Effect of an alpha-blocker (Nicergoline) and of a beta-blocker (Acebutolol) on the in vitro biosynthesis of vascular extracellular matrix.

The effect of an alpha-blocking agent and of a beta-blocking agent on the biosynthesis of extracellular matrix macromolecules of the arterial wall was investigated. Rabbit aorta explants were cultured up to 48 hours with radioactive proline, lysine or glucosamine. In presence of these drugs, at concentration shown to be effective for the inhibition of platelet-endothelial cell interactions (10(-7) M), the incorporation of 14C proline in total macromolecular proline was higher than in macromolecular hydroxyproline suggesting a relatively higher rate of biosynthesis of non-collagenous proteins as compared to collagens. The alpha-blocking increased the incorporation of 14C proline in collagenous and non-collagenous proteins after 18 hours of incubation. beta-blocking also increased the incorporation of proline in macromolecular proline and hydroxyproline as compared to control cultures. Both increased the incorporation of 3H glucosamine in newly synthesised glycosaminoglycans. beta-blocking increased mainly the neosynthesis of heparan sulphate, alpha-blocking that of hyaluronan. The incorporation of 14C-lysine in crosslinked, insoluble elastin was not modified. These experiments confirm that alpha and beta-blocking agents can influence not only the tonus of aortic smooth muscle cells but also the relative rates of biosynthesis of extracellular matrix macromolecules. This effect should be taken in consideration for the evaluation of the long range effect of alpha and beta-blocking drugs on the vascular wall.

Pseudointima in inflow and outflow conduits of a left ventricular assist system: possible role in clinical outcome.

Activation of blood coagulation and thromboemboli have been shown to present significant clinical risks in patients supported with an left ventricular assist system (LVAS). The interaction of pseudointima (PI) with blood in the conduits of the device could be involved in these clinical complications. Our aim was to study the morphology of the PI versus duration of circulatory support. Novacor N 100 PC LVASs were explanted from 10 men and 2 women after a mean of 209 days (range 23-560 days) of circulatory assistance. PI in the inflow and outflow conduits were investigated with immunohistochemical assays. In the inflow conduits, a loosely adherent PI had built up from collagen type I and III fibers growing into and between fibrin deposits. Disorganized collagenous matrix and longitudinally oriented collagen fibers included alpha-smooth muscle actin positive cells with random orientation. Macrophages were concentrated in the fibrin and were dispersed throughout the extracellular matrix. In the outflow conduits, a thin, adherent PI was composed of regular collagen type I and III layers. Collagen type I fibers had grown into the woven Dacron and alpha-smooth muscle actin positive cells were oriented in the axis of the blood flow. Macrophages were concentrated in the Dacron and reached the inner collagen layers. Venous blood flow in the inflow conduits allows the development of a non endothelialized irregular collagenous matrix intermingled with fibrin and invaded by macrophages. These persistent structural features progress with duration of circulatory assistance and reflect matrix degradation and remodeling. The potential to release thromboembolic fragments from the non stable, thrombogenic PI may be involved in the thromboembolic or neurologic complications sustained by 5 of 12 patients who were on circulatory support for as long as 200 days.

Effect of variation in systemic blood flow on plasma TNF-alpha in a pig model with left ventricular assist device.

Tumor necrosis factor-alpha (TNF-alpha) release has been implicated in a sepsis-like syndrome following cardiopulmonary bypass (CPB). This also may be important in patients who have had a left ventricular assist device (LVAD) implanted. This report investigates the effect of reducing systemic blood flow on hemodynamic response, mixed venous oxygen saturation (SvO(2)), and the release of TNF-alpha. LVADs were implanted in 9 pigs. The aorta was clamped, and thus the LVAD flow represented the entire systemic blood flow. Plasma TNF-alpha in the femoral artery (FA) and superior mesenteric vein (SMV) was measured at baseline and following systemic blood flow changes. Simultaneously, hemodynamic parameters and oxygen saturation in the pulmonary artery (SvO(2)) were measured. Following reductions in systemic blood flow, plasma TNF-alpha increased gradually to a maximum level at a systemic blood flow of 20%. There was no significant difference between TNF-alpha levels in the SMV and the FA. There was a significant (p < 0.05) correlation between cardiac index, stroke volume index, and TNF-alpha. The SvO(2) decreased significantly (p < 0.05) at a systemic blood flow of 30 and 20%. A rise in TNF-alpha occurred when the SvO(2) was less than 75%. The data demonstrate that a reduction in systemic blood flow causes an increase in plasma TNF-alpha. This can lead to the development of a sepsis-like syndrome in a group of patients who already are hemodynamically compromised. While weaning short-term LVAD support, rapid diminution of the cardiac output and the pump flow must be avoided.

