RESUMEN

Vaccine adjuvants increase the breadth of serum antibody responses, but whether this is due to the generation of antigen-specific B cell clones with distinct specificities or the maturation of memory B cell clones that produce broadly cross-reactive antibodies is unknown. Here, we longitudinally analyzed immune responses in healthy adults after two-dose vaccination with either a virus-like particle COVID-19 vaccine (CoVLP), CoVLP adjuvanted with AS03 (CoVLP+AS03), or a messenger RNA vaccination (mRNA-1273). CoVLP+AS03 enhanced the magnitude and durability of circulating antibodies and antigen-specific CD4+ T cell and memory B cell responses. Antigen-specific CD4+ T cells in the CoVLP+AS03 group at day 42 correlated with antigen-specific memory B cells at 6 months. CoVLP+AS03 induced memory B cell responses, which accumulated somatic hypermutations over 6 months, resulting in enhanced neutralization breadth of monoclonal antibodies. Furthermore, the fraction of broadly neutralizing antibodies encoded by memory B cells increased between day 42 and 6 months. These results indicate that AS03 enhances the antigenic breadth of B cell memory at the clonal level and induces progressive maturation of the B cell response.

Asunto(s)

COVID-19 , Vacunas contra la Influenza , Gripe Humana , Polisorbatos , Escualeno , alfa-Tocoferol , Adulto , Humanos , Células B de Memoria , Vacunas contra la COVID-19 , Anticuerpos Antivirales , COVID-19/prevención & control , Combinación de Medicamentos

RESUMEN

Respiratory viruses such as influenza are encountered multiple times through infection and/or vaccination and thus have the potential to shape immune cell phenotypes over time. In particular, memory T cell compartments may be affected, as both CD4+ and CD8+ T cell responses likely contribute to viral control. In this study, we assessed immune phenotypes using cytometry by time of flight in the peripheral blood of 22 humans with acute respiratory illness and 22 age-matched noninfected controls. In younger infected individuals (1-19 y of age), we found decreased B and NK cell frequencies and a shift toward more effector-like CD4+ and CD8+ T cell phenotypes, compared with young healthy controls. Significant differences between noninfected and infected older individuals (30-74 y of age) were not seen. We also observed a decrease in naive CD4+ T cells and CD27+CD8+ T cells as well as an increase in effector memory CD8+ T cells and NKT cells in noninfected individuals with age. When cell frequencies were regressed against age for infected versus noninfected subjects, significant differences in trends with age were observed for multiple cell types. These included B cells and various subsets of CD4+ and CD8+ T cells. We conclude that acute respiratory illness drives T cell differentiation and decreases circulating B cell frequencies preferentially in young compared with older individuals.

Asunto(s)

Linfocitos T CD8-positivos , Gripe Humana , Humanos , Diferenciación Celular , Activación de Linfocitos , Linfocitos B

Asunto(s)

Vacuna BNT162 , Vacunología , Humanos , Vacunas Sintéticas , Vacunas de ARNm

RESUMEN

The emergency use authorization of two mRNA vaccines in less than a year from the emergence of SARS-CoV-2 represents a landmark in vaccinology1,2. Yet, how mRNA vaccines stimulate the immune system to elicit protective immune responses is unknown. Here we used a systems vaccinology approach to comprehensively profile the innate and adaptive immune responses of 56 healthy volunteers who were vaccinated with the Pfizer-BioNTech mRNA vaccine (BNT162b2). Vaccination resulted in the robust production of neutralizing antibodies against the wild-type SARS-CoV-2 (derived from 2019-nCOV/USA_WA1/2020) and, to a lesser extent, the B.1.351 strain, as well as significant increases in antigen-specific polyfunctional CD4 and CD8 T cells after the second dose. Booster vaccination stimulated a notably enhanced innate immune response as compared to primary vaccination, evidenced by (1) a greater frequency of CD14+CD16+ inflammatory monocytes; (2) a higher concentration of plasma IFNγ; and (3) a transcriptional signature of innate antiviral immunity. Consistent with these observations, our single-cell transcriptomics analysis demonstrated an approximately 100-fold increase in the frequency of a myeloid cell cluster enriched in interferon-response transcription factors and reduced in AP-1 transcription factors, after secondary immunization. Finally, we identified distinct innate pathways associated with CD8 T cell and neutralizing antibody responses, and show that a monocyte-related signature correlates with the neutralizing antibody response against the B.1.351 variant. Collectively, these data provide insights into the immune responses induced by mRNA vaccination and demonstrate its capacity to prime the innate immune system to mount a more potent response after booster immunization.

