Ablation of VLA4 in multiple myeloma cells redirects tumor spread and prolongs survival.

Multiple myeloma (MM) is a cancer of bone marrow (BM) plasma cells, which is increasingly treatable but still incurable. In 90% of MM patients, severe osteolysis results from pathological interactions between MM cells and the bone microenvironment. Delineating specific molecules and pathways for their role in cancer supportive interactions in the BM is vital for developing new therapies. Very Late Antigen 4 (VLA4, integrin α4ß1) is a key player in cell-cell adhesion and signaling between MM and BM cells. We evaluated a VLA4 selective near infrared fluorescent probe, LLP2A-Cy5, for in vitro and in vivo optical imaging of VLA4. Furthermore, two VLA4-null murine 5TGM1 MM cell (KO) clones were generated by CRISPR/Cas9 knockout of the Itga4 (α4) subunit, which induced significant alterations in the transcriptome. In contrast to the VLA4+ 5TGM1 parental cells, C57Bl/KaLwRij immunocompetent syngeneic mice inoculated with the VLA4-null clones showed prolonged survival, reduced medullary disease, and increased extramedullary disease burden. The KO tumor foci showed significantly reduced uptake of LLP2A-Cy5, confirming in vivo specificity of this imaging agent. This work provides new insights into the pathogenic role of VLA4 in MM, and evaluates an optical tool to measure its expression in preclinical models.

New 4-Aminoproline-Based Small Molecule Cyclopeptidomimetics as Potential Modulators of α4ß1 Integrin.

Integrin α4ß1 belongs to the leukocyte integrin family and represents a therapeutic target of relevant interest given its primary role in mediating inflammation, autoimmune pathologies and cancer-related diseases. The focus of the present work is the design, synthesis and characterization of new peptidomimetic compounds that are potentially able to recognize α4ß1 integrin and interfere with its function. To this aim, a collection of seven new cyclic peptidomimetics possessing both a 4-aminoproline (Amp) core scaffold grafted onto key α4ß1-recognizing sequences and the (2-methylphenyl)ureido-phenylacetyl (MPUPA) appendage, was designed, with the support of molecular modeling studies. The new compounds were synthesized through SPPS procedures followed by in-solution cyclization maneuvers. The biological evaluation of the new cyclic ligands in cell adhesion assays on Jurkat cells revealed promising submicromolar agonist activity in one compound, namely, the c[Amp(MPUPA)Val-Asp-Leu] cyclopeptide. Further investigations will be necessary to complete the characterization of this class of compounds.

Integrin cross-talk modulates stiffness-independent motility of CD4+ T lymphocytes.

To carry out their physiological responsibilities, CD4+ T lymphocytes interact with various tissues of different mechanical properties. Recent studies suggest that T cells migrate upstream on surfaces expressing intracellular adhesion molecule-1 (ICAM-1) through interaction with leukocyte function-associated antigen-1 (αLß2) (LFA-1) integrins. LFA-1 likely behaves as a mechanosensor, and thus we hypothesized that substrate mechanics might affect the ability of LFA-1 to support upstream migration of T cells under flow. Here we measured motility of CD4+ T lymphocytes on polyacrylamide gels with predetermined stiffnesses containing ICAM-1, vascular cell adhesion molecule-1 (VCAM-1), or a 1:1 mixture of VCAM-1/ICAM-1. Under static conditions, we found that CD4+ T cells exhibit an increase in motility on ICAM-1, but not on VCAM-1 or VCAM-1/ICAM-1 mixed, surfaces as a function of matrix stiffness. The mechanosensitivity of T-cell motility on ICAM-1 is overcome when VLA-4 (very late antigen-4 [α4ß1]) is ligated with soluble VCAM-1. Last, we observed that CD4+ T cells migrate upstream under flow on ICAM-1-functionalized hydrogels, independent of substrate stiffness. In summary, we show that CD4+ T cells under no flow respond to matrix stiffness through LFA-1, and that the cross-talk of VLA-4 and LFA-1 can compensate for deformable substrates. Interestingly, CD4+ T lymphocytes migrated upstream on ICAM-1 regardless of the substrate stiffness, suggesting that flow can compensate for substrate stiffness.

