A.M. Dräger

Plasma concentration of C-reactive protein is increased in type I diabetic patients without clinical macroangiopathy and correlates with markers of endothelial dysfunction: evidence for chronic inflammation.

Summary Moderately increased plasma concentrations of C-reactive protein are associated with an increased risk of cardiovascular disease. C-reactive protein, its relation to a low degree of inflammatory activation and its association with activation of the endothelium have not been systematically investigated in Type I (insulin-dependent) diabetes mellitus. C-reactive protein concentrations were measured in 40 non-smoking patients with Type I diabetes without symptoms of macrovascular disease and in healthy control subjects, and in a second group of Type I diabetic patients (n = 60) with normo- (n = 20), micro- (n = 20) or macroalbuminuria (n = 20). Differences in glycosylation of α1-acid glycoprotein were assayed by crossed affinity immunoelectrophoresis. Activation of the endothelium was measured with plasma concentrations of endothelial cell markers. The median plasma concentration of C-reactive protein was higher in Type I diabetic patients compared with healthy control subjects [1.20 (0.06–21.64) vs 0.51 (0.04–9.44) mg/l; p < 0.02]. The Type I diabetic subjects had a significantly increased relative amount of fucosylated α1-acid glycoprotein (79 ± 12 % vs 69 ± 14 % in the healthy control subjects; p < 0.005), indicating a chronic hepatic inflammatory response. In the Type I diabetic group, log(C-reactive protein) correlated significantly with von Willebrand factor (r = 0.439, p < 0.005) and vascular cell adhesion molecule-1 (r = 0.384, p < 0.02), but not with sE-selectin (r = 0.008, p = 0.96). In the second group of Type I diabetic patients, increased urinary albumin excretion was associated with a significant increase of von Willebrand factor (p < 0.0005) and C-reactive protein (p = 0.003), which were strongly correlated (r = 0.53, p < 0.0005). Plasma concentrations of C-reactive protein were higher in Type I diabetic patients without (clinical) macroangiopathy than in control subjects, probably due to a chronic hepatic inflammatory response. The correlation of C-reactive protein with markers of endothelial dysfunction suggests a relation between activation of the endothelium and chronic inflammation. [Diabetologia (1999) 42: 351–357]

Proteoglycans guide SDF-1-induced migration of hematopoietic progenitor cells

Stromal cell‐derived factor‐1 (SDF‐1) is a chemoattractant involved in hematopoietic progenitor cell (HPC) trafficking to the bone marrow. We studied the role of bone marrow endothelial proteoglycans (PGs) in SDF‐1‐mediated migration of HPC using a transwell assay. A subclone of progenitor cell line KG‐1 (KG‐1v) was used, displaying CXCR4‐dependent transmigration. Cell surface PGs on bone marrow endothelial cell line 4LHBMEC did not mediate SDF‐1‐induced transendothelial migration. In contrast, transwell filters precoated with various glycosaminoglycans (GAGs) enhanced migration toward SDF‐1. SDF‐1‐induced migration was reduced by degradation of heparan sulfate in subendothelial matrix produced by 4LHBMEC. The stimulating effect of GAGs was caused by the formation of a stable haptotactic SDF‐1 gradient, as SDF‐1 bound to immobilized GAGs and triggered migration. Soluble heparan sulfate enhanced SDF‐1‐induced migration dose‐dependently, suggesting that SDF‐1‐heparan sulfate complexes optimized SDF‐1 presentation. In conclusion, we provide evidence that PGs in the subendothelial matrix establish an SDF‐1 gradient guiding migrating HPC into the bone marrow.

