Biomedical subjects
Publications and source records attributed to R Eisenstein.
The inner parts of the walls of large blood vessels do not normally contain intrinsic vasculature. In pathologic conditions such as arteriosclerosis or thrombosis, angiogenesis occurs, and may have significant clinical consequences. This review attempts to relate the little that is known about the factors specific to vascular walls which regulate angiogenesis to more general knowledge of the phenomenon.
The aqueous and vitreous humors are normally avascular and may be sites of altered inflammatory responses. Inhibitors of endothelial cell and lymphocyte growth as well as of angiogenesis have been isolated from vitreous and an inhibitor of lymphocyte mitogenesis has been found in aqueous. Data are presented here indicating that aqueous humor also inhibits endothelial cell growth. In addition, neutralizing antibodies against TGF beta block the effects of materials isolated from these tissues on endothelial cell and lymphocyte growth. Similar data were obtained from aorta, another normally avascular site. These observations suggest that the avascularity and altered inflammatory responses in these sites may be regulated in part by TGF beta.
Amino-terminal and carboxyl-terminal fragments of human plasma fibronectin, at nM concentrations, reversibly inhibit the growth of cultured bovine aortic endothelial cells. To define smaller active peptides, synthetic peptides corresponding to a carboxyl-terminal disulfide bonded loop segment of one of the fragments were tested for activity and found to be active at microM concentrations. The data suggest that the entire loop sequence is required for full expression of endothelial cell growth inhibitory activity in vitro.
Bovine vitreous body and aorta contain extractable leukocyte elastase inhibitors, which were purified by gel filtration and affinity chromatography on agarose-pancreatic elastase. The purified inhibitor preparation from aorta was resolved by polyacrylamide gel electrophoresis into a main band migrating slightly faster than commercial Trasylol and a more weakly stained band migrating close to chymotrypsinogen. The purified inhibitor preparation from both sources inhibited, in a competitive fashion, purified human leukocyte elastase and was ineffective against bovine trypsin and leukocyte cathepsin G or collagenase. These inhibitors from vitreous body and aorta were distinguishable by several criteria from serum inhibitors.
Heparin-binding fragments derived from the amino- and carboxyl-terminal regions of human plasma fibronectin appear to be at least relatively specific potent inhibitors of the growth of bovine aortic endothelial cells in culture by as yet unknown mechanisms. In order to understand better the sites which subserve this activity, we have compared the relative potency of other major fragments of fibronectin, most of which have dissimilar properties and do not bind heparin. We have also proteolytically digested and chemically modified the most potent of these fragments, the amino-terminal 29-kDa fragment, in order to test whether structural alterations that affect heparin-binding also affect the inhibitory property. Not all chemical modifications that abolished heparin-binding also abolished endothelial cell growth. Neither an amino-terminal 20-kDa nor a carboxyl-terminal 8-kDa subfragment of the 29-kDa fragment bound heparin; however, both were as inhibitory as native 29-kDa fragment. Reduction of the disulfides of the 20-kDa and 8-kDa fragments did not abolish inhibitory activity. We therefore conclude that the activity is not strictly conformation-dependent and that although the inhibitory activity is distributed throughout the 29-kDa segment, it can be expressed by an 8-kDa carboxyl-terminal segment containing residues of the last Type I loop structure.
A factor of nominal molecular weight 6K-10K Daltons, isolated from bovine aorta, has previously been shown to inhibit neovascularization and tumor growth in vivo and the growth of some tumor cells as well as endothelial cells in culture. This factor, termed A-10, was tested alone and in combination with Adriamycin against TA3Ha mammary adenocarcinoma cells in tissue culture. It was found to have cytotoxicity additive to that of Adriamycin in inhibiting the growth of these cells. In vitro and animal studies show that the sequence of Adriamycin----A-10 is superior to either agent alone in delaying the appearance of palpable tumors after subcutaneous injection of 10(5) pre-treated tumor cells in the tail of strain A mice. While the growth rate of the primary tumor was not affected by such treatment, survival was prolonged to a greater degree by the this sequence than by either of these agents used alone. A-10 treatment reduced the number of metastases to the adrenal gland but not to lung, liver, or lymph nodes. It did, however, reduce the size of metastases to para-aortic lymph nodes.
Bovine eyes were dissected and separate pools of lens, lens capsule, cornea and vitreous were extracted in guanidine, subjected to ultrafiltration, and examined for their effects on collagenolytic activity. Although lens extract was not inhibitory, the cornea and vitreous both contained inhibitors of collagenase. More inhibition was present in the filtrate of the vitreous than in the retentate, whereas the total amount of inhibition in the cornea was distributed almost equally between the two fractions. The inhibition observed was dose dependent. The partially purified inhibitors from cornea and vitreous blocked the activity of human skin and tadpole back skin collagenases, but they failed to inhibit the bacterial (Clostridium histolyticum) collagenase. The inhibitor was stable to heating to 60 degrees for 30 minutes and to trypsinization.
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The histologic distinction between mesotheliomas and adenocarcinomas on mesothelial surfaces is sometimes difficult. High concentrations of hyaluronic acid in pleural and peritoneal fluids of mesothelioma patients have been reported. This paper describes results of glycosaminoglycan electrophoresis of papain digests of 7 mesotheliomas and 27 other tumors. Either all or almost all demonstrable glycosaminoglycans in mesotherliomas was hyaluronic acid. Control tissues had variable electrophoretic patterns showing other glycosaminoglycans in addition to hyaluronic acid. In the clinical context of differentiating mesotheliomas and adenocarcinomas in tissue samples, this technique seems simple, economical, and relatively specific.