Identification of circulating cells interacted with integrin α4ß1 ligand peptides REDV or HGGVRLY.

Recently, artificial blood vessels modified by integrin α4ß1 ligand, such as REDV, showed endothelialization improvement and antithrombotic properties have been reported. Early endothelialization was affected by the type of circulating cells captured by the peptide in the initial transplantation state, however, it is still not clarified. In this study, we identified in vitro circulating cells bound with the peptides arginine-glutamic acid-aspartic acid-valine (REDV) or histidine-glycine-glycine-valine-arginine-leucine-tyrosine (HGGVRLY). The effect of free C- or N-terminal of HGGVRLY on the type of peptide-binding cells was also studied. The rat circulating cells were isolated from blood and incubated with 5(6)-carboxyfluorescein (5/6-FAM, F) labeled F-REDV (C-terminal free), F-HGGVRLY (C-terminal free), or HGGVRLY-F (N-terminal free). Furthermore, peptide-binding cells were identified by co-staining with various antibodies labeled with PE, PerCP/Cy5.5, or APC. N-terminal free HGGVRLY-F was found to bind to more circulating cells than C-terminal free F-REDV and F-HGGVRLY. The ratio of integrin α4ß1 positive cell bound with F-REDV, F-HGGVRLY, or HGGVRLY-F reached over 90 %, demonstrating that HGGVRLY is also a ligand of integrin α4ß1. Among identified cell types, we found that F-REDV mainly bounds with EPC and BMSC, while F-HGGVRLY with BMSC. HGGVRLY-F bounds with EPC and BMSC, exhibiting a higher EPC binding ratio than F-REDV and F-HGGVRLY.

Magnetic Resonance Imaging of Atherosclerotic Plaque at Clinically Relevant Field Strengths (1T) by Targeting the Integrin α4ß1.

Inflammation drives the degradation of atherosclerotic plaque, yet there are no non-invasive techniques available for imaging overall inflammation in atherosclerotic plaques, especially in the coronary arteries. To address this, we have developed a clinically relevant system to image overall inflammatory cell burden in plaque. Here, we describe a targeted contrast agent (THI0567-targeted liposomal-Gd) that is suitable for magnetic resonance (MR) imaging and binds with high affinity and selectivity to the integrin α4ß1(very late antigen-4, VLA-4), a key integrin involved in recruiting inflammatory cells to atherosclerotic plaques. This liposomal contrast agent has a high T1 relaxivity (~2 × 105 mM-1s-1 on a particle basis) resulting in the ability to image liposomes at a clinically relevant MR field strength. We were able to visualize atherosclerotic plaques in various regions of the aorta in atherosclerosis-prone ApoE-/- mice on a 1 Tesla small animal MRI scanner. These enhanced signals corresponded to the accumulation of monocyte/macrophages in the subendothelial layer of atherosclerotic plaques in vivo, whereas non-targeted liposomal nanoparticles did not demonstrate comparable signal enhancement. An inflammatory cell-targeted method that has the specificity and sensitivity to measure the inflammatory burden of a plaque could be used to noninvasively identify patients at risk of an acute ischemic event.

The α4ß1/EMILIN1 interaction discloses a novel and unique integrin-ligand type of engagement.

