Proteomic investigations into hypertension: what's new and how might it affect clinical practice?

Introduction: Hypertension is a multifactorial disease that has, thus far, proven to be a difficult target for pharmacological intervention. The application of proteomic strategies may help to identify new biomarkers for the early diagnosis and prompt treatment of hypertension, in order to control blood pressure and prevent organ damage. Areas covered: Advances in proteomics have led to the discovery of new biomarkers to help track the pathophysiological processes implicated in hypertension. These findings not only help to better understand the nature of the disease, but will also contribute to the clinical needs for a timely diagnosis and more precise treatment. In this review, we provide an overview of new biomarkers identified in hypertension through the application of proteomic techniques, and we also discuss the difficulties and challenges in identifying biomarkers in this clinical setting. We performed a literature search in PubMed with the key words 'hypertension' and 'proteomics', and focused specifically on the most recent literature on the utility of proteomics in hypertension research. Expert opinion: There have been several promising biomarkers of hypertension identified by proteomics, but too few have been introduced to the clinic. Thus, further investigations in larger cohorts are necessary to test the feasibility of this strategy for patients. Also, this emerging field would profit from more collaboration between clinicians and researchers.

[Cardiovascular risk study in patients with renin-angiotensin system blockade by means of the proteone of circulating extracellular vesicles]. / Estudio del riesgo cardiovascular en pacientes con bloqueo del sistema renina-angiotensina a través del proteoma de las vesículas extracelulares circulantes.

INTRODUCTION: Extracellular vesicles (EVs) are released to the bloodstream by certain cell types due to transport, activation and cell death processes. Blood count of EVs from platelet and endothelial origin has been proved to be a cardiovascular risk biomarker. Thus, EVs proteome might reflect the underlying cellular processes in hypertensive patients with albuminuria. MATERIAL AND METHODS: Protein content of circulating EVs was analyzed by liquid chromatography coupled to mass spectrometry. EVs were isolated by an ultracentrifugation protocol optimized in order to avoid contamination by blood plasma proteins. Purity of the isolated fraction was verified by electronic and confocal microscopy, and by flow cytometry. RESULTS: We hereby show a method to isolate circulating EVs from hypertensive patients with/without albuminuria with high yield and purity. Besides, we provide a reference proteome of the EVs of these patients, composed of 2,463 proteins, and prove that the proteins carried by these vesicles are associated with crucial processes involved in the inherent cardiovascular risk. CONCLUSION: The proteome of circulating EVs is an interesting source of indicators in the evaluation of cardiovascular risk in hypertensive patients with renin-angiotensin system blockage.

Proteomic characterization of EPCs and CECs "in vivo" from acute coronary syndrome patients and control subjects.

BACKGROUND: Circulating endothelial cells (CECs) and endothelial progenitor cells (EPCs) represent two scarce blood populations that are thought to play important roles in tissue vascularization. They have also been proposed as potential markers for more than a dozen pathologies. Moreover, EPCs have arisen as a new therapeutic option for cardiovascular disease. However nowadays there is certain controversy about their roles and a better understanding of EPC biology is required to develop new strategies for forthcoming therapies. METHODS: Flow cytometry analysis was performed on freshly isolated mononuclear cells from control subjects and Acute Coronary Syndrome (ACS) patients. EPCs and CECs for both groups were isolated and quantified. Statistical analyses were performed to test the potential biomarker usefulness of both populations in ACS together with the first "in vivo" proteomic characterizations of these populations. RESULTS: Our results do not show statistical differences in the quantification of CECs and EPCs in control subjects and ACS patients. The proteomic characterization allowed us to identify 673 proteins associated to CECs (389 in controls and 462 in ACS patients), and another 502 proteins in EPCs (350 in controls and 274 in ACS patients). CONCLUSIONS: Our data show the necessity to obtain a more accurate and specific phenotype of CECs and EPCs cells as well as a flow cytometry "golden standard" protocol, before they can be considered useful clinical markers. GENERAL SIGNIFICANCE: The proteomic data suggest a potential effect of ACS in the protein profiles of these cells.

An optimal protocol to analyze the rat spinal cord proteome.