Asunto(s)

Inmunidad Adaptativa , Anticuerpos Antivirales/inmunología , Vacunas contra la COVID-19/inmunología , COVID-19/inmunología , Inmunidad Innata , Linfocitos T/inmunología , Vacunología , Adulto , Anciano , Anticuerpos Neutralizantes/inmunología , Autoanticuerpos/inmunología , Vacuna BNT162 , Vacunas contra la COVID-19/administración & dosificación , Femenino , Humanos , Inmunización Secundaria , Masculino , Persona de Mediana Edad , Análisis de la Célula Individual , Glicoproteína de la Espiga del Coronavirus/inmunología , Transcripción Genética , Transcriptoma/genética , Adulto Joven

RESUMEN

The emergency use authorization of two COVID-19 mRNA vaccines in less than a year since the emergence of SARS-CoV-2, represents a landmark in vaccinology1,2. Yet, how mRNA vaccines stimulate the immune system to elicit protective immune responses is unknown. Here we used a systems biological approach to comprehensively profile the innate and adaptive immune responses in 56 healthy volunteers vaccinated with the Pfizer-BioNTech mRNA vaccine. Vaccination resulted in robust production of neutralizing antibodies (nAbs) against the parent strain and the variant of concern, B.1.351, but no induction of autoantibodies, and significant increases in antigen-specific polyfunctional CD4 and CD8 T cells after the second dose. The innate response induced within the first 2 days of booster vaccination was profoundly increased, relative to the response at corresponding times after priming. Thus, there was a striking increase in the: (i) frequency of CD14+CD16+ inflammatory monocytes; (ii) concentration of IFN- y in the plasma, which correlated with enhanced pSTAT3 and pSTAT1 levels in monocytes and T cells; and (iii) transcriptional signatures of innate responses characteristic of antiviral vaccine responses against pandemic influenza, HIV and Ebola, within 2 days following booster vaccination compared to primary vaccination. Consistent with these observations, single-cell transcriptomics analysis of 242,479 leukocytes demonstrated a ~100-fold increase in the frequency of a myeloid cluster, enriched in a signature of interferon-response transcription factors (TFs) and reduced in AP-1 TFs, one day after secondary immunization, at day 21. Finally, we delineated distinct molecular pathways of innate activation that correlate with CD8 T cell and nAb responses and identified an early monocyte-related signature that was associated with the breadth of the nAb response against the B1.351 variant strain. Collectively, these data provide insights into the immune responses induced by mRNA vaccines and demonstrate their capacity to stimulate an enhanced innate response following booster immunization.

RESUMEN

BACKGROUND: Ischemic cardiac damage is associated with upregulation of cardiac pro-inflammatory cytokines, as well as invasion of lymphocytes into the heart. Regulatory T cells (Tregs) are known to exert a suppressive effect on several immune cell types. We sought to determine whether the Treg pool is influenced by myocardial damage and whether Tregs transfer and deletion affect cardiac remodeling. METHODS AND RESULTS: The number and functional suppressive activity of Tregs were assayed in mice subjected to experimental myocardial infarction. The numbers of splenocyte-derived Tregs in the ischemic mice were significantly higher after the injury than in the controls, and their suppressive properties were significantly compromised. Compared with PBS, adoptive Treg transfer to mice with experimental infarction reduced infarct size and improved LV remodeling and functional performance by echocardiography. Treg deletion with blocking anti-CD25 antibodies did not influence infarct size or echocardiographic features of cardiac remodeling. CONCLUSION: Treg numbers are increased whereas their function is compromised in mice with that underwent experimental infarction. Transfer of exogeneous Tregs results in attenuation of myocardial remodeling whereas their ablation has no effect. Thus, Tregs may serve as interesting potential interventional targets for attenuating left ventricular remodeling.

Asunto(s)

Daño por Reperfusión Miocárdica/fisiopatología , Miocardio/inmunología , Linfocitos T Reguladores/metabolismo , Remodelación Ventricular/inmunología , Traslado Adoptivo , Animales , Células Cultivadas , Técnicas de Cocultivo , Modelos Animales de Enfermedad , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Daño por Reperfusión Miocárdica/inmunología , Miocardio/patología , Bazo/inmunología