Intestinal tissue oxygenation and tumor necrosis factor-alpha release during systemic blood flow changes in pigs with left ventricular assist devices.

We previously demonstrated that tumor necrosis factor-alpha (TNF-alpha) increased following a reduction in systemic blood flow to 60% or less of the original cardiac output using a left ventricular assist device (LVAD). The aim of this study was to investigate the effect of reducing systemic blood flow on tissue oxygenation in the gastrointestinal tract (GIT) and the consequences of this on TNF-alpha release. LVADs were implanted in 9 pigs. The aorta was clamped, and thus the LVAD flow represented the entire systemic blood flow. Plasma TNF-alpha of the superior mesenteric vein was measured at baseline and during systemic blood flow changes. Simultaneously, pH, lactate, oxygen delivery index (DO(2)I), oxygen consumption index (VO(2)I), and oxygen extraction (O(2)ER) in the GIT were measured. The pH decreased and the lactate level increased significantly (p < 0.05) at a systemic blood flow of 50% or less. The VO(2)I was positively correlated with DO(2)I. The O(2)ER increased significantly (p < 0.05) with reductions in systemic blood flow to 30% or less. There was a significant (p < 0.01) correlation between TNF-alpha and O(2)ER at levels higher than 55%. These data demonstrate that the GIT oxygenation is inadequate with a reduction in systemic blood flow to 50% and that GIT oxygenation becomes critical at a reduction of 30%. During LVAD weaning, careful attention must be given to the GIT. The pH and lactate may be good markers of the adequacy of tissue oxygenation in the GIT.

New agents for the treatment of infarcted myocardium.

Local delivery of angiogenic growth factors for the treatment of myocardial ischemia has been well documented in various animal models, and clinical trials are now in progress. Our strategy was radically different, based on selective protection of some of the growth factors naturally present within the injured tissue. This protection was obtained by applying a chemically defined substitute for Dextran called RGTA11 (for ReGeneraTing Agent). RGTA is a family of agents, which has properties mimicking those of heparan sulfates toward heparin-binding growth factors (HBGF) and which stimulate tissue repair and protection. Indeed, we have previously shown that RGTA prevents most of the damage resulting from acute skeletal muscle ischemia [FASEB J. (1999) 13, 761-766]. We now show that the same agent can be used for the treatment of myocardial infarction. Acute myocardial infarction was induced in pigs by ligation of the left circumflex artery. One hour later, a single injection of 10 mg of RGTA11 was made in the center of the infarcted area. Three weeks later we observed 1) recovery of 84% of the initial left ventricular ejection fraction (only 55% in saline-treated controls), 2) an almost 50% reduction in the infarct size, 3) a reduction in fibrotic tissue formation, 4) significant preservation of myocytes, and 5) an increase in the number of blood vessels. The treatment of ischemic heart disease with RGTA would have clear advantages over other therapies such as growth factor, gene, or cell transplants, based on a stable, simple, and easy-to-develop chemical product.

Structural changes in porcine bioprosthetic valves of a left ventricular assist system in human patients.

