Revascularization in the rabbit hindlimb: dissociation between capillary sprouting and arteriogenesis.

OBJECTIVE: Animal models of hindlimb ischemia are critical to our understanding of peripheral vascular disease and allow us to evaluate therapeutic strategies aimed to improve peripheral collateral circulation. To further elucidate the processes involved in revascularization following ischemia, we evaluated the temporal association between tissue ischemia, vascular endothelial cell growth factor (VEGF) release, angiogenesis (capillary sprouting), arteriogenesis (growth of the larger muscular arteries), and reserve blood flow (functional collateral flow). METHODS: New Zealand White rabbits (male 3-4 kg) were evaluated at specific days (0, 5, 10, 20 or 40) following femoral artery removal for measurement of hindlimb blood flow, skeletal muscle lactate production and VEGF content, capillary density (a marker of angiogenesis), and angiographic score (a marker of arteriogenesis). RESULTS: Maximal capillary sprouting occurred within 5 days of femoral artery removal and was temporally associated with reduced resting hindlimb blood flow, increased lactate release and detectable levels of skeletal muscle VEGF. The growth of larger angiographically visible collateral vessels occurred after 10 days and was not temporally associated with ischemia or skeletal muscle VEGF content, but did coincide with a large functional improvement in the reserve blood flow capacity of the limb. CONCLUSIONS: Following femoral artery removal in the rabbit, the time course of angiogenesis and arteriogenesis were clearly distinct. Tissue ischemia and/or VEGF may stimulate capillary sprouting, but this response does not translate to a significant improvement in collateral flow. The growth and development of the larger collateral vessels was correlated with a large functional improvement in collateral flow, and occurred at a time when VEGF levels were undetectable.

Gas-liquid chromatography of fecal neutral steriods.

A method is described for the analysis of fecal neutral steriods with a dual-column gas-liquid chromatography (GLC) system. After saponification of the fecal slurry, the neutral steroids were extracted with hexane. The GLC separation of the compounds and quantitation were achieved by simultaneous injection of the derivatized and derivatized aliquots of the extract onto dual colmuns under identical conditions. The neutral steroids of interest were than identified by matching the retention times with those of known standards, and identification was confirmed by use of an interfaced GLC high-resolution mass spectrometry system. The detection limit was 0.003 mg of steroid/g of fecal slurry. The pricision of the method is illustrated by a relative standard diviation of 2-10% and a recovery of neutral steroids from 73-96%. The method was applied to the determination of fecal neutral steroids in a "High protein diet in colon cancer study". A considerably larger level of coprostanone than of coprostanol was observed. Data on neutral steroids in fecal samples from subjects on different diets are the subject of a separate publication.

Effects of anesthesia and open-thorax surgery on coronary vascular reserve in swine.

The coronary reactive hyperemic response was examined in seven pigs under anesthetized and conscious conditions, (i.e., 5 days and 3 and 5 weeks after surgery). Tygon catheters were inserted in the descending aorta of five pigs; transonic flow probes and hydraulic occluders were placed on the left cranial descending and/or left circumflex coronary arteries. Two pigs underwent long-term implantation of similar instruments. The coronary reactive hyperemic response, expressed as repayment of flow deficit, was induced by brief complete coronary artery occlusion for 15 sec. Baseline mean arterial pressure, heart rate, and coronary blood flow were similar in the anesthetized and conscious pigs. There was also no significant difference in repayment of flow deficit between the anesthetized and conscious pigs 5 days after surgery. The repayment of flow deficit (709 +/- 144%) in conscious pigs 5 days after surgery tended to be greater, but was not statistically significant from that observed in the anesthetized pigs (510 +/- 79%). However, at 3 and 5 weeks after surgery, the reactive hyperemic flow and the repayment of flow deficit were numerically greater than those values observed in anesthetized pigs. The difference in reactive hyperemic flow between conscious and anesthetized pigs was statistically significant at week 3. The difference in repayment of flow deficit between conscious and anesthetized pigs was statistically significant at week 5. These results suggest that anesthesia, as well as recent surgery, attenuates coronary vascular reserve. The major factor in the attenuation of coronary reserve appears to be recent surgical manipulation, because repayment of flow deficit was still depressed in conscious pigs during the early phase of recovery from surgery.

Leptin gene therapy and daily protein administration: a comparative study in the ob/ob mouse.

We have compared the efficacy of daily injection of recombinant leptin protein (rh-leptin) with adenovirus-mediated delivery of the murine or human leptin gene (Ad-leptin) for treatment of obesity in the obese (ob/ob) mouse model. We demonstrate an improved correction profile for obesity and associated surrogate markers using the adenovirus delivery method. Rate of weight loss and percentage satiety were significantly greater in the mice treated with Adleptin. These findings were associated with lower peak serum leptin levels with Ad-leptin (22.9 +/- 2.6 ng/ml for the human gene, and 48.9 +/- 11.5 ng/ml for the murine gene) compared to rh-leptin (385.2 +/- 36.0 ng/ml). (Values are given as mean +/- standard error of the mean.) Importantly rh-leptin and ex vivo-expressed Ad-leptin were equivalently active in a functional cell-based assay. The primary difference in the two therapeutic approaches is the continuous chronic secretion of leptin mediated by gene delivery, versus the intermittent bolus delivery and rapid clearance of the daily injection of rh-leptin protein. Thus, in vivo findings suggest that leptin effects are better achieved at lower steady-state levels, a pharmacological feature attained here by gene therapy. These findings may have implications for the potential use of leptin in the treatment of obesity.