RESUMO
Low-level laser therapy is a clinically well established tool for enhancement of wound healing. In vitro studies have also shown that low level laser therapy has a biostimulatory effect on cells of different origin. The aim of this in vitro study was to investigate the initial effect of low-level laser therapy on growth and differentiation of human osteoblast-like cells. SaOS-2 cells were irradiated with laser doses of 1 J/cm2 and 2 J/cm2 using a diode laser with 670 nm wave length and an output power of 400 mW. Untreated cells were used as controls. At 24 h, 48 h and 72 h post irradiation, cells were collected and assayed for viability of attached cells and alkaline phosphatase specific activity. In addition, mRNA expression levels of osteopontin and collagen type I were assessed using semi-quantitative RT-PCR. Over the observation period, cell viability, alkaline phosphatase activity and the expression of osteopontin and collagen type I mRNA were slightly enhanced in cells irradiated with 1 J/cm2 compared with untreated control cells. Increasing the laser dose to 2 J/cm2 reduced cell viability during the first 48 h and resulted in persistently lower alkaline phosphatase activity compared with the other two groups. The expression of osteopontin and collagen type I mRNA slightly decreased with time in untreated controls and cells irradiated with 1 J/cm2, but their expression was increased by treatment with 2 J/cm2 after 72 h. These results indicate that low-level laser therapy has a biostimulatory effect on human osteoblast-like cells during the first 72 h after irradiation. Further studies are needed to determine the potential of low-level laser therapy as new treatment concept in bone regeneration.
Assuntos
Diferenciação Celular/efeitos da radiação , Divisão Celular/efeitos da radiação , Terapia com Luz de Baixa Intensidade , Osteoblastos/efeitos da radiação , RNA Mensageiro/genética , Fosfatase Alcalina/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos da radiação , Colágeno Tipo I/genética , Expressão Gênica/efeitos da radiação , Humanos , Técnicas In Vitro , Lasers Semicondutores , Osteopontina/genética , Dosagem Radioterapêutica , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
OBJECTIVE: A number of studies have shown that statins decrease morbidity and mortality in patients with cardiovascular diseases. The anti-inflammatory effects of statins have recently been implicated in the clinical benefit that can be obtained in the treatment of atherosclerosis. Little is known about the mechanisms by which statins counteract inflammation. METHODS AND RESULTS: In this study, we asked whether simvastatin can influence in vitro and in vivo production of the proinflammatory cytokines interleukin (IL)-6, IL-8, and monocyte chemoattractant protein-1. A total of 107 hypercholesterolemic patients were treated with simvastatin. As measured by ELISA, serum levels of cytokines significantly decreased after 6 weeks of treatment (P<0.05). Furthermore, simvastatin decreased the expression of IL-6, IL-8, and monocyte chemoattractant protein-1 mRNA in peripheral blood mononuclear cells. Similar results were obtained in vitro by using cultured human umbilical vein endothelial cells and peripheral blood mononuclear cells from healthy normolipemic donors. Exposure to simvastatin, atorvastatin, or cerivastatin caused downregulation of the expression of cytokine mRNA in a time- and dose-dependent manner. Furthermore, all statins tested were able to reduce the concentrations of cytokines in cellular and extracellular fractions of human umbilical vein endothelial cells (P<0.05). CONCLUSIONS: Our data show that simvastatin is anti-inflammatory through the downregulation of cytokines in the endothelium and leukocytes. These effects may explain some of the clinical benefits of these drugs in the treatment of atherosclerosis.
