Angiogenic synergism, vascular stability and improvement of hind-limb ischemia by a combination of PDGF-BB and FGF-2.

The establishment of functional and stable vascular networks is essential for angiogenic therapy. Here we report that a combination of two angiogenic factors, platelet-derived growth factor (PDGF)-BB and fibroblast growth factor (FGF)-2, synergistically induces vascular networks, which remain stable for more than a year even after depletion of angiogenic factors. In both rat and rabbit ischemic hind limb models, PDGF-BB and FGF-2 together markedly stimulated collateral arteriogenesis after ligation of the femoral artery, with a significant increase in vascularization and improvement in paw blood flow. A possible mechanism of angiogenic synergism between PDGF-BB and FGF-2 involves upregulation of the expression of PDGF receptor (PDGFR)-alpha and PDGFR-beta by FGF-2 in newly formed blood vessels. Our data show that a specific combination of angiogenic factors establishes functional and stable vascular networks, and provides guidance for the ongoing clinical trials of angiogenic factors for the treatment of ischemic diseases.

Vascular endothelial cell growth factor and fibroblast growth factor 2 expression in patients with critical limb ischemia.

BACKGROUND: For patients with critical limb ischemia (CLI), there is a great need for alternative treatment strategies. One option is therapeutic angiogenesis by administration of vascular growth factors. The lack of convincing clinical data supporting this strategy may be due to the ignorance of endogenous angiogenic processes in CLI. To evaluate the importance of vascular growth factors in the pathogenesis in CLI and provide information for clinical growth factor treatment trials, we determined the levels of vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF-2) in the ischemic legs of patients with this disease. METHODS: Skin and muscle biopsies from the calf and groin were gathered from 25 patients with CLI. Control samples came from 10 orthopedic patients and from 5 patients who were undergoing coronary artery bypass. The concentration of VEGF and FGF-2 in the biopsies was measured by enzyme-linked immunoassay, and to localize growth factor production, biopsied sections were immunostained. RESULTS: Patients with CLI had lower levels of VEGF in distal skin samples than in proximal ones (mean difference: 16.7 pg/mg total protein, 95% confidence interval: -1.0 to -32.3, P =.038), but these levels were similar to those in distal samples from control subjects (8.0, -4.6 to 20.5, P =.65). In muscle, VEGF concentrations were similar in calf and groin (5.4, -12.4 to 23.1, P =.55), but distal levels were higher than in distal samples from control subjects (23.7, 1.2 to 56.7, P =.028). Skin FGF levels tended to be higher in distal samples (45.3, 26.5 to 117.5, P =.090) and were higher than in skin from control subjects (106.2, -11.4 to 223.8, P =.050). Also in muscle, distal samples had higher levels of FGF-2 (35.6, -1.6 to 59.7, P =.006), but these levels were similar to what was found in control subjects (29.4., -16.3 to 81.2, P =.39). Growth factors were located in connective tissue between muscle fibers. In skin, the predominant FGF-2 staining was just below the epidermal layer, whereas VEGF appeared in the dermal layer. CONCLUSIONS: The results indicate that there are elevated concentrations of FGF-2 in calf muscle, whereas VEGF concentrations do not appear to be higher in the ischemic part of the leg in patients with CLI. These findings suggest that VEGF supplementation may be a more appropriate strategy for therapeutic angiogenesis to the calf area for CLI than FGF-2.

Vascular growth factor expression in a rat model of severe limb ischemia.

BACKGROUND: In ischemic tissue hypoxia induces production of vascular growth factors, especially VEGF, which initiate local angiogenesis. Collateralization-or arteriogenesis-occurs at a distance from the ischemic tissue and depends on different growth factors such as FGF-2. A spatial discrepancy in endogenous growth factor production in limb ischemia may have implications for therapeutic angiogenesis. The present study elucidates if such spatial and temporal variation occurs. MATERIALS AND METHODS: A two-staged procedure was performed to generate severe long-lasting limb ischemia in 60 rats. At 1, 7, 28, and 56 days, subgroups were subjected to perfusion assessment with laser Doppler imaging and angiography. Muscle samples and foot skin were gathered to measure growth factor expression and signs of angiogenesis using immunohistochemistry. RESULTS: There was an early twofold increase (P < 0.05) in both VEGF and FGF-2 levels in distal muscle from the ischemic leg, but no significant rise in the thigh. The concentrations decreased over time with an exception for VEGF in soleus and FGF-2 in anterior tibial muscle, which remained high. An increased capillarity was noted (P < 0.05) in soleus after 28 days, and the number of BrdU-positive ECs was elevated in all ischemic samples at 56 days. Collateral arteries were observed on the angiograms after 7 days. CONCLUSIONS: The results suggest that in limb ischemia any major increase in vascular growth factor production is limited to ischemic tissue. The spatial and temporal distribution patterns of growth factor production are complex and to a great extent influenced by inflammation.