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changes in membrane constituents, leading to activation. These alterations in membrane components may result in direct activation of some component of the membrane pathways described above or more likely may result from in an alteration in gating properties of certain ion channels. The so called stretch-activated channels (SACS)were first described in skeletal muscle almost simultaneously by two groups working independently. They noted that simply applying suction pressure (in the range of -10 to -50 mm Hg) to a patch clamp pipette, which is used to record channel activity, resulted in cation of selective currents. Unlike voltage or ligand-gated channels, SACSare gated by the expansion or distortion of the cell membrane, occurring with lessthan a 2% increasein membrane area. SACShave been located now in a variety of cells including ECs. Lansman et al9 have described in porcine aortic ECs a cation selectiveSAC, which is six times more permeable to calcium than to potassium. They suggest that the entry of calcium ions by this route could modulate certain cell functions. Olesen et a1.l’ performed patch-clamp recording on bovine aortic ECs exposed to varying shear stress and have noted a potassium selective, shear stress activated ion channel. Nakache and Gaul?’ have confirmed these findings in bovine aortic ECs using fluorescence membrane-potential dyes. Unlike the SACS described by Lansman et al.,9 opening these shear sensitive channels results in hyperpolarization of the cell and may play a role in the relaxation of underlying smooth muscle cells, which are electrically coupled to endothelium in smaller vessels. In summary, the intracellular mechanisms regulating EC adaptations to physical stress are still poorly understood, and quantitative data relating shear stressor cyclic deformation directly to cellular biosynthesis is just emerging. The impetus in the field has now shifted from a description of the alterations in phenotype of cellsexposed to external forces to a more mechanistic investigation of the cellular and biochemical machinery which link events at the cell surface to the nucleus. Such studies may contribute insights into the mechanisms by which tissues normally subjected to mechanical stressgrow, adapt and involute and should also enable us to develop strategies for preventing the detrimental effect of such a processwhen it occurs in pathologic conditions. E. Swnpio, MD, PhD Tale University Schol of Medicine West Haven Veterans Adminktrhn Medical Center New Haven, Corm. Bauer

REFERENCES Iba T, Sumpio BE. Morphologic evaluation of human endothelial cells subjected to repetitive stretch in vitro. (Submitted). Gilman AG. G Proteins and regulation of adenylyl cyclase. JAMA 1989;262:1819-25. Letsou GV, Resales 0, Maiu S, Vogt A, Surnpio BE. Stimulation of adenylate cyclase activity in cultured endothelial cells subjected to cyclic stretch. J Cardiovasc Surg 1990;31:634-9.

Journal of VASCULAR SURGERY

4. Mills I, Letsou G, Rabban J, Sumpio BE, Gewertz H. Mechanosensitive adenylate cyclase activity in coronary vascular smooth muscle cells. Biochem Biophys Res Comm 1990;171:143-7. 5. Reich KM, Gay CV, Frangos JA. Fluid shear stress as a mediator of osteoblast cyclic adenosine monophosphate production. J Cell Physiol 1990;143:100-4. 6. Berridge MJ. Inositol triphosphate, calcium, lithium and cell signalling. JAMA 1989;262:1834-41. 7. Resales 0, Maitz S, Vogt A, Iba T, Sumpio BE. Phosphatidylinositol turnover in endothelial cells subjected to cyclic stretching. Fed Proc 1990;4:415A. MU, E&in SG, McIntire LV. Shear stress increases 8. Nollert inositol triphosphate levels in human endothelial cells. Biothem Biophys Res Comm 1990;170:281-7. 9. Lansman JB, Hallam TJ, Rink TJ. Single stretch-activated ion channels in vascular endothelial cells as mechanotransducers? Nature 1987;325:811-3. 10. Olesen SP, Clapham DE, Davies PF. Haemodynamic shear stress activates a K’ current in vascular endothelial cells. Nature 1988;331:168-70. 11. Nakache M, Gaub He. Hydrodynamic hyperpolarization of endothelial cells. Proc Natl AcadSci USA 1988;85:1841-3.