RESUMEN

BACKGROUND: Ranibizumab (Lucentis®) is a Fab-antibody fragment developed from Bevacizumab, a full-length anti-VEGF antibody. Both compounds are used for treating age-related macular degeneration (AMD). The influence of bevacizumab and ranibizumab on genes involved in signal transduction and cell signaling downstream of VEGF were compared in order to detect possible differences in their mode of action, which are not related to their Fab-antibody fragments. METHODS: Human umbilical vein cell lines (EA.hy926) and retinal pigment epithelial cells (ARP-19) were exposed to oxidative stress. The cells were treated with therapeutic concentrations of bevacizumab (0.25 mg/mL) and ranibizumab (125 mg/mL) for 24 hours prior to all experiments, and their effects on gene expressions were determined by RT- PCR. RESULTS: After exposure to bevacizumab, more genes in the endothelial cells were up-regulated (KDR, NFATc2) and down-regulated (Pla2g12a, Rac2, HgdC, PRKCG) compared to non-treated controls. After exposure to ranibizumab, fewer genes were up-regulated (PTGS2) and down-regulated (NOS3) compared to controls. In comparison between drugs, more genes were up-regulated (NFATc2 and KDR) and more were down-regulated (Pla2g12a, Pla2g1b, Ppp3r2, Rac2) by bevacizumab than by ranibizumab. In RPE cells, NOS3 and PGF were up-regulated and Pla2g12b was down-regulated after exposure to ranibizumab, while PIK3CG was up-regulated and FIGF was down-regulated after exposure to bevacizumab, but the differences in gene expression were minor between drugs (PIK3CGand PGF were down-regulated more by ranibizumab than by bevacizumab). CONCLUSIONS: The different gene expressions after exposure to ranibizumab and bevacizumab in endothelial and RPE cells may indicate a somewhat different biological activity of the two compounds.

Asunto(s)

Anticuerpos Monoclonales Humanizados/farmacología , Perfilación de la Expresión Génica , Epitelio Pigmentado de la Retina/efectos de los fármacos , Transducción de Señal , Venas Umbilicales/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/metabolismo , Bevacizumab , Línea Celular , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Humanos , Estrés Oxidativo , Ranibizumab , Reacción en Cadena en Tiempo Real de la Polimerasa , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/metabolismo , Venas Umbilicales/citología , Venas Umbilicales/metabolismo , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/genética

RESUMEN

AIMS: MicroRNAs (miRNAs, miR) are endogenous short RNA sequences that regulate a wide range of physiological and pathophysiological processes. Several miRNAs control the formation of new blood vessels either by increasing or by inhibiting angiogenesis. Here, we investigated the possible role of the miR-106b∼25 cluster in postnatal neovascularization and in regulation of the angiogenic properties of adult bone marrow-derived stromal cells. METHODS AND RESULTS: To study the effect of miR-106b∼25 deletion on neovascularization, we used a miR-106b∼25 knockout (KO) mouse model. After inducing hindlimb ischaemia, we showed that vascularization in ischaemic mice devoid of miR-106b∼25 is impaired, as evident from the reduced blood flow on laser Doppler perfusion imaging. The miR-106b∼25 cluster was also shown here to be an essential player in the proper functioning of bone marrow-derived stromal cells through its regulation of apoptosis, matrigel tube formation capacity, cytokine secretion, and expression of the stem-cell marker Sca-1. In addition, we showed that capillary sprouting from miR-106b∼25 KO aortic rings is diminished. CONCLUSION: These results show that the miR-106b∼25 cluster regulates post-ischaemic neovascularization in mice, and that it does so in part by regulating the function of angiogenic bone marrow-derived stromal cells and of endothelial cells.

Asunto(s)

MicroARNs/fisiología , Neovascularización Fisiológica/fisiología , Antígeno AC133 , Animales , Antígenos CD/metabolismo , Antígenos Ly/metabolismo , Aorta/fisiología , Apoptosis/fisiología , Velocidad del Flujo Sanguíneo/fisiología , Células de la Médula Ósea/fisiología , Movimiento Celular/fisiología , Proliferación Celular/fisiología , Supervivencia Celular/fisiología , Células Endoteliales/fisiología , Femenino , Glicoproteínas/metabolismo , Miembro Posterior/irrigación sanguínea , Isquemia/fisiopatología , Proteínas de la Membrana/metabolismo , Ratones Noqueados , MicroARNs/metabolismo , Fosfohidrolasa PTEN/metabolismo , Comunicación Paracrina/fisiología , Péptidos/metabolismo , Proteínas Proto-Oncogénicas c-kit/metabolismo , Células del Estroma/fisiología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo

RESUMEN

BACKGROUND: Vitamin D has been shown to induce beneficial effects on cardiovascular and renal morbidity by regulating inflammation and tissue fibrosis. OBJECTIVES: To evaluate the effect of vitamin D analogues on cardiac function and fibrosis in an animal model of cardiorenal syndrome. METHODS: Unilateral nephrectomy was performed and myocardial infarction induced in rats. The rats were treated with vitamin D receptor activator (VDRA, paricalcitol, 40 ng/250 g x 3/week) versus a vehicle. A third group of animals, which served as the control, underwent sham surgery and received no treatment. After 4 weeks of treatment, cardiac function and fibrosis were assessed by trans-thoracic echo and histology, respectively. As a parameter of systemic inflammation, previously shown to be altered in acute coronary syndrome, T regulatory (Treg) cell levels were measured by flow cytometry. Renal dysfunction was documented by standard laboratory tests. RESULTS: After 4 weeks of treatment, no significant improvement in cardiac function parameters was noted following VDRA administration. VDRA treatment did not significantly alter Treg cell systemic levels. Consistently, despite a trend toward less extent of myocardial fibrosis, we found no clear beneficial effects of VDRA on myocardial tissue inflammation and remodeling. CONCLUSIONS: Vitamin D treatment showed no beneficial effects on cardiac function parameters and fibrosis in an animal model of cardiorenal syndrome.