BACKGROUND AND AIM OF THE STUDY: Porcine, specially manufactured bioprosthetic valves regulate blood flow from the left ventricle to pump sac (inflow valve) and from the pump to the aorta (outflow valve) in a wearable, electrically powered left ventricular support system (LVAS, Novacor). The increased need for long-term circulatory assistance requires information on the evolution of these valves when exposed to specific hemodynamic conditions and inflammatory reactions in the device. The study aim was to examine structural changes in valves from explanted LVASs. METHODS: Thirteen patients (11 males, two females; mean age 42 years (range: 17-64 years) were supported for a mean of 285 days (range: 37-1,293 days) with LVAS. Histologic sections from explanted inflow and outflow valves were studied immunohistochemically using peroxidase-labeled antibodies and avidin-biotinylated peroxidase complex for detection. RESULTS: In the macroscopically normal inflow valves (11/13), the outflow surface (facing the pump) was covered with a discontinuous deposit of fibrin, macrophages and granulocyte elastase. Fibrinogen, IgG, complement proteins C1q and C3 had infiltrated the extracellular matrix (ECM) between 37 and 1,293 days. The crevices were enlarged during circulatory support, and fibrinogen/fibrin insudations were detected in the spongiosa. The collagen layers in the fibrosa were disrupted after 293 days, and eroded on the inflow surface in the ventricularis after 1,293 days. In a deteriorated valve from a patient with endocarditis, Gram-positive bacteria and metalloproteinases were concentrated in the ECM. In the macroscopically normal (11/13) outflow valves, fibrin and complement proteins had penetrated the ECM from the inflow side (facing the pump), while macrophages and granulocytes were localized mainly on the outflow surface. IgG and complement proteins were detected on and beneath the cusp surface up to 200 days and covered the disrupted ECM as implant time progressed. CONCLUSIONS: Structural changes appear to progress more rapidly in the inflow than in the outflow of bioprosthetic valves. This difference indicates that the effects of biological factors are modulated by mechanical stress.

Pseudointima in inflow conduits of left ventricular assist devices.

BACKGROUND: Explant analysis of left ventricular assist systems (LVAS) should permit a better evaluation of long-term evolution of materials and tissue healing in patients supported by mechanical devices and a precise understanding of embolic phenomena, observed clinically. METHODS: Five Novacor LVAS and their conduits have been explanted after 156 days (range 61-226 days) of mechanical support. The pseudo-intima (PI) developed in the inflow and outflow conduits was characterized microscopically, using monoclonal antibodies. RESULTS: The morphological aspects of PI were quite different in the inflow and outflow conduits. Blood coagulation between the basal surface of the PI and the Dacron tube, irregular collagen type I matrix with plasma infiltration, macrophages, and neutrophil granulocyte elastase characterized the nonadherent, loose, and potentially thrombogenic PI growth in the inflow conduit. The PI from collagen types I and IV with circumferentially oriented alpha-smooth muscle cell actin-positive cells was anchored to the outflow conduits. CONCLUSIONS: The observations, which have to be confirmed by a more extensive study on a larger number of specimens, suggest the role of the biomaterial itself, as well as the configuration, physical characteristics, and rheology in the conduit. They also suggest that thromboembolic complications of LVAS may eventually be related to this host tissue response.

Inflammatory response to cardiopulmonary bypass using two different types of heparin-coated extracorporeal circuits.

Previous reports have highlighted the disparity in biocompatibility of two differently engineered heparin coatings during the cardiopulmonary bypass (CPB) procedure. The aim of this prospective study was to evaluate the impact of the difference in haemocompatibility provided by either the Duraflo II equipment or the Carmeda equipment in the terminal inflammatory response observed after coronary artery surgery. Thirty patients were randomly allocated to two groups to be operated on using either Duraflo II equipment (group I) or Carmeda equipment (group 2) for extracorporeal circulation (ECC). Initial inflammatory response was assessed by terminal complement complex activation (SC5b-9). The late inflammatory response observed in the postoperative period was assessed by measuring cytokine production (tumour factor necrosis (TNF alpha), interleukin IL-6, interleukin IL-8) and circulating concentrations of adhesion molecules (ELAM-1, ICAM-1). The release of SC5b-9 after CPB and after protamine administration was lower in group 2 than in group 1 (p = 0.0002 and p = 0.006, respectively). A significant production of cytokines was detected in both groups with peak values observed within the time range of 4-6 h after the start of CPB.

