Biomechanical Assessment of Macro-Calcification in Human Carotid Atherosclerosis and Its Impact on Smooth Muscle Cell Phenotype.

Intimal calcification and vascular stiffening are predominant features of end-stage atherosclerosis. However, their role in atherosclerotic plaque instability and how the extent and spatial distribution of calcification influence plaque biology remain unclear. We recently showed that extensive macro calcification can be a stabilizing feature of late-stage human lesions, associated with a reacquisition of more differentiated properties of plaque smooth muscle cells (SMCs) and extracellular matrix (ECM) remodeling. Here, we hypothesized that biomechanical forces related to macro-calcification within plaques influence SMC phenotype and contribute to plaque stabilization. We generated a finite element modeling (FEM) pipeline to assess plaque tissue stretch based on image analysis of preoperative computed tomography angiography (CTA) of carotid atherosclerotic plaques to visualize calcification and soft tissues (lipids and extracellular matrix) within the lesions. Biomechanical stretch was significantly reduced in tissues in close proximity to macro calcification, while increased levels were observed within distant soft tissues. Applying this data to an in vitro stretch model on primary vascular SMCs revealed upregulation of typical markers for differentiated SMCs and contractility under low stretch conditions but also impeded SMC alignment. In contrast, high stretch conditions in combination with calcifying conditions induced SMC apoptosis. Our findings suggest that the load bearing capacities of macro calcifications influence SMC differentiation and survival and contribute to atherosclerotic plaque stabilization.

Solubilization and Purification of α5ß1 Integrin from Rat Liver for Reconstitution into Nanodiscs.

Transmembrane proteins (or integral membrane proteins) are synthesized in the endoplasmic reticulum where most of them are core glycosylated prior to folding and in some cases assembly into quaternary structures. Correctly glycosylated, folded, and assembled transmembrane proteins are then shuttled to the Golgi apparatus for additional posttranslational modifications such as complex-type glycosylations, sulfation or proteolytic clipping. At the plasma membrane, the glycosylated extracellular domains are key to communicate with the cellular environment for a variety of functions, such as binding to the extracellular matrix for cell adhesion and migration, to neighboring cells for cell-cell interaction, or to extracellular components for nutrient uptake and cell signaling. Intracellular domains are essential to mediate signaling cascades, or to connect to cytosolic adaptors for internalization and intracellular compartmentalization. Despite its importance for the understanding of molecular mechanisms of transmembrane protein function, the determination of their structures has remained a challenging task. In recent years, their reconstitution in lipid nanodiscs in combination with high resolution cryo-electron microscopy has provided novel avenues to render this process more accessible. Here, we describe detailed protocols for the solubilization of heavily glycosylated α5ß1 integrin from rat livers, its purification and reconstitution into nanodiscs. At the plasma membrane of many cells, including tumor metastases, this essential heterodimeric transmembrane protein mediates the communication between extracellular matrix and cytosolic cytoskeleton in processes of cell adhesion and migration. We expect that the protocols that are described here will provide new opportunities for the determination of the full structure of α5ß1 integrin, as well as for the understanding of how interacting partners can regulate function and activity of this transmembrane protein.

Integrin-Mediated Adhesion Promotes Centrosome Separation in Early Mitosis.

Integrin-mediated adhesion to the extracellular matrix is a key regulator of the cell cycle, as demonstrated for the passage of the G1/S checkpoint and the completion of cytokinetic abscission. Here, integrin-dependent regulation of the cell cycle in G2 and early M phases was investigated. The progression through the G2 and M phases was monitored by live-cell imaging and immunofluorescence staining in adherent and non-adherent fibroblast cells. Non-adherent cells, as well as adherent cells lacking FAK activity due to suppressed expression or pharmacological inhibition, exhibited a prolonged G2 phase and severely defect centrosome separation, resulting in delayed progress through the early mitotic stages. The activation of the critical mitotic regulator PLK1 and its indirect target Eg5, a kinesin-family motor protein driving the centrosome separation, were reduced in the cells lacking FAK activity. Furthermore, the absence of integrin adhesion or FAK activity destabilized the structural integrity of centrosomes and often caused detachment of pericentriolar material from the centrioles. These data identify a novel adhesion-dependent mechanism by which integrins via FAK and PLK1 contribute to the regulation of the cell cycle in the G2 and early M phases, and to the maintenance of genome integrity.