Assuntos
Quimiocina CCL2/antagonistas & inibidores , Hipercolesterolemia/metabolismo , Interleucina-6/antagonistas & inibidores , Interleucina-8/antagonistas & inibidores , Monócitos/efeitos dos fármacos , Sinvastatina/farmacologia , Idoso , Anticolesterolemiantes/farmacologia , Anticolesterolemiantes/uso terapêutico , Células Cultivadas , Quimiocina CCL2/biossíntese , Quimiocina CCL2/sangue , Regulação para Baixo/efeitos dos fármacos , Endotélio Vascular/química , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Feminino , Humanos , Hipercolesterolemia/sangue , Hipercolesterolemia/tratamento farmacológico , Interleucina-6/biossíntese , Interleucina-6/sangue , Interleucina-8/biossíntese , Interleucina-8/sangue , Leucócitos/química , Leucócitos/efeitos dos fármacos , Leucócitos/metabolismo , Metabolismo dos Lipídeos , Lipídeos/sangue , Masculino , Monócitos/química , Monócitos/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/metabolismo , Sinvastatina/uso terapêutico , Veias Umbilicais/citologiaRESUMO
OBJECTIVE: The intercellular adhesion molecule-1 (ICAM-1/CD54) and its ligand, CD11a/CD18, mediate endothelial adhesion of leukocytes and their consecutive transmigration. Anti-inflammatory effects of statins are considered to be exerted in part through inhibition of leukocyte-endothelial interactions. We investigated the in vivo effects of simvastatin treatment in hypercholesterolemic patients and the influence of various statins on expression of cellular adhesion molecules in vitro. METHODS AND RESULTS: A total number of 107 hypercholesterolemic patients were treated with 20 mg (n=52) or 40 mg (n=55) of simvastatin daily. After 6 weeks of treatment, peripheral blood mononuclear cells (PBMCs) expressed lower amounts of CD54-, CD18-, and CD11a-mRNA compared with pretreatment values. Surface expression of CD54 and CD18/CD11a on CD14+-monocytes also decreased significantly in both groups of patients. Moreover, simvastatin, atorvastatin, and cerivastatin were found to downregulate tumor necrosis factor (TNF)-alpha-induced expression of CD54 and CD18/CD11a in isolated PBMCs obtained from normal donors as well as TNF-alpha-dependent expression of these CAMs in cultured human umbilical vein endothelial cells (HUVECs). Furthermore, all three statins were found to reduce the binding of PBMCs to TNF-alpha-stimulated HUVECs in vitro. CONCLUSIONS: Statin-induced inhibition of expression of CD54 and CD18/CD11a in PBMCs and HUVECs with consecutive loss of adhesive function may contribute to the anti-inflammatory effects of these drugs and some of their beneficial clinical activities.
Assuntos
Moléculas de Adesão Celular/efeitos dos fármacos , Hipercolesterolemia/tratamento farmacológico , Hipercolesterolemia/metabolismo , Monócitos/metabolismo , Sinvastatina/administração & dosagem , Fator de Necrose Tumoral alfa/metabolismo , Idoso , Antígenos CD11/metabolismo , Antígenos CD18/metabolismo , Moléculas de Adesão Celular/metabolismo , Células Cultivadas , Regulação para Baixo/efeitos dos fármacos , Endotélio Vascular/metabolismo , Feminino , Humanos , Molécula 1 de Adesão Intercelular/biossíntese , Molécula 1 de Adesão Intercelular/metabolismo , Lipoproteínas LDL/sangue , Lipoproteínas LDL/efeitos dos fármacos , Antígeno-1 Associado à Função Linfocitária/biossíntese , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/efeitos dos fármacos , Veias UmbilicaisRESUMO
Recent data suggest that the statins, apart from their lipid-lowering activity, exhibit profound anti-inflammatory effects. Basophils are major proinflammatory effector cells in diverse pathologic reactions. We have examined the in vitro effects of five different statins on primary human basophils, their progenitors, and the basophil cell line KU-812. Preincubation of blood basophils with cerivastatin or atorvastatin (0.1-100 microM) for 24 h reduced their capacity to release histamine on immunoglobulin E (IgE)-dependent stimulation in a dose-dependent manner. These statins also inhibited IgE-dependent up-regulation of the basophil-activation antigen CD203c. Moreover, both statins suppressed interleukin-3-induced differentiation of basophils from their progenitors as well as (3)H-thymidine uptake in KU-812 cells. All inhibitory effects of cerivastatin and atorvastatin were reversed by mevalonic acid (200 microM). The other statins tested (lovastatin, simvastatin, pravastatin) did not show significant inhibitory effects on basophils. Together, these data identify cerivastatin and atorvastatin as novel inhibitors of growth and activation of human basophils.
