Gunnar Fager

Multivariate analyses of serum apolipoproteins and risk factors in relation to acute myocardial infarction.

In 25 middle-aged infarction survivors and 76 corresponding controls, representative for a well-defined community, multivariate analysis was used to evaluate whether serum apolipoproteins were better discriminators of infarction survivors than serum lipids and other risk factors. Levels of serum cholesterol and triglycerides, alphalipoprotein cholesterol, apolipoproteins A-l, A-ll, B, and D, as well as tobacco smoking and other risk factors, were included. In descending order, serum apo A-ll levels (tb = −3.12, p = 0.002), tobacco consumption (tb = 2.64, p = 0.010), and serum triglycerides (tb = 2.06, p = 0.042) contributed significantly to the multiple regression on myocardial infarction (R = 0.53, p = 0.00001). When entered into a discriminant function, these three variables gave a good separation between survivors and controls. Of the survivors, 50% were above the 90th percentlle in the control group. The relative prevalence of infarction increased continuously with increasing values of the function from zero to more than 6 times the average. Serum apo A-ll levels alone were almost as good in separating cases and controls. From this study, we concluded that, among apolipoproteins, apo A-ll seems to be a more sensitive discriminator of infarction survivors than other risk factors.

Characterization of Heparin and Heparan Sulfate Domains Binding to the Long Splice Variant of Platelet-derived Growth Factor A Chain

Platelet-derived growth factors (PDGFs) are homo- or heterodimers of two related polypeptides, known as A and B chains. The A chain exists as two splice variants due to the alternative usage of exons 6 (PDGF-AL, longer) and 7 (PDGF-AS, shorter). Exon 6 encodes an 18-amino acid sequence rich in basic amino acid residues, which has been implicated as a cell retention signal. Several lines of evidence indicate that the retention is due to binding of PDGF-AL to glycosaminoglycans, especially to heparan sulfate. We have analyzed the saccharide domains of smooth muscle cell-derived heparan sulfate involved in this interaction. Furthermore, we have employed selectively modified heparin oligosaccharides to elucidate the dependence of the binding on different sulfate groups and on fragment length. The shortest PDGF-AL binding domain consists of 6-8 monosaccharide units. Studies using selectively desulfated heparins and heparin fragments suggest that N-, 2-O-, and 6-O-sulfate groups all contribute to the interaction. Structural comparison of heparan sulfate oligosaccharides separated by affinity chromatography on immobilized PDGF-AL showed that the bound pool was enriched in -IdceA(2-OSO3)-GlcNSO3(6-OSO3)- disaccharide units. Furthermore, analogous separation of a partially O-desulfated heparin decamer preparation, using a highly selective nitrocellulose filter-trapping system, yielded a PDGF-AL-bound fraction in which more than half of the disaccharide units had the structure -IdceA(2-OSO3)-GlcNSO3(6-OSO3)-. Our results suggest that the interaction between PDGF-AL and heparin/heparan sulfate is mediated via N-sulfated saccharide domains containing both 2-O- and 6-O-sulfate groups.

Alphalipoprotein cholesterol levels in relation to acute myocardial infarction and its risk factors.

During 1975-1977 twenty-nine males surviving acute myocardial infarction at an age between 40-44 years were registered in Gothenburg, Sweden. Twenty-five of these were studied and compared with two control groups. One group, the reference group (RG, n = 76), was randomly selected from the male population from which the acute myocardial infarction (AMI) group was derived. A second group, the matched control group (MC, n = 47), consisted of men with no history of coronary heart disease, matched with patients for age, serum cholesterol and body weight index. Serum triglyceride levels were higher and alphalipoprotein cholesterol lower in the AMI group than in RG. Prior to infarction, patients had a higher degree of physical activity at work and a higher tobacco consumption than RG. When AMI cases were compared with MC subjects lower alphalipoprotein cholesterol levels were found in AMI, and they also had a higher tobacco consumption prior to infarction. There was a negative correlation between alphalipoprotein cholesterol levels and tobacco consumption in the RG. The differences in alphalipoprotein cholesterol levels between AMI cases and controls could not attributed to smoking habits, but smoking may at least to some extent exert its effect as a risk factor through influence on alphalipoprotein cholesterol levels.

Serum apolipoprotein levels in relation to acute myocardial infarction and its risk factors: Apolipoprotein A-I levels in male survivors of myocardial infarction

The significance of high density lipoproteins in the etiology of clinical complications to atherosclerosis has recently received increased attention. The levels of the major apolipoprotein in high density lipoproteins, apoA-I, have been determined in patients who had had an acute myocardial infarction, and compared with a cholesterol-matched and a randomly selected control group. ApoA-I levels were lower in the patients than in the control groups. ApoA-I levels were also lower in smokers than in non-smokers. The difference between patients and control groups persisted even when the groups were stratified according to smoking habits. This suggests that low levels of apo-A-I as well as alphalipoprotein cholesterol are additional characteristics of the infarction patients, even when the established risk factors, smoking and hyperlipidemia are taken into account.