Proteoglycans on bone marrow endothelial cells bind and present SDF-1 towards hematopoietic progenitor cells

Recognition events between hematopoietic progenitor cells (HPC) and bone marrow endothelial cells (BMEC) initiate homing of HPC to the bone marrow. The chemokine SDF-1 is present on BMEC and plays a crucial role in bone marrow engraftment. We studied the role of proteoglycans (PGs) on BMEC in binding and presentation of SDF-1. SDF-1 mRNA was present in three human BMEC cell lines. Competition experiments showed that 125I-SDF-1α binding to the BMEC cell line 4LHBMEC was inhibited by heparins, heparan sulfate (HS) intestinal mucosa, chondroitin and dermatan sulfate (CS/DS), but not by HS bovine kidney. Pretreatment of 4LHBMEC with glycosaminoglycan (GAG)-degrading enzymes or sodium chlorate demonstrated that SDF-1 bound to both HSPGs and CS/DSPGs in a sulfation-dependent manner, as determined with an SDF-1 antibody recognizing the CXCR4-binding site. 4LHBMEC bound four-fold more SDF-1 than HUVEC. Isolated endothelial PGs did not bind SDF-1 in a filter or microplate-binding assay, suggesting the necessity of membrane association. In flow adhesion experiments, endothelial arrest of CXCR4+ KG-1 and not of CXCR4− KG-1a cells increased significantly when SDF-1 was presented on 4LHBMEC. In conclusion, SDF-1 is produced by BMEC and binds to the BMEC cell surface via HS and CS/DS-GAGs, thereby presenting its CXCR4 binding site to HPC contributing to their arrest.

Extensive early apoptosis in frozen–thawed CD34-positive stem cells decreases threshold doses for haematological recovery after autologous peripheral blood progenitor cell transplantation

Stem cell doses necessary for engraftment after myelo-ablative therapy as defined for fresh transplants vary largely. Loss of CD34+ cell quality after cryopreservation might contribute to this variation. With a new early apoptosis assay including the vital stain Syto16, together with the permeability marker 7-AAD, CD34+ cell viability in leucapheresis samples of 49 lymphoma patients receiving a BEAM regimen was analysed. After freeze–thawing large numbers of non-viable, early apoptotic cells appeared, leading to only 42% viability compared to 72% using 7-AAD only. Based on this Syto16 staining in the frozen–thawed grafts, threshold numbers for adequate haematological recovery of 2.8–3.0 × 106 CD34+ cells/kg body weight determined for fresh grafts, now decreased to 1.2–1.3 × 106 CD34+ cells/kg. In whole blood transplantation of lymphoma patients (n = 45) receiving a BEAM-like regimen, low doses of CD34+ cells were sufficient for recovery (0.3–0.4 × 106CD34+ cells/kg). In contrast to freeze–thawing of leucapheresis material, a high viability of CD34+ cells was preserved during storage for 3 days at 4°C, leaving threshold doses for recovery unchanged. In conclusion, the Syto16 assay reveals the presence of many more non-functional stem cells in frozen–thawed transplants than presumed thus far. This led to a factor 2.3-fold adjustment downward of viable CD34+ threshold doses for haematological recovery.Bone Marrow Transplantation (2002) 29, 249–255. doi:10.1038/sj.bmt.1703357

Early Apoptosis Largely Accounts for Functional Impairment of CD34+ Cells in Frozen-Thawed Stem Cell Grafts

Quality assessment of stem cell grafts is usually performed by flow cytometric CD34(+) enumeration or assessment of clonogenic output of fresh material. Previously, we identified the occurrence of early apoptosis, not detectable with the permeability marker 7-amino actinomycin D (7-AAD), in purified frozen-thawed CD34(+) cells, using the vital stain Syto16. Syto(high)/7-AAD(-) cells were defined as viable, Syto16(low)/7-AAD(-) cells as early apoptotic and Syto16(low)/7-AAD(+) as dead. This was confirmed in a subsequent study using frozen-thawed transplants of lymphoma patients. In the present study on grafts from multiple myeloma and lymphoma patients, we investigated the functional consequences of the early apoptotic process. The mean Syto16-defined viability was 41 and 42%, respectively, for both graft groups, compared to 78% and 72%, respectively, using 7-AAD only. The established early apoptosis marker annexin V missed roughly 50% of the early apoptosis detected with Syto16. In contrast, viability of CD34(+) cells in nonmanipulated whole blood transplants from a matched group of lymphoma patients, after 72 h of storage at 4 degrees C, was more than 90%, even with the Syto16 assay. CFU recovery (median 26-33%) after cryopreservation matched CD34(+) recovery after Syto16, but not 7-AAD correction. In contrast, colony-forming unit (CFU) recovery in the whole blood transplant was close to 100%. Furthermore, early apoptotic CD34(+) cells had lost migratory ability toward stromal cell derived factor-1alpha (SDF-1alpha). The establishment of a Syto16(high)/7-AAD(-) proportion of CD34(+) cells offers a new approach for a more correct determination of the number of viable nonapoptotic CD34(+) cells in stem cell grafts. Further development of this assay should allow its incorporation into the routine CD34(+) assessment of post-thawed samples in clinical flow cytometry laboratories.