ELASTIC PROTEIN-BASED POLYMERS AS CELL ATTACHMENT MATRICES The repeating amino acid sequencesof elastin provide the basis of a group of synthetic elastic polymers having considerable potential for construction of vascularprostheses.’ The goal is to develop long-term vascular prostheses that would be colonized by the cellsof the vascularwall and gradually be functionally integrated into the vasculature. The elastomeric materials (matrixes) to be used are formed by y-irradiation cross-linking of the elastic polymers. Such matrices have the following advantages: (a) their elastic characteristicsare in line with those of the vascularwall, and specificelasticitiesmay be built in for specificapplications; (b) they can be ‘cast’ into tubular shapes, and such tubes may have multilayered walls; and (c) as implants, they appear biocompatible and slowly biodegradable2-4. A fundamental requirement of these materials for use in their proposed roles as temporary functional scaffoldings is that they support cell adhesion and cell migration. In theory it seems possible that the matrixes could be modified to possessspecific cell adhesion properties by the synthetic incorporation of specific amino acid sequences from adhesion molecules. In the work reported herein, we describe part of our continuing investigation of the cell adhesion supporting properties of the 20 Mrad y-irradiation cross-linked polypentamer matrix, i.e., X2’-poly(GVGVP) with and without selected sequence insertions. The cell attachment sequence of fibronectin,’ GRGDSP, was synthesized and incorporated into copolymers with GVGVP in ratios of 1: 100 and 1: 500 to give X2’-poly[lOO(GVGVP), (GRGDSP)] and X2’-poly [500(GVGVP), (GRGDSP)]. Also, YIGSR, a putative cell attachment sequence of laminin,” was synthesized and incorporated as X2’-poly(lO[GVGVP], [YIGSR]). Bovine aortic endothelial (BAB) cells were cultured in Ml99 containing 25 mmol/L HEPES, 10%

Volume 13 Number 5 May 1991

fetal bovine serum, 5% Ryan’s growth supplement, 100 units/ml penicillin and 100 kg/ml streptomycin. Bovine ligamenturn nuchae fibroblasts (LNF) were cultured in DMEM containing 10% fetal bovine serum, 2 mrnol/L L-glutamine, 0.1 mmol/L non-essential amino acids, 100 units/ml penicillin and 100 Kg/ml streptomycin. Both were incubated in a humidified incubator at 37” C and 5% CO,. Cells were used for experiment at early confluency and at passages3 to 6. After two rinseswith Hank’s balanced salt solution cells were dissociated using 0.05% trypsinJ0.53 mmol/L EDTA. The cells were treated with soybean trypsin inhibitor, washed, then counted using a hemocytometer and suspended at 0.32 x lo5 cells/ml of medium without serum but with 1% bovine serum albumin (BSA). The high dimensional sensitivity of the matrixes to temperature change in the range 20” to 40” C necessitatedthe use of the following cell adhesion assay.This set-up allows for dimensional change in the matrix while restricting the area of plating of the cells and maintaining a planar matrix surface. The matrixes, formed assheets,were cut into disks using a punch. Disks were equilibrated with protein-free media by incubation overnight and then placed into the chambers of multiwell plates. A glasscylinder (Bellco cloning cylinder) was then placed onto each disk and 50 mm3 of assaymedium added. After re-equilibration, 50 mm3 of cell suspension prepared as above was added to each glasscylinder and the multi-well plate incubated to allow cell attachment and spreading. Following incubation the glass cylinders were removed and unattached and very loosely attached cells were removed from the matrixes by gently rinsing with phosphate buffered saline. The attached cells were fixed in 3.5% formaldehyde in phosphate buffered saline pH 7.2. Adherent cells were classified and quantitated by phasecontrast microscopy. Generally, five areason each of at least three matrix disks were counted for each matrix type. Adherent cellswere classifiedinto three categories, (a) rounded cells, (b) poorly spread cells, which includes all cellshaving both refractile cell bodies and visible attachment processes,and (c) well-spread cells, generally possessingvisible nuclei. BAE cells were plated onto disks of X2’-poly (GVGVP), X2’-poly(lO[GVGVP], [YIGSR]), X2’-poly (SOO[GVGVP], [GRGDSP]), X2’-poly (lOO[GVGVP], [GRGDSP]), and onto fibronectin coated substratum in BSA containing medium. The cellswere allowed to attach and spread for 3 hours. Neither X2’-poly[GVGVP] nor X2’-poly(lO[GVGVP], (YIGSR)] was able to support any significant cell attachment. X2’-poly (500[GVGVP], [GRGDSP]) gave marginally increased attachment. X2’poly( lOO[GVGVP], [GRGDSP]) however, with its higher proportion of attachment sequences,was found to support a substantial amount of BAE cell attachment, and the bulk of cells attached were well spread. The total number of cells adhering to Xzo-poly(lOO[GVGVP], [GRGDSP]) was - 30% of the maximum possible, as indicated by the fibronectin coated substratum positive control. Similarly, LNF cellswere plated onto the samematrixes and allowed to attach and spread for 3 hours. As was found