Since the function of the spinal cord depends on the proteins found there, better defing the normal Spinal Cord Proteome is an important and challenging task. Although brain and cerebrospinal fluid samples from patients with different central nervous system (CNS) disorders have been studied, a thorough examination of specific spinal cord proteins and the changes induced by injury or associated to conditions such as neurodegeneration, spasticity and neuropathies has yet to be performed. In the present study, we aimed to describe total protein content in the spinal cord of healthy rats, employing different proteomics tools. Accordingly, we have developed a fast, easy, and reproducible sequential protocol for protein extraction from rat spinal cords. We employed conventional two dimensional electrophoresis (2DE) in different pH ranges (eg. 4-7, 3-11 NL) combined with identification by mass spectrometry (MALDI-TOF/TOF), as well as first dimension protein separation combined with Liquid Chromatography Mass Spectrometry/Mass Spectrometry (LC-MS/MS) to maximise the benefits of this technology. The value of these techniques is demonstrated here by the identification of several proteins known to be associated with neuroglial structures, neurotransmission, cell survival and nerve growth in the central nervous system. Furthermore this study identified many spinal proteins that have not previously been described in the literature and which may play an important role as either sensitive biomarkers of dysfunction or of recovery after Spinal Cord Injury.

Proteomic Biomarkers of Atherosclerosis.

SUMMARY: Biomarkers provide a powerful approach to understanding the spectrum of cardiovascular diseases. They have application in screening, diagnostic, prognostication, prediction of recurrences and monitoring of therapy. The "omics" tool are becoming very useful in the development of new biomarkers in cardiovascular diseases. Among them, proteomics is especially fitted to look for new proteins in health and disease and is playing a significant role in the development of new diagnostic tools in cardiovascular diagnosis and prognosis. This review provides an overview of progress in applying proteomics to atherosclerosis. First, we describe novel proteins identified analysing atherosclerotic plaques directly. Careful analysis of proteins within the atherosclerotic vascular tissue can provide a repertoire of proteins involved in vascular remodelling and atherogenesis. Second, we discuss recent data concerning proteins secreted by atherosclerotic plaques. The definition of the atheroma plaque secretome resides in that proteins secreted by arteries can be very good candidates of novel biomarkers. Finally we describe proteins that have been differentially expressed (versus controls) by individual cells which constitute atheroma plaques (endothelial cells, vascular smooth muscle cells, macrophages and foam cells) as well as by circulating cells (monocytes, platelets) or novel biomarkers present in plasma.

Development of a low-cost, insect larvae-derived recombinant subunit vaccine against RHDV.

Vaccine antigens against rabbit hemorrhagic disease virus (RHDV) are currently derived from inactivated RHDV obtained from livers of experimentally infected rabbits. Several RHDV-derived recombinant immunogens have been reported. However, their application in vaccines has been restricted due to their high production costs. In this paper, we describe the development of an inexpensive, safe, stable vaccine antigen for RHDV. A baculovirus expressing a recombinant RHDV capsid protein (VP60r) was used to infect Trichoplusia ni insect larvae. It reached an expression efficiency of 12.5% of total soluble protein, i.e. approximately 2 mg of VP60r per larva. Preservation of the antigenicity and immunogenicity of the VP60r was confirmed by immunological and immunization experiments. Lyophilized crude larvae extracts, containing VP60r, were stable, at room temperature, for at least 800 days. In all cases, rabbits immunized with a single dose of VP60r by the intramuscular route were protected against RHDV challenge. Doses used were as low as 2 microg of VP60r in the presence of adjuvant or 100 microg without one. Orally administered VP60r in the absence of an adjuvant gave no protection. The potential costs of an RHDV vaccine made using this technology would be reduced considerably compared with producing the same protein in insect cells maintained by fermentation. In conclusion, the larva expression system may provide a broad-based strategy for production of recombinant subunit antigens (insectigens) for human or animal medicines, especially when production costs restrain their use.

Expresión diferencial de proteínas en el corazón de ratas espontáneamente hipertensas con hipertrofia cardíaca / Differential protein expression in hearts of spontaneously hypertensive rats with cardiac hypertrophy

Introducción. La hipertensión arterial afecta a alrededor del 20% de la población del mundo occidental. A pesar de la disponibilidad de excelentes fármacos antihipertensivos, la repercusión orgánica sobre los órganos diana (corazón, cerebro y riñón) sigue siendo elevada. La hipertrofia ventricular izquierda es un hallazgo común en pacientes hipertensos. Aunque se han asociado diversos patrones transcripcionales celulares y genéticos con la hipertrofia cardíaca, sus mecanismos siguen siendo desconocidos. Los objetivos de este trabajo son: a) estudiar el perfil proteico de ratas hipertensas con hipertrofia cardíaca en comparación con animales normotensos, y b) identificar las proteínas de interés. Materiales y métodos. Los estudios se realizaron en ratas macho espontáneamente hipertensas (SHR) y se utilizaron como controles ratas normotensas Wistar Kyoto (WKY). Después de 48 semanas de vida se sacrificó a los animales y se les extrajo el corazón. Para detectar cambios proteicos en la hipertensión grave, analizamos el patrón de expresión de las proteínas cardíacas por electroforesis bidimensional. Resultados. De los más de 1.000 spots resueltos en el rango de pH 4 a 7 del ventrículo izquierdo, nos centramos en 432 bien definidos. En la comparación con los corazones normotensos, de los 432 spots analizados 360 permanecieron invariables y 72 se encontraron alterados en el corazón de las ratas SHR. La identificación de los spots alterados se está realizando mediante espectrometría de masas (MALDI-TOF). Por el momento se han identificado algunos spots tales como la tropomiosina 1-α y el polipéptido Va de la citocromo C oxidasa. Conclusiones. A través de un enfoque proteómico hemos analizado las diferencias entre los proteomas del corazón hipertrófico de animales hipertensos y de controles sanos. En el corazón hipertrófico se observó un cierto número de proteínas sobre o infraexpresadas. Estos datos ilustran un uso inexplorado de la proteómica como herramienta para el descubrimiento de nuevas dianas terapéuticas (AU)

