Fiber type-specific differential expression of angiogenic factors in response to chronic hindlimb ischemia

Abstract

Alterations in endogenous levels of the angiogenic proteins basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) were assessed in rabbit hindlimb muscles subjected to 1, 5, or 21 days of ischemia. In the glycolytic [tibialis anterior (TA)] and the oxidative [soleus (SOL)] muscles from the ischemic and contralateral (control) hindlimb, bFGF and VEGF protein expression was determined by ELISA and immunoblot analysis. Total VEGF protein expression was greater in oxidative than in glycolytic muscles after 5 days of hindlimb ischemia. In SOL muscle, total VEGF detected by ELISA in ischemic limbs was increased to 137, 300, and 220% of control at 1, 5, and 21 days, respectively. However, in TA, total VEGF expression by ELISA was increased only at 1 and 5 days of ischemia to 140 and 134% of control, respectively. By immunoblotting, the expression of the 165-amino acid isoform (VEGF(165)) was initially decreased to 55% of control in ischemic SOL at 1 day but was increased to 250% of control at day 5 and remained at 155% at day 21. In TA, VEGF(165) was increased to 260% of control at 1 day of ischemia but only to 150% of control by day 5. The only significant change in bFGF expression in either the oxidative or glycolytic muscles was a small increase (129% of control) at 21 days in SOL. This study demonstrates that the magnitude and direction of change in VEGF protein expression depend on VEGF subtype, muscle fiber type, and duration of ischemia. These findings suggest that strategies in therapeutic angiogenesis may need to differ depending on muscle fiber type.

Engineered Zinc Finger–Activating Vascular Endothelial Growth Factor Transcription Factor Plasmid DNA Induces Therapeutic Angiogenesis in Rabbits With Hindlimb Ischemia

“…By previously described methods, 10,19 rabbits (mean weight, 3.1 kg) were anesthetized with ketamine (50 mg/kg) and xylazine (5 mg/kg). A longitudinal incision was made along the left medial thigh to allow proper isolation, ligation, and excision of the femoral artery from its origin just above the inguinal ligament to its bifurcation at the origin of the saphenous and popliteal arteries.…”

“…For protein determination, muscle samples were weighed, homogenized, and centrifuged, and the protein content of the supernatant was determined by Bradford assay by previously described methods. 10,19,20 …”

“…8,9 All of these VEGF isoforms exist in vivo, and the various isoforms appear to be differentially regulated in the setting of ischemia. 10 The delivery of a single isoform of VEGF has been used in a number of uncontrolled human clinical trials that reported exciting clinical results, although significant edema developed in the VEGF-treated limb. [11][12][13] VEGF has been used in only 1 placebo-controlled clinical trial in patients with PAOD.…”

In Mice With Type 2 Diabetes, a Vascular Endothelial Growth Factor (VEGF)-Activating Transcription Factor Modulates VEGF Signaling and Induces Therapeutic Angiogenesis After Hindlimb Ischemia

“…VEGF-A is known to be present in mammalian striated skeletal muscle and is transcribed as a single mRNA that undergoes differential splicing to result in expression of at least four splice variants (VEGF 121 , VEGF 165 , VEGF 189 , and VEGF 206 in humans) that vary with respect to their solubility and binding to extracellular matrix proteoglycans (9,10). Expression of the different VEGF-A isoforms (hereafter referred to as VEGF) appears to be differentially regulated in the setting of ischemia (11). Preclinical models of PAOD have studied the effects of the delivery of a single isoform with different results (12,13).…”

Mouse models to study angiogenesis in the context of cardiovascular diseases

“…Because muscle atrophy is a common phenomenon in the hindlimb ischemia models, determination of the number of "capillaries per muscle fiber" may be more accurate than the number of "capillaries per millimeter squared" (23-25). Analyses of muscle fiber type, fibrosis, inflammation degree, cell death and proliferation are easy to achieve (26). pO 2 measurement could be asserted by using a pO 2 probe inserted in the ischemic muscle, by electron paramagnetic resonance (EPR), oximetry and use of fiber-optic probes(27-49)-the surgery typically results in oxygen saturations as low as 64% measured at day 0 post operation -or immunohistochemical techniques (Hydroxyprobe Kit, Chemicon International) to visualize gradients of hypoxia.…”