Assuntos
Basófilos/efeitos dos fármacos , Ácidos Heptanoicos/farmacologia , Liberação de Histamina/efeitos dos fármacos , Diester Fosfórico Hidrolases/efeitos dos fármacos , Piridinas/farmacologia , Pirofosfatases/efeitos dos fármacos , Pirróis/farmacologia , Antígenos CD/análise , Atorvastatina , Basófilos/citologia , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Humanos , Imunoglobulina E/imunologia , Interleucina-3/fisiologia , Diester Fosfórico Hidrolases/biossíntese , Pirofosfatases/biossínteseRESUMO
The presence of hepatitis C virus (HCV) in normal cervical smears (CS) obtained from 22 HCV-seropositive and 50 HCV-seronegative patients was assessed by reverse-transcriptase-polymerase chain reaction (RT-PCR). The presence of HCV in serum was established by use of enzyme-linked immunosorbent assay, Western blot test, and RT-PCR. HCV was detected in 36.4% (n=8) of CS cells recovered from 22 HCV-seropositive patients, but not in CS samples obtained from 50 HCV-seronegative patients. Furthermore, cells from the CS of 2 seropositive/smear-positive patients and 1 seropositive/smear-negative patient were isolated; HCV RNA was detectable in the cervical lymphocytes of the 2 smear-positive patients, but not in epithelial cells or granulocytes. HCV RNA is detectable in the CS of some HCV-seropositive women. The clinical importance of these data requires further study.
Assuntos
Colo do Útero/virologia , Hepacivirus/isolamento & purificação , Hepatite C/transmissão , RNA Viral/análise , Adulto , Feminino , Hepacivirus/genética , Hepatite C/virologia , Humanos , RNA Viral/sangue , Testes Sorológicos , Infecções Sexualmente Transmissíveis/virologia , Esfregaço VaginalRESUMO
Activation of the endothelium, platelets and leukocytes has been shown to play an important role in the aetiology of deep venous thrombosis (DVT) in in-vitro experiments, resulting in the release of soluble cell adhesion molecules (sCAMs). We therefore assessed the value of soluble intracellular adhesion molecule-1 (sICAM-1), soluble vascular cell adhesion molecule-1 (sVCAM-1), soluble E-selectin and soluble P-selectin for the diagnostic process in 69 consecutive patients with suspected DVT. Final diagnosis was based on the results of Duplex sonography or ascending venography. Thirty-seven patients (53.6%) finally suffered from DVT. Mean levels of sVCAM-1 were 589 +/- 530 ng/ml for controls and 587 +/- 328 ng/ml for patients. Corresponding levels concerning sICAM-1 were 316 +/- 161 and 342 +/- 186 ng/ml, those concerning soluble E-selectin were 54 +/- 38 and 42 +/- 18 ng/ml, and those concerning soluble P-selectin were 94 +/- 37 and 99 +/- 36 ng/ml (all P > 0.05). There was no significant correlation of the thrombus extension (all P > 0.05) or the duration of symptoms with sCAMs (all P > 0.05). In conclusion, we detected no significant differences concerning the concentration of four major sCAMs between patients with DVT and controls, so their assessment does not add any useful information for the diagnostic process of DVT.
Assuntos
Moléculas de Adesão Celular/fisiologia , Trombose Venosa/diagnóstico , Trombose Venosa/etiologia , Adulto , Idoso , Biomarcadores/sangue , Estudos de Casos e Controles , Moléculas de Adesão Celular/sangue , Selectina E/sangue , Feminino , Humanos , Molécula 1 de Adesão Intercelular/sangue , Masculino , Pessoa de Meia-Idade , Selectina-P/sangue , Flebografia , Solubilidade , Ultrassonografia Doppler Dupla , Molécula 1 de Adesão de Célula Vascular/sangueRESUMO
The objective of the present study was to evaluate the role of skin tissue cholesterol (SkinTc) in predicting the presence of atherosclerosis. SkinTc concentrations were determined in 318 consecutive patients by using the non-invasive PREVU POC Skin Sterol Test. Additionally, a complete lipid status and cardiovascular risk profile according to the PROCAM and Framingham scores as well as an evaluation by carotid duplex sonography and ankle-brachial blood pressure index testing was obtained from all patients. SkinTc concentrations did not differ significantly among patients suffering from cerebrovascular disease (CVD) and peripheral arterial disease (PAD) compared to the corresponding control groups and among patients with a calculated cardiovascular risk > 10% in 10 years compared to patients with a risk < 10% (all p > 0.05). Additionally, SkinTc concentrations were not significantly higher in the 245 patients with at least one documented atherosclerotic disease compared with the remaining 73 patients without evidence of atherosclerosis. In conclusion, SkinTc concentrations determined by the PREVU POC Skin SterolTest are not related to the presence of CVD and PAD or to an elevated cardiovascular risk, indicating that this parameter cannot be used as a reliable indicator of atherosclerosis.
