Adenoviral-mediated gene transfer of a constitutively active form of the retinoblastoma gene product attenuates neointimal thickening in experimental vein grafts.

PURPOSE: Inappropriate or excessive vascular smooth muscle cell proliferation leads to the development of occlusive lesions in up to 50% of vein grafts. The purpose of this study was to test the hypothesis that induced overexpression of a cytostatic nonphosphorylatable form of the retinoblastoma protein (DeltaRb) would attenuate neointimal thickening in experimental vein grafts. METHODS: A replication-deficient adenovirus vector that encoded a nonphosphorylatable, constitutively active form of DeltaRb was constructed (AdDeltaRb) and contained an NH2-terminal epitope tag from the influenza hemagglutinin molecule (HA). Forty-eight male New Zealand white rabbits underwent surgical exposure of the external jugular vein for transfection with either 3 x 10(10) plaque-forming units/mL AdDeltaRb (n = 16), 3 x 10(10) plaque-forming units/mL control adenovirus (AdBglII, n = 15), or vehicle (n = 17) for 10 minutes at 120 mm Hg. After vector exposure, the vein was excised and interposed end-to-end into the carotid circulation. After 5 days, 12 grafts (four from each group) were excised and assayed for genomic DeltaRb DNA with the polymerase chain reaction or for hemagglutinin molecule expression and localization with immunohistochemistry. The remainder of the grafts (n = 36) were perfusion-fixed after 4 weeks, and 5 microm sections prepared for digital planimetric analysis. RESULTS: Polymerase chain reaction results identified the DeltaRb gene only in the grafts that were transfected with AdDeltaRb. Immunohistochemical analysis results revealed transgene expression in most of the endothelial cells and in many of the smooth muscle cells. After 4 weeks, the grafts that were exposed to AdDeltaRb exhibited a 22% reduction in neointimal thickness (vehicle, 77 +/- 7 microm; AdBglII, 75 +/- 5 microm; AdDeltaRb, 60 +/- 5 microm; P =.05), and medial thickness, luminal diameter, and other parameters were unchanged (medial thickness: vehicle, 72 +/- 10 microm; AdBglII, 85 +/- 7 microm; AdDeltaRb, 69 +/- 9 microm; P = NS; luminal diameter: vehicle, 4.5 +/- 0.2 mm; AdBglII, 4.4 +/- 0.2 mm; AdDeltaRb, 4.7 +/- 0.1 mm; P = NS). CONCLUSION: With this delivery system, adenoviral-mediated gene transfer is highly efficient and induced overexpression of DeltaRb leads to a reduction in vein graft neointimal thickening.

Molecular cloning of FOG-2: a modulator of transcription factor GATA-4 in cardiomyocytes.

GATA transcription factors are important regulators of both hematopoiesis (GATA-1/2/3) and cardiogenesis (GATA-4) in mammals. The transcriptional activities of the GATA proteins are modulated by their interactions with other transcription factors and with transcriptional coactivators and repressors. Recently, two related zinc finger proteins, U-shaped (USH) and Friend of GATA-1 (FOG) have been reported to interact with the GATA proteins Pannier and GATA-1, respectively, and to modulate their transcriptional activities in vitro and in vivo. In this report, we describe the molecular cloning and characterization of a third FOG-related protein, FOG-2. FOG-2 is an 1,151 amino acid nuclear protein that contains eight zinc finger motifs that are structurally related to those of both FOG and USH. FOG-2 is first expressed in the mouse embryonic heart and septum transversum at embryonic day 8.5 and is subsequently expressed in the developing neuroepithelium and urogenital ridge. In the adult, FOG-2 is expressed predominately in the heart, brain, and testis. FOG-2 associates physically with the N-terminal zinc finger of GATA-4 both in vitro and in vivo. This interaction appears to modulate specifically the transcriptional activity of GATA-4 because overexpression of FOG-2 in both NIH 3T3 cells and primary rat cardiomyocytes represses GATA-4-dependent transcription from multiple cardiac-restricted promoters. Taken together, these results implicate FOG-2 as a novel modulator of GATA-4 function during cardiac development and suggest a paradigm in which tissue-specific interactions between different FOG and GATA proteins regulate the differentiation of distinct mesodermal cell lineages.