Peter Vanderslice

Cell Adhesion Antagonists

Chronic obstructive pulmonary disease (COPD) and asthma are inflammatory diseases of the lung where a hallmark feature is excessive leukocyte infiltration that leads to tissue injury. Cell adhesion molecules (e.g. selectins and integrins) play a key role in cell trafficking, and in the lung they regulate leukocyte extravasation, migration within the interstitium, cellular activation, and tissue retention. All selectin family members (including L-selectin, P-selectin, and E-selectin) and many of the β1 and β2 integrins appear to be important therapeutic targets, as numerous animal studies have demonstrated essential roles for these cell adhesion molecules in lung inflammation. Not surprisingly, these families of adhesion molecules have been under intense investigation by the pharmaceutical industry for the development of novel therapeutics. Integrins are validated drug targets, as drugs that antagonize integrin αIIbβ3 (e.g. abciximab), integrin αLβ2 (efalizumab), and integrin α4β1 (natalizumab) are currently US FDA-approved for acute coronary syndromes, psoriasis, and multiple sclerosis, respectively. However, none has been approved for indications related to asthma or COPD. Here, we provide an overview of roles played by selectins and integrins in lung inflammation. We also describe recent clinical results (both failures and successes) in developing adhesion molecule antagonists, with specific emphasis on those targets that may have potential benefit in asthma and COPD. Early clinical trials using selectin and integrin antagonists have met with limited success. However, recent positive phase II clinical trials with a small-molecule selectin antagonist (bimosiamose) and a small-molecule integrin α4β1 antagonist (valategrast [R411]), have generated enthusiastic anticipation that novel strategies to treat asthma and COPD may be forthcoming.

Novel inhibitors of α4β1 integrin receptor interactions through library synthesis and screening

Abstract A library of 2302 small molecule β-turn mimetics was screened for inhibition of the α4β1 integrin-CS1 splice variant binding interaction. Preliminary data revealed several active ligands, and validation with purified material culminated in the identification of some of the first small molecule ligands (1, IC50 = 5 μM, and 2, IC50 = 8 μM) to be reported for this class of integrins.

Angiogenesis induced by tumor necrosis factor-agr; is mediated by α4 integrins

Tumor necrosis factor-α (TNF-α) and fibroblast growth factor-2 (FGF-2 or bFGF) are potent stimulators of angiogenesis. TNF-α, but not FGF-2, can induce the expression of vascular cell adhesion molecule-1 (VCAM-1) on the surface of endothelial cells. The soluble form of VCAM-1 has recently been demonstrated to function as an angiogenic mediator. Here we demonstrate that monoclonal antibodies directed against VCAM-1 or its α4 integrin counter-receptor inhibited TNF-α-induced endothelial cell migration in vitro. Angiogenesis induced in vivo in rat corneas by TNF-α was inhibited by a neutralizing antibody directed against the rat α4 integrin subunit. A peptide antagonist of the a4 integrins blocked TNF-α-induced endothelial cell migration in vitro and angiogenesis in rat corneas in vivo. No inhibition by the antibodies or peptide antagonist was observed either in vitro or in vivo when FGF-2 was used as the stimulus. The peptide antagonist did not inhibit TNF-a binding to its receptor nor did it block the function of αvβ3, an integrin previously implicated in TNF-a and FGF-2 mediated angiogenesis. These results demonstrate that angiogenic processes induced by TNF-α are mediated in part by agr;4 integrins possibly by a mechanism involving the induction of soluble VCAM-1.

Contrasting Roles for Domain 4 of VCAM-1 in the Regulation of Cell Adhesion and Soluble VCAM-1 Binding to Integrin α4β1

Cell adhesion mediated by the interaction between integrin α4β1 and VCAM-1 is important in normal physiologic processes and in inflammatory and autoimmune disease. Numerous studies have mapped the α4β1 binding sites in VCAM-1 that mediate cell adhesion; however, little is known about the regions in VCAM-1 important for regulating soluble binding. In the present study, we demonstrate that 6D VCAM-1 (an alternatively spliced isoform of VCAM-1 lacking Ig-like domain 4) binds α4β1 with a higher relative affinity than does the full-length form of VCAM-1 containing 7 Ig-like extracellular domains (7D VCAM-1). In indirect binding assays, the EC50 of soluble 6D VCAM-1 binding to α4β1 on Jurkat cells (in 1 mM MnCl2) was 2 × 10−9 M, compared with 7D VCAM-1 at 11 × 10−9 M. When used in solution to inhibit α4β1 mediated cell adhesion, the IC50 of 6D VCAM-1 was 13 × 10−9 M, compared with 7D VCAM-1 measured at 150 × 10−9 M. Removal of Ig-like domains 4, 5, or 6, or simply substituting Asp328 in domain 4 of 7D VCAM-1 with alanine, caused increased binding of soluble 7D VCAM-1 to α4β1. In contrast, cells adhered more avidly to 7D VCAM-1 under shear force, as it induced cell spreading at lower concentrations than did 6D VCAM-1. Finally, soluble 6D VCAM-1 acts as an agonist through α4β1 by augmenting cell migration and inducing cell aggregation. These results indicate that the domain 4 of VCAM-1 plays a contrasting role when VCAM-1 is presented in solution or as a cell surface-expressed adhesive substrate.

