RESUMEN
Pleiotrophin (PTN) is a growth factor with both pro-angiogenic and limited pro-tumorigenic activity. We evaluated the potential for PTN to be used for safe angiogenic gene therapy using the full length gene and a truncated gene variant lacking the domain implicated in tumorigenesis. Mouse myoblasts were transduced to express full length or truncated PTN (PTN or T-PTN), along with a LacZ reporter gene, and injected into mouse limb muscle and myocardium. In cultured myoblasts, PTN was expressed and secreted via the Golgi apparatus, but T-PTN was not properly secreted. Nonetheless, no evidence of uncontrolled growth was observed in cells expressing either form of PTN. PTN gene delivery to myocardium, and non-ischemic skeletal muscle, did not result in a detectable change in vascularity or function. In ischemic hindlimb at 14 days post-implantation, intramuscular injection with PTN-expressing myoblasts led to a significant increase in skin perfusion and muscle arteriole density. We conclude that (1) delivery of the full length PTN gene to muscle can be accomplished without tumorigenesis, (2) the truncated PTN gene may be difficult to use in a gene therapy context due to inefficient secretion, (3) PTN gene delivery leads to functional benefit in the mouse acute ischemic hindlimb model.
Asunto(s)
Proteínas Portadoras/genética , Citocinas/genética , Terapia Genética , Isquemia/terapia , Músculo Esquelético/irrigación sanguínea , Fragmentos de Péptidos/genética , Animales , Proteínas Portadoras/metabolismo , Movimiento Celular , Transformación Celular Neoplásica/metabolismo , Células Cultivadas , Vasos Coronarios/patología , Citocinas/metabolismo , Retículo Endoplásmico/metabolismo , Estrés del Retículo Endoplásmico , Miembro Posterior/irrigación sanguínea , Miembro Posterior/patología , Células Endoteliales de la Vena Umbilical Humana/fisiología , Humanos , Masculino , Ratones , Ratones Desnudos , Ratones SCID , Músculo Esquelético/patología , Mioblastos/metabolismo , Mioblastos/trasplante , Miocardio/patología , Miocitos del Músculo Liso/fisiología , Neovascularización Fisiológica , Fragmentos de Péptidos/metabolismoRESUMEN
Delivery of bone marrow cells (BMCs) to the heart has substantially improved cardiac function in most rodent models of myocardial infarction (MI), but clinical trials of BMC therapy have led to only modest improvements. Rodent models typically involve intramyocardial injection of BMCs from distinct donor individuals who are healthy. In contrast, autologous BMCs from individuals after MI are used for clinical trials. Using BMCs from donor mice after MI, we discovered that recent MI impaired BMC therapeutic efficacy. MI led to myocardial inflammation and an increased inflammatory state in the bone marrow, changing the BMC composition and reducing their efficacy. Injection of a general anti-inflammatory drug or a specific interleukin-1 inhibitor to donor mice after MI prevented this impairment. Our findings offer an explanation of why human trials have not matched the success of rodent experiments and suggest potential strategies to improve the success of clinical autologous BMC therapy.
Asunto(s)
Trasplante de Médula Ósea/métodos , Interleucina-1/metabolismo , Infarto del Miocardio/inmunología , Infarto del Miocardio/terapia , Donantes de Tejidos , Animales , Ecocardiografía , Citometría de Flujo , Masculino , Ratones , Ratones Endogámicos C57BL , Infarto del Miocardio/patologíaRESUMEN
We compared therapeutic benefits of intramyocardial injection of unfractionated bone marrow cells (BMCs) versus BMC extract as treatments for myocardial infarction (MI), using closed-chest ultrasound-guided injection at a clinically relevant time post-MI. MI was induced in mice and the animals treated at day 3 with either: (i) BMCs from green fluorescent protein (GFP)-expressing mice (n = 14), (ii) BMC extract (n = 14), or (iii) saline control (n = 14). Six animals per group were used for histology at day 6 and the rest followed to day 28 for functional analysis. Ejection fraction was similarly improved in the BMC and extract groups versus control (40.6 +/- 3.4 and 39.1 +/- 2.9% versus 33.2 +/- 5.0%, P < 0.05) with smaller scar sizes. At day 6 but not day 28, both therapies led to significantly higher capillary area and number of arterioles versus control. At day 6, BMCs increased the number of cycling cardiomyocytes (CMs) versus control whereas extract therapy resulted in significant reduction in the number of apoptotic CMs at the border zone (BZ) versus control. Intracellular components within BMCs can enhance vascularity, reduce infarct size, improve cardiac function, and influence CM apoptosis and cycling early after therapy following MI. Intact cells are not necessary and death of implanted cells may be a major component of the benefit.
Asunto(s)
Células de la Médula Ósea/fisiología , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Corazón/fisiología , Infarto del Miocardio/terapia , Animales , Apoptosis , Células de la Médula Ósea/metabolismo , Ecocardiografía , Masculino , Ratones , Ratones Endogámicos C57BL , Miocitos Cardíacos/citología , Miocitos Cardíacos/fisiologíaRESUMEN
The major vascular endothelial growth factor (VEGF) isoforms are splice variants from a single gene that differ in their extent of heparin affinity due to the absence of the heparin binding domain in the smallest isoform (mouse VEGF120, human VEGF121). A long-held assumption that has guided the use of VEGF isoforms clinically has been that their differences in heparin binding dictate their ability to diffuse through tissue, with VEGF121 moving most freely and that the distribution of recombinant VEGF would have therapeutically relevant consequences. To test this assumption, we delivered the genes encoding these isoforms by myoblast-mediated gene transfer, a means of delivering genes to highly localized sites within muscle. Surprisingly, all isoforms induced comparable extremely localized physiological effects. Significantly, irrespective of the isoform delivered, the vessels passing within several micrometers of muscle fibers expressing VEGF displayed sharply delineated changes in morphology. The induction of capillary wrapping around VEGF-producing fibers, and of vascular malformations in the muscle at high levels, did not differ among isoforms. These results indicate that heparin binding is not essential for the localization of VEGF in adult tissue and suggest that the preferential delivery of VEGF121 cDNA for clinical applications may not have a physiological basis.