RESUMEN
Autologous CD34(+) cells are widely used for vascular repair; however, in individuals with diabetes and microvascular disease these cells are dysfunctional. In this study, we examine expression of the clock genes Clock, Bmal, Per1, Per2, Cry1, and Cry2 in CD34(+) cells of diabetic and nondiabetic origin and determine the small encoding RNA (miRNA) profile of these cells. The degree of diabetic retinopathy (DR) was assessed. As CD34(+) cells acquired mature endothelial markers, they exhibit robust oscillations of clock genes. siRNA treatment of CD34(+) cells revealed Per2 as the only clock gene necessary to maintain the undifferentiated state of CD34(+) cells. Twenty-five miRNAs targeting clock genes were identified. Three of the miRNAs (miR-18b, miR-16, and miR-34c) were found only in diabetic progenitors. The expression of the Per2-regulatory miRNA, miR-92a, was markedly reduced in CD34(+) cells from individuals with DR compared with control subjects and patients with diabetes with no DR. Restoration of miR-92a levels in CD34(+) cells from patients with diabetes with DR reduced the inflammatory phenotype of these cells and the diabetes-induced propensity toward myeloid differentiation. Our studies suggest that restoring levels of miR-92a could enhance the usefulness of CD34(+) cells in autologous cell therapy.
Asunto(s)
Diferenciación Celular , Retinopatía Diabética/patología , Células Progenitoras Endoteliales/patología , Endotelio Vascular/patología , MicroARNs/metabolismo , Proteínas Circadianas Period/metabolismo , Antígeno AC133 , Antígenos CD/metabolismo , Antígenos CD34/sangre , Antígenos CD34/metabolismo , Biomarcadores/sangre , Biomarcadores/metabolismo , Proteínas CLOCK/antagonistas & inhibidores , Proteínas CLOCK/sangre , Proteínas CLOCK/genética , Proteínas CLOCK/metabolismo , Cadherinas/metabolismo , Células Cultivadas , Estudios de Cohortes , Diabetes Mellitus/sangre , Diabetes Mellitus/inmunología , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patología , Retinopatía Diabética/sangre , Retinopatía Diabética/inmunología , Retinopatía Diabética/metabolismo , Regulación hacia Abajo , Células Progenitoras Endoteliales/inmunología , Células Progenitoras Endoteliales/metabolismo , Endotelio Vascular/inmunología , Endotelio Vascular/metabolismo , Perfilación de la Expresión Génica , Glicoproteínas/metabolismo , Humanos , Persona de Mediana Edad , Péptidos/metabolismo , Proteínas Circadianas Period/antagonistas & inhibidores , Proteínas Circadianas Period/sangre , Proteínas Circadianas Period/genética , Interferencia de ARN , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismoRESUMEN
Previously, we showed that transient inhibition of TGF- ß1 resulted in correction of key aspects of diabetes-induced CD34(+) cell dysfunction. In this report, we examine the effect of transient inhibition of plasminogen activator inhibitor-1 (PAI-1), a major gene target of TGF-ß1 activation. Using gene array studies, we examined CD34(+) cells isolated from a cohort of longstanding diabetic individuals, free of microvascular complications despite suboptimal glycemic control, and found that the cells exhibited reduced transcripts of both TGF-ß1 and PAI-1 compared to age, sex, and degree of glycemic control-matched diabetic individuals with microvascular complications. CD34(+) cells from diabetic subjects with microvascular complications consistently exhibited higher PAI-1 mRNA than age-matched non-diabetic controls. TGF- ß1 phosphorodiamidate morpholino oligo (PMO) reduced PAI-1 mRNA in diabetic (p<0.01) and non-diabetic (p=0.05) CD34(+) cells. To reduce PAI-1 in human CD34(+) cells, we utilized PAI-1 siRNA, lentivirus expressing PAI-1 shRNA or PAI-1 PMO. We found that inhibition of PAI-1 promoted CD34(+) cell proliferation and migration in vitro, likely through increased PI3(K) activity and increased cGMP production. Using a retinal ischemia reperfusion injury model in mice, we observed that recruitment of diabetic CD34(+) cells to injured acellular retinal capillaries was greater after PAI-1-PMO treatment compared with control PMO-treated cells. Targeting PAI-1 offers a promising therapeutic strategy for restoring vascular reparative function in defective diabetic progenitors.