Asunto(s)

Síndrome Cardiorrenal/tratamiento farmacológico , Ergocalciferoles/farmacología , Inflamación/tratamiento farmacológico , Linfocitos T Reguladores/metabolismo , Animales , Síndrome Cardiorrenal/fisiopatología , Modelos Animales de Enfermedad , Fibrosis , Citometría de Flujo , Inflamación/fisiopatología , Masculino , Ratas , Ratas Endogámicas Lew , Resultado del Tratamiento

RESUMEN

BACKGROUND: Atherosclerosis is a well-established inflammatory disease in which T helper 1 (Th1) cells play a key role. Regulatory T (Treg) cells drive a shift from Th1 to Th2 response and were shown to be reduced in atherosclerosis. ST2/interleukin (IL)-33 signal was found to promote Th2 response, attenuating atherosclerotic plaque progression. OBJECTIVES: To evaluate the effect of IL-33 on Treg cell number. METHODS: We employed flow cytometry to determine Treg cell number, as well as ST2 levels, among splenocytes of C57BL/61 vs ApoE-/- mice. Soluble ST2 (sST2) levels were detected by enzyme-linked immunosorbent assay. RESULTS: IL-33 contributed to an increase in Treg cells, but this association was attenuated in ApoE knockout (ApoE-/-) atherosclerotic mice. As a possible mechanism we demonstrated a reduction in the levels of CD4+ST2+ cells by flow cytometry, which is cotemporary to the previously described decrease in Treg cells in ApoE-/- mice. Additionally, the serum level of the soluble ST2 (sST2) decoy receptor was higher in ApoE-/- mice than in control animals. CONCLUSIONS: Our results suggest that a repressed ST2/ IL-33 signaling may contribute to the decrease in Treg cells observed in atherosclerosis.

Asunto(s)

Aterosclerosis/inmunología , Inmunidad Celular , Interleucinas/sangre , Linfocitos T Reguladores/inmunología , Animales , Aterosclerosis/sangre , Aterosclerosis/patología , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Interleucina-33 , Recuento de Linfocitos , Masculino , Ratones , Ratones Endogámicos C57BL , Linfocitos T Reguladores/metabolismo , Linfocitos T Reguladores/patología

RESUMEN

BACKGROUND: Cardiac events are the main cause of death among patients with end-stage renal failure. Even a mild renal disease is currently considered a major risk factor for cardiovascular complications following myocardial infarction (MI). The aim of the present study was to detect histological, sera and urine characteristics of kidney injury in cardiorenal syndrome (CRS) compared to chronic kidney disease (CKD) with an intact cardiac function. METHODS: We employed a rat model for CRS, in which an acute MI (AMI) was induced 4 weeks after establishment of subtotal nephrectomy. Four weeks later, left ventricular function was assessed by echocardiography and changes in renal performance were examined using histological and biochemical parameters. RESULTS: Increased interstitial fibrosis as well as renal inflammation were observed in renal sections derived from CRS rats, compared to subtotal nephrectomy (CKD)-only animals. Moreover, we found that even though AMI on the background of CKD was not associated with a further decrease in creatinine clearance or a further increase in sera BUN levels compared to CKD only, a significant long-term elevation in urine neutrophil gelatinase-associated lipocalin (Ngal) levels was detectable post-MI induction. CONCLUSIONS: AMI in the CKD setting is associated with accelerated renal fibrosis and long-term elevated urine Ngal values, suggesting that cardiac dysfunction contributes to accelerated intrinsic kidney injury in CKD. The data indicate that elevated urine Ngal may potentially serve as an early non-invasive laboratory parameter for a left ventricular dysfunction-related renal injury.