Human arterial smooth muscle cells in culture. Effects of platelet-derived growth factor and heparin on growth in vitro.

Human arterial smooth muscle cells (hASMC) were cultured from explants of the inner media of uterine arteries obtained at hysterectomy. The presence of alpha-actin and smooth muscle-specific actin isoforms and the microscopic appearance of the cells in secondary culture established their smooth muscle origin. The hASMC were diploid and had no signs of transformation. Plasma-derived serum failed to stimulate their proliferation in vitro. Their rate of proliferation was, however, proportional to the concentration of whole blood serum in the medium. Anti-PDGF IgG at high concentrations inhibited the stimulatory effect of whole blood serum on cell proliferation. This suggests that hASMC depend on exogenous PDGF for their growth. In PDS or bovine serum albumin cell numbers remained constant for 7 days in culture and the thymidine index was below 1% per 24 h. When reexposed to whole blood serum these cells started to proliferate within 2 days. This indicates that hASMC when deprived of PDGF enter a quiescent state that is fully reversible upon rexposure to the mitogen. Heparin is a powerful growth inhibitor for SMC. In our system, heparin caused a dose-dependent inhibition of cell proliferation despite optimal concentrations of whole blood serum. This inhibition was reversible upon withdrawal of heparin. At heparin concentrations which caused a half-maximal inhibition it was also competed for by increasing concentrations of whole blood serum. Quiescent hASMC expressed the PDGF receptor on their surface as judged from immunofluorescence with a monoclonal antibody. This was true irrespective of whether growth arrest was achieved by serum depletion or by the addition of heparin to serum-containing medium. Cells growing in the presence of whole blood serum did not, however, express the receptor antigen. These observations suggest that heparin may interfere with PDGF or with its binding and further processing at the level of the cell-surface receptor.

Human arterial smooth muscle cells in culture: Inverse relationship between proliferation and expression of contractile proteins

SummaryHuman arterial smooth muscle cells (hASMC) from explants of the inner media of uterine arteries were studied in secondary culture. We had previously found that these cells depend on exogenous platelet-derived growth factor (PDGF) for proliferation in vitro. Deprivation of the serum mitogen(s) by culture in plasma-derived serum or bovine serum albumin (BSA) caused a true growth arrest that was reversible upon reexposure to the mitogen(s). When added to serum-containing medium, heparin caused a reversible growth arrest which could be competed for by increasing concentrations of serum. In the current study we used a set of smooth muscle-specific actin and myosin, antibodies to study the expression of contractile proteins in stress fibers under indirect immunofluorescence on hASMC in culture. Even in sparse culture, grwoth-arrested hASMC expressed stress fibers containing these actin and myosin epitopes. This was true irrespective of whether growth arrest was achieved by culture in media containing only BSA or a combination of heparin and whole blood serum. hASMC proliferating in whole blood serum in sparse culture did not express such strees fibers, as judged by immunofluorescent staining. This was true also for cells that were restimulated to proliferate in serum after a growth arrest. Utilizing a monoclonal antibody against a nuclear antigen expressed in proliferating human cells, we were able to demonstrate an inverse relationship between the expression of this antigen and the SMC-specific contractile proteins, respectively. Under these culture conditions, the reversible transition between defifferentiated and differentiated hASMC was almost complete and terminated about 1 wk after the change in culture condition. We conclude that hASMC in vitro respond, to exogenous PDGF by proliferation and dedifferetiation as a single population of cells. We also conclude that this modulation is reversible, because the cells become uniformly quiescent and differentiated when the mitogenic stimulus is blocked or removed.

Cholesterol Reduction and Clinical Benefit Are There Limits to Our Expectations

Encouraging intervention trials drive our expectations toward more aggressive cholesterol-lowering therapies, lower target levels, and less severe hypercholesterolemia. Available studies may predict which patients, degrees of total cholesterol (TC) reduction, and baseline and target levels of TC provide the most clinical benefit. Data were pooled from seven primary and nine secondary controlled trials with major coronary heart disease (CHD) events as primary endpoints. The analysis showed that we can expect large reductions in CHD from TC reduction in primary and secondary prevention. However, the reduction is much larger in subjects with high TC and/or previous CHD events. The percent reduction in CHD increased exponentially with increasing percent TC reductions, which predicted > 70% of the change in CHD. Consequently, we cannot expect cost-effective clinical benefits from mean reductions in TC > 15 (LDL cholesterol > 20%). The TC level at the study endpoint correlated with CHD incidence irrespective of the study group and explained almost 45% of CHD incidence. The relationship was progressive and leveled off at a TC level below about 150 mgdL (3.9 mmol/L) (LDL cholesterol approximately equal to 110 mg/dl [approximately equal to 2.8 mmol/L]). Little extra clinical benefit can be expected from further reductions. We can expect an average 2% reduction in CHD events per percent reduction in TC. We can also expect a 2-fold greater clinical benefit among subjects with high initial TC levels than among those with low levels. Finally, we can expect that the cholesterol-attributable risk is reset to that predicted by the TC level achieved within 4 to 6 years.