EMILIN1, a homo-trimeric adhesive ECM glycoprotein, interacts with the α4ß1 integrin through its gC1q domain. Uniquely among the C1q family members, the EMILIN1 gC1q presents only nine-stranded ß-sandwich fold and the missing strand is substituted by a disordered 19-residue long segment spanning from Y927 to G945 at the apex of the gC1q domain. This unstructured loop exposes to the solvent the acidic residue E933, which plays a key role in the α4ß1 integrin mediated interaction. Here, we experimentally determined that the three E933 residues (one from each monomer) are all required for ligand binding. By docking the NMR structure of the gC1q to a virtual α4ß1 crystal structure based on the known structures of α4ß7 and α5ß1 integrins we built a model of α4ß1-gC1q complex where three E933 residues are smoothly forced to coordinate the Mg2+ ion at the ßI MIDAS site of the integrin. By bringing the three E933 close in space, the trimeric supramolecular organization of gC1q allows the formation of a proper 3D geometry and suggests a quaternary-structure-dependent mode of interaction. Furthermore, we experimentally identified R904 as a synergistic residue for cell adhesion. Accordingly, the model showed that this residue is able to form potential stabilizing intra-chain salt bridges with residues E928 and E930. This mode of interaction likely accounts for a more stable and durable α4ß1-gC1q interaction in comparison with the prototypic CS1 ligand. To our knowledge, this is the first report describing the simultaneous involvement of all the three acidic residues of a trimeric ligand in the formation of a dimeric complex with the integrin ßI domain.

Neutrophil elastase cleavage of the gC1q domain impairs the EMILIN1-α4ß1 integrin interaction, cell adhesion and anti-proliferative activity.

The extracellular matrix glycoprotein EMILIN1 exerts a wide range of functions mainly associated with its gC1q domain. Besides providing functional significance for adhesion and migration, the direct interaction between α4ß1 integrin and EMILIN1-gC1q regulates cell proliferation, transducing net anti-proliferative effects. We have previously demonstrated that EMILIN1 degradation by neutrophil elastase (NE) is a specific mechanism leading to the loss of functions disabling its regulatory properties. In this study we further analysed the proteolytic activity of NE, MMP-3, MMP-9, and MT1-MMP on EMILIN1 and found that MMP-3 and MT1-MMP partially cleaved EMILIN1 but without affecting the functional properties associated with the gC1q domain, whereas NE was able to fully impair the interaction of gC1q with the α4ß1 integrin by cleaving this domain outside of the E933 integrin binding site. By a site direct mutagenesis approach we mapped the bond between S913 and R914 residues and selected the NE-resistant R914W mutant still able to interact with the α4ß1 integrin after NE treatment. Functional studies showed that NE impaired the EMILIN1-α4ß1 integrin interaction by cleaving the gC1q domain in a region crucial for its proper structural conformation, paving the way to better understand NE effects on EMILIN1-cell interaction in pathological context.

Secreted Phospholipase A2 Type IIA (sPLA2-IIA) Activates Integrins in an Allosteric Manner.

Secreted phospholipase A2 type IIA (sPLA2-IIA) is a well-established pro-inflammatory protein and has been a major target for drug discovery. However, the mechanism of its signaling action has not been fully understood. We previously found that sPLA2-IIA binds to integrins αvß3 and α4ß1 in human and that this interaction plays a role in sPLA2-IIA's signaling action. Our recent studies found that sPLA2-IIA activates integrins in an allosteric manner through direct binding to a newly identified binding site of integrins (site 2), which is distinct from the classical RGD-binding site (site 1). The sPLA2-IIA-induced integrin activation may be related to the signaling action of sPLA2-IIA. Since sPLA2-IIA is present in normal human tears in addition to rheumatoid synovial fluid at high concentrations the sPLA2-IIA-mediated integrin activation on leukocytes may be involved in immune responses in normal and pathological conditions.

Tissue-engineered acellular small diameter long-bypass grafts with neointima-inducing activity.

Researchers have attempted to develop efficient antithrombogenic surfaces, and yet small-caliber artificial vascular grafts are still unavailable. Here, we demonstrate the excellent patency of tissue-engineered small-caliber long-bypass grafts measuring 20-30 cm in length and having a 2-mm inner diameter. The inner surface of an acellular ostrich carotid artery was modified with a novel heterobifunctional peptide composed of a collagen-binding region and the integrin α4ß1 ligand, REDV. Six grafts were transplanted in the femoral-femoral artery crossover bypass method. Animals were observed for 20 days and received no anticoagulant medication. No thrombogenesis was observed on the luminal surface and five cases were patent. In contrast, all unmodified grafts became occluded, and severe thrombosis was observed. The vascular grafts reported here are the first successful demonstrations of short-term patency at clinically applicable sizes.