Asunto(s)

Proteínas de Fase Aguda/orina , Síndrome Cardiorrenal/orina , Lipocalinas/orina , Infarto del Miocardio/orina , Proteínas Proto-Oncogénicas/orina , Insuficiencia Renal Crónica/orina , Animales , Biomarcadores/orina , Síndrome Cardiorrenal/epidemiología , Síndrome Cardiorrenal/patología , Modelos Animales de Enfermedad , Fibrosis/epidemiología , Fibrosis/patología , Fibrosis/orina , Riñón/fisiología , Lipocalina 2 , Masculino , Infarto del Miocardio/epidemiología , Infarto del Miocardio/patología , Nefrectomía , Ratas , Ratas Endogámicas Lew , Insuficiencia Renal Crónica/epidemiología , Insuficiencia Renal Crónica/patología , Factores de Riesgo , Disfunción Ventricular Izquierda/epidemiología , Disfunción Ventricular Izquierda/patología , Disfunción Ventricular Izquierda/orina

RESUMEN

OBJECTIVE: The LIM-homeobox transcription factor Isl1 plays a crucial role during heart embryogenesis and later on gives rise to adult resident cardiac stem cells. In this study, we aimed to discover new extra cardiac populations of Isl1 stem cells. We then investigated endogenous Isl1 kinetics after myocardial infarction (MI), and the effect of intra-myocardial gene transfer of naked DNA encoding Isl1 on functional recovery after MI. METHODS: We used the transgenic mice Isl1/cre/Z/EG for lineage tracing of extra cardiac Isl1 stem cells. Non transgenic mice were used to study Isl1 kinetics post-MI by RT-PCR and FACS analysis. MI was induced in non transgenic mice by permanent ligation of the left anterior descending coronary artery (LAD). Naked DNA encoding Isl1 was injected to the peri-infarct region. Evaluation of cardiac performance was conducted by echocardiogram. Analysis of myocardial fibrosis and number of vessels was performed on histological cryosections. RESULTS AND CONCLUSIONS: Isl1 gives rise to subpopulations of progenitors in both the bone marrow and spleen, and is re-expressed in the spleen and left ventricle following MI. Intramyocardial gene transfer of Isl1 to the border zone of the infarcted hearts resulted in partial salvage of left ventricular function, enhanced vascularization, and reduced myocardial fibrosis. The Isl1 gene appears to be an attractive reparative target for future management of myocardial dysfunction.

Asunto(s)

Terapia Genética , Proteínas con Homeodominio LIM/metabolismo , Infarto del Miocardio/terapia , Miocardio/metabolismo , Células Madre/metabolismo , Factores de Transcripción/metabolismo , Animales , Células de la Médula Ósea/metabolismo , Linaje de la Célula , Separación Celular/métodos , Células Cultivadas , Modelos Animales de Enfermedad , Fibrosis , Citometría de Flujo , Técnicas de Transferencia de Gen , Inyecciones , Cinética , Proteínas con Homeodominio LIM/genética , Masculino , Ratones , Ratones Transgénicos , Infarto del Miocardio/diagnóstico por imagen , Infarto del Miocardio/genética , Infarto del Miocardio/metabolismo , Infarto del Miocardio/fisiopatología , Miocardio/patología , Neovascularización Fisiológica , Reacción en Cadena de la Polimerasa , Recuperación de la Función , Bazo/metabolismo , Células Madre/patología , Factores de Transcripción/genética , Transfección , Ultrasonografía , Función Ventricular Izquierda

RESUMEN

The LIM-homeobox transcription factor islet-1 (Isl1) marks a cell population which gives rise to myocardial, pacemaker, endothelial and smooth muscle cells, which are derived from the secondary heart field during heart embryogenesis. Isl1+ precursors have the potential of self-renewal and differentiation into endothelial, cardiomyocyte and smooth muscle lineages. The primary objective of this study was to determine whether retroviral gene delivery of Isl1 to endothelial cells and mesenchymal stem cells (MSCs) could promote angiogenic and vasculogenic properties. To this end, endothelial cells and rat MSCs were retrovirally transduced to express Isl1. Isl1 expression in endothelial cells resulted in enhanced proliferation and adhesion to fibronectin. In addition, increased IL-1b and VEGF secretion was evident in Isl1 transduced endothelial cells, concomitant with increased migratory and tube formation properties of the endothelial cells. Isl1 expression in MSCs promoted their vasculogenic properties and resulted in enhanced in vitro tube formation. Finally, Isl1 expressing endothelial cells induced enhanced in vivo vascularisation in C57BL/6J mice. These data suggest, for the first time, that Isl1 promotes postnatal angiogenesis and vasculogenesis by improving the angiogenic properties of endothelial cells and MSCs.