Septin and Ras regulate cytokinetic abscission in detached cells.

BACKGROUND: Integrin-mediated adhesion is normally required for cytokinetic abscission, and failure in the process can generate potentially oncogenic tetraploid cells. Here, detachment-induced formation of oncogenic tetraploid cells was analyzed in non-transformed human BJ fibroblasts and BJ expressing SV40LT (BJ-LT) ± overactive HRas. RESULTS: In contrast to BJ and BJ-LT cells, non-adherent BJ-LT-Ras cells recruited ALIX and CHMP4B to the midbody and divided. In detached BJ and BJ-LT cells regression of the cytokinetic furrow was suppressed by intercellular bridge-associated septin; after re-adhesion these cells divided by cytofission, however, some cells became bi-nucleated because of septin reorganization and furrow regression. Adherent bi-nucleated BJ cells became senescent in G1 with p21 accumulation in the nucleus, apparently due to p53 activation since adherent bi-nucleated BJ-LT cells passed through next cell cycle and divided into mono-nucleated tetraploids; the two centrosomes present in bi-nucleated BJ cells fused after furrow regression, pointing to the PIDDosome pathway as a possible mechanism for the p53 activation. CONCLUSIONS: Several mechanisms prevent detached normal cells from generating tumor-causing tetraploid cells unless they have a suppressed p53 response by viruses, mutation or inflammation. Importantly, activating Ras mutations promote colony growth of detached transformed cells by inducing anchorage-independent cytokinetic abscission in single cells.

Development and validation of an in vitro model system to study peripheral sensory neuron development and injury.

The ability to discriminate between diverse types of sensation is mediated by heterogeneous populations of peripheral sensory neurons. Human peripheral sensory neurons are inaccessible for research and efforts to study their development and disease have been hampered by the availability of relevant model systems. The in vitro differentiation of peripheral sensory neurons from human embryonic stem cells therefore provides an attractive alternative since an unlimited source of biological material can be generated for studies that specifically address development and injury. The work presented in this study describes the derivation of peripheral sensory neurons from human embryonic stem cells using small molecule inhibitors. The differentiated neurons express canonical- and modality-specific peripheral sensory neuron markers with subsets exhibiting functional properties of human nociceptive neurons that include tetrodotoxin-resistant sodium currents and repetitive action potentials. Moreover, the derived cells associate with human donor Schwann cells and can be used as a model system to investigate the molecular mechanisms underlying neuronal death following peripheral nerve injury. The quick and efficient derivation of genetically diverse peripheral sensory neurons from human embryonic stem cells offers unlimited access to these specialised cell types and provides an invaluable in vitro model system for future studies.

CD93 promotes ß1 integrin activation and fibronectin fibrillogenesis during tumor angiogenesis.

Tumor angiogenesis occurs through regulation of genes that orchestrate endothelial sprouting and vessel maturation, including deposition of a vessel-associated extracellular matrix. CD93 is a transmembrane receptor that is upregulated in tumor vessels in many cancers, including high-grade glioma. Here, we demonstrate that CD93 regulates ß1 integrin signaling and organization of fibronectin fibrillogenesis during tumor vascularization. In endothelial cells and mouse retina, CD93 was found to be expressed in endothelial filopodia and to promote filopodia formation. The CD93 localization to endothelial filopodia was stabilized by interaction with multimerin-2 (MMRN2), which inhibited its proteolytic cleavage. The CD93-MMRN2 complex was required for activation of ß1 integrin, phosphorylation of focal adhesion kinase (FAK), and fibronectin fibrillogenesis in endothelial cells. Consequently, tumor vessels in gliomas implanted orthotopically in CD93-deficient mice showed diminished activation of ß1 integrin and lacked organization of fibronectin into fibrillar structures. These findings demonstrate a key role of CD93 in vascular maturation and organization of the extracellular matrix in tumors, identifying it as a potential target for therapy.