A low molecular weight fraction of bovine aortic extract inhibited corneal vascularization and edema in rabbits when administered either subconjunctivally or topically as long as 48 hours after injury. The extract also appeared to enhance the regression of newly formed corneal vessels. Topical administration for as long as two months had no deleterious ocular side effects. Tissue culture experiments showed that analagous fractions prepared from bovine vitreous inhibit endothelial cell growth. The major growth inhibitor of corneal neovascularization was not the Kunitz bovine protease inhibitor.
The individual and combined effects of PGD2, PGI2 and an aortic proteoglycan on human platelet aggregation and plasma clotting were studied. PGI2 was at least 10 times more potent than PGD2 in inhibiting platelet aggregation. Small doses of prostaglandins inhibited ADP- and thrombin-induced aggregation, but only prolonged aggregation time without affecting the extent of arachidonic acid (AA)-induced aggregation. Small doses of prostaglandins did not affect thrombin-induced clotting of PRP. Large doses of prostaglandins abolished platelet aggregation and prolonged the onset of thrombin-induced clotting. The aortic proteoglycan (APG) had no appreciable effect on ADP- or AA-induced aggregation. Small doses of APG abolished thrombin-induced clotting, while large doses of APG suppressed both clotting and aggregation induced by thrombin. PGI2 and PGD2 showed additive inhibition of platelet aggregation regardless how the aggregation was induced. APG and prostaglandins showed additive inhibition of only thrombin-induced aggregation. APG, but not any of the prostaglandins, prolonged clotting time of PPP. This prolongation was not potentiated by PGI2 or PGD2.
The behavior of bovine aortic endothelial cell cultures indicates that they interact with the extracellular materials they produce. In nonconfluent cultures the basal part of the cell is oriented on the plastic of the culture dish with the apex facing the culture medium. At confluence, fibrillar and ruthenium red-stained material, partly proteoglycan in nature, appears beneath the cells. In older cultures, a second layer of endothelial cells grows beneath the original one next to the plastic. This layer is oriented differently, being inverted in that the apex of the cells points to the plastic and the base toward secreted extracellular materials. Because of this orientation, cells tend to peel off the dish. The morphologic sequences thus suggest that polarity of endothelial cells in culture depends on its environment, with a hierarchy of polarity. In the absence of matrix materials, orientation is determined by the plastic. When extracellular materials form, endothelial cells then orient on them. Thus, the cells appear to manufacture and secrete the molecules on which their topography depends. Smooth muscle cells from the same bovine aortas do not seem to have this polarity. Glycosaminoglycans synthesized by the two cell types also differ.
If at all successful, tis paper will emphasized the perspective that the current understanding of the interactions between arterial walls and the flowing blood, the major events leading to the development of arterial diseases, still has many obvious gaps. Even as superficial and selective a discussion as this one points out the truism that any hypothesis for the pathogenesis of atherosclerosis which considers only one of the structural elements in the artery wall is almost certainly incomplete. Decades ago, one of the great conceptual insights developed in the pioneering studies of intermediary metabolism was that well-integrated biochemical pathways have multiple interrelated controls. The many extracellular molecules present in aorta with both structural biochemistry and functions as yet only partially understood may well be components of analogous control systems.
Studies on the effects of bovine aorta extracts on endothelial and smooth muscle cells cultured from the same tissue indicate they contain at least four separate molecular species capable of inhibiting cell growth. Two, heparin and a recently characterized dermatan sulfate-chondroitin sulfate proteoglycan, are large polyanions and inhibit the growth of both smooth muscle and endothelial cells, but require large amounts to induce this effect. The other two are low molecular weight molecules which inhibit only the growth of endothelial cells, one of which is a protease inhibitor which has been purified to homogeneity. The fourth and most potent is an as yet unidentified fraction distinguishable from the protease inhibitor. Heparin, as reported by others, and the proteoglycan also enhance collagenase activity. It would therefore appear that aorta contains several molecular species which have the potential to affect both invasive and proliferative processes.
A low-molecular-weight fraction prepared from extracts of bovine aorta inhibits the growth of a transplantable mammary tumor and a fibrosarcoma in mice when injected systemically. It also inhibits the growth of the fibrosarcoma in cell culture. The effect on the fibrosarcoma is much more marked than on the mammary tumor. Since the extract is more effective against the fibrosarcoma and is known to inhibit the growth of endothelial cells, it appears that the enhanced effect on this tumor is due to its activity on the endothelial cells of the host and the tumor cells themselves. The material injected is enriched in an antiproteinase we have previously isolated, which has anticollagneolytic activity and is presumed to be the effector molecule.
A single subconjunctival injection of 250 microgram of a partially purified extract of bovine aorta, administered immediately before or after silver nitrate injury to the cornea, markedly inhibited corneal vascularization in the rabbit eye. We believe the active molecule is a protease inhibitor that prevents the potential source of new vessels from proliferating and invading the diseased cornea.
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Areas of medial alteration (Erdheim cystic medionecrosis) in an aortic root aneurysm surgically removed from a patient with Marfan syndrome were studied with an electron microscope. The single striking morphologic abnormality was the disorganized, haphazard architecture of the media in these areas. This lesion is interpreted as being an abortive reparative response to hemodynamic stress in a condition with a molecular defect in the structural proteins of connective tissue.