Biomedical subjects

Guttorm Haraldsen

Publications and source records attributed to Guttorm Haraldsen.

18 recordsLinked to original sources

PubMed2007

The HUVEC/Matrigel assay: an in vivo assay of human angiogenesis suitable for drug validation.

The future ability to manipulate the growth of new blood vessels (angiogenesis) holds great promise for treating ischemic disease and cancer. Several models of human in vivo angiogenesis have been described, but they seem to depend on transgenic support and have not been validated in a therapeutic context. Here, we describe an in vivo assay that mimics human angiogenesis in which native human umbilical vein-derived endothelial cells are suspended in a liquid laminin/collagen gel (Matrigel), injected into immunodeficient mice, and develop into mature, functional vessels that vascularize the Matrigel plug in the course of 30 d. Moreover, we demonstrate how to target this process therapeutically by sustained delivery of the angiogenesis inhibitor endostatin from subcutaneously implanted microosmotic pumps.

Angiogenesis Inducing Agents↗

PubMed2006

Coy decoy with a new ploy: interceptor controls the levels of homeostatic chemokines.

A new subfamily of chemokine receptors is emerging that do not signal along classical G-protein-mediated pathways. Instead, these "silent" receptors efficiently internalize their cognate chemokine ligands, hence their suggested name, "chemokine interceptors", for internalizing receptors. Two of these interceptors, DARC and D6, possess intriguing patterns of tissue expression and are believed to be involved in controlling the local levels of proinflammatory chemokines. In this issue of the European Journal of Immunology, the biochemical properties of a third silent chemokine receptor, CCX-CKR, have been characterized and it is suggested that it may act as a scavenger for homeostatic chemokines, pointing to a broad and significant role for this group of chemokine binding molecules in chemokine biology.

Animals↗

PubMed2006

Human complement-activating immunoglobulin (Ig)G3 antibodies are essential for porcine endothelial cell activation.

BACKGROUND: Complement-activating naturally occurring anti-porcine endothelial cell antibodies (Abs) are responsible for hyperacute rejection in porcine-to-primate transplantation, whereas the role of complement in acute vascular rejection, characterized by type II endothelial cell activation, is less well understood. We previously demonstrated a correlation between porcine type II endothelial cell activation, as detected by E-selectin expression, and human immunoglobulin (Ig)G3 anti-Gal alpha1-3Gal (Gal) Abs, which was not seen for IgG1, IgG2 or IgG4. The present study was undertaken to investigate whether there is a causal relationship between human anti-porcine IgG3 Abs and porcine endothelial cell activation. METHODS: IgG3 was isolated employing a Protein A column to 98.3% purity. Porcine endothelial cells were incubated with isolated human IgG3 or the combination of IgG1, IgG2 and IgG4. E-selectin expression and complement activation were investigated by flow cytometry and Western blotting, respectively. RESULTS: Purified IgG3, in contrast to the other IgG subclasses, induced a substantial increase in E-selectin expression. This activation was accompanied by complement activation as detected by C3 cleavage, and was abolished by heat inactivation or by adding the complement inhibitor FUT-175. Depletion of anti-Gal Abs reduced E-selectin expression by 60%, consistent with the presence of complement-activating anti-porcine non-Gal Abs of the IgG3 subclass. CONCLUSIONS: Collectively, these data strengthen the hypothesis that human anti-porcine endothelial cell Abs of the IgG3 subclass are essential for endothelial cell activation in porcine-to-human species grafts and demonstrate such activation to be partly independent of Gal epitopes.

Animals↗

PubMed2005

Characterization of a novel chemokine-containing storage granule in endothelial cells: evidence for preferential exocytosis mediated by protein kinase A and diacylglycerol.