The E-cadherin/AmotL2 complex organizes actin filaments required for epithelial hexagonal packing and blastocyst hatching.

Epithelial cells connect via cell-cell junctions to form sheets of cells with separate cellular compartments. These cellular connections are essential for the generation of cellular forms and shapes consistent with organ function. Tissue modulation is dependent on the fine-tuning of mechanical forces that are transmitted in part through the actin connection to E-cadherin as well as other components in the adherens junctions. In this report we show that p100 amotL2 forms a complex with E-cadherin that associates with radial actin filaments connecting cells over multiple layers. Genetic inactivation or depletion of amotL2 in epithelial cells in vitro or zebrafish and mouse in vivo, resulted in the loss of contractile actin filaments and perturbed epithelial packing geometry. We further showed that AMOTL2 mRNA and protein was expressed in the trophectoderm of human and mouse blastocysts. Genetic inactivation of amotL2 did not affect cellular differentiation but blocked hatching of the blastocysts from the zona pellucida. These results were mimicked by treatment with the myosin II inhibitor blebbistatin. We propose that the tension generated by the E-cadherin/AmotL2/actin filaments plays a crucial role in developmental processes such as epithelial geometrical packing as well as generation of forces required for blastocyst hatching.

Integrin signaling via FAK-Src controls cytokinetic abscission by decelerating PLK1 degradation and subsequent recruitment of CEP55 at the midbody.

Adhesion to extracellular matrix is required for cell cycle progression through the G1 phase and for the completion of cytokinesis in normal adherent cells. Cancer cells acquire the ability to proliferate anchorage-independently, a characteristic feature of malignantly transformed cells. However, the molecular mechanisms underlying this escape of the normal control mechanisms remain unclear. The current study aimed to identify adhesion-induced reactions regulating the cytokinesis of non-transformed human fibroblasts.The adhesion-dependent control of cytokinesis was found to occur at a late stage close to the abscission, during which the endosomal sorting complex required for transport (ESCRT) severs the thin intercellular bridge connecting two nascent daughter cells. CEP55, a key protein involved in the abscission process, was localized at the midbody in both adherent and non-adherent fibroblasts, but it was unable to efficiently recruit ALIX, TSG101, and consequently the ESCRT-III subunit CHMP4B was missing in the non-adherent cells. PLK1, a kinase that prevents premature recruitment of CEP55 to the midbody, disappeared from this site more rapidly in the non-adherent cells. A FAK-Src signaling pathway downstream of integrin-mediated cell adhesion was found to decelerate both PLK1 degradation and CEP55 accumulation at the midbody. These data identify the regulation of PLK1 and CEP55 as steps where integrins exert control over the cytokinetic abscission.

G-Protein-Coupled Lysophosphatidic Acid Receptors and Their Regulation of AKT Signaling.

A hallmark of G-protein-coupled receptors (GPCRs) is their ability to recognize and respond to chemically diverse ligands. Lysophospholipids constitute a relatively recent addition to these ligands and carry out their biological functions by activating G-proteins coupled to a large family of cell-surface receptors. This review aims to highlight salient features of cell signaling by one class of these receptors, known as lysophosphatidic acid (LPA) receptors, in the context of phosphatidylinositol 3-kinase (PI3K)-AKT pathway activation. LPA moieties efficiently activate AKT phosphorylation and activation in a multitude of cell types. The interplay between LPA, its receptors, the associated Gαi/o subunits, PI3K and AKT contributes to the regulation of cell survival, migration, proliferation and confers chemotherapy-resistance in certain cancers. However, detailed information on the regulation of PI3K-AKT signals induced by LPA receptors is missing from the literature. Here, some urgent issues for investigation are highlighted.