Asunto(s)

Células Endoteliales/metabolismo , Proteínas de Homeodominio/biosíntesis , Células Madre Mesenquimatosas/metabolismo , Neovascularización Fisiológica , Animales , Adhesión Celular , Línea Celular , Movimiento Celular , Proliferación Celular , Medios de Cultivo Condicionados/metabolismo , Células Endoteliales/trasplante , Fibronectinas/metabolismo , Vectores Genéticos , Proteínas de Homeodominio/genética , Interleucina-1beta/metabolismo , Proteínas con Homeodominio LIM , Masculino , Ratones , Ratones Endogámicos C57BL , Fenotipo , Ratas , Ratas Wistar , Retroviridae/genética , Factores de Tiempo , Factores de Transcripción , Transducción Genética , Factor A de Crecimiento Endotelial Vascular/metabolismo

RESUMEN

BACKGROUND: Myocarditis is an inflammatory disorder of the heart in which T lymphocytes have a central role. No effective treatment is currently at hand for management of the myocarditis. Lymphocyte function requires the active signal transducer Ras. We thus hypothesized that S-farnesylthiosalicylic acid (FTS), a synthetic small molecule that detaches Ras from the inner cell membrane and induces its rapid degradation, will attenuate experimental autoimmune myocarditis (EAM). METHODS AND RESULTS: Two groups of Lewis rats were induced to develop EAM by immunization with porcine cardiac myosin. Group A received 5 mg/kg of FTS, and group B received phosphate-buffered saline (PBS) according to two protocols: FTS or PBS was given 2 days before myosin immunization in protocol 1 and FTS or PBS was given 14 days after myosin immunization in protocol 2. FTS significantly suppressed myocarditis, and this effect was accompanied by a reduction in myosin-specific cellular and humoral immune responses. In the longer regimen, FTS treatment for 6 weeks was associated with preservation of myocardial function made evident by echocardiography. In vitro, FTS significantly attenuated the proliferation of lymphocytes from untreated myocarditic rats to myosin. CONCLUSIONS: FTS is effective in suppressing the progression of EAM and its consequent functional myocardial dysfunction. The effect may be mediated by suppression of the cellular and humoral responses to myosin.

Asunto(s)

Enfermedades Autoinmunes/tratamiento farmacológico , Inhibidores Enzimáticos/farmacología , Farnesol/análogos & derivados , Miocarditis/tratamiento farmacológico , Salicilatos/farmacología , Proteínas ras/antagonistas & inhibidores , Animales , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/patología , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Farnesol/farmacología , Corazón/efectos de los fármacos , Ganglios Linfáticos/efectos de los fármacos , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/patología , Activación de Linfocitos/efectos de los fármacos , Miocarditis/inmunología , Miocarditis/patología , Miocardio/inmunología , Miocardio/patología , Miosinas/inmunología , Ratas , Ratas Endogámicas Lew , Transducción de Señal/efectos de los fármacos , Porcinos/inmunología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T/patología

RESUMEN

Myocardial injury, developed after a period of ischemia/reperfusion (I/R) results in the destruction of functional heart tissue, this being replaced by scar tissue. Intracellular signaling pathways mediating cardiomyocyte death are partially understood and involve the activation of Ras. p38-MAPK, JNK and Mst-1 are downstream effectors of Ras protein. We hypothesized that S-farnesylthiosalicylic acid (FTS), a synthetic small molecule that detaches Ras from the inner cell membrane, consequently inhibiting Ras activity, reduces I/R myocardial injury in vitro and in vivo. Wistar rat hearts were isolated, mounted on the Langendorff apparatus and subjected to ischemia (30 min, 37 degrees C) and reperfusion. During the reperfusion period, the hearts were perfused with FTS (1 microM) solution or control buffer. Left anterior descending (LAD) ligation and subsequent reperfusion was performed in two groups of Wistar rats. Rats received 5mg/kg FTS or PBS according to two protocols: (A) FTS or PBS were administered daily 7 days prior, immediately before and 14 days (every other day) after LAD occlusion or (B) every other day for 14 days post-I/R. Hearts from FTS-treated rats (Langendorff) and FTS-treated rats (protocol A) showed a significant improvement in myocardial performance and smaller scar tissue compared with the PBS group. Infarct size in the FTS-treated group was 12.7+/-2% vs. 23.7+/-4% in the PBS-treated (in vitro) group and 17.3+/-2.5% vs. 36+/-7% compared with control I/R rats (in vivo) p<0.05. These effects may be associated with the down regulation of JNK as a short-term effector and with Mst-1 in the long-term remodeling process.

Asunto(s)

Farnesol/análogos & derivados , Daño por Reperfusión Miocárdica/prevención & control , Miocardio/metabolismo , Salicilatos/farmacología , Proteínas ras/antagonistas & inhibidores , Animales , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Farnesol/administración & dosificación , Farnesol/farmacología , Pruebas de Función Cardíaca , Técnicas In Vitro , Masculino , Daño por Reperfusión Miocárdica/metabolismo , Ratas , Ratas Endogámicas Lew , Ratas Wistar , Salicilatos/administración & dosificación