Assuntos
Aterosclerose/diagnóstico , Colesterol/análise , Pele/metabolismo , Idoso , Análise de Variância , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Estudos Prospectivos , Análise de Regressão , Medição de Risco , Fatores de Risco , Sensibilidade e Especificidade , Pele/química , Testes CutâneosRESUMO
Vascular endothelial growth factor plays a pivotal role in the progression of atherosclerotic lesions and causes instability of atherosclerotic plaques by inducing neoangiogenesis inside the current plaque. The pro-inflammatory cytokine interleukin (IL-) 6 induces vascular endothelial growth factor in smooth muscle cells (SMC). HMG-CoA reductase inhibitors (statins), display beside their lipid-lowering potency various pleiotropic effects. Such pleiotropic effects include improvement of endothelial dysfunction, increased nitric oxide bioavailability, antioxidant properties, inhibition of inflammatory responses, and stabilization of atherosclerotic plaques. In this study we investigate the influence of statin treatment on the serum levels of VEGF in hypercholesterolemic patients. One hundred and seven hypercholesterolemic patients were treated with 20 (n = 52) or 40 mg (n = 55) simvastatin daily. Six weeks of treatment resulted in a significant decrease of VEGF from 1017.1 +/- 297.8 pg/mL at baseline to 543.5 +/- 317.4 pg/mL after 6 weeks (-47.7%) and to 211.8 +/- 155.3 pg/mL after 6 months (-79.7%; all P < 0.001). IL-6 induced the expression of vascular endothelial growth factor in human SMC as analyzed by rt-PCR and flow cytometry. Statins decreased the stimulatory effect of IL-6 on mRNA and protein levels. This effect could be inhibited by co-incubation with mevalonate acid. This study contributes in understanding the pleiotropic effects of statins particularly with regard to their use in treatment and prevention of cardiovascular disease.
Assuntos
Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Hipercolesterolemia/tratamento farmacológico , Sinvastatina/farmacologia , Fator A de Crescimento do Endotélio Vascular/sangue , Adulto , Idoso , Quimiocinas/sangue , Citocinas/sangue , Feminino , Humanos , Hipercolesterolemia/sangue , Interleucina-6/antagonistas & inibidores , Lipídeos/sangue , Masculino , Pessoa de Meia-Idade , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , RNA Mensageiro/análise , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Endothelial nitric oxide synthase (eNOS), VEGF, and hypoxia-inducible factor 1-alpha (HIF-1alpha) are important regulators of endothelial function, which plays a role in the pathophysiology of heart failure (HF). PGE1 analog treatment in patients with HF elicits beneficial hemodynamic effects, but the precise mechanisms have not been investigated. We have investigated the effects of the PGE1 analog alprostadil on eNOS, VEGF, and HIF-1alpha expression in human umbilical vein endothelial cells (HUVEC) using RT-PCR and immunoblotting under normoxic and hypoxic conditions. In addition, we studied protein expression by immunohistochemical staining in explanted hearts from patients with end-stage HF, treated or untreated with systemic alprostadil. Alprostadil causes an upregulation of eNOS and VEGF protein and mRNA expression in HUVEC and decreases HIF-1alpha. Hypoxia potently increased eNOS, VEGF, and HIF-1alpha synthesis. The alprostadil-induced upregulation of eNOS and VEGF was prevented by inhibition of MAPKs with PD-98056 or U-0126. Consistently, the expression of eNOS and VEGF was increased, and HIF-1alpha was reduced in failing hearts treated with alprostadil. The potent effects of alprostadil on endothelial VEGF and eNOS synthesis may be useful for patients with HF where endothelial dysfunction is involved in the disease process.