HAdV-2-suppressed growth of SV40 T antigen-transformed mouse mammary epithelial cell-induced tumours in SCID mice.

Human adenovirus (HAdV) vectors are promising tools for cancer therapy, but the shortage of efficient animal models for productive HAdV infections has restricted the evaluation of systemic effects to mainly immunodeficient mice. Previously, we reported a highly efficient replication of HAdV-2 in a non-tumorigenic mouse mammary epithelial cell line, NMuMG. Here we show that HAdV-2 gene expression and progeny formation in NMuMG cells transformed with the SV40 T antigen (NMuMG-T cells) were as efficient as in the parental NMuMG cells. Injection of HAdV-2 into tumours established by NMuMG-T in SCID mice caused reduced tumour growth and signs of intratumoural lesions. HAdV-2 replicated within the NMuMG-T-established tumours, but not in interspersed host-derived tissues within the tumours. The specific infection of NMuMG-T-derived tumours was verified by the lack of viral DNA in kidney, lung or spleen although low levels of viral DNA was occasionally found in liver.

Histidine-rich glycoprotein blocks collagen-binding integrins and adhesion of endothelial cells through low-affinity interaction with α2 integrin.

The plasma protein histidine-rich glycoprotein (HRG) affects the morphology and function of both endothelial cells (ECs) and monocytes/macrophages in cancer. Here, we examined the mechanism of action of HRG's effect on ECs. HRG suppressed adhesion, spreading and migration of ECs specifically on collagen I (COL I) whereas ECs seeded on other extracellular matrix proteins were insensitive to HRG. HRG did not bind specifically to COL I or to the α-integrin binding site on collagen, GFOGER. Furthermore, HRG's inhibition of EC adhesion was not dependent upon heparan sulfate (HS) moieties as heparitinase-treated ECs remained sensitive to HRG. C2C12 cells expressing α2 integrin, the major collagen-binding α-integrin subunit in ECs, showed increased binding of HRG compared with wild type C2C12 cells lacking the α2 subunit. Recombinant α2 I-domain protein bound HRG and to a higher extent when in active conformation. However, the α2 I-domain bound weakly to HRG compared with COL I and the purified α2ß1 ectodomain complex failed to retain HRG. We conclude that HRG binds to α2 integrin through low-affinity interactions in a HS-independent manner, thereby blocking EC-adhesion to COL I.

Fibronectin assembly in the crypts of cytokinesis-blocked multilobular cells promotes anchorage-independent growth.

Anchorage-independent growth is a characteristic feature of cancer cells. However, it is unclear whether it represents a cause or a consequence of tumorigenesis. For normal cells, integrin-mediated adhesion is required for completion of the G1 and cytokinesis stages of the cell cycle. This study identified a mechanism that can drive anchorage-independent growth if the G1 checkpoint is suppressed. Cells with defective G1 checkpoint progressed through several rounds of the cell cycle in suspension in spite of uncompleted cytokinesis, thereby forming bi- and multilobular cells. Aurora B and CEP55 were localized to midbodies between the lobes, suggesting that the cytokinesis process reached close to abscission. Integrin-mediated re-attachment of such cells induced cytokinesis completion uncoupled from karyokinesis in most cells. However, a portion of the cells instead lost the constriction and became binucleated. Also, long-term suspension culture in soft agar produced colonies where the cytokinesis block was overcome. This process was fibronectin-dependent since fibronectin-deficient cells did not form colonies unless fibronectin was expressed or exogenously added. While fibronectin normally is not deposited on non-adherent single cells, bi/multilobular cells accumulated fibronectin in the intussusceptions. Based on our data we conclude: 1) Suppression of the G1 checkpoint allows multiple rounds of the cell cycle in detached cells and thereby enables matrix formation on their surface. 2) Uncompleted cytokinesis due to cell detachment resumes if integrin interactions are re-formed, allowing colony formation in soft agar 3) Such delayed cell division can generate binucleated cells, a feature known to cause chromosomal instability.

The role of mechanical force and ROS in integrin-dependent signals.