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 β-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.

Clustering T-cell GM1 lipid rafts increases cellular resistance to shear on fibronectin through changes in integrin affinity and cytoskeletal dynamics

Lipid rafts are small laterally mobile microdomains that are highly enriched in lymphocyte signaling molecules. GM1 gangliosides are a common lipid raft component and have been shown to be important in many T‐cell functions. The aggregation of specific GM1 lipid rafts can control many T‐cell activation events, including their novel association with T‐cell integrins. We found that clustering GM1 lipid rafts can regulate β1 integrin function. This was apparent through increased resistance to shear flow‐dependent detachment of T cells adherent to the α4β1 and α5β1 integrin ligand fibronectin (FN). Adhesion strengthening as a result of clustering GM1 enriched lipid rafts correlated with increased cellular rigidity and morphology through the localization of cortical F‐actin, the resistance to shear‐induced cell stretching, and an increase in the surface area and symmetry of the contact area between the cell surface and adhesive substrate. Furthermore, clustering GM1 lipid rafts could initiate integrin ‘inside‐out’ signaling mechanisms. This was seen through increased integrin–cytoskeleton associations and enhanced soluble binding of FN and VCAM‐1, suggesting the induction of high‐affinity integrin conformations. The activation of these adhesion‐strengthening characteristics appears to be specific for the aggregation of GM1 lipid rafts as the aggregation of the heterogeneous raft‐associated molecule CD59 failed to activate these functions. These findings indicate a novel mechanism to signal to β1 integrins and to activate adhesion‐strengthening processes.

Treatment of hind limb ischemia using angiogenic peptide nanofibers

For a proangiogenic therapy to be successful, it must promote the development of mature vasculature for rapid reperfusion of ischemic tissue. Whole growth factor, stem cell, and gene therapies have yet to achieve the clinical success needed to become FDA-approved revascularization therapies. Herein, we characterize a biodegradable peptide-based scaffold engineered to mimic VEGF and self-assemble into a nanofibrous, thixotropic hydrogel, SLanc. We found that this injectable hydrogel was rapidly infiltrated by host cells and could be degraded while promoting the generation of neovessels. In mice with induced hind limb ischemia, this synthetic peptide scaffold promoted angiogenesis and ischemic tissue recovery, as shown by Doppler-quantified limb perfusion and a treadmill endurance test. Thirteen-month-old mice showed significant recovery within 7 days of treatment. Biodistribution studies in healthy mice showed that the hydrogel is safe when administered intramuscularly, subcutaneously, or intravenously. These preclinical studies help establish the efficacy of this treatment for peripheral artery disease due to diminished microvascular perfusion, a necessary step before clinical translation. This peptide-based approach eliminates the need for cell transplantation or viral gene transfection (therapies currently being assessed in clinical trials) and could be a more effective regenerative medicine approach to microvascular tissue engineering.

Integrin antagonists as therapeutics for inflammatory diseases.

Integrins are a family of heterodimeric cell surface receptors that mediate adhesion events crucial to cellular migration, proliferation and activation. Although critical to a normal immune response, integrins can also facilitate the progression of many inflammatory and autoimmune disorders. As such, they have attracted the attention of the pharmaceutical industry. Several humanised monoclonal antibodies directed against integrin targets have proven to be successful in clinical trials and have been approved for use in humans. This has not only resulted in effective therapies for patients, but also has provided important proof-of-concept studies for the development of small-molecule antagonists. This review focuses on those integrin subclasses that are being evaluated for their potential role in pulmonary, dermatological, gastrointestinal or rheumatic diseases. These include the α4 and β2 integrins, as well as an emerging group of targets from the collagen-binding family of integrins. Interfering with integrin signalling pathways represents a future area of interest. The rationale for pursuing these targets, as well as the drugs presently under development, are discussed.

