Hemolysate Induces Tyrosine Phosphorylation and Collagen-Lattice Compaction in Cultured Fibroblasts Anita Patlolla,* Kotaro Ogihara,* Kazuya Aoki,* Alexander Zubkov,* Eva Bengten,† Andrew D. Parent,* and John H. Zhang* *Department of Neurosurgery and †Department of Microbiology, University of Mississippi Medical Center, Jackson, Mississippi 39216 Received July 20, 1999 Hemolysate, a proposed causative agent for cerebral vasospasm after subarachnoid hemorrhage, produces contraction of cerebral arteries by activation of ty- rosine kinases. In addition, hemolysate increases fibroblast-collagen compaction that could play a role in cerebral vasospasm. We studied the effect of hemo- lysate on tyrosine phosphorylation and fibroblast- collagen compaction in cultured canine basilar and human dermal fibroblasts using tyrosine kinase inhib- itors and tyrosine antibodies. Hemolysate enhanced tyrosine phosphorylation of two proteins, 64 and 120 kDa, in cultured canine basilar artery and human der- mal fibroblast cells. The effect of hemolysate was time- dependent and concentration-dependent. Oxyhemo- globin and ATP, the two major components of hemolysate, produced similar tyrosine phosphoryla- tion, however, with a different time course. Tyrosine kinase inhibitors genistein and tyrphostin A51 abol- ished the effect of hemolysate in both cerebral and dermal fibroblasts. Hemolysate increased fibroblast- populated collagen-lattice compaction and tyrosine kinase inhibitors genistein and tyrphostin A51 atten- uated the effect of hemolysate. We conclude that he- molysate activates tyrosine kinase that leads to the increase of fibroblast compaction. This effect of hemo- lysate may contribute to cerebral vasospasm. © 1999 Academic Press Key Words: tyrosine phosphorylation; hemolysate; collagen-lattice compaction; cerebral vasospasm. Cerebral vasospasm, a persisted narrowing of major cerebral arteries, is a major cause of morbidity and mortality following subarachnoid hemorrhage (SAH) (11, 13, 15). The etiological factors for cerebral vaso- spasm are subarachnoid blood clots, especially the ly- sate of erythrocyte (2, 17, 18). However, the pathogen- esis of cerebral vasospasm and the signal transduction pathways responding to the spasmogens remain un- clear (7, 15). Recently, tyrosine kinases and their sub- strates have been suggested involved in hemolysate- induced elevation of intracellular Ca 2+ (9), in contraction of cerebral arteries (12, 24, 30), and in a canine model of cerebral vasospasm (4). Even though cerebral vasospasm is regarded as a prolonged contraction of major cerebral arteries, there is a line of evidence suggesting additional or alterna- tive mechanisms (3, 21). First, cerebral vasospasm has been resistant to all known vasodilators. Second, the most striking histological feature of the spastic vessels is the thickening of the sub-endothelial layer called subintimal cellular proliferation. Third, the cellular nature of those proliferative areas has the properties similar to myofibroblasts. Furthermore, cerebrospinal fluid obtained from patients with ruptured aneurysm significantly accelerated collagen-lattice contraction, especially when the patient developed symptomatic va- sospasm (21, 25). This suggests that non-muscle com- ponents can produce and maintain vascular constric- tion (10, 19, 26). Thus, we studied the effect of hemolysate and its com- ponents on tyrosine phosphorylation and fibroblast- collagen compaction in cultured canine basilar artery and human dermal fibroblasts. MATERIALS AND METHODS Cell culture. Neonatal normal human dermal fibroblast cells (NHDF-Neo) were purchased from Clonetics (San Diego, CA). Cells were cultured in FBM medium, supplemented with 2% fetal bovine serum, 1 ng/ml basic human fibroblast growth factor, antibiotics (gentamycin 50 g/ml, amphotericin-B 50 ng/ml) and 5 g/ml insulin in a 5% CO 2 incubator. Canine basilar arterial fibroblast cells were obtained using explant methods (9) and cultured in Dulbecco’s modified Eagle’s medium (Gibco Brl, Grand Island, NY) with 10% fetal bovine serum. These cells were stained negative to factor VIII and -smooth muscle actin. Cells from the 3rd and 8th passages were used. Preparation of hemolysate. Hemolysate was prepared from fresh arterial dog blood in our laboratory as described (9). Briefly, dog red blood cells were washed with cold saline solution and lysed by adding 5 mM cold sodium phosphate buffer. The membrane debris was Biochemical and Biophysical Research Communications 264, 100 –107 (1999) Article ID bbrc.1999.1383, available online at http://www.idealibrary.com on 100 0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.