Proinflammatory secreted phospholipase A2 type IIA (sPLA-IIA) induces integrin activation through direct binding to a newly identified binding site (site 2) in integrins αvß3, α4ß1, and α5ß1.

Integrins are activated by signaling from inside the cell (inside-out signaling) through global conformational changes of integrins. We recently discovered that fractalkine activates integrins in the absence of CX3CR1 through the direct binding of fractalkine to a ligand-binding site in the integrin headpiece (site 2) that is distinct from the classical RGD-binding site (site 1). We propose that fractalkine binding to the newly identified site 2 induces activation of site 1 though conformational changes (in an allosteric mechanism). We reasoned that site 2-mediated activation of integrins is not limited to fractalkine. Human secreted phospholipase A2 type IIA (sPLA2-IIA), a proinflammatory protein, binds to integrins αvß3 and α4ß1 (site 1), and this interaction initiates a signaling pathway that leads to cell proliferation and inflammation. Human sPLA2-IIA does not bind to M-type receptor very well. Here we describe that sPLA2-IIA directly activated purified soluble integrin αvß3 and transmembrane αvß3 on the cell surface. This activation did not require catalytic activity or M-type receptor. Docking simulation predicted that sPLA2-IIA binds to site 2 in the closed-headpiece of αvß3. A peptide from site 2 of integrin ß1 specifically bound to sPLA2-IIA and suppressed sPLA2-IIA-induced integrin activation. This suggests that sPLA2-IIA activates αvß3 through binding to site 2. sPLA2-IIA also activated integrins α4ß1 and α5ß1 in a site 2-mediated manner. We recently identified small compounds that bind to sPLA2-IIA and suppress integrin-sPLA2-IIA interaction (e.g. compound 21 (Cmpd21)). Cmpd21 effectively suppressed sPLA2-IIA-induced integrin activation. These results define a novel mechanism of proinflammatory action of sPLA2-IIA through integrin activation.

The chemokine fractalkine can activate integrins without CX3CR1 through direct binding to a ligand-binding site distinct from the classical RGD-binding site.

The chemokine domain of fractalkine (FKN-CD) binds to the classical RGD-binding site of αvß3 and that the resulting ternary complex formation (integrin-FKN-CX3CR1) is critical for CX3CR1 signaling and FKN-induced integrin activation. However, only certain cell types express CX3CR1. Here we studied if FKN-CD can activate integrins in the absence of CX3CR1. We describe that WT FKN-CD activated recombinant soluble αvß3 in cell-free conditions, but the integrin-binding defective mutant of FKN-CD (K36E/R37E) did not. This suggests that FKN-CD can activate αvß3 in the absence of CX3CR1 through the direct binding of FKN-CD to αvß3. WT FKN-CD activated αvß3 on CX3CR1-negative cells (K562 and CHO) but K36E/R37E did not, suggesting that FKN-CD can activate integrin at the cellular levels in a manner similar to that in cell-free conditions. We hypothesized that FKN-CD enhances ligand binding to the classical RGD-binding site (site 1) through binding to a second binding site (site 2) that is distinct from site 1 in αvß3. To identify the possible second FKN-CD binding site we performed docking simulation of αvß3-FKN-CD interaction using αvß3 with a closed inactive conformation as a target. The simulation predicted a potential FKN-CD-binding site in inactive αvß3 (site 2), which is located at a crevice between αv and ß3 on the opposite side of site 1 in the αvß3 headpiece. We studied if FKN-CD really binds to site 2 using a peptide that is predicted to interact with FKN-CD in site 2. Notably the peptide specifically bound to FKN-CD and effectively suppressed integrin activation by FKN-CD. This suggests that FKN-CD actually binds to site 2, and this leads to integrin activation. We obtained very similar results in α4ß1 and α5ß1. The FKN binding to site 2 and resulting integrin activation may be a novel mechanism of integrin activation and of FKN signaling.

How natalizumab binds and antagonizes α4 integrins.