Cells are exposed to several types of integrin stimuli, which generate responses generally referred to as "integrin signals", but the specific responses to different integrin stimuli are poorly defined. In this study, signals induced by integrin ligation during cell attachment, mechanical force from intracellular contraction, or cell stretching by external force were compared. The elevated phosphorylation levels of several proteins during the early phase of cell attachment and spreading of fibroblast cell lines were not affected by inhibition of ROCK and myosin II activity, i.e. the reactions occurred independently of intracellular contractile force acting on the adhesion sites. The contraction-independent phosphorylation sites included ERK1/2 T202/Y204, AKT S473, p130CAS Y410, and cofilin S3. In contrast to cell attachment, cyclic stretching of the adherent cells induced a robust phosphorylation only of ERK1/2 and the phosphorylation levels of the other investigated proteins were not or only moderately affected by stretching. No major differences between signaling via α5ß1 or αvß3 integrins were detected. The importance of mitochondrial ROS for the integrin-induced signaling pathways was investigated using rotenone, a specific inhibitor of complex I in the respiratory chain. While rotenone only moderately reduced ATP levels and hardly affected the signals induced by cyclic cell stretching, it abolished the activation of AKT and reduced the actin polymerization rate in response to attachment in both cell lines. In contrast, scavenging of extracellular ROS with catalase or the vitamin C analog Asc-2P did not significantly influence the attachment-derived signaling, but caused a selective and pronounced enhancement of ERK1/2 phosphorylation in response to stretching. In conclusion, the results showed that "integrin signals" are composed of separate sets of reactions triggered by different types of integrin stimulation. Mitochondrial ROS and extracellular ROS had specific and distinct effects on the integrin signals induced by cell attachment and mechanical stretching.

Characterization of heparanase-induced phosphatidylinositol 3-kinase-AKT activation and its integrin dependence.

Heparanase functions as a heparan sulfate-degrading enzyme and as a ligand for an unidentified signaling receptor(s). Here, several reactions involved in the activation of the PI3K-AKT pathway by latent heparanase were characterized. Protein suppression using specific siRNAs revealed that heparanase-induced phosphorylation of AKT at Ser-473 was RICTOR-mTOR-dependent, whereas ILK and PAK1/2 were dispensable. p110α was the PI3K catalytic isoform preferred by heparanase for AKT activation and cell proliferation because the p110α inhibitor YM024 blocked these processes. Heparanase-induced AKT phosphorylation was low in mouse embryonic fibroblast cells expressing a RAS interaction-defective p110α compared with wild type cells, indicating that RAS has an important role in the PI3K-AKT activation. The response to heparanase was also inefficient in suspension cultures of several cell lines, suggesting a requirement of integrins in this pathway. Adhesion via either αVß3 or α5ß1 promoted heparanase-induced AKT phosphorylation, and a stronger effect was seen when both integrins were engaged. Simultaneous inhibition of FAK and PYK2 using a chemical inhibitor, or suppression of their expression, inhibited heparanase-induced AKT activation and cell proliferation. Stimulation of cells with heparanase enhanced their resistance against oxidative stress- or growth factor starvation-induced apoptosis. These results demonstrate that there is an intimate cross-talk between the heparanase receptor(s) and integrins during induction of the prosurvival PI3K-AKT pathway by heparanase.

A mouse mammary epithelial cell line permissive for highly efficient human adenovirus growth.

Although a few immunocompetent animal models to study the immune response against human adenoviruses (HAdV) are available, such as Syrian hamsters and cotton rats, HAdV replication is several logs lower compared to human control cells. We have identified a non-transformed mouse epithelial cell line (NMuMG) where HAdV-2 gene expression and progeny formation was as efficient as in the highly permissive human A549 cells. HAdV from species, D and E (HAdV-37 and HAdV-4, respectively) also caused a rapid cytopathic effect in NMuMG cells, while HAdV from species A, B1, B2 and F (HAdV-12, HAdV-3, HAdV-11 and HAdV-41, respectively) failed to do so. NMuMG cells might therefore be useful in virotherapy research and the analysis of antiviral defense mechanisms and the determination of toxicity, biodistribution and specific antitumour activity of oncolytic HAdV vectors.