We have recently shown that several proinflammatory chemokines can be stored in secretory granules of endothelial cells (ECs). Subsequent regulated exocytosis of such chemokines may then enable rapid recruitment of leukocytes to inflammatory sites. Although IL-8/CXCL8 and eotaxin-3/CCL26 are sorted to the rod-shaped Weibel-Palade body (WPB), we found that GROalpha/CXCL1 and MCP-1/CCL2 reside in small granules that, similarly to the WPB, respond to secretagogue stimuli. In the present study, we report that GROalpha and MCP-1 colocalized in 50- to 100-nm granules, which occur throughout the cytoplasm and at the cell cortex. Immunofluorescence confocal microscopy revealed no colocalization with multimerin or tissue plasminogen activator, i.e., proteins that are released from small granules of ECs by regulated exocytosis. Moreover, the GROalpha/MCP-1-containing granules were Rab27-negative, contrasting the Rab27-positive, WPB. The secretagogues PMA, histamine, and forskolin triggered distinct dose and time-dependent responses of GROalpha release. Furthermore, GROalpha release was more sensitive than IL-8 release to inhibitors and activators of PKA and PKC but not to an activator of Epac, a cAMP-regulated GTPase exchange factor, indicating that GROalpha release is regulated by molecular adaptors different from those regulating exocytosis of the WPB. On the basis of these findings, we designated the GROalpha/MCP-1-containing compartment the type 2 granule of regulated secretion in ECs, considering the WPB the type 1 compartment. In conclusion, we propose that the GROalpha/MCP-1-containing type 2 granule shows preferential responsiveness to important mediators of EC activation, pointing to the existence of selective agonists that would allow differential release of selected chemokines.

Cells, Cultured↗

PubMed2005

Low mannose-binding lectin and increased complement activation correlate to allograft vasculopathy, ischaemia, and rejection after human heart transplantation.

AIMS: Transplant-associated coronary artery disease (TxCAD) is a major cause of post-transplant graft failure. The aim of this study was to investigate a possible role of mannose-binding lectin (MBL) deficiency and complement activation in TxCAD. METHODS AND RESULTS: In a prospective study of heart transplant recipients (n=38) with a follow-up of 5.3+/-1.3 years (range: 0.9-6.6), angiographically verified TxCAD (n=6) was correlated to plasma MBL, complement activation, and endothelial activation (soluble E-selectin). MBL deficiency (<100 ng/mL) was detected in 3/6 patients with TxCAD and in 3/32 with non-TxCAD (Kaplan-Meier, P=0.020). Furthermore, one or more acute rejection episodes were observed in 6/6 of the MBL-deficient patients and in 15/32 of the MBL-sufficient patients (chi(2); P=0.016). Complement activation (C4bc) correlated with soluble E-selectin (r=0.36; P=0.027), both being significantly higher in patients with ischaemia detected in the first biopsy (C4bc: 13.4+/-6.1 AU/mL; E-selectin: 96+/-13 ng/mL) than in those without ischaemia (C4bc: 6.3+/-0.5; E-selectin: 51+/-6; P=0.037 and 0.002). Finally, terminal complement complex correlated closely with mortality (P=0.002). CONCLUSION: Low MBL was related to the development of TxCAD and acute rejection and increased complement activation correlated to histopathologic ischaemia and mortality after heart transplantation.

Adult↗

PubMed2005

Disparate lymphoid chemokine expression in mice and men: no evidence of CCL21 synthesis by human high endothelial venules.

T-cell homing to secondary lymphoid tissues generally depends on chemokine-induced firm adhesion in high endothelial venules (HEVs) and is primarily mediated through the CC chemokine receptor 7 (CCR7) on lymphocytes. The CCR7 ligand designated CCL21 is considered the most important trigger because it appears constitutively expressed by murine HEVs. Surprisingly, when we analyzed human tissues, no CCL21 mRNA could be detected in HEVs. In fact, CCL21 mRNA was only expressed in extravascular T-zone cells and lymphatics, whereas immunostaining revealed CCL21 protein within HEVs. This suggests that T-cell recruitment to human lymphoid tissues depends on the transcytosis of lymphoid chemokines through HEV cells because there is at present no evidence of alternative chemokine production in these cells that could explain the attraction of naive T lymphocytes.

Animals↗

PubMed2005

A role for CCR4 in development of mature circulating cutaneous T helper memory cell populations.