Differences in sulfation patterns of heparan sulfate derived from human bone marrow and umbilical vein endothelial cells

OBJECTIVE Heparan sulfates (HS), the polysaccharide side chains of HS proteoglycans, differ in structure and composition of sulfated domains among various tissue types, resulting in selective protein binding. HS proteoglycans on bone marrow endothelial cells (BMEC) could contribute to tissue specificity of the bone marrow endothelium and play a role in the presentation of chemokines such as stromal cell-derived factor-1 (SDF-1) and adhesion of hematopoietic progenitor cells after stem cell transplantations. We characterized differences in HS structure and SDF-1 binding between BMEC and human umbilical vein endothelial cells (HUVEC). MATERIALS AND METHODS Expression of HS proteoglycans on human bone marrow microvessels was investigated by immunohistochemical staining. Comparison of three human BMEC cell lines with HUVEC and an HUVEC cell line was studied by flow cytometry using antibodies against different epitopes of the HS polysaccharide chain. HS proteoglycans were biochemically characterized after isolation from metabolically labeled cultures of the BMEC cell line 4LHBMEC and HUVEC. Binding of radiolabeled SDF-1 to 4LHBMEC and HUVEC and competition with heparins were investigated. RESULTS Bone marrow microvessels constitutively expressed HS proteoglycans. Flow cytometric experiments showed differences in HS chain composition between BMEC and HUVEC. Biochemical characterization revealed more O-sulfation of the N-sulfated domains present in cell-associated HS glycosaminoglycans in 4LHBMEC compared to HUVEC. Binding experiments showed that 4LHBMEC bound more 125[I]-SDF-1 per cell than HUVEC. This could be inhibited largely by heparin and O-sulfated heparin and to a lesser extent by N-sulfated heparin. CONCLUSIONS Cellular HS from BMEC differs in composition from HUVEC. We postulate that the presence of highly sulfated domains in the HS chains from BMEC contributes to tissue specificity of bone marrow endothelium in which HS may be involved in SDF-1 presentation and adhesion of hematopoietic progenitor cells.

Changes in L-selectin expression on CD34-positive cells upon cryopreservation of peripheral blood stem cell transplants

Several studies have pointed out that L-selectin on CD34-positive cells plays a role in haematopoietic reconstitution after peripheral blood stem cell (PBSC) transplantation. Since it is known that a decrease in L-selectin expression in lymphocytes and granulocytes can be induced by a variety of stress situations, we have investigated in this study whether the freeze–thawing procedure, used in PBSC transplantation, would affect L-selectin expression on CD34+ stem cells. Flow cytometry was performed by labelling the cells with anti-CD34 (HPCA2 PE) and anti-CD62L (FMC46 FITC). The leucapheresis procedure itself caused a slight decrease of L-selectin expression on CD34 cells in 11 out of 12 cases (mean decrease of the percentage of positive cells 11.9; range 6–23). A much larger decrease was found upon freeze–thawing: a mean of 39% (range 4–78% in 27 cases) compared to fresh material. To determine if L-selectin expression might be up-regulated after cryopreservation, thawed transplant samples (n = 11) were incubated at 37°C in RPMI with 10% FCS at 5% CO2. Already early in the course of incubation two CD34-positive populations appeared in the blast region, characterized by either a low or high forward scatter. Simultaneous viability staining with the DNA dye 7-Amino Actinomycin D and the DNA/RNA dye Syto16 revealed that the population with low forward scatter was apoptotic while the population with the high forward scatter was non-apoptotic. The latter population is considered to be most relevant for transplantation. In this population an increase of L-selectin expression after overnight incubation was observed in 8/11 samples up to values of 46–120% of the values of the fresh non-frozen samples. In addition, the mean fluorescence intensity was significantly increased in 10/11 cases. Kinetic experiments with shorter incubation times revealed that only part of the leucapheresis samples (two out of 8) showed an increase of L-selectin expression within 4 h. In addition, a decrease of L-selectin expression was found upon CD34 purification from fresh leucapheresis material by magnetic isolation (decrease ranging from 59 to 92%, n = 5). In contrast to frozen samples, L-selectin reappearance was seen already within 4 h of incubation in all samples. Both the loss of L-selectin expression on CD34 cells occurring upon freeze–thawing, the emergence of apoptosis, as well as the recovery of L-selectin expression on non-apoptotic cells varies largely between individual leucapheresis samples, and therefore it is concluded that such processes should be considered when correlations with clinical outcome after transplantation are made.