Heparin-like glycosaminoglycans influence growth and phenotype of human arterial smooth muscle cells in vitro. I: Evidence for reversible binding and inactivation of the platelet-derived growth factor by heparin

SummaryWe have investigated the effects of interactions between growth factors and heparin-like glycosaminoglycans on untransformed human arterial smooth muscle cells (hASMC) in vitro. The results indicate that heparin in the presence of serum mitogens prevents the cells from entering the S phase of the cell cycle by binding and inactivating reversibly some serum mitogen(s). Our results suggest that platelet-derived growth factor (PDGF) is one of them and that it is the most potent stimulator of hASMC growth in vitro. Thymidine incorporation as well as increase in DNA content was inhibited not only by the presence of heparin in serum-containing medium but also when serum was chromatographed on Heparin-Sepharose at physiologic salt concentrations before exposure to the cells. The mitogenic activity of the unretained serum fraction was restored by the addition of PDGF AA, AB, or BB dimers or of a fraction (RF I) that dissociated from Heparin-Sepharose at 0.2 to 0.6M NaCl. Radiolabeled recombinant PDGF (c-sis) dissociated from Heparin-Sepharose within a concentration range of NaCl similar to that of RF I. Neither the unretained material nor the RF I or PDGF dimers were effective alone. The effect of RF I was significantly decreased by the addition of an anti-PDGF IgG that is known to neutralize the PDGF mitogenic activity partially. Addition of heparin abolished DNA-synthesis when the PDGF dimers or RF I were combined with the unretained fraction. A second fraction (RF II) bound strongly to Heparin-Sepharose and eluted between 1.1 and 1.6M NaCl. The RF II also induced DNA synthesis but was neither as efficient as RF I nor depending on other serum fractions for growth promotion and it was not inhibited by anti-PDGF IgG. A similar strong affinity for Heparin-Sepharose was found for labeled basic fibroblast growth factor and we cannot exclude the possibility that RF II represent fibroblast growth factor. Under these culture conditions, inhibition of hASMC proliferation was directly correlated with the expression of smooth muscle specific alpha actin isoforms in stress fibers and the suppression of a proliferating cell-specific nuclear antigen. Conversely, stimulation of hASMC proliferation was associated with the opposite phenomenon. We conclude that heparin-like glycosaminoglycans influence growth and phenotype of hASMCs in vitro by binding and inactivating PDGF. Inasmuch as heparin-like substances constitute a significant proportion of the proteoglycan-associated glycosaminoglycans of the arterial wall, such mechanisms might be important for the development of atherosclerotic lesions.

Serum lipids and apolipoprotein levels in women with acute myocardial infarction.

In this study covering more than 150,000 person-years from women younger than 55 years of age, 61 survived a first acute myocardial infarction (AMI). Of these, 59 were compared with a random sample from the same population regarding serum lipids and apolipoproteins (apo) A-I, A-II, B, and E, as well as several other cardiovascular risk factors. Mean values of serum cholesterol, triglycerides, apo B, and apo E were significantly higher and high density lipoprotein cholesterol and apo A-I were significantly lower among patients with infarction than among controls. Those who sustained and survived an AMI more often had a history of hypertension and of tobacco smoking than did the controls. Cigarette smoking, a history of hypertension, age, high serum triglycerides and apo E, as well as low levels of apo A-I, were independently and significantly associated with infarction. Sixty percent of the cases and 11% of the controls were distributed in the highest quartile of risk. A major contribution to the association with AMI was accounted for by the conventional risk factors, cigarette smoking and hypertension, as well as high serum triglycerides. In this group of relatively young women, high serum triglycerides were strongly associated with infarction, while levels of serum cholesterol were not.

Differential binding of platelet-derived growth factor isoforms to glycosaminoglycans

The platelet-derived growth factor (PDGF) family comprises disulfide-bonded dimeric isoforms and plays a key role in the proliferation and migration of mesenchymal cells. Traditionally, it consists of homo- and heterodimers of A and B polypeptide chains that occur as long (AL and BL) or short (AS and BS) isoforms. Short isoforms lack the basic C-terminal extension that mediates binding to heparin. In the present study, we show that certain PDGF isoforms bind in a specific manner to glycosaminoglycans (GAGs). Experiments performed with wild-type and mutant Chinese hamster ovary cells deficient in the synthesis of GAGs revealed that PDGF long isoforms bind to heparan sulfate and chondroitin sulfate, while PDGF short isoforms only bind to heparan sulfate. This was confirmed by digestion of cell surface GAGs with heparitinase and chondroitinase ABC and by incubation with sodium chloride to prevent GAG sulfation. Furthermore, exogenous GAGs inhibited the binding of long isoforms to the cell membrane more efficiently than that of short isoforms. Additionally, we performed surface plasmon resonance experiments to study the inhibition of PDGF isoforms binding to low molecular weight heparin by GAGs. These experiments showed that PDGF-AAL and PDGF-BBS isoforms bound to GAGs with the highest affinity. In conclusion, PDGF activity at the cell surface may depend on the expression of various cellular GAG species.