Expression of the chemokine receptor CCR4 is strongly associated with trafficking of specialized cutaneous memory T helper (Th) lymphocytes to the skin. However, it is unknown whether CCR4 itself participates in the development of cutaneous Th populations. We have addressed this issue via competitive bone marrow (BM) reconstitution assays; equal numbers of BM cells from CCR4(+/+) and CCR4(-/-) donors were allowed to develop side-by-side within RAG-1(-/-) hosts. Cells from both donor types developed equally well into B cells, naive CD8 T cells, naive CD4 T cells, interferon-gamma(+) Th1 cells, and interleukin-4(+) Th2 cells. In marked contrast, circulating cutaneous memory Th cells (i.e., E-selectin ligand(+) [E-lig(+)]) were more than fourfold more likely to be derived from CCR4(+/+) donors than from CCR4(-/-) donors. Most of this effect resides within the CD103(+) subset of the E-lig(+) Th population, in which donor CCR4(+/+) cells can outnumber CCR4(-/-) cells by >12-fold. No similar effect was observed for alpha4beta7(+) intestinal memory Th cells or CD103(+)/E-lig(-) Th cells. We conclude that CCR4 expression provides a competitive advantage to cutaneous Th cells, either by participating in their development from naive Th cells, or by preferentially maintaining them within the memory population over time.

Animals↗

PubMed2005

Tissue transglutaminase-mediated formation and cleavage of histamine-gliadin complexes: biological effects and implications for celiac disease.

Celiac disease is an HLA-DQ2-associated disorder characterized by an intestinal T cell response. The disease-relevant T cells secrete IFN-gamma upon recognition of gluten peptides that have been deamidated in vivo by the enzyme tissue transglutaminase (transglutaminase 2 (TG2)). The celiac intestinal mucosa contains elevated numbers of mast cells, and increased histamine secretion has been reported in celiac patients. This appears paradoxical because histamine typically biases T cell responses in the direction of Th2 instead of the Th1 pattern seen in the celiac lesions. We report that histamine is an excellent substrate for TG2, and it can be efficiently conjugated to gluten peptides through TG2-mediated transamidation. Histamine-peptide conjugates do not exert agonistic effects on histamine receptors, and scavenging of biologically active histamine by gluten peptide conjugation can have physiological implications and may contribute to the mucosal IFN-gamma response in active disease. Interestingly, TG2 is able to hydrolyze the peptide-histamine conjugates when the concentrations of substrates are lowered, thereby releasing deamidated gluten peptides that are stimulatory to T cells.

Amino Acid Sequence↗

PubMed2005

Primary antitumor immune response mediated by CD4+ T cells.

Gene-targeted mice have recently revealed a role for lymphocytes and interferon-gamma (IFNgamma) in conferring protection against cancer, but the mechanisms remain unclear. Here, we have characterized a successful primary antitumor immune response initiated by naive CD4+ T cells. Major histocompatibility complex class II (MHC-II)-negative myeloma cells injected subcutaneously into syngeneic mice were surrounded within 3 days by macrophages that captured tumor antigens. Within 6 days, naive myeloma-specific CD4+ T cells became activated in draining lymph nodes and subsequently migrated to the incipient tumor site. Upon recognition of tumor-derived antigenic peptides presented on MHC-II by macrophages, the myeloma-specific CD4+ T cells were reactivated and started to secrete cytokines. T cell-derived IFNgamma activated macrophages in close proximity to the tumor cells. Tumor cell growth was completely inhibited by such locally activated macrophages. These data indicate a mechanism for immunosurveillance of MHC-II-negative cancer cells by tumor-specific CD4+ T cells through collaboration with macrophages.

Animals↗

PubMed2004

Endostatin dramatically inhibits endothelial cell migration, vascular morphogenesis, and perivascular cell recruitment in vivo.