Introduction. Arterial hypertension affects approximately 20% of the Western world. Despite the availability of excellent antihypertensive drugs, damage to target organs (heart, brain and kidney) remains significant. Left ventricular hypertrophy is a common finding in hypertensive patients. Although different cellular and gene transcription patterns have been associated with cardiac hypertrophy, the molecular mechanisms of this process remain mostly unknown. The aims of the present work were: a) to analyze the differential cardiac protein expression in spontaneously hypertensive rats (SHR) with cardiac hypertrophy compared with normotensive rats, and b) identify proteins of interest. Materials and methods. Studies were conducted in male spontaneously hypertensive rats (SHR), with normotensive Wistar-Kyoto rats (WKY) used as controls. At 48 weeks of age, rats were euthanized and hearts removed on block. Analysis of protein expression patterns by two-dimensional polyacrylamide gel electrophoresis (2-DE) was performed to detect changes in heart proteins associated to severe hypertension. Results. From the more than 1000 spots resolved in the 4-7 pH range by 2-DE of myocardial tissue from WKY and SHR rats, we focused on 432 well-defined spots. In comparison with those obtained in normotensive rat hearts, 360 spots remained invariable, 43 increased and 29 decreased or were absent. The identification by mass spectrometry (MALDI-TOF) of altered spots is under study. To date, few spots such as alphatropomyosin and the Va polypeptide of cytochrome c-oxidase have been identified. Conclusions. Via a proteomic approach, we analyzed the difference between proteomes of hypertrophic hearts of hypertensive and normotensive rats and observed a number of over or under expressed proteins in damaged hearts. These data illustrate a hitherto unexplored use of proteomics to discover new therapeutic targets for antihypertensive drugs (AU)

Antigenic and immunogenic properties of a chimera of two immunodominant African swine fever virus proteins.

A chimera of the two immunodominant African swine fever (ASF) virus proteins p54 and p30 was constructed by insertion of the gene CP204L into a Not I restriction site of E183L gene. The resulting chimeric protein p54/30, expressed by a recombinant baculovirus in insect cells and in Trichoplusia ni larvae, retained antigenic determinants present in both proteins and reacted in Western blot with a collection of sera from inapparent ASF virus carrier pigs. Remarkably, pigs immunized with the chimeric protein developed neutralizing antibodies and survived the challenge with a virulent African swine fever virus, presenting a reduction of about two logs in maximum viremia titers with respect to control pigs. In conclusion, this study revealed that the constructed chimeric protein may have utility as a serological diagnostic reagent and for further immunological studies that may provide new insights on mechanisms of protective immunity to ASFV.

Serodiagnosis of African swine fever using the recombinant protein p30 expressed in insect larvae.

African swine fever (ASF) has a substantial economic impact in many African developing countries and its eradication is based only on an efficient diagnosis program because of the absence of an available vaccine. Previous data suggested the convenience of using the highly antigenic virus protein p30 as ELISA antigen for serological diagnosis of this disease. A simple and efficient method is described for producing the recombinant protein p30 from ASF virus in Trichoplusia ni larvae (cabbage looper) in order to facilitate the large-scale production of this recombinant protein in the absence of fermentation procedures. A baculovirus encoding the virus protein p30 was used to infect insect larvae, showing that recombinant protein production had a sharp optimal peak with a time of occurrence dependent on the initial virus dose inoculated to the larvae. Crude lysates of infected larvae were used without further purification as coating antigen in ELISA to analyse a limited number of sera from natural or experimentally ASF virus infected pigs. Remarkably, the recombinant protein obtained from a single infected larva was sufficient for serological diagnosis of at least 3750 serum samples. Recombinant p30 obtained by this procedure was also used in a confirmatory immunoblotting, reacting with all positive sera tested previously by ELISA. In conclusion, production of the recombinant ASF virus protein p30 in larvae should be applicable to large-scale production of diagnostic reagents for this disease in developing countries, eliminating the need for specialised facilities for tissue culture.