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for BAE cells, the LNF cellsdid not attach significantly to X2’-poly[GVGVP],X”-poly(lO[GVGVP], [YIGSR]) or X2’-poly(500[GVGVP], [GRGDSP]) but did so to X2’poly(lOO[GVGVP], [GRGDSP]) where the total number of cells adhering was -25% of the fibronectin positive control. The lack of cell adhesion to the YIGSR containing matrix appears to be a result of the y-irradiation crosslinking process because substrata coated with the noncross-linked poly(lO[GVGVP], [YIGSR]) were found to support the adhesion of LNF cellsplated for 3 hours. The total number of cells adherent to poly(lO[GVGVP], [YIGSR]) was -87% of the fibronectin positive control whereas the number adherent to BSA coated substratum was -21% of the fibronectin control. Preliminary results for 20-hour LNF cell attachment incubations indicated that in the presence of exogenous adhesive factors from 10% fetal bovine serum or if the poly(GVGVP) was preabsorbed with fibronectin then cell attachment and spreading was supported. The requirement for exogenous adhesive factors seems clear because even after 20 hours in BSA containing medium, the cells were unable to attach to untreated poly(GVGVP) The synthetic protein-based elastomeric material X2’poly(GVGVP) has been demonstrated not to support the adhesion of BAE or LNF cells.However, incorporation of GRGDSP sequences at 1% into the matrix or the absorption of adhesive factors including fibronectin onto the matrix have been shown to promote cell adhesion. Sincecell adhesion is a critical requirement, these results are an important advance toward the successfuldevelopment of the elastin-based elastomeric matrices as vascular prosthetic materials. We are working toward increasing the level of cell adhesion by increasing the proportion of cell attachment sequences and by incorporating other fibronectin peptide sequences known to play roles in mediating cell adhesion.‘~’ To this end we are nearing completion of X2’-poly(20[GVGVP], [GRGDSP]) and have synthesized part of the fibronectin heparin binding region known to enhance cell adhesion. One area of concern is the apparent selectivedestruction of some amino acid sequencesby the cross-linking y-irradiation. Tyrosine is one of severalamino acidsknown to be more sensitiveto y-irradiation. It is possible that this explains the inactivation of YIGSR in the cross-linked matrix, and thus for YIGSR and some other sequencesalternative methods of matrix incorporation may be required. Other areasof high priority for future researchinclude investigation of whether the endothelial cell adhesion to the matrix is sufficiently strong to resistthe shearof arterial flow and whether or not the matrix surfaces are thrombogenic in the absence of endothelial cell coverage. Ahstair Nicol, PhD Cbanne Gowdu, PhD Dan W. Uny, PhD Universi~ of Alabama School of Medicine Birmingham, Ala.

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REFERENCES

1. Urry DW, Prasad KU, Long MM, Harris RD. Elastomeric polypeptides aspotential vascular prosthetic materials. Polymer Material Sci Eng 1988;59:684-9. 2. Wood SA, Lemons JE, Prasad KU, Urry DW. In vitro calcification and in vivo biocompatibility of the cross-linked polypentapeptide of elastin. J Biomed Mater Res 1986;20, 315-35. 3. HoUinger JO, Schmitz JP, Yaskovich R, Long MM, Prasad KU, Urry DW. A synthetic polypentapeptide of elastin for initiating calcification. CaIcif Tissue Int 1988;42, 231-6. 4. Lemons JE, Iqbal MA, Urry DW. Biocompatibility comparisons of Dacron, Dexon and radiation cross-linked poly(Val’Pro’-Gly3-Val*-Glf ). (Submitted for publication). 5. Pierschbacher MD, Ruoslahti E. CelI attachment activity of fibronectin can be duplicated by small synthetic fragments of the molecule. Nature 1984;309:30-3. 6. Graf J, Iwamoto Y, Sasaki M, et al. Identification of an amino acid sequence in laminin mediating ceil attachment, chemotaxis, and receptor binding. Ceil 1987;48: 989-96. 7. Rusolahti E. Fibronectin and its receptors. Ann Rev Biochem 1988;57:375-413. 8. McCarthy JB, Skubia APN, Zhao Q, Yi X, Mickelson DJ, Klein DJ, Furcht LT: RGD-independent ceU adhesion to the carboxy-terminal heparin-binding fragment of fibronectin involves heparin-dependent and independent activities. J Cell Biol 1990;110:777-87.