Endostatin is a proteolytic fragment of collagen XVIII that inhibits endothelial cell migration in vitro and experimental tumor growth in vivo. To determine how endostatin affects the in vivo behavior of endothelial cells, we took advantage of a surrogate model of human angiogenesis, in which human endothelial cells are transferred to immunodeficient mice and develop into complex vessels in the course of 30 days. Systemic delivery of human yeast-derived endostatin (serum levels of 30-35 ng/mL) inhibited the number of human vessels dramatically (95% at day 20), as most endothelial cells remained suspended as single cells. The fraction of cells with a migratory phenotype (F-actin-positive, extending pseudopods) was strongly reduced (from 50% to 13% at day 10), while the number of apoptotic and mitotic cells remained unchanged. Endostatin also hampered the recruitment of alpha-smooth muscle actin-expressing perivascular cells and thus reduced the number of mature vessels (from 64.3% to 28.6% at day 30). Moreover, transcripts of pericyte-recruiting platelet-derived growth factor-B (PDGFB) were strongly reduced in endothelial cells of endostatin-treated mice. Our results are strong evidence that endostatin inhibits angiogenesis at several levels in vivo, including perivascular cell recruitment.

Angiogenesis Inhibitors↗

PubMed2004

Monocyte-like and mature macrophages produce CXCL13 (B cell-attracting chemokine 1) in inflammatory lesions with lymphoid neogenesis.

The homeostatic chemokine CXCL13 (also called B cell-attracting chemokine 1 [BCA-1] or B-lymphocyte chemoattractant [BLC]) is constitutively expressed in secondary lymphoid tissue and initiates lymphoid neogenesis when expressed aberrantly in mice. CXCL13 has also been detected in chronic inflammation associated with human lymphoid neogenesis, suggesting a pathogenic role. Follicular dendritic cells (FDCs) are generally considered to be the major source of CXCL13 both in normal and aberrant lymphoid tissue. We show here, instead, that most CXCL13-expressing cells in rheumatoid arthritis and ulcerative colitis are of monocyte/macrophage lineage. They are located in irregular lymphoid aggregates within an FDC network, but also within and near smaller collections of B cells in diseased tissue where no FDCs are detected. Some of these CXCL13-expressing cells are CD14(+), suggesting derivation from recently extravasated monocytes. Interestingly, monocytes from healthy donors stimulated in vitro with lipopolysaccharide secrete CXCL13. This induced production is enhanced after in vitro maturation of the monocytes toward macrophages but markedly decreased after maturation toward dendritic cells. Together, our findings strongly suggest that newly recruited monocytes/macrophages play a role for lymphoid neogenesis in human inflammatory diseases. Circulating monocytes are therefore potential candidates for future targeted therapy of chronic inflammation.

Adult↗

PubMed2004

Rapid chemokine secretion from endothelial cells originates from 2 distinct compartments.

The neutrophil-attracting chemokine interleukin 8 (IL-8) is stored in the Weibel-Palade body (WPB) of endothelial cells (ECs) from which it can be rapidly released after exposure to the secretagogues histamine or thrombin. In this manner, IL-8 may enable rapid recruitment of leukocytes to inflammatory sites. To explore the possible storage of EC-derived chemokines that may attract other subsets of leukocytes, we examined the intracellular localization and secretagogue responsiveness of growth-related oncogene alpha (GROalpha), monocyte chemoattractant protein-1 (MCP-1), eotaxin-3, interferon-gamma-inducible protein 10 (IP-10), and regulated on activation, normal T-cell expressed and secreted (RANTES). While eotaxin-3, GROalpha, and MCP-1 were rapidly released from ECs, the release of the T-cell attractors RANTES and IP-10 was not sensitive to the secretagogues. Moreover, of the 3 former chemokines, only eotaxin-3 was stored in WPBs. GROalpha and MCP-1 resided mainly in smaller vesicles compatible with sorting to a different, histamine-responsive compartment, which has been described in ECs although not reported to contain chemokines. In conclusion, we propose that rapid release of chemokines is restricted to those primarily recruiting leukocytes of the innate immune system, and that their storage in ECs is not restricted to the WPB compartment.

Carcinogens↗

PubMed2004

Plasticity of endothelial cells: rapid dedifferentiation of freshly isolated high endothelial venule endothelial cells outside the lymphoid tissue microenvironment.