Asunto(s)
Antígenos CD34/metabolismo , Diabetes Mellitus/genética , Leucocitos Mononucleares/metabolismo , Inhibidor 1 de Activador Plasminogénico/genética , Adulto , Animales , Movimiento Celular/genética , Proliferación Celular , Células Cultivadas , Estudios de Cohortes , GMP Cíclico/metabolismo , Diabetes Mellitus/sangre , Diabetes Mellitus/metabolismo , Angiopatías Diabéticas/sangre , Angiopatías Diabéticas/genética , Angiopatías Diabéticas/metabolismo , Humanos , Leucocitos Mononucleares/citología , Ratones , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidor 1 de Activador Plasminogénico/sangre , Interferencia de ARN , Daño por Reperfusión/metabolismo , Vasos Retinianos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcriptoma/genética , Factor de Crecimiento Transformador beta1/sangre , Factor de Crecimiento Transformador beta1/genéticaRESUMEN
The nef gene products encoded by human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus type 1 (SIV-1) increase viral loads in infected hosts and accelerate clinical progression to AIDS. Nef exhibits a spectrum of biological activities, including the ability to downregulate surface expression of CD4 and major histocompatibility complex (MHC) class I antigens, to alter the state of T-cell activation, and to enhance the infectivity of viral particles. To determine which of these in vitro functions most closely correlates with the pathogenic effects of Nef in vivo, we constructed recombinant HIV-1 NL4-3 viruses carrying mutations within the nef gene that selectively impair these functions. These mutant viruses were evaluated for pathogenic potential in severe combined immunodeficiency (SCID) mice implanted with human fetal thymus and liver (SCID-hu Thy/Liv mice), in which virus-mediated depletion of thymocytes is known to be Nef dependent. Disruption of the polyproline type II helix (Pxx)4 within Nef (required for binding of Hck and p21-activated kinase-like kinases, downregulation of MHC class I, and enhancement of HIV-1 infectivity in vitro but dispensable for CD4 downregulation) did not impair thymocyte depletion in virus-infected Thy/Liv human thymus implants. Conversely, three separate point mutations in Nef that compromised its ability to downregulate CD4 attenuated thymocyte depletion while not diminishing viral replication. These findings indicate that the functional ability of Nef to downregulate CD4 and not MHC class I downregulation, Hck or PAK binding, or (Pxx)4-associated enhancement of infectivity most closely correlates with Nef-mediated enhancement of HIV-1 pathogenicity in vivo. Nef-mediated CD4 downregulation merits consideration as a new target for the development of small-molecule inhibitors.
Asunto(s)
Antígenos CD4/análisis , Productos del Gen nef/fisiología , VIH-1/patogenicidad , Alelos , Animales , Antígenos CD4/fisiología , Línea Celular , Regulación hacia Abajo , Antígenos de Histocompatibilidad Clase I/análisis , Humanos , Ratones , Ratones SCID , Productos del Gen nef del Virus de la Inmunodeficiencia HumanaRESUMEN
The ability of HIV-1 to evade the host immune response leads to the establishment of chronic infection. HIV-1 has been reported to up-regulate MHC I molecules on the surface of thymocytes from HIV-1-infected thymus. We demonstrate in this study that HIV-1 up-regulates MHC I on both HIV-1-infected and uninfected thymocytes in a manner that is independent of Nef, proportional to viral replication, and entirely mediated by IFN-alpha. IL-3Ralpha+ type 2 predendritic cells (preDC2) resident in the thymic medulla secrete IFN-alpha, which acts on IFN-alphabetaR-expressing immature thymocytes to induce MHC I expression. Furthermore, thymic preDC2 are permissive for HIV-1 infection and positive for intracellular p24. These data demonstrate the ability of IFN-alpha secreted by preDC2 to induce MHC I up-regulation in the HIV-1-infected human thymus.