Receptor-specific mechanisms regulate phosphorylation of AKT at Ser473: role of RICTOR in ß1 integrin-mediated cell survival.

A tight control over AKT/PKB activation is essential for cells, and they realise this in part by regulating the phosphorylation of Ser473 in the "hydrophobic motif" of the AKT carboxy-terminal region. The RICTOR-mTOR complex (TORC2) is a major kinase for AKT Ser473 phosphorylation after stimulation by several growth factors, in a reaction proposed to require p21-activated kinase (PAK) as a scaffold. However, other kinases may catalyse this reaction in stimuli-specific manners. Here we characterised the requirement of RICTOR, ILK, and PAK for AKT Ser473 phosphorylation downstream of selected family members of integrins, G protein-coupled receptors, and tyrosine-kinase receptors and analysed the importance of this phosphorylation site for adhesion-mediated survival. siRNA-mediated knockdown in HeLa and MCF7 cells showed that RICTOR-mTOR was required for phosphorylation of AKT Ser473, and for efficient phosphorylation of the downstream AKT targets FOXO1 Thr24 and BAD Ser136, in response to ß1 integrin-stimulation. ILK and PAK1/2 were dispensable for these reactions. RICTOR knockdown increased the number of apoptotic MCF7 cells on ß1 integrin ligands up to 2-fold after 24 h in serum-free conditions. ß1 integrin-stimulation induced phosphorylation of both AKT1 and AKT2 but markedly preferred AKT2. RICTOR-mTOR was required also for LPA-induced AKT Ser473 phosphorylation in MCF7 cells, but, interestingly, not in HeLa cells. PAK was needed for the AKT Ser473 phosphorylation in response to LPA and PDGF, but not to EGF. These results demonstrate that different receptors utilise different enzyme complexes to phosphorylate AKT at Ser473, and that AKT Ser473 phosphorylation significantly contributes to ß1 integrin-mediated anchorage-dependent survival of cells.

Mechanism of estradiol-induced block of voltage-gated K+ currents in rat medial preoptic neurons.

The present study was conducted to characterize possible rapid effects of 17-ß-estradiol on voltage-gated K(+) channels in preoptic neurons and, in particular, to identify the mechanisms by which 17-ß-estradiol affects the K(+) channels. Whole-cell currents from dissociated rat preoptic neurons were studied by perforated-patch recording. 17-ß-Estradiol rapidly (within seconds) and reversibly reduced the K(+) currents, showing an EC(50) value of 9.7 µM. The effect was slightly voltage dependent, but independent of external Ca(2+), and not sensitive to an estrogen-receptor blocker. Although 17-α-estradiol also significantly reduced the K(+) currents, membrane-impermeant forms of estradiol did not reduce the K(+) currents and other estrogens, testosterone and cholesterol were considerably less effective. The reduction induced by estradiol was overlapping with that of the K(V)-2-channel blocker r-stromatoxin-1. The time course of K(+) current in 17-ß-estradiol, with a time-dependent inhibition and a slight dependence on external K(+), suggested an open-channel block mechanism. The properties of block were predicted from a computational model where 17-ß-estradiol binds to open K(+) channels. It was concluded that 17-ß-estradiol rapidly reduces voltage-gated K(+) currents in a way consistent with an open-channel block mechanism. This suggests a new mechanism for steroid action on ion channels.

PI3-kinase p110α mediates ß1 integrin-induced Akt activation and membrane protrusion during cell attachment and initial spreading.

