Small molecule agonist of very late antigen-4 (VLA-4) integrin induces progenitor cell adhesion.

Activation of the integrin family of cell adhesion receptors on progenitor cells may be a viable approach to enhance the effects of stem cell-based therapies by improving cell retention and engraftment. Here, we describe the synthesis and characterization of the first small molecule agonist identified for the integrin α4ß1 (also known as very late antigen-4 or VLA-4). The agonist, THI0019, was generated via two structural modifications to a previously identified α4ß1 antagonist. THI0019 greatly enhanced the adhesion of cultured cell lines and primary progenitor cells to α4ß1 ligands VCAM-1 and CS1 under both static and flow conditions. Furthermore, THI0019 facilitated the rolling and spreading of cells on VCAM-1 and the migration of cells toward SDF-1α. Molecular modeling predicted that the compound binds at the α/ß subunit interface overlapping the ligand-binding site thus indicating that the compound must be displaced upon ligand binding. In support of this model, an analog of THI0019 modified to contain a photoreactive group was used to demonstrate that when cross-linked to the integrin, the compound behaves as an antagonist instead of an agonist. In addition, THI0019 showed cross-reactivity with the related integrin α4ß7 as well as α5ß1 and αLß2. When cross-linked to αLß2, the photoreactive analog of THI0019 remained an agonist, consistent with it binding at the α/ß subunit interface and not at the ligand-binding site in the inserted ("I") domain of the αL subunit. Co-administering progenitor cells with a compound such as THI0019 may provide a mechanism for enhancing stem cell therapy.

Atorvastatin preserves the integrity of endothelial adherens junctions by inhibiting vascular endothelial cadherin tyrosine phosphorylation.

Vascular endothelial cadherin (VE-cad) tyrosine (Tyr) phosphorylation has been implicated in the disruption of adherens junctions (AJs) induced by inflammatory reactions. The impacts of statins on integrity of AJs and VE-cad Tyr phosphorylation have not been explored. The effects of atorvastatin on IL-1ß and monocyte-induced VE-cad Tyr phosphorylation in human umbilical vein endothelial cells (ECs) were studied. In ECs treated with interleukin (IL)-1ß for 30 min, VE-cad Tyr phosphorylation, dissociation of the VE-cad/ß-catenin complex and transendothelial migration (TEM) of monocytes were increased. These processes were mediated by activation of HRas and RhoA that leads to phosphorylation of myosin light chain (MLC). Atorvastatin inhibited IL-1ß-induced Tyr phosphorylation of VE-cad by inhibiting RhoA and by dephosphorylating MLC. The attenuating effect of atorvastatin on VE-cad Tyr phosphorylation was reversed when RhoA was activated or MLC phosphatase was inhibited. Furthermore, inhibiting farnesyl transferase or geranylgeranyl transferase reproduced the inhibitory effects of atorvastatin on VE-cad Tyr phosphorylation. In addition, atorvastatin inhibited monocyte-induced VE-cad Tyr phosphorylation in ECs and attenuated IL-1ß-induced TEM of monocytes. Our study introduces a novel pleiotropic effect of atorvastatin and suggests that statins protect the integrity of AJs in ECs by inhibiting RhoA-mediated Tyr phosphorylation of VE-cad.

LFA-1 activation enriches tumor-specific T cells in a cold tumor model and synergizes with CTLA-4 blockade.

