Madeleine Moczar

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 μg 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.

Age Dependence of the Biosynthesis of Intercellular Matrix Macromolecules of Rabbit Aorta in Organ Culture and Cell Culture

The age dependence of the relative rate of biosynthesis of intercellular matrix macromolecules was studied in organ culture and cell culture obtained from aortas of newborn, young and adult rabbits. In organ culture there was a strong decrease with age of the rate of incorporation of (14C)-lysine and (3H)-glucosamine in all macromolecular fractions. Neosynthesis of elastin could be demonstrated by the isolation of labelled demosine at all ages. In cell cultures derived from newborn and adult aortas, no decrease in total incorporation was noticed. The pattern of synthesis and secretion of glycosaminoglycans and glycoproteins did however change with age. These results suggest the existence of matrix-dependent and of a matrix-independent regulation of the relative rate of synthesis of matrix macromolecules.

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 (TNFα), 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. However, no difference was observed between the groups except for the IL-8 level in group 2, which was lower 2 h after the start of CPB (p = 0.01). Plasma levels of adhesion molecules were similar in both groups within the investigation period. Although the Carmeda equipment was more effective in reducing complement activation, the late inflammatory response was similar using either the Duraflo II or Carmeda equipment for extracorporeal circulation as reflected by the changes of cytokine and circulating adhesion molecule levels.

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. 4mm) (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 α-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.

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.

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 residues 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.

Composition of glycopeptides obtained by proteolytic digestion of the media of porcine aorta.

Abstract Glycopeptides were isolated from the pronase-collagenase digested polymeric fibrous stroma of porcine aortic media. The glycopeptides were separated by gel filtration and high voltage electrophoresis and their sugar and aminoacid composition was determined. Two major types of glycopeptides could be isolated: one (type I) similar to the heteropolysaccharides isolated from structural glycoproteins of other connective tissues containing galactose, glucose, mannose, glucosamine, galactosamine, fucose and sialic acid. The other (type II) of low molecular weight corresponding to the hydroxylysylgalactoside and hydroxylysylgalactosido-glucoside, was previously isolated from several purified collagen preparations. Type I glycopeptides turned out to be very heterogenous and the seven fractions separated by high voltage electrophoresis all had different carbohydrate compositions. One fraction was devoid of sialic acid and fucose. The sialic acid/fucose ratio increased regularly with the anodic mobility of these fractions. The following chemical parameters were estimated from these results and are considered to be characteristic of the morphological pattern of aortic media: hexose content of the polymeric stroma of aorta, 6.6%; ratio of hydroxylysylglycopeptide/ heteropolysaccharides (ratio of glycopeptides of type II/type I), ∼ 1; ratio of hydroxylysyl mono- and disaccharide ( Hyl-Gal Hyl-GaIGIc ), ∼1. These chemical parameters were previously shown to be closely related to the morphological parameters (as fiber diameter) of the fibrous stroma of different connective tissues.

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 μg/cm2) prior to cell seeding. At 24 hours after low density cell seeding and 20 μg/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 (105 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.

Peptides Obtained From Elastin by Hydrolysis with Aqueous Ethanolic Potassium Hydroxide

Elastin from bovine ligamentum nuchae was hydrolyzed with 1 M KOH in 80% ethanol at 37°C for 0.5 to 72 h. Sephadex G 100 gel filtration separated the coacervable fractions from the retarded peptides (MW: 16,000) unable to form coacervates. Their ratio changed with progressing hydrolysis. The maximal yield fo coacervable peak was obtained after hydrolysis for about 6 h, whereas after hydrolysis from 18 to 72 h, only a nonco-acervable peak was recovered. The noncoacervable Sephadex peak was eluted at an identical position after hydrolysis for 4 and 72 h with an apparent molecular weight of 16,000 dalton on polyacrylamide SDS gel electrophoresis. The noncoacervable Sephadex peaks (MW: 16,000) obtained after hydrolysis for 4 and 72 h were submitted to isoelectric focusing in Servalyt in the pH range 2.5-11. The major crosslinked peptides were refocused in the pH range 2.5-6.In the major purified crosslinked peptides, recovered after hydrolysis for 4 h, the Ala to Gly ratio (1:1) was similar to that found in e...

N-oleoyl heparin inhibits the amidolytic activity of plasmin and urokinase

N-desulphated heparin, partially N-acylated on average with three oleoyl chains per molecule, inhibits the amidolytic activity of plasmin (IC50 16 nM) and urokinase (IC50 10mM) when assayed on N-p-tosyl-Gly-Pro-Lys-4-nitroanilide and benzoyl-Ala-Gly-Arg-4-nitroanilide substrates respectively. N-desulphated heparin is not inhibitory. This effect requires the covalent binding of oleoyl residues to heparin and it decreases with increasing concentration of Tris-HCl and non-ionic detergents.