In vitro model for hematopoietic progenitor cell homing reveals endothelial heparan sulfate proteoglycans as direct adhesive ligands

Proteoglycans (PGs) play a dominant role within the bone marrow (BM), but their role in homing of transplanted hematopoietic progenitor cells (HPC) is unknown. In this study, the role of heparan sulfate (HS) PGs on BM endothelium as adhesive structures was investigated. HPC (primary CD34+ cells and cell line KG‐1a) were able to bind fractionated heparin, which could be competed by highly sulfated heparin/HS‐glycosaminoglycans (GAGs). Under flow conditions, HPC adhered to immobilized heparin after rolling over E‐selectin. Rolling of KG‐1a on BM endothelial cell (EC) line 4LHBMEC was completely E selectin‐dependent. Addition of heparin/HS‐GAGs, endothelial treatment with chlorate, or anti‐HS all partially inhibited firm adhesion. Moreover, enzymatic removal of endothelial HS‐GAGs reduced initial adhesion. Finally, HPC‐bound PGs isolated from 4LHBMEC, which was largely inhibited by enzymatic HS‐degradation. In summary, we identified sulfated structures on BM endothelium, most likely HSPGs, as a novel class of glycoconjugates involved in the multistep homing cascade of HPC.

The phenotypic profile of CD34-positive peripheral blood stem cells in different mobilization regimens

The type of regimen used might result in mobilization of phenotypically and functionally different CD34+ cells. We compared the phenotype of CD34+ cells in leukapheresis products of three homogeneous groups: I, healthy individuals treated with granulocyte colony‐stimulating factor (G‐CSF) alone (n = 13); II, patients mobilized with G‐CSF following chemotherapy (n = 16); and III, patients mobilized with G‐CSF after high‐dose chemotherapeutic pretreatment (n = 24). Multiparameter flow cytometry was performed for CD34+ subpopulation analysis and focused on adhesion molecules, differentiation markers and megakaryocytic markers relevant for stem cell homing, with special reference to the importance of L‐selectin expression. Regimens I and II led to higher numbers of mobilized CD34+ cells (mean 468 × 106 and 491 × 106 CD34+ cells per leukapheresis procedure respectively) than regimen III (mean 41 × 106 CD34+ cells per leukapheresis procedure). Both the expression of L‐selectin and CD54 on CD34+ cells was significantly lower in group III, as was the percentage of megakaryocytic (CD41+) progenitors. A higher percentage of primitive (CD38− and/or HLA−DR−) CD34+ cells was found in group III, correlating with a higher clonogenicity of the CD34+ cells. However, when comparing the CD34+ subpopulations that were also positive for L‐selectin, there was no significant difference between the three regimens. A similar approach for the megakaryocytic CD34+ population resulted in an even worse quality of regimen III: 5·1% of CD34+ being CD41+/L‐selectin+ compared with 9·2% and 8·9% in regimens I and II respectively. We concluded that the phenotypes of the CD34+ cells in the G‐CSF (group I) and G‐CSF–chemotherapy (group II) regimens are similar, whereas the phenotype of the CD34+ cells mobilized in the high‐dose regimen (group III) displayed features that might negatively influence homing of the cells. Future studies will be directed towards regimens that will lead to the mobilization of a higher amount of CD34+ cells with a phenotypically favourable phenotype.