Endothelial cells display remarkable heterogeneity in different organs and vascular beds. Although many studies suggest that tissues "speak" to endothelial cells, endothelial cell diversity remains poorly characterized at the molecular level. Here, we describe a novel strategy to characterize tissue-specific endothelial cell phenotypes and to identify endothelial cell genes that are under the control of the local microenvironment. By comparing post-capillary high endothelial venule endothelial cells (HEVECs), freshly isolated from human tonsils without any cell culture step, with HEVECs cultured for 2 days, we found that HEVECs rapidly lost their specialized characteristics when isolated from the lymphoid tissue microenvironment. Striking changes occurred as early as after 48 hours, with complete loss of the postcapillary venule-specific Duffy antigen receptor for chemokines (DARCs) and the HEV-specific fucosyltransferase Fuc-TVII. DNA microarray analysis identified several other candidate HEV genes that were rapidly down-regulated ex vivo, including type XV collagen, which we characterized as a novel, abundant HEV transcript in situ. Together, our results demonstrate that blood vessel type-specific and tissue-specific characteristics of endothelial cells are under the control of their microenvironment. Therefore, even short-term primary cultures of human endothelial cells may not adequately mimic the differentiated endothelial cell phenotypes existing in vivo.

Cell Differentiation↗

PubMed2003

Endothelial cell diversity revealed by global expression profiling.

The vascular system is locally specialized to accommodate widely varying blood flow and pressure and the distinct needs of individual tissues. The endothelial cells (ECs) that line the lumens of blood and lymphatic vessels play an integral role in the regional specialization of vascular structure and physiology. However, our understanding of EC diversity is limited. To explore EC specialization on a global scale, we used DNA microarrays to determine the expression profile of 53 cultured ECs. We found that ECs from different blood vessels and microvascular ECs from different tissues have distinct and characteristic gene expression profiles. Pervasive differences in gene expression patterns distinguish the ECs of large vessels from microvascular ECs. We identified groups of genes characteristic of arterial and venous endothelium. Hey2, the human homologue of the zebrafish gene gridlock, was selectively expressed in arterial ECs and induced the expression of several arterial-specific genes. Several genes critical in the establishment of left/right asymmetry were expressed preferentially in venous ECs, suggesting coordination between vascular differentiation and body plan development. Tissue-specific expression patterns in different tissue microvascular ECs suggest they are distinct differentiated cell types that play roles in the local physiology of their respective organs and tissues.

Cells, Cultured↗

PubMed2003

Molecular characterization of NF-HEV, a nuclear factor preferentially expressed in human high endothelial venules.

Lymphocyte homing to secondary lymphoid tissue and lesions of chronic inflammation is directed by multi-step interactions between the circulating cells and the specialized endothelium of high endothelial venules (HEVs). In this study, we used the PCR-based method of suppression subtractive hybridization (SSH) to identify novel HEV genes by comparing freshly purified HEV endothelial cells (HEVECs) with nasal polyp-derived microvascular endothelial cells (PMECs). By this approach, we cloned the first nuclear factor preferentially expressed in HEVECs, designated nuclear factor from HEVs (NF-HEV). Virtual Northern and Western blot analyses showed strong expression of NF-HEV in HEVECs, compared to human umbilical vein endothelial cells (HUVECs) and PMECs. In situ hybridization and immunohistochemistry revealed that NF-HEV mRNA and protein are expressed at high levels and rather selectively by HEVECs in human tonsils, Peyers's patches, and lymph nodes. The NF-HEV protein was found to contain a bipartite nuclear localization signal, and was targeted to the nucleus when ectopically expressed in HUVECs and HeLa cells. Furthermore, endogenous NF-HEV was found in situ to be confined to the nucleus of tonsillar HEVECs. Finally, threading and molecular modeling studies suggested that the amino-terminal part of NF-HEV (aa 1-60) corresponds to a novel homeodomain-like Helix-Turn-Helix (HTH) DNA-binding domain. Similarly to the atypical homeodomain transcription factor Prox-1, which plays a critical role in the induction of the lymphatic endothelium phenotype, NF-HEV may be one of the key nuclear factors that controls the specialized HEV phenotype.

Amino Acid Sequence↗

PubMed2002

CD38 is a marker of human lacteals.

