RESUMEN
Critical limb ischemia (CLI) is the most severe presentation of peripheral arterial disease. We developed cell-based therapy entailing intra-arterial injection of autologous venous endothelial cells (ECs) modified to express angiopoietin 1, combined with autologous venous smooth muscle cells (SMCs) modified to express vascular endothelial growth factor. This combination promoted arteriogenesis in animal models and was safe in patients with limiting claudication. In an open-label, phase Ib study, we assessed the safety and efficacy of this therapy in CLI patients who failed or were unsuitable for surgery or intravascular intervention. Of 23 patients enrolled, 18 with rest pain or non-healing ulcers (Rutherford categories 4 and 5) were treated according to protocol, and 5 with significant tissue loss (Rutherford 6) were treated under compassionate treatment. Patients were assigned randomly to receive 1 × 107 or 5 × 107 (EC-to-SMC ratio, 1:1) of the cell combination. One-year amputation-free survival rate was 72% (13/18) for Rutherford 4 and 5 patients; all 5 patients with Rutherford 6 underwent amputation. Of the 12 with unhealing ulcers at dosing, 6 had complete healing and 2 others had >66% reduction in ulcer size. Outcomes did not differ between the dose groups. No severe adverse events were observed related to the therapy.
Asunto(s)
Proteínas Angiogénicas/genética , Terapia Genética , Isquemia/genética , Isquemia/terapia , Extremidad Inferior/irrigación sanguínea , Anciano , Anciano de 80 o más Años , Terapia Combinada , Células Endoteliales/metabolismo , Femenino , Expresión Génica , Técnicas de Transferencia de Gen , Terapia Genética/efectos adversos , Terapia Genética/métodos , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Humanos , Isquemia/diagnóstico , Isquemia/etiología , Masculino , Persona de Mediana Edad , Miocitos del Músculo Liso/metabolismo , Calidad de Vida , Retroviridae/genética , Transducción Genética , Transgenes , Resultado del TratamientoRESUMEN
RATIONAL AND OBJECTIVES: Activation of fully differentiated vascular cells using angiogenic genes can lead to phenotypic changes resulting in formation of new blood vessels. We tested whether Ang-1 gene transfer to endothelial cells (EC) activates these cells. METHODS AND RESULTS: EC and SMC were transduced using retroviral or adenoviral vectors to produce Ang-1 or vascular endothelial growth factor (VEGF). EC Tie-2 receptor was phosphorilated by autologous secretion of Ang-1. Transduced EC and SMC sprouting capacity was tested using collagen embedded spheroids assay and capacity to produce arteriogenesis was tested in a hind limb model of ischemia. EC expressing Ang-1 in the presence of SMC expressing VEGF exhibited high levels of sprouting of the two cell types. Flow and numbers of arteries were increased after transduced cells implantation in vivo. CONCLUSIONS: Autologous secretion of Ang-1 by transduced EC resulted in Tie-2 activation and in the presence of SMC expressing VEGF resulted in coordinated sprouting in vitro and increase in flow and number of arteries in vivo.
Asunto(s)
Angiopoyetina 1/biosíntesis , Angiopoyetina 1/genética , Arterias/metabolismo , Células Endoteliales/citología , Técnicas de Transferencia de Gen , Adenoviridae/genética , Animales , Arterias/patología , Biopsia , ADN Complementario/metabolismo , Células Endoteliales/metabolismo , Humanos , Miocitos del Músculo Liso/citología , Neovascularización Patológica , Fosforilación , Retroviridae/genética , Porcinos , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Ceramide, the basic structural unit of sphingolipids, controls the balance between cell growth and death by inducing apoptosis. We have previously shown that accumulation of ceramide, triggered by hydrogen peroxide (H(2)O(2)) or by short-chain ceramide analogs, induces apoptosis of lung epithelial cells. Here we elucidate the link between caspase-3 activation, at the execution phase, and ceramide accumulation, at the commitment phase of apoptosis in A549 human lung adenocarcinoma cells. The induction of ceramide accumulation by various triggers of ceramide generation, such as H(2)O(2), C(6)-ceramide, or UDP-glucose-ceramide glucosyltransferase inhibitor dl-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol, triggered the activation of caspase-3. This ceramide elevation also induced the cleavage of the death substrate poly(ADP-ribose) polymerase and was followed by apoptotic cell death. Ceramide-mediated apoptosis was blocked by a general caspase inhibitor, Boc-d-fluoromethylketone, and by overexpression of the antiapoptotic protein Bcl-2. Notably, overexpression of Bcl-2 reduced the basal cellular levels of ceramide and prevented the induction of ceramide generation by C(6)-ceramide, which implies ceramide generation as a possible target for the antiapoptotic effects of Bcl-2.