RESUMEN

AIM: Clopidogrel is a widely used anti-thrombotic for the prevention of stent thrombosis and cardiovascular events in patients with coronary atherosclerosis. Clopidogrel has been shown to exhibit anti-inflammatory effects that are related to the attenuated activation of platelets. Atherosclerosis is a complex process in which the immune system and the endothelium appear to play a prominent role. Herein, we tested the hypothesis that clopidogrel will influence plaque size and composition in the atherosclerosis prone apolipoprotein E knockout (apoE KO) mouse model. METHODS AND RESULTS: Eight week old mice were fed daily with either PBS, 1 mg or 2 mg of clopidogrel for 10 weeks. Plaque size was evaluated in the aortic sinus and cellular and humoral responses were studied as well as splenic and bone marrow endothelial progenitors by FACS. Treatment with either 1 mg and 2 mg of clopidogrel significantly reduced plaque size and augmented its stability by increasing atheromatous fibrous area. Whereas antigen specific oxLDL immune response was not influenced by clopidogrel feeding, the number of atheroprotective regulatory CD4+CD25+ T cells was significantly increased. Moreover, clopidogrel treatment resulted in a prominent rise in splenic but not bone marrow derived Sca-1+/flk-1+ endothelial progenitors. CONCLUSION: Clopidogrel significantly reduces atheroma burden and stabilizes aortic sinus plaques in apoE KO mice. These effects may partially be mediated by upregulation of the regulatory T cell pool and splenic endothelial progenitor cells. These findings may expand the potential applications of clopidogrel in human subjects.

Asunto(s)

Apolipoproteínas E , Aterosclerosis/tratamiento farmacológico , Inhibidores de Agregación Plaquetaria/farmacología , Seno Aórtico/efectos de los fármacos , Ticlopidina/análogos & derivados , Animales , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Aterosclerosis/patología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Clopidogrel , Modelos Animales de Enfermedad , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Femenino , Fibrosis/inducido químicamente , Fibrosis/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Seno Aórtico/patología , Bazo/citología , Bazo/efectos de los fármacos , Células Madre/citología , Células Madre/efectos de los fármacos , Linfocitos T Reguladores/patología , Ticlopidina/farmacología

RESUMEN

BACKGROUND: Lymphocytes play an important role in the progression of atherosclerosis. Recently, hypoxia inducible factor-1 (HIF-1) was found to attenuate inflammation by regulating T cell activation and cytokine production. We studied the effects of overexpression of HIF-1alpha in ApoE knockout murine lymphocytes, on experimental atherosclerosis. METHODS AND RESULTS: ApoE-/- mice were submitted to intravenous hydrodynamic injection of empty plasmid or HIF-1alphaP564A (HIF-1alpha mutated stabilized construct). Robust expression of HIF-1alpha was evident in spleen cells of recipient animals. Increased expression of IL-10 as well as decreased expression of IFN-gamma was measured in splenocytes of HIF-1alpha-treated mice by RT-PCR. One week postinjection, antibody array analysis revealed a pattern consistent with a T helper 1 to T helper 2 shift. On sacrifice, assessment of aortic sinus lesions revealed a significant reduction in plaque size in HIF-1alpha injected mice. A reduced expression of IFN-gamma was evident in CD4+ spleen-derived lymphocytes and aortas of HIF-1alpha-injected mice. CONCLUSIONS: HIF-1alpha expression in mouse lymphocytes is associated with a reduced IFN-gamma expression and attenuation of experimental atherosclerosis.

Asunto(s)

Aterosclerosis/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Interferón gamma/metabolismo , Animales , Aorta/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Células Cultivadas , Modelos Animales de Enfermedad , Humanos , Masculino , Ratones , Bazo/metabolismo , Células Th2/metabolismo

RESUMEN

OBJECTIVE: While both play a role in the transcriptional response of hypoxic endothelial cells (ECs), hypoxia-inducible factor-1alpha (HIF-1alpha) and HIF-2alpha differ in their transactivation sites, pointing at potentially different target genes. We studied the discrete and common effects of HIF-1alpha and HIF-2alpha on the cytokine expression and vasculogenic properties of ECs. METHODS AND RESULTS: H5V and bovine aortic ECs were transfected to express HIF-1alpha, HIF-2alpha or both. Overexpression of HIF-1alpha or HIF-2alpha and, to a greater extent, cotransfection of HIF-1alpha and HIF-2alpha resulted in EC activation, as revealed by analysis of the adhesion capacities and adhesion molecule surface expression of ECs. From the paracrine aspect, conditioned medium from HIF-expressing ECs was found to promote the migration and tube formation capacity of wild-type ECs, mostly following HIF-1alpha and HIF-2alpha coexpression. Antibody arrays revealed altered expression of multiple cytokines, pointing at consistent additive effects of HIF-1alpha and HIF-2alpha on angiogenic protein expression. Finally, HIF-1alpha and HIF-2alpha additively promoted vessel formation in vivo, as demonstrated by a Matrigel angiogenesis assay. CONCLUSION: Our results further clarify the functional roles of HIF-1alpha and HIF-2alpha in ECs and for the first time demonstrate a common contribution of HIF-1alpha and HIF-2alpha to vasculogenesis.