CD38 is a type II transmembrane glycoprotein involved in signaling and adhesion which is expressed mainly by immature hematopoietic cells and activated lymphoid cells. Central lymphatic channels of human small intestinal villi, the so-called lacteals, were coincidentally found to express CD38. Gastric and large intestinal mucosae, pancreas, liver, lung, nasal mucosa, kidney, thymus, palatine tonsil, Peyer's patches, appendix, and mesenteric lymph nodes, and rodent intestinal mucosa were subsequently examined for lymphatic expression of CD38. Cryosections prepared from biopsy or surgical resection specimens were immunostained with four different antibodies to CD38 combined with antibodies to von Willebrand factor and CD31 to differentiate lymphatics from blood vessels, or with antibody to lysosomal protein. Sections were evaluated by ordinary and confocal immunofluorescence microscopy. Jejunal cryosections were subjected to in situ hybridization for CD38. All CD38 antibodies decorated human lacteals, and some of these were positive for CD38 mRNA. Lymphatics draining Peyer's patches and appendix as well as afferent lymphatics of mesenteric lymph nodes expressed CD38 weakly. CD38 was not detected on lymphatics in other organs or in rodent lacteals. We propose that CD38 is a novel marker of human small intestinal lymphatic vessels.

ADP-ribosyl Cyclase↗

PubMed2002

Vascular morphogenesis and differentiation after adoptive transfer of human endothelial cells to immunodeficient mice.

To establish a model for adoptive transfer of endothelial cells, we transferred human umbilical vein endothelial cells (HUVECs) to immunodeficient mice (Rag 2(-/-)). HUVECs were suspended as single cells in Matrigel and injected subcutaneously in the abdominal midline. Within 10 days after injection, HUVECs expressed pseudopod-like extensions and began to accumulate in arrays. By day 20, we observed human vessels that contained erythrocytes, and on day 30 we observed perivascular cells that expressed smooth muscle actin, thus resembling mature vessels. Throughout the experimental period, HUVECs bound Ulex europaeus lectin and expressed CD31, VE-cadherin, von Willebrand factor, as well as ICAM-2. A fraction of the cells also expressed the proliferation marker Ki67. Moreover, the sialomucin CD34, which is rapidly down-regulated in cultured HUVECs, was reinduced in vivo. However, we found no reinduction of CD34 in cells cultured inside or on top of Matrigel in vitro. We also injected cells suspended in Matrigel around the catheter tip of implanted osmotic pumps. Delivery of recombinant human interferon-gamma by this route led to strong induction of MHC class II and ICAM-1 on the human vessels. In conclusion, isolated human endothelial cells can integrate with the murine vascular system to form functional capillaries and regain in vivo properties.

Actins↗

PubMed2002

Human serum-induced porcine endothelial cell E-selectin expression is associated with IgG3 and IgM anti-Gal antibodies.

Naturally occurring anti-Galalpha1-3Gal (anti-Gal) antibodies and complement induce hyperacute rejection (HAR) of porcine organs transplanted to primates. If the hyperacute reaction is prevented, an acute vascular rejection (AVR) occurs within hours to few days. Antibodies are important for the development of AVR, whereas the role of complement is still not clarified. AVR is characterized by protein synthesis-dependent endothelial cell (EC) activation. In the present study we investigated the relation between EC activation as measured by E-selectin expression, and the concentrations of anti-Gal antibodies of IgM, IgG and IgG subclasses in sera from 80 healthy blood donors selected on the basis of sex and age. There was a significant correlation between E-selectin expression and the concentration of IgG3 anti-Gal (r=0.39; P=0.019), which was not seen for the other IgG subclasses or for total IgG anti-Gal. A modest, but significant correlation was found between the concentration of IgM anti-Gal and E-selectin expression (r=0.38; P=0.040), but not between IgM and IgG3 anti-Gal. There was a large interindividual variation in anti-Gal antibodies, 50-fold for IgM and 70-fold for IgG. Females had significantly higher concentrations of IgM anti-Gal than males (P=0.0006), which was explained by a substantial increase in IgM anti-Gal concentration in younger women. The concentration of IgG anti-Gal and the degree of E-selectin expression did not differ between sex or age groups. In conclusion, the close correlation between anti-Gal antibodies of the potent complement activating IgG3 subclass and porcine EC activation, may imply that these antibodies play a role in EC activation characteristic of AVR.

Adult↗