External biodegradable supporting conduit protects endothelium in vein graft in arterial interposition.

The prevention of circumferential distension could reduce structural damage in arteriovenous grafts. We studied the effect of an external biodegradable supporting conduit on the endothelium and extracellular matrix in vein graft in a pig model. Cephalic vein control grafts (Group I) and jugular veins wrapped in a vicryl mesh tube (I.D. 4 mm) (Group II) were implanted into autologous carotid arteries (n = 14). The grafts were explanted after 1 and 24 hours and at 1 and 3 weeks and evaluated by ELISA for endothelial DNA synthesis and by immunohistoenzymic assays for cells and extracellular matrix. In group I an initial loss of endothelial and smooth muscle cells along with elastin breakdown was followed by an impaired endothelial regeneration and significant graft wall thickening. The elastic tissue was replaced by collagen type I and chondroitin sulfate accumulations, which included a disarray of alpha-smooth muscle actin positive cells. The endothelium was preserved in group II. After 3 weeks the circumferential elastin layers were densified, distended and separated from the endothelium by a neointimal growth of irregular thickness. Biodegradable perivenous conduit minimized endothelial injury and allowed the partial preservation of elastin fibers and smooth muscle cells in the arteriovenous graft. It did not however, prevent myofibroblastic cell proliferation and triggered a macrophagic reaction.

Immunoglobulins and complement deposits in mitroflow pericardial bioprosthetic heart valves--a contributing factor to structural deterioration.

BACKGROUND AND AIMS OF THE STUDY: Twenty-two bovine pericardial Mitroflow prostheses were explanted after 73-114 months from either the aortic or mitral position because of clinical failure. All the samples exhibited cuspal tears and foldings. Eleven prostheses were calcified. The aim was to study biological factors involved in the structural deterioration. METHODS: Histologic and biochemical assays were carried out on the deteriorated, non-calcified and on the calcified leaflets. Labelled antibodies and avidinbiotinyilated peroxydase complex were used to detect plasma proteins and cells in the cusps. RESULTS: Fibrin covered the cuspal surface and accumulated in the deep disrupted layers (19/22). Scattered fibronectin filaments were seen across the transversal sections (20/22). IgG, complement fractions C1q, C3, C4 (20/22), macrophages (sixteen) and cells containing granulocyte elastase were revealed in the altered matrix. These plasma proteins and cells were detected in the disintegrated matrix of non-calcified and of calcified leaflets. IgA was present in amorphous cuspal thickenings with lipid infiltration (12/22). Western blot analysis of the PBS-2% SDS extracts from the leaflets indicated the breakdown of fibrinogen/fibrin, fibronectin and of complement proteins C3, C4 and C5. CONCLUSIONS: The results suggest the activation of the complement by the non-hemocompatible, chemically processed bovine pericardium. The bioactive peptides generated in this process can stimulate monocyte migration, phagocytosis and exocytosis of proteases able to degrade the glutaraldehyde cross-linked macromolecular matrix. These biological factors can contribute, together with the mechanical stress, to the structural deterioration of the bioprosthesis.

Complement activation is involved in the structural deterioration of bovine pericardial bioprosthetic heart valves.

Disintegrated collagen fibers surrounded with protein deposits are a morphologic feature in torn, folded, and disrupted cusps of pericardial prostheses explanted for clinical dysfunction. New technologies for valve bioprostheses with improved durability require further investigation of molecular mechanisms initiating the deterioration of bioprosthetic valves. The authors' aim was to obtain experimental evidence of biologic factors contributing to the degradation of the bioprosthetic matrix. Clinically failed Mitroflow (22), Hancock (3), Ionescu-Shiley (2), and Sorin (1) valves were explanted after 69-170 months. Non calcific deterioration of the prosthetic matrix was studied with labeled antibodies to plasma proteins and cells. IgG, and complement proteins C1q, C3, and C4 were accumulated close to dissociated collagen bundles (26/28) throughout the prostheses. Fibrin was identified on the cuspal surface and in the deep disrupted areas. The fibrin peptides and proteolytic breakdown products of the complement components, the latter consistent with complement activation and chemotaxis for monocytes, were shown by immunoenzymic assay on Western blots from the valve extracts. The complement activation triggered by the IgG aggregates generates bioactive peptide signals that can activate macrophages (22/28) and neutrophil granulocyte elastase (22/24) able to cooperate with the mechanical stress in the breakdown of the chemically processed, non hemocompatible, and non-self macromolecular matrix.