Integrin-mediated cell adhesion activates several signaling effectors, including phosphatidylinositol 3-kinase (PI3K), a central mediator of cell motility and survival. To elucidate the molecular mechanisms of this important pathway the specific members of the PI3K family activated by different integrins have to be identified. Here, we studied the role of PI3K catalytic isoforms in ß1 integrin-induced lamellipodium protrusion and activation of Akt in fibroblasts. Real-time total internal reflection fluorescence imaging of the membrane-substrate interface demonstrated that ß1 integrin-mediated attachment induced rapid membrane spreading reaching essentially maximal contact area within 5-10 min. This process required actin polymerization and involved activation of PI3K. Isoform-selective pharmacological inhibition identified p110α as the PI3K catalytic isoform mediating both ß1 integrin-induced cell spreading and Akt phosphorylation. A K756L mutation in the membrane-proximal part of the ß1 integrin subunit, known to cause impaired Akt phosphorylation after integrin stimulation, induced slower cell spreading. The initial ß1 integrin-regulated cell spreading as well as Akt phosphorylation were sensitive to the tyrosine kinase inhibitor PP2, but were not dependent on Src family kinases, FAK or EGF/PDGF receptor transactivation. Notably, cells expressing a Ras binding-deficient p110α mutant were severely defective in integrin-induced Akt phosphorylation, but exhibited identical membrane spreading kinetics as wild-type p110α cells. We conclude that p110α mediates ß1 integrin-regulated activation of Akt and actin polymerization important for survival and lamellipodia dynamics. This could contribute to the tumorigenic properties of cells expressing constitutively active p110α.

Stabilins are expressed in bone marrow sinusoidal endothelial cells and mediate scavenging and cell adhesive functions.

Bone marrow sinusoidal endothelial cells have a specific function as a site of transmigration of hematopoietic stem and progenitor cells and mature blood cells between bone marrow and blood stream. However, the specific characteristics of bone marrow sinusoidal endothelial cells are still largely unclear. We here report that these cells express stabilin-1 and stabilin-2, which in liver sinusoidal endothelial cells have been identified as endocytic scavenger receptors for several ligands, including SPARC and hyaluronan. We show here that intravenously injected formaldehyde-treated serum albumin, advanced glycation end-products, and collagen I alpha-chains were taken up by bone marrow sinusoidal endothelial cells, showing that these cells have a scavenging function and thereby may modulate bone marrow vascular stem cell niches. Importantly, we show hyaluronan mediated adhesion of hematopoietic stem and progenitor cells to stabilin-2-transfected cells, suggesting that stabilin-2 contributes to adhesion and homing of circulating stem and progenitor cells to bone marrow.

Mechanistic proposal for the formation of specific immunogenic complexes via a radical pathway: a key step in allergic contact dermatitis to olefinic hydroperoxides.

The widespread use of scented products causes an increase of allergic contact dermatitis to fragrance compounds in Western countries today. Many fragrance compounds are prone to autoxidation, forming hydroperoxides as their primary oxidation products. Hydroperoxides are known to be strong allergens and to form specific immunogenic complexes. However, the mechanisms for the formation of the immunogenic complexes are largely unknown. We have investigated this mechanism for (5R)-5-isopropenyl-2-methyl-2-cyclohexene-1-hydroperoxide (Lim-2-OOH) by studying the formation of adducts in the reaction between this hydroperoxide and 5,10,15,20-tetraphenyl-21H,23H-porphine iron(III) chloride (Fe(III)TPPCl) in the presence of protected cysteine (NAc-Cys-OMe) or glutathione (GSH). Isolated adducts originate from the addition of the thiol group of NAc-Cys-OMe over the carbon-carbon double bonds of carvone. Furthermore, adducts between NAc-Cys-OMe and carveol as well as between GSH and carvone have been identified. The formation of these adducts most likely proceeds via the radical thiol-ene mechanism. The addition of a terpene moiety to cysteine offers an explanation of the specificity of the immune response to structurally different hydroperoxides. These results also explain the lack of cross-reactivity between carvone and Lim-2-OOH. In conclusion, we propose that immunogenic complexes of olefinic hydroperoxides can be formed via the radical thiol-ene mechanism. These complexes will be specific for the individual olefinic hydroperoxides due to the inclusion of a terpene moiety derived from the hydroperoxide.