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THE COUNCIL FOR TOBACCO RESEARCH-U.S.A., INC. September 30. 1983 Memorandum To: Dr. S.C. Sommers and Staff From D.H. Ford and D. Stone Re: Site visit with Dr. E. Roberts and staff at the City of Hope Hospital in Duarte, CA, Sept. 20, 1983 Site Visitors: Drs. D.H. Ford, D. Stone J. Feldman and R. Bing Grant No. 1474 entitled "Effects of nicotine on the cerebrovasculature In Vitro." Before the initiation of support for this program there were questions as to whether or not Dr. Roberts and his colleagues could successfully culture endothelial cells from brain capillaries and large brain vessels which would maintain demonstrable endothelial "marker" characteristics of such cells. Prior to commencing support, we were informed that this had been successfully accomplished. It p$@a.Ks that: the: first•9 months•o€, this~ proqram has been direeted A ` ,~ t~wa~~;c~~ fdmpitiric}?~~o~d~ve~a lqat even . , 3.t ~. ase of the study, their results are preliminary anc~-riot4 conclusxve The only feature which so far identifies these cultured cell's asbeing endothelial in character is the presence of Factor VIII. Thus, the various experiments on effect of nicotine which they discussed at this visit are of questionable value. Summary of nicotine effects on endothelial cells. Plasminogen activator; No effect with a level of 10-5M nicotine in the culture media as assayed with NZGGA (N-CBZ-GlyclGlycyl arganine- 4-methyl-coumarinyl-7-amide) in endothg~ial cells grown for 4 days. Measurements of pinocytosis using the C-sucrose method of Davies also showed no significant change when nicotine was incorporated in the culture medium. HPLC analysis of free amino acids in cerebral capillary endothelium showed no real change in the presence of nicotine, except perhaps for H-taurine. Other studies with free amino acids from aortic and cerebral artery endothelium or glial cells were also inconclusive. They have determined the number of g/k~nt cells in their cultures and compared then with those reported by Booyse and observed them to be decreased rather than increased, with aortic endothelium. Capillary endothelium had no giant cell formation and was not effected by nicotine. When Booyse created wounded areas in his cultures, he observed them to become filled with giant cells. Roberts' group observed that wounded areas were filled in only with normal cells. It might be noted that Roberts' used younger cultures with fewer passes, and this may make a difference. In culture Roberts' group observed a higher plating density of cells than did Booyse, which again may be due to the difference in the age of the cells (30-45 passes with Booyse and 8-12 with Roberts)~' Also, the media were not quite identical, Growth of cells: A dose response curve was determined for nicotine effects on endotheliual cell growth in culture. Cortical and caudate capillary endotheli,um and g~ial cel~s showed no change with nicotine concerntrations of from 10 to 10 M.

2 Aortic capillary endothelium showed an increase in cell number with increase in nM~tine, w4ile pial artery endothelium showed°„"increase between 10 and 10 bi nicotine. All of these were for short periods of culture. When brain capillary endothelail cells were grown in a plasm clot, cord-like formations were formed which superficially looked like small vessels. However, it is not yet known whether or not these cords have a lumen. PGI2 (prostacyclin)4formatign: In a small pilot study, nicotine at doses of 10 to 10 M appears to decrease PGI2 formation in capillary endothelial cultures and to possibly increase it in cerebral artery endothelium. Perhaps the most interesting observation made to date is the presence of a glial growth factor arising from cultured cerebral endothelail cells. Whether this is truely a "new" growth factor or not (as distinct from NGF and other brain growth factors), and whether it is produced by endothelial cells in vivo is unknown. Further observations on this factor, other than it appears to be a protein, have yet to be undertaken (see abstract). Studies on nicotine receptors on endothelial cells still associated with their vessels'wall have not yet been undertaken. ,. . Comment.~ w.,hl~l~ th~e~ob~er,y,at,4on$Y`on:the'.cultured endothelial ~~ celis ; still. : renia~ir~,~omewhat: "stispect inasmuch as it is not' clear to what extent these cells are biologically similar-to those of the parent vessels and to what degree there are differences between cells with different vessels origins. Thus, the initial prenis that there are biochemical differences between endothelials cells from capillaries of brain and those from other sources remains still to be established in this cultured cell model. Given that there are morphological and physiological differences in vivo, to what degre ~`these differences (presence or absence of pinocytosis, presence of tight junctions between capillary endothelial cells, presence of monoamine oxidase and Dopadecarboxylase) present in the cultured cells? It would seem that until Roberts can establish .-that he can culture. endothelial cells which maintain the characteristics of the parent vessels, he is still in the pilot study stage of attempting to develop his model. Recommendation: Would renew for one year only and on a terminal basis. Any consideration of further support for a new proposal would then depend on the successful creation of his model. If continued work confirms the presence of a glial growth factor, which can be derived from capillary endothelium in vivo (would depend on histocytochemical demonstration of the protein in situ in~ the~ end'o-thelium b#'intact cerPl--,r vessels), there might be some interest in this aspect of the work from the standpoint of a connection with Parkinson's disease. (Apparently, in the normal brain, neurons of the substantia nigra are in direct apposition with the capillaries, as occurs in the supraoptic nucleus, but no other brain area, :.

3 In Parkinson patients, it has been observed that a number of glial process become interposed between the neuron and the capillary and may possibly interfere with the normal metabolic support of the neurons, leading to their death...to be followed by gliosis in the non post encephalitic variety. Roberts feels that it is conceivable that localized increases in an endothelail derived glial growth factor might be the cause of the morphologic change which appears to occur in the capillary- glial-neuronal inter-relationship in the substantia nigra and thus initiate Parkinsonism. However, any interest from CTR in such a Parkinson project would seem to first hinge on the establishment that there is indeed a specific glial growth factor derived from capillary endothelium in vivo as well as in culture. DHF AND DS/mla