The superiority of hollow fiber membrane over bubble oxygenator in a perfusion circuit for the evaluation of small caliber endothelialized arterial prostheses.

A perfusion circuit was constructed from a pneumatic ventricular assist device, 2 compliance chambers, 4 small-diameter silicone tubes (ID 4 mm) simulating shear inducing vascular prostheses, and an oxygenator with a heat exchanger. A bubble oxygenator (in a BO circuit) and a hollow fiber membrane oxygenator (in an MO circuit) were studied. The circuits were perfused with 30% human serum containing culture medium for 7 days at 37 degrees C. The pH, Po2, PCo2, Na+, K+, Ca2+, Cl, glucose, and total protein concentrations remained the same in BO and MO circuits during the 7 days of perfusion. The differences between the values measured in the perfusion medium and in the medium maintained in the static conditions of cell culture were not significant. In the BO circuit, the amount of cholesterol and triglyceride concentrations decreased whereas the relative amounts of albumin, alpha 1, alpha 2, beta, and gamma globulins remained stable in the perfusion medium. The medium from the BO circuit did not promote the proliferation of cultured human saphenous vein endothelial cells. In the medium from the MO circuit, the cholesterol and triglyceride concentrations did not change with perfusion time; the proliferation rate and anticoagulant function of endothelial cells were maintained. The hollow fiber membrane oxygenator preserves the biological characteristics of the cell culture medium in a perfusion circuit. The MO circuit permits the performance of relevant studies on shear stress resistance and functional activity of human endothelial cells seeded onto vascular prostheses.

Effects of cryopreservation on the proliferation and anticoagulant activity of human saphenous vein endothelial cells.

Human saphenous veins were cryopreserved in 4% human albumin and 10% dimethyl sulfoxide. The effect of cryopreservation on endothelial cells was studied in terms of the anticoagulant activity of thrombomodulin and in terms of cell proliferation. After storage for 2 weeks at -150 degrees C, 0.45 +/- 0.07 x 10(5) endothelial cells/cm2 were detected in cryopreserved veins and 1.03 +/- 0.04 x 10(5) endothelial cells/cm2 in fresh veins (p < 0.01). The thrombin-catalyzed activation of protein C decreased after cryopreservation, indicating altered thrombomodulin activity in the endothelial cells. On a cell number basis, the release of soluble thrombomodulin was three times higher from the cryopreserved endothelium than from the fresh endothelium (p < 0.05). The amount of spontaneous release of von Willebrand factor from the endothelial surface was not significantly different between fresh and cryopreserved veins. Endothelial cells were cultured from fresh veins and from their cryopreserved counterparts. On plating of endothelial cells in primary culture, the number of adhered cells was 0.9 +/- 0.09 x 10(3) cells/cm2 from fresh veins and 0.25 +/- 0.03 x 10(3) cells/cm2 from cryopreserved veins (p < 0.01). The positive immunohistochemical stain for von Willebrand factor indicated that the endothelial cell character was maintained after cryopreservation. The endothelial desquamation with loss of anticoagulant function and the slow proliferation of surviving cells in vitro suggest an impaired endothelial healing in vivo. The loss of anticoagulant activity complicates the problems of the exposure of thrombogenic subendothelial matrix to blood in implanted cryopreserved veins.

Heparin and its derivatives modulate serine proteinases (SERPS) serine proteinase inhibitors (SERPINS) balance. Physiopathological relevance.

