Fredrik J. Olson

Recombinant MUC1 mucin with a breast cancer-like O-glycosylation produced in large amounts in Chinese-hamster ovary cells.

We have developed an expression system for the production of large quantities of recombinant MUC1 mucin in CHO-K1 (Chinese-hamster ovary K1) cells. The extracellular part of human MUC1, including 16 MUC1 tandem repeats, was produced as a fusion protein with murine IgG Fc, with an intervening enterokinase cleavage site for the removal of the Fc tail. Stable MUC1-IgG-producing CHO-K1 clones were generated and were found to secrete MUC1-IgG into the culture medium. After adaptation to suspension culture in protein-free medium in a bioreactor, the fusion protein was secreted in large quantities (100 mg/l per day) into the culture supernatant. From there, MUC1 could be purified to homogeneity using a two-step procedure including enterokinase cleavage and ion-exchange chromatography. Capillary liquid chromatography MS of released oligosaccharides from CHO-K1-produced MUC1 identified the main O-glycans as Galbeta1-3GalNAc (core 1) and mono- and di-sialylated core 1. The glycans occupied on average 4.3 of the five potential O-glycosylation sites in the tandem repeats, as determined by nano-liquid chromatography MS of partially deglycosylated Clostripain-digested protein. A very similar O-glycan profile and site occupancy was found in MUC1-IgG produced in the breast carcinoma cell line T47D, which has O-glycosylation typical for breast cancer. In contrast, MUC1-IgG produced in another breast cancer cell line, MCF-7, showed a more complex pattern with both core 1- and core 2-based O-glycans. This is the first reported production of large quantities of recombinant MUC1 with a breast cancer-like O-glycosylation that could be used for the immunotherapy of breast cancer.

Differences in Lesion Severity and Cellular Composition between in vivo Assessed Upstream and Downstream Sides of Human Symptomatic Carotid Atherosclerotic Plaques

Background: The heterogeneous structure of carotid atherosclerotic plaques may be better understood if it is related to blood flow variations, influencing gene expression and cellular functions. Upstream of the maximum stenosis there is laminar blood flow and high shear stress, downstream there is turbulence and low shear stress. We studied if these variations were associated with differences in plaque morphology and composition between sites located up- and downstream of the maximum stenosis in symptomatic carotid plaques. Methods: Patients with symptomatic carotid stenosis were examined with magnetic resonance angiography to localize the maximum stenosis in-vivo, prior to endarterectomy. In 41 endarterectomized specimens, transverse tissue sections prepared up- and downstream of the maximum stenosis were compared using histopathology and immunohistochemistry. Results: The location of maximum stenosis relative the carotid bifurcation varied considerably between plaques. Compared with the downstream side, the upstream side of the stenosis had higher incidence of severe lesions with cap rupture and intraplaque hemorrhage, more macrophages, less smooth muscle cells and more collagen. Conclusions: The up- and downstream sides of symptomatic carotid plaques differed in plaque morphology and composition. This implies that the intraplaque location of sampling sites may be a confounding factor in studies of atherosclerotic plaques.

Two glycosylation alterations of mouse intestinal mucins due to infection caused by the parasite Nippostrongylus brasiliensis.

The glycosylation alterations of mouse small intestinal mucins during a 12-day infectious cycle caused by the parasite Nippostrongylus brasiliensis have been studied. The guanidinium chloride insoluble mucins were isolated at day 0 to 12 from the small intestine of infected and non-infected C57BL/6 mice. The O-linked oligosaccharides were released by reductive β-elimination from the mucins and separated into neutral, sialylated and sulfated fractions. All fractions were analyzed by monosaccharide composition analysis and the neutral oligosaccharides were structurally characterized by gas chromatography/mass spectrometry. Two oligosaccharides containing blood group H-type epitopes (Fucα1-2Gal-) were transiently expressed with a maximum at day 6. Additional oligosaccharides with the common structure HexNAc-Gal-3GalNAcol were transiently induced with a maximum at day 10. Northern blot analysis on total RNA showed a transient expression at day 4–6 of the Fut2 gene encoding a Fucα1-2 fucosyltransferase, probably responsible for the detected blood group H-type epitopes. Comparisons with the corresponding infection in rat studied previously, revealed structurally different alterations, although occurring as transient events in both species. Both showed an induced blood group-type transferase halfway through the infection (a blood group A transferase in rat) and an induced transferase adding a terminal GalNAc (to a sialic acid- containing epitope in rat) towards the end of the infection. These differences between closely related species suggest rapid evolutionary alterations in glycosyltransferase expression.