Natalizumab antibody to α4-integrins is used in therapy of multiple sclerosis and Crohn's disease. A crystal structure of the Fab bound to an α4 integrin ß-propeller and thigh domain fragment shows that natalizumab recognizes human-mouse differences on the circumference of the ß-propeller domain. The epitope is adjacent to but outside of a ligand-binding groove formed at the interface with the ß-subunit ßI domain and shows no difference in structure when bound to Fab. Competition between Fab and the ligand vascular cell adhesion molecule (VCAM) for binding to cell surface α4ß1 shows noncompetitive antagonism. In agreement, VCAM docking models suggest that binding of domain 1 of VCAM to α4-integrins is unimpeded by the Fab, and that bound Fab requires a change in orientation between domains 1 and 2 of VCAM for binding to α4ß1. Mapping of species-specific differences onto α4ß1 and α4ß7 shows that their ligand-binding sites are highly conserved. Skewing away from these conserved regions of the epitopes recognized by current therapeutic function-blocking antibodies has resulted in previously unanticipated mechanisms of action.

Selective activation of cannabinoid receptor 2 in leukocytes suppresses their engagement of the brain endothelium and protects the blood-brain barrier.

Cannabinoid receptor 2 (CB2) is highly expressed in immune cells and stimulation decreases inflammatory responses. We tested the idea that selective CB2 activation in human monocytes suppresses their ability to engage the brain endothelium and migrate across the blood-brain barrier (BBB), preventing consequent injury. Intravital videomicroscopy was used to quantify adhesion of leukocytes to cortical vessels in lipopolysaccharide-induced neuroinflammation, after injection of ex vivo CB2-activated leukocytes into mice; CB2 agonists markedly decreased adhesion of ex vivo labeled cells in vivo. In an in vitro BBB model, CB2 activation in monocytes largely attenuated adhesion to and migration across monolayers of primary human brain microvascular endothelial cells and diminished BBB damage. CB2 stimulation in monocytes down-regulated active forms of integrins, lymphocyte function-associated antigen 1 (LFA-1), and very late antigen 4 (VLA-4). Cells treated with CB2 agonists exhibited increased phosphorylation levels of inhibitory sites of the actin-binding proteins cofilin and VASP, which are upstream regulators of conformational integrin changes. Up-regulated by relevant stimuli, Rac1 and RhoA were suppressed by CB2 agonists in monocytes. CB2 stimulation decreased formation of lamellipodia, which play a key role in monocyte migration. These results indicate that selective CB2 activation in leukocytes decreases key steps in monocyte-BBB engagement, thus suppressing inflammatory leukocyte responses and preventing neuroinflammation.

PI3-kinase γ promotes Rap1a-mediated activation of myeloid cell integrin α4ß1, leading to tumor inflammation and growth.

Tumor inflammation, the recruitment of myeloid lineage cells into the tumor microenvironment, promotes angiogenesis, immunosuppression and metastasis. CD11b+Gr1lo monocytic lineage cells and CD11b+Gr1hi granulocytic lineage cells are recruited from the circulation by tumor-derived chemoattractants, which stimulate PI3-kinase γ (PI3Kγ)-mediated integrin α4 activation and extravasation. We show here that PI3Kγ activates PLCγ, leading to RasGrp/CalDAG-GEF-I&II mediated, Rap1a-dependent activation of integrin α4ß1, extravasation of monocytes and granulocytes, and inflammation-associated tumor progression. Genetic depletion of PLCγ, CalDAG-GEFI or II, Rap1a, or the Rap1 effector RIAM was sufficient to prevent integrin α4 activation by chemoattractants or activated PI3Kγ (p110γCAAX), while activated Rap (RapV12) promoted constitutive integrin activation and cell adhesion that could only be blocked by inhibition of RIAM or integrin α4ß1. Similar to blockade of PI3Kγ or integrin α4ß1, blockade of Rap1a suppressed both the recruitment of monocytes and granulocytes to tumors and tumor progression. These results demonstrate critical roles for a PI3Kγ-Rap1a-dependent pathway in integrin activation during tumor inflammation and suggest novel avenues for cancer therapy.