The inability of CD8+ effector T cells (Teffs) to reach tumor cells is an important aspect of tumor resistance to cancer immunotherapy. The recruitment of these cells to the tumor microenvironment (TME) is regulated by integrins, a family of adhesion molecules that are expressed on T cells. Here, we show that 7HP349, a small-molecule activator of lymphocyte function-associated antigen-1 (LFA-1) and very late activation antigen-4 (VLA-4) integrin cell-adhesion receptors, facilitated the preferential localization of tumor-specific T cells to the tumor and improved antitumor response. 7HP349 monotherapy had modest effects on anti-programmed death 1-resistant (anti-PD-1-resistant) tumors, whereas combinatorial treatment with anti-cytotoxic T lymphocyte-associated protein 4 (anti-CTLA-4) increased CD8+ Teff intratumoral sequestration and synergized in cooperation with neutrophils in inducing cancer regression. 7HP349 intratumoral CD8+ Teff enrichment activity depended on CXCL12. We analyzed gene expression profiles using RNA from baseline and on treatment tumor samples of 14 melanoma patients. We identified baseline CXCL12 gene expression as possibly improving the likelihood or response to anti-CTLA-4 therapies. Our results provide a proof-of-principle demonstration that LFA-1 activation could convert a T cell-exclusionary TME to a T cell-enriched TME through mechanisms involving cooperation with innate immune cells.

Use of a small molecule integrin activator as a systemically administered vaccine adjuvant in controlling Chagas disease.

The development of suitable safe adjuvants to enhance appropriate antigen-driven immune responses remains a challenge. Here we describe the adjuvant properties of a small molecule activator of the integrins αLß2 and α4ß1, named 7HP349, which can be safely delivered systemically independent of antigen. 7HP349 directly activates integrin cell adhesion receptors crucial for the generation of an immune response. When delivered systemically in a model of Chagas disease following immunization with a DNA subunit vaccine encoding candidate T. cruzi antigens, TcG2 and TcG4, 7HP349 enhanced the vaccine efficacy in both prophylactic and therapeutic settings. In a prophylactic setting, mice immunized with 7HP349 adjuvanted vaccine exhibited significantly improved control of acute parasite burden in cardiac and skeletal muscle as compared to vaccination alone. When administered with vaccine therapeutically, parasite burden was again decreased, with the greatest adjuvant effect of 7HP349 being noted in skeletal muscle. In both settings, adjuvantation with 7HP349 was effective in decreasing pathological inflammatory infiltrate, improving the integrity of tissue, and controlling tissue fibrosis in the heart and skeletal muscle of acutely and chronically infected Chagas mice. The positive effects correlated with increased splenic frequencies of CD8+T effector cells and an increase in the production of IFN-γ and cytolytic molecules (perforin and granzyme) by the CD4+ and CD8+ effector and central memory subsets in response to challenge infection. This demonstrates that 7HP349 can serve as a systemically administered adjuvant to enhance T cell-mediated immune responses to vaccines. This approach could be applied to numerous vaccines with no reformulation of existing stockpiles.

Myosin light chain phosphorylation facilitates monocyte transendothelial migration by dissociating endothelial adherens junctions.

AIMS: Transendothelial migration (TEM) of monocytes is a crucial step in inflammatory processes such as atherogenesis. Tyrosine phosphorylation of vascular endothelial cadherin (VE-cad) has been implicated in the dissociation of adherens junctions and the increased paracellular permeability of endothelial cells (ECs) that occur during TEM of monocytes. However, the underlying molecular mechanism has not been determined. We tested the hypothesis that the phosphorylation of myosin light chain (MLC) in ECs is crucial for the dissociation of adherens junctions during TEM of monocytes. METHODS AND RESULTS: Using a combination of biochemical and cellular techniques, we provide evidence for the signal transduction pathways that regulate tyrosine phosphorylation of VE-cad in ECs after the attachment of monocytes. Our findings indicate that after interaction of integrins on THP-1 cells with adhesion molecules on ECs, the induction of the HRas\Raf\MEK\ERK signalling cascade leads to the phosphorylation of MLC. This results in the recruitment of Src to the VE-cad complex and tyrosine phosphorylation of VE-cad, which leads to dissociation of ß-catenin from the VE-cad complex, formation of gaps between ECs, and enhancement of THP-1 cell TEM. CONCLUSION: Our studies suggest that monocyte-induced phosphorylation of MLC in ECs enhances TEM of monocytes through dissociation of EC adherens junctions.