Expression of chemokine (C–C motif) ligand 18 in human macrophages and atherosclerotic plaques

OBJECTIVE Using gene expression profiling, we aimed to identify genes that are predominantly expressed in human carotid atherosclerotic plaques. Such genes may be important in atherogenesis and pathophysiology of the plaque, and genes that encode for secreted proteins may be potential biomarkers for atherosclerosis and cardiovascular disease. METHODS DNA microarray generated expression profiles of human carotid atherosclerotic plaques were compared to expression profiles of 80 different human tissues and cell types, to identify plaque-specific genes. RESULTS We identified the chemokine (C-C motif) ligand 18 (CCL18) as predominantly expressed in human carotid plaque. Immunohistochemistry showed that CCL18 protein was localized to a subset of macrophages in carotid plaques. Monocyte-derived macrophages from subjects with atherosclerosis had threefold higher expression of CCL18 than macrophages from control subjects (p=0.012). Subjects with A/G genotype of the rs2015086 SNP in the promoter region of the CCL18 gene had threefold higher macrophage expression of CCL18 than subjects with A/A genotype (p=0.049), but we found no association of this SNP with an increased risk of coronary heart disease. We also compared serum levels of CCL18 from subjects with symptomatic carotid artery disease with control subjects. There were no differences in serum levels of CCL18 between the two groups, however CCL18 correlated with measurements of adiposity. CONCLUSION CCL18 is predominantly expressed in human atherosclerotic plaques and may participate in the atherosclerotic plaque formation.

Soluble urokinase-type plasminogen activator receptor forms in plasma as markers of atherosclerotic plaque vulnerability.

OBJECTIVES : To test if circulating forms of the soluble urokinase-type plasminogen activator receptor (suPAR) are potential biomarkers of plaque vulnerability. DESIGN AND METHODS : Plasma concentrations of suPAR(I-III), suPAR(II-III) and uPAR(I) were measured by time-resolved fluorescence immunoassays in Caucasian patients operated for symptomatic carotid atherosclerosis (n=255). Local suPAR release from plaques into the circulation was assessed in plasma passing retrogradely over the plaque in the carotid artery, collected during surgery (n=7). RESULTS : The suPAR(I-III) (P=0.03) and suPAR(II-III) (P=0.006) concentrations were higher after ischemic strokes and transient ischemic attacks, i.e., clinical subgroups associated with poorer prognosis and a less stable plaque phenotype, than after amaurosis fugax. Slightly elevated suPAR(I-III) levels were found in plasma from the carotid lesion. However, refuting the hypothesis, the concentrations of the suPAR forms were not higher in patients with short intervals between clinical event and blood sampling compared with those with long intervals. Age, inflammatory markers and diabetes were confounding factors independently associated with suPAR forms. CONCLUSION : Circulating suPAR forms are probably not useful biomarkers of atherosclerotic plaque vulnerability.

Circulating matrix metalloproteinase 9 levels in relation to sampling methods, femoral and carotid atherosclerosis.

Objectives.  To examine whether circulating levels of matrix metalloproteinase 9 (MMP‐9) were associated with ultrasound‐assessed intima‐media thickness (IMT) and echolucent plaques in the carotid and femoral arteries. To examine preanalytical sources of variability in MMP‐9 concentrations related to sampling procedures.

Blood group A glycosyltransferase occurring as Alleles with high sequence difference is transiently induced during a Nippostrongylus Brasiliensis parasite infection

Neutral mucin oligosaccharides from the small intestine of control rats and rats infected with the parasiteNippostrongylus brasiliensis were released and analyzed by gas chromatography-mass spectrometry. Infected animals expressed seven blood group A-like structures that were all absent in the control animals. The blood group A nature of these epitopes was confirmed by blood group A reactivity of the prepared mucins, of which Muc2 was one. Transferase assays and Northern blotting on small intestines from infected animals showed that an α-N-acetylgalactosaminyltransferase similar to the human blood group A glycosyltransferase had been induced. The expression was a transient event, with a maximum at day 6 of the 13-day-long infection. The rat blood group A glycosyltransferase was cloned, revealing two forms with an amino acid similarity of 95%. Both types had blood group A transferase activity and were probably allelic because none of 12 analyzed inbred strains carried both types. The second type was found in outbred rats and in one inbred strain. First generation offspring of inbred rats of each type were heterozygous, further supporting the allelic hypothesis. The transient induction and the large allelic variation could suggest that glycosyltransferases are part of a dynamic system altering mucins and other glycoconjugates as a protecting mechanism against microbial challenges.

Increased vascularization of shoulder regions of carotid atherosclerotic plaques from patients with diabetes.