Endothelial-Like Progenitor Cells Engineered to Produce Prostacyclin Rescue Monocrotaline-Induced Pulmonary Arterial Hypertension and Provide Right Ventricle Benefits

Background— Intravenous prostacyclin is approved for treating pulmonary arterial hypertension (PAH), but it has a short half-life and must be delivered systemically via an indwelling intravenous catheter. We hypothesize that localized jugular vein delivery of prostacyclin-producing cells may provide sustained therapeutic effects without the limitations of systemic delivery. Methods and Results— We generated a vector expressing a human cyclooxygenase isoform 1 and prostacyclin synthase fusion protein that produces prostacyclin from arachidonic acid. Endothelial-like progenitor cells (ELPCs) were transfected with the cyclooxygenase isoform 1-prostacyclin synthase plasmid and labeled with lentivirus expressing nuclear-localized red fluorescent protein (nuRFP). The engineered ELPCs (expressing cyclooxygenase isoform 1-prostacyclin synthase and nuRFP) were tested in rats with monocrotaline (MCT)-induced PAH. In PAH prevention studies, treatment with engineered ELPCs or control ELPCs (expressing nuRFP alone) attenuated MCT-induced right ventricular systolic pressure increase, right ventricular hypertrophy, and pulmonary vessel wall thickening. Engineered ELPCs were more effective than control ELPCs in all variables evaluated. In PAH reversal studies, engineered ELPCs or control ELPCs increased the survival rate of rats with established PAH and decreased right ventricular hypertrophy. Engineered ELPCs provided a survival benefit 2 weeks earlier than did control ELPCs. Microarray-based gene ontology analysis of the right ventricle revealed that a number of MCT-altered genes and neurotransmitter pathways (dopamine, serotonin, and &ggr;-aminobutyric acid) were restored after ELPC-based prostacyclin gene therapy. Conclusions— Cyclooxygenase isoform 1-prostacyclin synthase–expressing ELPCs reversed MCT-induced PAH. A single jugular vein injection offered survival benefits for at least 4 weeks and may provide a promising option for PAH patients.

CD34⁺/M-cadherin⁺ bone marrow progenitor cells promote arteriogenesis in ischemic hindlimbs of ApoE⁻/⁻ mice.

Background Cell-based therapy shows promise in treating peripheral arterial disease (PAD); however, the optimal cell type and long-term efficacy are unknown. In this study, we identified a novel subpopulation of adult progenitor cells positive for CD34 and M-cadherin (CD34+/M-cad+ BMCs) in mouse and human bone marrow. We also examined the long-lasting therapeutic efficacy of mouse CD34+/M-cad+ BMCs in restoring blood flow and promoting vascularization in an atherosclerotic mouse model of PAD. Methods and Findings Colony-forming cell assays and flow cytometry analysis showed that CD34+/M-cad+ BMCs have hematopoietic progenitor properties. When delivered intra-arterially into the ischemic hindlimbs of ApoE−/− mice, CD34+/M-cad+ BMCs alleviated ischemia and significantly improved blood flow compared with CD34+/M-cad− BMCs, CD34−/M-cad+ BMCs, or unselected BMCs. Significantly more arterioles were seen in CD34+/M-cad+ cell-treated limbs than in any other treatment group 60 days after cell therapy. Furthermore, histologic assessment and morphometric analyses of hindlimbs treated with GFP+ CD34+/M-cad+ cells showed that injected cells incorporated into solid tissue structures at 21 days. Confocal microscopic examination of GFP+ CD34+/M-cad+ cell-treated ischemic legs followed by immunostaining indicated the vascular differentiation of CD34+/M-cad+ progenitor cells. A cytokine antibody array revealed that CD34+/M-cad+ cell-conditioned medium contained higher levels of cytokines in a unique pattern, including bFGF, CRG-2, EGF, Flt-3 ligand, IGF-1, SDF-1, and VEGFR-3, than did CD34+/M-cad− cell-conditioned medium. The proangiogenic cytokines secreted by CD34+/M-cad+ cells induced oxygen- and nutrient-depleted endothelial cell sprouting significantly better than CD34+/M-cad− cells during hypoxia. Conclusion CD34+/M-cad+ BMCs represent a new progenitor cell type that effectively alleviates hindlimb ischemia in ApoE−/− mice by consistently improving blood flow and promoting arteriogenesis. Additionally, CD34+/M-cad+ BMCs contribute to microvascular remodeling by differentiating into vascular cells and releasing proangiogenic cytokines and growth factors.