expression of PDI isoforms was reduced by 17beta-oestradiol. In summary, 17beta-oestradiol modified the expression of isoforms of proteins associated with smooth muscle cell proliferation (alpha-enolase, vimentin and HSP-60), cell contraction (vimentin and caldesmon) and cell redox modulation (PDI). These findings confirm that 17beta-oestradiol may modulate a wide range of signalling pathways in vascular smooth muscle cells.

Endothelial nitric oxide synthase/soluble guanylate cyclase system in human nasal polyps.

The aim of our study was to analyze the level of expression of the endothelial nitric oxide synthase (eNOS)/soluble guanylate cyclase (sGC) system in nasal polyps and control nasal mucosae. The study was performed in polyps from 15 patients and nasal mucosae from 11 subjects operated on the nasal septum (control group). The expression of endothelial nitric oxide synthase (eNOS) and soluble guanylate cyclase (sGC) was determined in nasal mucosae. Western blot analysis demonstrated that eNOS protein was overexpressed in the nasal polyps with respect to control nasal mucosae. Immunohistochemistry also demonstrated that the vascular endothelium of nasal polyps contained higher amounts of eNOS protein than control nasal mucosae. Moreover, the beta(1) subunit of sGC was also overexpressed in the nasal polyps, which was associated with an increased content of cyclic GMP in the nasal polyps with respect to nasal control mucosae. In human nasal polyposis, there is an overexpression of the eNOS/sGC system. Further studies are needed to assess whether this overexpression is involved in the genesis of nasal polyposis.

Endothelial hypoxic preconditioning in rat hypoxic isolated aortic segments.

Our aim was to analyse endothelial hypoxic preconditioning after hypoxia-reperfusion (HR). Endothelial functionality was analysed through the vasorelaxation responses to acetylcholine (Ach) and the level of serine1177 phosphorylated endothelial nitric oxide synthase (eNOS) (ser1177-eNOS) measured by Western blot in in vitro hypoxic preconditioned (P + HR) isolated rat aortic segments. Relaxation in response to Ach was reduced in phenylephrine-precontracted aortic segments after HR (control: IC50, 5 +/- 2.5 x 10(-8) mol l(-1); HR: IC50, 3 +/- 1.2 x 10(-7) mol l(-1); P < 0.05). Ach-dependent vasodilatation was improved by P + HR. The content of ser1177-eNOS in the HR segments was 1.5-fold lower than in P + HR. Confocal microscopy showed an increased content of both superoxide anion and peroxynitrite in the vascular wall of HR aortic segments, which it was reduced by P + HR. Geldanamycin (10 microg ml(-1)), an agent known to inhibit heat shock protein 90 (hsp90), reduced the level of ser1177-eNOS in P + HR aortic segments. However in the presence of geldanamycin, endothelial hypoxic preconditioning persisted. We conclude that short periods of hypoxia induced endothelial hypoxic preconditioning that was accompanied by enhanced levels of ser1177-eNOS in the vascular wall. The fact that endothelial hypoxic preconditioning persisted in the presence of geldanamycin suggests that other molecular mechanisms are involved in the endothelial adaptation to HR injury.

Effect of C-reactive protein on Fcgamma receptor II in cultured bovine endothelial cells.

The major CRP (C-reactive protein) receptor on leucocytes has been identified as the low-affinity IgG receptor Fcgamma receptor II (CD32). Our aim was to assess whether inflammation may modify the presence of the CD32 receptor in BAEC (bovine aortic endothelial cells). Confocal microscopy experiments showed a weak expression of the CD32 receptor in control BAEC that was slightly increased by 10 microg/ml CRP. Incubation of BAEC with TNF-alpha (tumour necrosis factor-alpha) did not modify the fluorescence signal of CD32. Addition of CRP to TNF-alpha-incubated BAEC enhanced the fluorescence signal of the CD32 receptors. The CD32 receptors showed a perinuclear cytoplasmic localization in BAEC. An alteration of the NO (nitric oxide)-dependent vasorelaxation has been defined as endothelial dysfunction. Endothelial dysfunction has been associated with the presence of superoxide anion and with a reduction in the expression of the eNOS (endothelial NO synthase). A concentration of CRP similar to that detected in patients with cardiovascular risk (10 microg/ml) failed to modify the generation of superoxide anion stimulated by TNF-alpha. Western blot experiments showed that TNF-alpha decreased the expression of the eNOS protein, which was partially protected by treatment with 10 microg/ml CRP. The protective effect of 10 microg/ml CRP on eNOS expression in TNF-alpha-incubated BAEC was prevented by an antibody against CD32 receptors. In conclusion, the present results suggest that, although CRP has been associated with inflammation, CRP may protect the expression of eNOS protein against pro-inflammatory mediators such as TNF-alpha.

Proteomic analysis of plasma from patients during an acute coronary syndrome.

OBJECTIVES: The aim of this study was to analyze modifications in the plasma protein map during an acute coronary syndrome (ACS) using proteomics. BACKGROUND: Proteomics is a new technology that allows the detection and identification of several proteins at a given time in a sample. METHODS: Plasma from 19 patients, 11 with acute myocardial infarction (AMI) and 8 with unstable angina (UA), was investigated. The control group included nine age-matched volunteers. RESULTS: In two-dimensional electrophoresis using a pH range of 4 to 7, constant differences were found in at least four different areas within the plasma protein map. In area 1, we identified the presence of seven alpha(1)-antitrypsin (AAT) isoforms in plasma from control subjects. alpha(1)-antitrypsin isoform 1 was undetectable in plasma from UA and AMI patients. The AAT isoforms 5, 6, and 7 were reduced in plasma from AMI patients when compared with UA patients. Three fibrinogen gamma chain isoforms were identified in area 2. Fibrinogen gamma chain isoforms 1 and 2 were increased in AMI patients with respect to UA patients. Five apolipoprotein A-I isoforms were identified in area 3. All of them were reduced in plasma from AMI patients with respect to UA patients. In area 4, the gamma-immunoglobulin heavy chains were detected and were found increased in plasma from ACS patients. CONCLUSIONS: Plasma proteomic analysis makes it possible to develop a map of the protein isoforms that are expressed in plasma during an ACS.