BIORBSORBABLE MATERIALS AND MACROPHAGE INTBRACTIONS The intuitively obvious presumption that a vascular graft need be strong and permanent neglects the possibility that the permanence of the foreign body, if not totally “biocompatible,” may render the prosthesis/tissue complex unstable by virtue of chronic foreign body reactions and/or suboptimal biomechanical properties. Biomaterials placed into the vascular system are not biologically inert. Restoration of circulation is followed instantaneously by a complex sequence of interactions at the interface with blood, surrounding tissues, and adjacent arterial segments. Such interactions include protein adsorption via passive and active processes, platelet adhesion and aggregation, neutrophil and monocyte deposition, macrophage activation, and endothelial cell and smooth muscle cell migration and proliferation. In the case of small diameter vascular grafts implanted into relatively low flow environments, it is the elicited tissue reactions that determine clinical efficacy. Experience in several laboratories has suggested that bioresorbable lactide/glycolide copolymeric vascular grafts implanted into animal models elicit more efficacious tissue reactions then do currently available nonresorbable materials, reactions mediated in part by macrophage activation and release of growth factors stimulating endothelial cell and smooth muscle cell proliferation. The rate of resorption of the lactide/glycolide family of copolymers is determined in part by the ratio of 1actide:glycolide rings. The higher the ratio, the slower the resorption. Resorption occurs primarily by hydrolysis, which occurs largely within the cytoplasm of phagocytizing macrophages and giant cells. The rate of tissue ingrowth

Journal of VASCULAR SURGERY

parallels the kinetics of macrophage mediated prosthetic resorption.le3 Macrophage phagocytosis of the prosthetic material begins as early as 1 week after implantation of rapidly resorbed materials such as polyglycolic acid (PGA) or polyglactin 910 (PG910) and is followed by an extensive increase in the myofibroblast population and neovascularization of the inner capsules.‘,* The source of both the myofibroblasts and the neocapillarization is a transinterstitial ingrowth from the surrounding tissues4 Such ingrowing capillaries likely provide the endothelium, which is seen repopulating the blood-contacting surfaces.2~4 Progressive tissue deposition stops after resorption of the prosthetic material at which time the macrophage population is no longer present. The tissue deposition results in a myofibroblast and collagen-laden wall 300 to 500 microns thick.‘-4 Autoradiographic analysis by use of tritiated thymidine demonstrate a significantly increased mitotic index within inner capsular myofibroblasts, that mitotic index paralleling the source of prosthetic resorption.” PG910, for example elicits in a mitotic index of 20.1% f 16.6% 3 weeks after implantation, progressively decreasing to 1.2% ? 1.3% after 12 weeks. The more slowly resorbed polydioxanone prostheses demonstrate a persistently elevated mitotic index, 7.1% ? 3.8%, 12 weeks after implantation, a time in which the prosthetic material is still being resorbed. By contrast Dacron never yields greater than a 1.2 r 1.3 mitotic index.5 These mitotic indexes correlate closely with the slopes of the inner capsule thickening curves suggesting that the myofibroblast proliferation contributes heavily to this tissue deposition. The labeled cells are found in the deeper zones of the inner capsule in proximity to the macrophages and the prosthetic material in the area in which the myofibroblasts demonstrate ultrastructurally synthetic phenotypes,5 a phenotype more commonly found in cells actively cycling.’ These myofibroblasts are likely the source of the collagen, the content of which also parallels the kinetics of tissue deposition and prosthetic resorption. Collagen content, determined by hydroxyproline spectrophotometric analysis, increases in direct relation to the rate of prosthetic resorption. In 1 month explants, PG910 inner capsules contain 3.55 kg collagen/mg dry weight compared to 2.22 and 2.59 for polydioxanone and Dacron respectively. In three month explants, collagen increases to 5.7 yg/mg in PG910,4.7 ug/rng in PDS, but no increase is found within Dacron explants. The resultant regenerated vascular conduit thus consists of abundant myofibroblasts and collagen beneath an endothelialized blood-contacting surface. This surface is capable of producing prostacyclin in response to exogenous arachidonic acid.6 After the resorption of the prosthetic material, regenerated tissues are capable of withstanding 2000 mm Hg pulsatile pressure ex vivo. Furthermore the compliance of these tissues does not decrease as a function of time as is seen after implantation of nonresorbable materials (as a result of fibroblastic reactions), but in fact slightly increases,’ a function of both prosthetic resorption and the myofibroblast ingrowth. Patency rates among bioresorbable grafts in rabbit and

Elastic protein-based polymers as cell attachment matrices.

746 Specid cotwwnicattin changes in membrane constituents, leading to activation. These alterations in membrane components may result in direct acti...
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