Asunto(s)

Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Células Endoteliales/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Neovascularización Fisiológica , Proteínas Angiogénicas/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Bovinos , Adhesión Celular , Moléculas de Adhesión Celular/metabolismo , Hipoxia de la Célula , Movimiento Celular , Células Cultivadas , Medios de Cultivo Condicionados/metabolismo , Citocinas/metabolismo , Células Endoteliales/inmunología , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Comunicación Paracrina , Transfección

RESUMEN

BACKGROUND: Circulating CD34+ endothelial progenitor cells (EPCs) are capable of differentiating into mature endothelial cells to assist in angiogenesis and vasculogenesis. We sought to quantify the numbers of apoptotic progenitors in patients with congestive heart failure. METHODS AND RESULTS: Peripheral blood mononuclear cells were isolated by Ficoll density-gradient from 58 patients with various degrees of heart failure and 23 matched controls. Apoptosis in progenitor CD34+ cells was assessed using the Annexin V-PE/PI detection kit, and FACS analysis was performed with triple staining for CD34, annexin-V and propidium iodide. The percentage of early and late apoptotic progenitor cells was determined in the subject groups and was correlated with clinical characteristics. While there was no significant difference in total CD34 positive cells or early apoptotic progenitors between control subjects and CHF patients (p = 0.42) or between severe and mild/moderate CHF groups (p = 0.544), there was an elevated number of late apoptotic progenitors in the severe CHF group compared with the mild/moderate CHF group (p = 0.03). Late apoptotic progenitors were significantly increased in CHF patients as compared to matched controls. There was also an inverse correlation between late apoptotic progenitors and ejection fraction (r = -0.252, p = 0.028) as well as a positive association with NYHA class (r = 0.223, p = 0.046). CONCLUSION: Severe heart failure patients exhibited higher numbers of late apoptotic progenitors, and this was positively associated with NYHA class and negatively correlated with ejection fraction. This finding may shed light on the numerous factors governing the pathophysiology of CHF.

Asunto(s)

Apoptosis , Insuficiencia Cardíaca/sangre , Insuficiencia Cardíaca/patología , Células Madre/citología , Adulto , Anciano , Anciano de 80 o más Años , Antígenos CD34/biosíntesis , Diferenciación Celular , Femenino , Humanos , Leucocitos Mononucleares/citología , Masculino , Persona de Mediana Edad , Modelos Biológicos , Neovascularización Patológica

RESUMEN

Bone marrow stromal cells (BMSCs) contain progenitors capable of participating in postnatal angiogenesis. Hypoxia-inducible factors (HIFs) mediate endothelial activation by driving the expression of multiple angiogenic factors. We explored the potential of HIF-1alpha and HIF-2alpha modification in BMSCs, as a tool to improve cell-based angiogenic therapy. BMSCs were retrovirally transduced to express stable forms of HIF-1alpha and HIF-2alpha. HIF-1alpha and, to a greater extent, HIF-2alpha overexpression promoted differentiation of BMSCs to the endothelial lineage, evident by CD31 and Tie-2 expression and improved adhesive properties. Whereas chemotaxis toward stromal-derived factor 1 was higher in both HIF-alpha-expressing BMSCs, enhanced migration toward vascular endothelial growth factor was found only following overexpression of HIF-2alpha, supported by a robust expression of its receptor, Flk-1. HIF-alpha expression was associated with upregulation of angiogenic proteins and improved tube formation. Cytokine arrays of endothelial cells stimulated by medium collected from HIF-alpha-expressing BMSCs revealed further angiogenic activation and improved adhesive capacity. Eventually, delivery of HIF-2alpha-transduced BMSCs induced a more robust angiogenic response, compared with sham-transduced or HIF-1alpha-transduced BMSCs in the corneal micropocket angiogenesis model. Our results support the use of HIF-alpha genes, particularly HIF-2alpha, to augment the efficacy of future cell-based therapy. Disclosure of potential conflicts of interest is found at the end of this article.

Asunto(s)

Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Células de la Médula Ósea/citología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Neovascularización Fisiológica , Células del Estroma/metabolismo , Animales , Células de la Médula Ósea/enzimología , Adhesión Celular , Diferenciación Celular , Movimiento Celular , Células Cultivadas , Quimiocina CXCL12/metabolismo , Células Endoteliales/citología , Células Endoteliales/metabolismo , Humanos , Masculino , Ratones , Comunicación Paracrina , Ratas , Ratas Wistar , Receptores CXCR4/metabolismo , Retroviridae , Células del Estroma/citología , Células del Estroma/enzimología , Transducción Genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo