RESUMO
Several clinical trials are exploring therapeutic effect of human CD34(+) cells in ischemic diseases, including myocardial infarction. Unfortunately, most of the cells die few days after delivery. Herein we show that lysophosphatidic acid (LPA)-treated human umbilical cord blood-derived CD34(+) cells cultured under hypoxic and serum-deprived conditions present 2.2-fold and 1.3-fold higher survival relatively to non-treated cells and prostaglandin E2-treated cells, respectively. The pro-survival effect of LPA is concentration- and time-dependent and it is mediated by the activation of peroxisome proliferator-activator receptor γ (PPARγ) and downstream, by the activation of pro-survival ERK and Akt signaling pathways and the inhibition of mitochondrial apoptotic pathway. In hypoxia and serum-deprived culture conditions, LPA induces CD34(+) cell proliferation without maintaining the their undifferentiating state, and enhances IL-8, IL-6 and G-CSF secretion during the first 12 h compared to non-treated cells. LPA-treated CD34(+) cells delivered in fibrin gels have enhanced survival and improved cardiac fractional shortening at 2 weeks on rat infarcted hearts as compared to hearts treated with placebo. We have developed a new platform to enhance the survival of CD34(+) cells using a natural and cost-effective ligand and demonstrated its utility in the preservation of the functionality of the heart after infarction.
Assuntos
Antígenos CD34/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Isquemia/metabolismo , Lisofosfolipídeos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Caspase 9/metabolismo , Diferenciação Celular/efeitos dos fármacos , Hipóxia Celular , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Transplante de Células-Tronco de Sangue do Cordão Umbilical , Citocinas/biossíntese , Modelos Animais de Doenças , Sangue Fetal/citologia , Células-Tronco Hematopoéticas/citologia , Humanos , Masculino , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/mortalidade , Infarto do Miocárdio/fisiopatologia , Infarto do Miocárdio/terapia , PPAR gama/metabolismo , Ratos , Transdução de Sinais/efeitos dos fármacos , Resultado do Tratamento , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
Herein, we report that VEGF-functionalized dextran (dexOx-VEGF) is comparatively superior to free VEGF in prolonging the phosphorylation of VEGF receptor 2 (VEGFR-2). Both dexOx-VEGF and free VEGF activate VEGFR-2, and the complexes are internalized into early endosomes (EEA1(+) vesicles) and then transported to lysosomes (Rab7(+) vesicles). However, after cell activation, dexOx-VEGF is preferentially colocalized in early endosomes where VEGF signaling is still active while free VEGF is preferentially transported to late endosomes or lysosomes. We further show that dexOx-VEGF after phosphorylation of VEGF receptor 2 induces an increase of intracellular Ca(2+) and activates VEGF downstream effectors such as Akt and extracellular signal-regulated kinase (ERK1/2) proteins. Under specific conditions, the activation level is different from the one observed for free VEGF, thus suggesting mechanistic differences, which is illustrated by cell migration and cord-like formation studies. DexOx-VEGF can be cross-linked with adipic acid dihydrazide to form a degradable gel, which in turn can be incorporated in a fibrin gel containing endothelial cells (ECs) to modulate their activity. We envision that these constructs might be beneficial to extend the pro-angiogenic activity of VEGF in ischemic tissues and to modulate the biological activity of vascular cells.
Assuntos
Dextranos/química , Endossomos/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Lisossomos/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/química , Cálcio/metabolismo , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Endocitose/efeitos dos fármacos , Endossomos/metabolismo , Expressão Gênica/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Hidrogéis , Lisossomos/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação/efeitos dos fármacos , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/farmacologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/farmacologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/química , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Hemoglobinopathies are priority genetic diseases for prevention programs in at-risk populations. We implemented an accurate and simple methodology to identify hemoglobin (Hb) variants and to quantify HbA2 and HbF in capillary blood samples stored at room temperature for up to 7 days after collection. This methodology is particularly indicated for screening for carriers in primary care medical centers in which facilities for collecting venous blood are not available.