A fusion protein of hepatocyte growth factor for immobilization to collagen.

We describe here a fusion protein consisting of hepatocyte growth factor (HGF; an angiogenic factor) and a collagen-binding domain (CBD) polypeptide of fibronectin (FN). This fusion protein (CBD-HGF), produced by a baculovirus expression system, exhibited much stronger collagen binding activity than native HGF in the range of 0.4-6.4microg/ml. Its binding at the lowest concentration exceeded that of HGF at the highest concentration. In addition, the collagen-bound CBD-HGF promoted growth of endothelial cells (ECs) to a greater degree at least 4 days longer than HGF added to the culture medium; about 5-fold greater increase in cell number after 10 days. These findings suggest that the fused CBD moiety not only helped immobilize HGF on collagen but also helped stabilize the fusion molecule, resulting in prolonged activity. The angiogenic activity of CBD-HGF in animal tissues was examined by subcutaneously implanting collagen sponges containing bound CBD-HGF. Blood vessel formation in the sponges after 7 days was 4-6-fold extensive as compared to the control sponges without sample. Implanted sponges with native HGF did not show significant difference from control. These results indicate that CBD-HGF is suitable for in vitro culture of ECs, and that this fusion protein can be used to confer HGF activity on biomaterials for use in tissue engineering.

Culture of human umbilical vein endothelial cells on immobilized vascular endothelial growth factor.

Vascular endothelial growth factor (VEGF) was immobilized on substrata in photoreactive gelatin to control the adhesion and growth of vascular endothelial cells. The gelatin and VEGF were mixed in water and cast on a polystyrene dish or a silane-coated glass plate. The surface was then photoirradiated in the presence or absence of a photomask and washed. Toughness of the immobilized material was confirmed by ethanol treatment. Human umbilical vein endothelial cells (HUVECs) grew on the immobilized VEGF but not on a nontreated surface. Growth of HUVEC increased significantly with an increase in the amount of immobilized VEGF, and the effects were inhibited by treatment with anti-VEGF antibody. Thus, immobilized VEGF specifically interacted with HUVECs to permit growth in culture. Micropatterning of HUVEC cultures was also achieved using micropattern-immobilized VEGF. This patterning technique may be useful for the formation of blood vessel networks in vitro.

Embolic brain infarction related to posttraumatic occlusion of vertebral artery resulting from cervical spine injury: a case report.

INTRODUCTION: The frequency of vertebrobasilar ischemia in patients with cervical spine trauma had been regarded as low in many published papers. However, some case reports have described cervical spine injury associated with blunt vertebral artery injury. Many aspects of the management of vertebral artery injuries still remain controversial, including the screening criteria, the diagnostic modality, and the optimal treatment for various lesions. The case of a patient who had a brain infarction due to recanalization of his occluded vertebral artery following open reduction of cervical spinal dislocation is presented here. CASE PRESENTATION: A 41-year-old Asian man presented with C4 to C5 distractive flexion injury manifesting with quadriplegia and anesthesia below his C3 cord level (including phrenic nerve paralysis), and bowel and bladder dysfunction. Magnetic resonance angiography and computed tomography angiography showed left extracranial vertebral artery occlusion and patent contralateral vertebral artery. He was observed without antiplatelet and/or anticoagulation therapy, and underwent surgery (open reduction and internal fusion of C4 to C5, and tracheostomy) 8 hours after the injury. After surgery, supraspinal symptoms such as left horizontal nystagmus and left homonymous hemianopsia led to cranial computed tomography and magnetic resonance imaging, which showed left-side cerebellar infarction in his posterior inferior cerebellar artery territory and right-side posterior cerebral artery infarction. Magnetic resonance angiography and computed tomography angiography demonstrated patent bilateral vertebral artery (but hypoplastic right vertebral artery) and occluded right posterior cerebral artery. His injured vertebral artery was treated conservatively, which did not cause any other ischemic complications. CONCLUSIONS: The management of asymptomatic vertebral artery injury is controversial with several treatment options available, including observation alone, antiplatelet therapy, anticoagulation therapy, or invasive intervention. Although there are some reports in which management with observation alone is described as safe, we should pay serious attention to the vertebral artery injury caused by cervical spine trauma.

Distal embolic brain infarction due to recanalization of asymptomatic vertebral artery occlusion resulting from cervical spine injury: a case report.

OBJECTIVE: The purpose of this case report is to describe a patient with brain infarction due to recanalization of an occluded vertebral artery (VA) following closed reduction and open fixation of cervical spinal dislocation and to discuss the management of asymptomatic VA injuries associated with spine trauma. CLINICAL FEATURES: A 41-year-old Asian man experienced a C4-5 distractive-flexion injury manifesting with quadriplegia and anesthesia below the C3 cord level (including phrenic nerve paralysis), and bowel and bladder dysfunction. Magnetic resonance angiography and computed tomography angiography showed left extracranial VA (V2) occlusion and a patent contralateral VA. INTERVENTION AND OUTCOME: The patient was observed without antiplatelet and/or anticoagulation therapy and underwent open reduction and internal fusion of C4/5 and tracheostomy 8 hours after the injury. After surgery, supraspinal symptoms such as left horizontal nystagmus and left homonymous hemianopsia led to cranial computed tomography and magnetic resonance imaging, which showed left-side cerebellar infarction in the posterior inferior cerebellar artery territory and right-side posterior cerebral artery infarction. Magnetic resonance angiography and computed tomography angiography demonstrated patent bilateral VA (but hypoplastic right VA) and occluded right posterior cerebral artery. The injured VA was treated conservatively, and there were no other ischemic complications. CONCLUSION: The management of asymptomatic VA injury is controversial, with several treatment options available, including observation alone, antiplatelet therapy, anticoagulation therapy, or invasive intervention. Although there are some reports described where management with observation alone seems safe, serious attention should be given to the VA injury caused by cervical spine trauma.

Delivery of a growth factor fusion protein having collagen-binding activity to wound tissues.

Recently, we established a collagen-binding growth factor consisting of epidermal growth factor and the fibronectin collagen-binding domain (FNCBD-EGF). FNCBD-EGF is a biologically active fusion protein that could stably bind to collagen materials, and exert its growth factor activity even after collagen binding. In this study, we investigated the concept that FNCBD moiety with high collagen affinity may enhance the effective local concentration of EGF at the site of administration in the following tissues: skin wounds, catheter-injured arteries, and hind limb muscles. In an animal model of impaired wound healing, application of FNCBD-EGF in combination with collagen gel induced granulation tissue formation in the wounds due to its sustained retention. In the injured artery, infused FNCBD-EGF remained bound to collagen exposed on the injured tissues even after blood circulation was restored. Injection of the fusion protein into the hind limbs revealed that our delivery system was effective for direct administration to muscular tissue.