OBJECTIVE Increased vascularization is considered an important contributing factor for plaque vulnerability. Microvascular proliferative disease in patients with diabetes results in renal damage and visual loss. We assessed the hypothesis that vascularization in carotid atherosclerotic tissue is increased in diabetic patients, especially in the critical shoulder regions of the plaque. METHODS Carotid endarterectomy specimens, clinical data, and blood samples were collected from patients with symptomatic carotid artery stenosis (median 85 days after clinical event) and pharmacologic treatment for diabetes (n = 26) or no diabetes (n = 85). Plaques were fixed in formalin and transverse tissue sections prepared. Histopathology and immunohistochemistry were performed for detection of endothelial cells (anti-CD34), macrophages (anti-CD68), vascular endothelial growth factor (VEGF), and its receptor (VEGFR-2). Neovascularization was assessed as CD34(+) neovessel density in the entire section area and by the presence or absence of CD34(+) vessels in the shoulder and cap regions of the plaques. RESULTS The patient groups did not differ significantly in neovascularization in the entire transverse sections (2.0 vs 2.1 vessels/mm(2); P = .61) or in the fibrous cap (52% of the patients in both groups; P = .95). Neovascularization of the plaque shoulder regions was observed in 52% of the diabetic patients and in 26% of the nondiabetic patients (P = .028). VEGF-stained areas were similar in the two patient groups (0.4% and 0.2% of shoulder area; P = .61). Patients with diabetes had more VEGFR-2 (1.0% vs 0.2% of shoulder area; P < .016) and less CD68 staining (0.4% vs 3.6% of shoulder area; P < .008). Time from clinical event to surgery was positively associated with neovascularization of the plaque shoulder regions (≤90 days, 18% of patients; >90 days, 50% of patients; P = .002), independently of diabetes status. CONCLUSIONS Diabetes was associated with increased vascularization of the shoulder regions in patients with symptomatic carotid atherosclerotic plaques. This was accompanied by increased expression of VEGFR-2. The increased vascularization of the plaque shoulder regions may help explain why patients with diabetes are at increased risk of atherosclerotic complications.

Consistent differences in protein distribution along the longitudinal axis in symptomatic carotid atherosclerotic plaques.

Identifying proteins associated with a complicated atherosclerotic plaque phenotype would provide potential biomarkers for detection of patients at elevated risk for clinically overt disease. We hypothesized that the protein content of carotid atherosclerotic tissue differs between complicated segments located in the internal carotid artery (ICA) and more stable segments in the common carotid artery (CCA). Using differential proteomics, we aimed to identify proteins differentially expressed between these segments of symptomatic carotid plaques. Ten snap-frozen human endarterectomies were divided into ICA and CCA segments and compared using two-dimensional differential gel electrophoresis and liquid chromatography-mass spectrometry. This study setup allowed pair-wise comparison of complicated and more stable atherosclerotic tissue from the same individual. We identified 19 proteins with differential distribution between ICA and CCA segments. Among the proteins more abundant in ICA were S100A10, ferritin light chain and fibrinogen. Among the proteins more abundant in CCA were ApoE, actin and l-lactate dehydrogenase B. Immunohistochemical staining revealed that S100A10 was expressed in endothelial cells, in clusters of macrophages and foam cells, and co-localized with the urokinase-type plasminogen activator receptor, uPAR. In conclusion, the results support the concept of comparing segments within plaques. The identified proteins constitute potential markers of complicated atherosclerotic lesions. The previously reported function of S100A10 to regulate plasmin activity affecting both angiogenesis and macrophage invasion, together with our observation of its accumulation in complicated plaque segments, warrants further studies of its potential role as a drug target for treatment of advanced atherosclerosis.

Adipose tissue is not an important source for matrix metalloproteinase-9 in the circulation.

Abstract Objectives: Matrix metalloproteinase 9 (MMP-9) is overexpressed in atherosclerotic plaques and in many cancers, and has emerged as a potential circulating biomarker for such diseases. However, adipose tissue (AT) might also produce circulating MMP-9, thereby reducing the value of MMP-9 as a biomarker. The aim of this study was to evaluate the impact of AT on circulating MMP-9, and if the metabolic syndrome might have a modifying effect. Methods: Gene expression of MMP-9 was measured in AT, isolated adipocytes, atherosclerotic plaques, macrophages and various other human tissues using real-time PCR. Relationships between plasma MMP-9 (ELISA), adiposity, and metabolic syndrome were analyzed in a population-based cohort of 61-year-old men (n=513). Both AT mRNA levels and circulating levels of MMP-9 were measured in obese subjects (n=40) with and without the metabolic syndrome, treated with a weight-reducing diet. Results: Bone marrow, atherosclerotic plaques and macrophages had considerably higher MMP-9 mRNA than subcutaneous AT and isolated adipocytes. Among the 61-year-old men, active plasma MMP-9 concentrations were associated with several metabolic syndrome factors, and inflammatory markers, but not body mass index (BMI). In obese patients with, but not without metabolic syndrome AT mRNA levels and circulating MMP-9 declined during weight reduction, but there was no association between changes in plasma MMP-9 and BMI. Conclusion: The results show that adipose tissue per se is not associated with circulating MMP-9. Components of the metabolic syndrome, such as circulating insulin and glucose were related to plasma MMP-9 both in the observation and dietary weight loss studies.