Heparin and heparan sulfate, exhibiting wide biological interactions, are constituted of block structures. A defined pentasaccharide motif was found responsible for the enhancement of the rate of inactivation of factor Xa by antithrombin III. Heparin also interacts with other serine proteinase inhibitors as protease nexin I, and thus possibly modulates extracellular matrix proteolysis by serine proteinases in the pericellular environment. Human neutrophil elastase (HNE) activity is inhibited by heparin with Ki = 75 pM. This strong interaction is electrostatic, involving HNE/arginine residues disposed in a "cluster shoe" arrangement on the surface of the molecule and mainly OSO3- groups of heparin. HNE-heparin interactions also interfere with HNE associations with its natural inhibitors: it decreases the rate of association of HNE with alpha 1 proteinase inhibitor (alpha 1 P(i)) by 3 orders of magnitude, while increasing kass between HNE and mucus bronchial inhibitor (MBI) by > 10 fold. In vivo experiments demonstrated that heparin fragments lacking anticoagulant activity were able to nearly completely abolish emphysematous lesions induced in mice by a single intratracheal administration of 200 micrograms HNE. Long chain unsaturated fatty acids peptide conjugates were described as competitive HNE inhibitors (Hornebeck W. et al. 1985). We synthesized N-oleoyl heparin derivative (3 oleoyl groups/one molecule of heparin); such a lipophilic glycosaminoglycan (LipoGAG), although acting as an elastin protecting agent, possessed lower HNE inhibitory capacity as compared with heparin. In contrast, however, it was able to inhibit other serine proteinases such as urokinase, plasmin, porcine pancreatic apha-chymotrypsin and elastase. Such Lipo GAG's can be therefore useful to control matrix metalloproteinases (MMPs) during tissue remodeling or tumor invasion.

Congenital soft tissue dysplasias: a morphological and biochemical study.

The term congenital soft tissue dysplasias (CSTDs) regroups some localized malformations of covering soft tissues in children, presenting as various clinical entities, either recognized as particular syndromes (e.g., Parkes-Weber, Klippel-Trenaunay, Proteus) or, most often, appearing less stereotyped (e.g., segmental hypertrophy or gigantism, lymphedema, angiodysplasia, phakomatosis), with a common histopathological lesion, the hamartoma. The aim of this paper is to report a morphological and biochemical study of the extracellular matrix of skin and subcutaneous tissue in children with CSTD. For every patient, pathological tissues were compared with contralateral, symmetrical tissues, taken as controls. In all CSTDs, pathological samples were characterized by an increase in water and total glycosaminoglycan (GAG) content with a decrease in collagen content. Other results lead the authors to distinguish two main entities, segmental dysplasia (SeD) and neuroectodermal dysplasia (NeD). Elastic fiber content was increased in SeD and decreased in NeD. Hyaluronic acid (HA) and dermatan sulfate (DS) were increased in NeD, whereas in SeD, HA was decreased with an increase in the DS/HA ratio. Cultured fibroblasts from dysplastic skin had slower proliferation in vitro than fibroblasts from control skin, whereas their biosynthetic activity concerning collagen and GAGs was greater. The difference in the composition of extracellular matrix supports the clinical classification of CSTDs in two main groups: segmental dysplasia with or without gigantism and neuroectodermal dysplasia (in von Recklinghausen's disease and nevi).

Deterioration of bioprosthetic heart valves.

The aim of this study was to detect biologic factors in the structural deterioration of bioprosthetic heart valves. Prostheses were removed from patients after 4-8 years of implantation and submitted to biochemical and morphologic assays. Successive staining of biologic sections revealed colocalization of lipids and glycosaminoglycans underneath calcifications in the disintegrated extracellular matrix. On biochemical assays, the amidolysis of synthetic peptide substrates indicated thrombin, plasmin, and tissue plasminogen activator activities in the nonhemocompatible leaflets; 0.15 mol NaCl, 0.05 mol Tris, and 5 mmol CaCl2 extracts from the prostheses cleaved the peptide substrate for collagenase and lysed gelatin gels. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate disclosed the presence of low molecular mass polypeptides in extracts of the deteriorated prostheses. The detection of plasmin and collagenolytic enzyme(s), and the known broad proteolytic activity of plasmin, may point to the role of activation of the fibrinolytic system in the proteolytic degradation of bioprosthetic valves.