Synthesis and evaluation of 4,5-dihydro-5-methylisoxazolin-5-carboxamide derivatives as VLA-4 antagonists.

A novel set of compounds containing a 4,5-dihydro-5-methylisoxazoline have been successfully designed as VLA-4 receptor antagonists. Compound (14p) had a high receptor binding affinity of 4 nM and also found to be metabolically stable in vitro.

Glycogen synthase kinase 3ß inhibition prevents monocyte migration across brain endothelial cells via Rac1-GTPase suppression and down-regulation of active integrin conformation.

Glycogen synthase kinase (GSK) 3ß has been identified as a regulator of immune responses. We demonstrated previously that GSK3ß inhibition in human brain microvascular endothelial cells (BMVECs) reduced monocyte adhesion/migration across BMVEC monolayers. Herein, we tested the idea that GSK3ß inhibition in monocytes can diminish their ability to engage the brain endothelium and migrate across the blood-brain barrier. Pretreatment of primary monocytes with GSK3ß inhibitors resulted in a decrease in adhesion (60%) and migration (85%), with similar results in U937 monocytic cells. Monocyte-BMVEC interactions resulted in diminished barrier integrity that was reversed by GSK3ß suppression in monocytic cells. Because integrins mediate monocyte rolling/adhesion, we detected the active conformational form of very late antigen 4 after stimulation with a peptide mimicking monocyte engagement by vascular cell adhesion molecule-1. Peptide stimulation resulted in a 14- to 20-fold up-regulation of the active form of integrin in monocytes that was suppressed by GSK3ß inhibitors (40% to 60%). Because small GTPases, such as Rac1, control leukocyte movement, we measured active Rac1 after monocyte activation with relevant stimuli. Stimulation enhanced the level of active Rac1 that was diminished by GSK3ß inhibitors. Monocytes treated with GSK3ß inhibitors showed increased levels of inhibitory sites of the actin-binding protein, cofilin, and vasodilator-stimulated phosphoprotein-regulating conformational changes of integrins. These results indicate that GSK3ß inhibition in monocytes affects active integrin expression, cytoskeleton rearrangement, and adhesion via suppression of Rac1-diminishing inflammatory leukocyte responses.

Directing mesenchymal stem cells to bone to augment bone formation and increase bone mass.

Aging reduces the number of mesenchymal stem cells (MSCs) that can differentiate into osteoblasts in the bone marrow, which leads to impairment of osteogenesis. However, if MSCs could be directed toward osteogenic differentiation, they could be a viable therapeutic option for bone regeneration. We have developed a method to direct MSCs to the bone surface by attaching a synthetic high-affinity and specific peptidomimetic ligand (LLP2A) against integrin α4ß1 on the MSC surface to a bisphosphonate (alendronate, Ale) that has a high affinity for bone. LLP2A-Ale induced MSC migration and osteogenic differentiation in vitro. A single intravenous injection of LLP2A-Ale increased trabecular bone formation and bone mass in both xenotransplantation studies and in immunocompetent mice. Additionally, LLP2A-Ale prevented trabecular bone loss after peak bone acquisition was achieved or as a result of estrogen deficiency. These results provide proof of principle that LLP2A-Ale can direct MSCs to the bone to form new bone and increase bone strength.

Potent in vivo suppression of inflammation by selectively targeting the high affinity conformation of integrin α4ß1.

The development of antagonists to the α4 integrin family of cell adhesion molecules has been an active area of pharmaceutical research to treat inflammatory and autoimmune diseases. Presently being tested in human clinical trials are compounds selective for α4ß1 (VLA-4) as well as several dual antagonists that inhibit both α4ß1 and α4ß7. The value of a dual versus a selective small molecule antagonist as well as the consequences of inhibiting different affinity states of the α4 integrins have been debated in the literature. Here, we characterize TBC3486, a N,N-disubstituted amide, which represents a unique structural class of non-peptidic, small molecule VLA-4 antagonists. Using a variety of adhesion assay formats as well as flow cytometry experiments using mAbs specific for certain activation-dependent integrin epitopes we demonstrate that TBC3486 preferentially targets the high affinity conformation of α4ß1 and behaves as a ligand mimetic. The antagonist is capable of blocking integrin-dependent T-cell co-activation in vitro as well as proves to be efficacious in vivo at low doses in two animal models of allergic inflammation. These data suggest that a small molecule α4 integrin antagonist selective for α4ß1 over α4ß7 and, specifically, selective for the high affinity conformation of α4ß1 may prove to be an effective therapy for multiple inflammatory diseases in humans.

