James M. Kneebone

Vascular endothelial growth factor and basic fibroblast growth factor in children with cyanotic congenital heart disease

OBJECTIVE Vascular endothelial growth factor and basic fibroblast growth factor are potent stimulators of angiogenesis. Children with cyanotic congenital heart disease often experience the development of widespread formation of collateral blood vessels, which may represent a form of abnormal angiogenesis. We undertook the present study to determine whether children with cyanotic congenital heart disease have elevated serum levels of vascular endothelial growth factor and basic fibroblast growth factor. METHODS Serum was obtained from 22 children with cyanotic congenital heart disease and 19 children with acyanotic heart disease during cardiac catheterization. Samples were taken from the superior vena cava, inferior vena cava, and a systemic artery. Vascular endothelial growth factor and basic fibroblast growth factor levels were measured in the serum from each of these sites by enzyme-linked immunosorbent assay. RESULTS Vascular endothelial growth factor was significantly elevated in the superior vena cava (P =.04) and systemic artery (P =.02) but not in the inferior vena cava (P =.2) of children with cyanotic congenital heart disease compared to children with acyanotic heart disease. The mean vascular endothelial growth factor level, determined by averaging the means of all 3 sites, was also significantly elevated (P =.03). Basic fibroblast growth factor was only significantly elevated in the systemic artery (P =.02). CONCLUSION Children with cyanotic congenital heart disease have elevated systemic levels of vascular endothelial growth factor. These findings suggest that the widespread formation of collateral vessels in these children may be mediated by vascular endothelial growth factor.

Effect of cryopreservation and histocompatibility on type I procollagen gene expression in aortic valve grafts

BACKGROUND Allograft valves are excellent substitutes for diseased or absent valves but undergo primary tissue degeneration. Fibroblast viability may determine resistance to valve deterioration. This study evaluated gene expression for procollagen by valve grafts and studied the effects of cryopreservation and histocompatibility on this property. METHODS AND RESULTS Fresh and cryopreserved rat aortic valves were implanted heterotopically into syngeneic or allogeneic recipients. Nonviable, cryothermally injured valves were used as negative controls. The grafts and native aortic roots were excised 3 days after implantation. Northern hybridization with a human procollagen alpha 1 (I) complementary DNA probe was used to assess the expression of type I procollagen mRNA. The content of procollagen mRNA relative to 18S ribosomal RNA was evaluated by means of scanning densitometry. In situ hybridization was used to locate the areas of procollagen mRNA expression in the grafts. Both fresh and cryopreserved grafts exhibited greater expression than the native valve. This increase in expression was observed in both syngeneic and allogeneic grafts, but not in the negative control group. In situ hybridization showed a strong signal for procollagen in the aortic wall and a weak signal in the leaflet and myocardium in the viable grafts and in native tissues. CONCLUSIONS Regardless of preservation or allogenicity, fibroblast viability in aortic valve grafts persists after implantation. Increased gene expression for procollagen suggests a capacity for repair and regeneration.

Procollagen production in fresh and cryopreserved aortic valve grafts

Long-term durability of aortic valve allografts may be enhanced by cellular capacities for regeneration and repair. To evaluate aortic valve graft production of an important structural protein, rat aortic roots were implanted heterotopically into the abdominal aorta of recipient rats. Grafts were either syngeneic or strongly allogeneic, were implanted either fresh or after cryopreservation, and were left in place 2 to 21 days after implantation. A total of 80 aortic valve grafts and the corresponding native aortic valves were examined. The grafts were retrieved and immunocytochemically stained for the presence of procollagen, a precursor to collagen. Regardless of histocompatibility or preservation, grafts exhibited consistent procollagen presence that equaled or exceeded that seen in the corresponding native valves. Positive procollagen staining was predominantly in the aortic wall. The most prominent staining was near the hinge point of the valve leaflets, with no staining in the free portion of the leaflets. Staining with alpha-actin demonstrated vascular smooth muscle in sites remote from the areas positive for procollagen, which suggests that vascular smooth muscle was not responsible for the procollagen production. These findings indicate that cryopreservation is compatible with persistent fibroblast viability and in vivo protein synthesis by both syngeneic and allogeneic aortic valve grafts.

Angiogenic proteins in the lungs of children after cavopulmonary anastomosis

Methods: Lung specimens were obtained from 13 children after cavopulmonary anastomosis and from 6 control subjects. Specimens were stained with antibodies against vascular endothelial growth factor and its receptor (flk-1/KDR), basic fibroblast growth factor, α-smooth muscle actin, CD31, collagen IV, fibronectin, and proliferating cell nuclear antigen. Staining was graded on a scale of 0 to 3. Vessels positive for proliferating cell nuclear antigen were counted in 10 fields per specimen, and the results were averaged.