Human vascular endothelial cells on expanded PTFE precoated with an engineered protein adhesion factor.

To elucidate the role of the molecular structure of adhesive proteins in an endothelialization of synthetic vascular prosthesis in vitro, a recombinant fibronectin-like engineered adhesion factor (FP) constructed from the specific Arg-Gly-Asp cell adhesion repeats was assayed as adhesive substrate to culture human saphenous vein endothelial cells on ePTFE. ePTFE samples (1 cm2) inserted into cell culture chambers were coated by incubation with increasing amounts of FP (up to 100 micrograms/cm2) prior to cell seeding. At 24 hours after low density cell seeding and 20 micrograms/ml/cm2 FP concentration, the number of adhered cells reached a plateau and the adhered cells did not proliferate up to 6 days of culture. At 24 hours after high density seeding (10(5) cells/cm2), the number of adhered cells was significantly higher on ePTFE with preadsorbed FP than on fibronectin coated PTFE. About 55% of the initially adhered cells survived up to 7 days on FP, whereas cell debris and free nuclei were predominant on fibronectin coated PTFE. In the investigated model the engineered RGD polymer potentialized a short-term adhesion of vascular endothelial cells to PTFE, nevertheless it did not ensure proliferation and long-term survival of these cells.

Accumulation of heparan sulfate in the culture of human melanoma cells with different metastatic ability.

Glycosaminoglycans were metabolically labeled in subconfluent cultures of highly metastatic 7Gp122 and poorly metastatic IC8 variants and of the low metastatic parental M4Be human melanoma cell line. Proteoglycans were separated by DEAE Trisacryl chromatography from the culture medium, from the heparin extract of the cell layer and from the heparin-extracted cell residue lyzed with detergents. Glycosaminoglycans were released from the proteoglycans by reductive alkaline hydrolysis and heparan sulfate (HS) was detected by deaminative cleavage with nitrous acid. Expressed on cell protein basis, the labeled HS content in the medium and in the cell layer decreased with increasing metastatic ability. The extraction of HS with heparin from the 7Gp122 cells indicated that this variant was enriched in (polypeptide bound) HS non inserted into the plasma membrane, compared with the low metastatic IC8 and M4Be cells. The HS fraction in heparin extract and in the heparin-extracted cell residue exhibited molecular mass heterogeneity on gel permeation chromatography and it contained HS fragments. Scission with nitrous acid followed by molecular sieve chromatography of the degradation products indicated that the tetra- and disaccharide repeats separated by the N-sulfated glucosamine residue were present in about equal amounts and constituted 60% of the HS chains in the IC8 and M4Be cells. HS from 7Gp122, IC8 and M4Be cells did not bind antithrombin III with high affinity but it was capable of binding bFGF in in vitro assay.

Stimulation of cell proliferation by hyaluronidase during in vitro aging of human skin fibroblasts.

The effect of the degradation of extracellular hyaluronan on the proliferation of human skin fibroblasts in serial cultures during in vitro aging was investigated. Human skin fibroblasts at different time intervals from 3rd to 36th passages were exposed after plating to bovine testicular hyaluronidase. The enzyme treatment resulted in an increase in cell proliferation (cell number vs. time) as compared to the untreated control fibroblasts. The effect was dose dependent, reversible, and was independent of the type of the glycosidic linkage cleaved in hyaluronan. The increased proliferation was observed at all passages when untreated cells underwent mitosis. The degradation of hyaluronan induced cell proliferation up to the presenescent phase. Depletion of hyaluronan did not induce proliferation of postmitotic fibroblasts. The incorporation of 3H-glucosamine into hyaluronan decreased with increasing cell passages (increase of the number of population doublings). Twenty-fourth passage fibroblasts accumulated about two time less hyaluronan in the medium than ninth passage cultures. Following hyaluronidase treatment, the amount of newly synthesized, labeled hyaluronan increased in the medium. Accordingly, the fibroblasts restored the degraded hyaluronan even in the declining phase of proliferation (phase III according to Hayflick).