Essential roles of VLA-4 in the hematopoietic system.

Integrins are one of the major families of adhesion molecules and make various kinds of biological effects by mediating cell-cell and cell-matrix interactions. Among integrins, VLA-4 is expressed on many types of hematopoietic cells including stem/progenitor cells and it is considered as a critical regulator of adult hematopoiesis. Recent studies revealed that VLA-4 is not necessarily required for the development or maintenance of adult hematopoietic cells. On the other hand, it was proved that VLA-4 is essential for homeostasis of distribution of hematopoietic stem/progenitor cells (HSPCs) and mature lymphocytes in the body. The dynamic regulation of VLA-4 function is mediated by its conformational change, which is strictly linked to the interaction between alpha and beta cytoplasmic domains. The study using knockin mice showed that GFFKR sequence, a well-preserved motif in the alpha cytoplasmic domain of VLA-4, is critical for binding of alpha and beta cytoplasmic domains as well as regulation of hematopoietic cell distribution. Small molecules targeting this cytoplasmic interaction or ligand-VLA-4 interaction may become good candidates of new drugs for mobilization of hematopoietic stem cells. Several studies have suggested the impact of VLA-4 on chemotherapy sensitivity and prognosis in hematological malignancies, which awaits further investigations.

Effects of supported lipid monolayer fluidity on the adhesion of hematopoietic progenitor cell lines to fibronectin-derived peptide ligands for alpha5beta1 and alpha4beta1 integrins.

Mimicking the in vivo stem cell niche to increase stem cell expansion will likely require the presentation of multiple ligands. Presenting ligands in fluid-supported lipid monolayers (SLMs) or bilayers (SLBs) allows for ligand diffusion to complement the arrangement of cell receptors as well as cell-mediated ligand rearrangement and clustering. Cells in tissues interact with ligands presented by other cells and the extracellular matrix (ECM), so it will likely be beneficial to present both cell-associated and ECM-derived ligands. A number of investigators have incorporated cell-membrane-associated ligands within fluid surfaces, and several groups have shown that these ligands cluster beneath the cells. However, few studies have investigated cell adhesion to ECM-derived ligands in fluid surfaces. Fibronectin is an important ECM component in many tissues, including the hematopoietic stem cell niche. We examined the adhesion of the M07e and THP-1 hematopoietic progenitor cell lines to fibronectin-derived peptide ligands for the alpha5beta1 (cyclic and linear RGD) and alpha4beta1 (cyclic LDV) integrins as well as the heparin-binding domain (HBD) presented as lipopeptides in fluid and gel SLMs. M07e cells adhered more avidly than THP-1 cells to all of the lipopeptides in fluid and gel surfaces. The adhesion of both cell lines to all peptides was less avid in fluid versus gel SLMs. Adhesion to cyclic LDV (cLDV) and cRGD was similar on gel SLMs for both cell lines. In contrast, adhesion to cLDV was less extensive than to cRGD in fluid SLMs, especially for M07e cells. Adhesion to linear RGD was less avid than to cRGD or cLDV and decreased to a greater extent in fluid SLMs. Human aortic endothelial cells adhered to cRGD in fluid SLMs and remained viable for at least 24 h but did not spread. We also showed additive THP-1 cell adhesion to cLDV and linear RGD lipopeptides presented in a fluid SLM. Although DOPC (dioleoyl phosphatidyl choline) SLMs are not sufficiently stable for long-term cell culture studies, our results and those of others suggest that fluid SLMs are likely to be useful for presenting multiple ligands and for mimicking short-term interactions in the stem cell niche.