Assuntos
Alprostadil/análogos & derivados , Alprostadil/farmacologia , Células Endoteliais/metabolismo , Fator A de Crescimento do Endotélio Vascular/biossíntese , Vasodilatadores/farmacologia , Western Blotting , Capilares/efeitos dos fármacos , Capilares/enzimologia , Capilares/metabolismo , Cardiomiopatia Dilatada/enzimologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/enzimologia , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/biossíntese , Imuno-Histoquímica , Técnicas In Vitro , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Gravidez , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Cordão Umbilical/citologia , Cordão Umbilical/efeitos dos fármacos , Cordão Umbilical/enzimologiaRESUMO
OBJECTIVE: Statins are potent inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase and widely used to treat hyperlipidaemia. Apart from their direct lipid-lowering effects, statins may also influence lipid metabolism through modulation of low-density lipoprotein (LDL) receptors. Basophils and mast cells have been reported to express LDL receptors and have been implicated in atherogenesis. The aim of this study was to investigate the effects of statins on the interactions of 125I-LDL with purified primary human blood basophils, a human basophil cell line, KU812, and a human mast cell line, HMC-1. METHODS: Direct binding experiments were carried out with the primary basophils and KU812 as well as HMC-1 cells before and after pretreatment of the cells with atorvastatin, simvastatin, or cerivastatin. The effects of these three statins on the LDL-uptake and degradation as well as on thymidine incorporation in the cells were also studied. RESULTS: Primary basophils, HMC-1 and KU812 cells expressed two classes of LDL binding sites. Exposure to atorvastatin, simvastatin or cerivastatin increased significantly ( P<0.05) the number of 125I-LDL binding sites on primary basophils and HMC-1 as well as KU812 cells. The effects of the statins were dose dependent. The statins also enhanced the uptake and degradation of LDL in primary basophils, HMC-1 and KU812 cells. The increase in the number of LDL binding sites induced by statins was abolished by mevalonic acid (200 micromol/l). Statins had no effect on the thymidine incorporation into the cells in an unstimulated condition. CONCLUSION: Our results provide evidence for the upregulation of LDL binding sites on human basophils and mast cells by statins. We hypothesise that effects of statins on the lipid metabolism might also involve basophils and mast cells.
Assuntos
Basófilos/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Mastócitos/metabolismo , Receptores de LDL/biossíntese , Basófilos/efeitos dos fármacos , Sítios de Ligação , Linhagem Celular , Relação Dose-Resposta a Droga , Humanos , Mastócitos/efeitos dos fármacos , Ensaio Radioligante , Receptores de LDL/metabolismo , Timidina/metabolismo , Regulação para CimaRESUMO
BACKGROUND: Mast cells (MCs) are multifunctional effector cells of the immune system. These cells originate from pluripotent hemopoietic progenitors. In contrast to basophils and other leukocytes, MCs exhibit a remarkably long life span (years) in vivo. Although a role for stem cell factor (SCF) and SCF receptor (KIT) in long-term survival of MCs has been proposed, the underlying biochemical mechanisms remain unknown. MATERIALS AND METHODS: We have examined expression of 'survival-related' molecules of the bcl-2 family including bcl-2 and bcl-x(L), in primary human MCs and the human MC line HMC-1. Primary MCs were isolated from dispersed lung tissue by cell sorting using an antibody against KIT. mRNA expression was analyzed by RT-PCR and Northern blotting. RESULTS: As assessed by RT-PCR, purified unstimulated lung MCs (>98% pure) exhibited KIT- and bcl-x(L) mRNA, but did not express bcl-2 mRNA. However, exposure of lung MCS to SCF (100 ng/ml) for 8 h resulted in expression of bcl-2 mRNA. Corresponding results were obtained by immunocytochemistry. In fact, exposure of MC to SCF resulted in expression of the bcl-2 protein whereas unstimulated MCs displayed only the bcl-x(L) protein without expressing the bcl-2 protein. The human MC leukemia cell line HMC-1, which contains a mutated and intrinsically activated SCF receptor, showed constitutive expression of both bcl-2 and bcl-x(L) at the mRNA and protein level. CONCLUSION: Our data show that human MCs can express members of the bcl-2 family. It is hypothesized that bcl-x(L) plays a role in KIT-independent growth of MCs, whereas bcl-2 may be involved in KIT-dependent functions of MCs.