An image-driven micromechanical approach to characterize multiscale remodeling in infarcted myocardium.

Myocardial infarction (MI) is accompanied by the formation of a fibrotic scar in the left ventricle (LV) and initiates significant alterations in the architecture and constituents of the LV free wall (LVFW). Previous studies have shown that LV adaptation is highly individual, indicating that the identification of remodeling mechanisms post-MI demands a fully subject-specific approach that can integrate a host of structural alterations at the fiber-level to changes in bulk biomechanical adaptation at the tissue-level. We present an image-driven micromechanical approach to characterize remodeling, assimilating new biaxial mechanical data, histological studies, and digital image correlation data within an in-silico framework to elucidate the fiber-level remodeling mechanisms that drive tissue-level adaptation for each subject. We found that a progressively diffused collagen fiber structure combined with similarly disorganized myofiber architecture in the healthy region leads to the loss of LVFW anisotropy post-MI, offering an important tissue-level hallmark for LV maladaptation. In contrast, our results suggest that reductions in collagen undulation are an adaptive mechanism competing against LVFW thinning. Additionally, we show that the inclusion of subject-specific geometry when modeling myocardial tissue is essential for accurate prediction of tissue kinematics. Our approach serves as an essential step toward identifying fiber-level remodeling indices that govern the transition of MI to systolic heart failure. These indices complement the traditional, organ-level measures of LV anatomy and function that often fall short of early prognostication of heart failure in MI. In addition, our approach offers an integrated methodology to advance the design of personalized interventions, such as hydrogel injection, to reinforce and suppress native adaptive and maladaptive mechanisms, respectively, to prevent the transition of MI to heart failure. STATEMENT OF SIGNIFICANCE: Biomechanical and architectural adaptation of the LVFW remains a central, yet overlooked, remodeling process post-MI. Our study indicates the biomechanical adaptation of the LVFW post-MI is highly individual and driven by altered fiber network architecture and collective changes in collagen fiber content, undulation, and stiffness. Our findings demonstrate the possibility of using cardiac strains to infer such fiber-level remodeling events through in-silico modeling, paving the way for in-vivo characterization of multiscale biomechanical indices in humans. Such indices will complement the traditional, organ-level measures of LV anatomy and function that often fall short of early prognostication of heart failure in MI.

A Micro-anatomical Model of the Infarcted Left Ventricle Border Zone to Study the Influence of Collagen Undulation.

Myocardial infarction (MI) results in cardiac myocyte death and often initiates the formation of a fibrotic scar in the myocardium surrounded by a border zone. Myocyte loss and collagen-rich scar tissue heavily influence the biomechanical behavior of the myocardium which could lead to various cardiac diseases such as systolic heart failure and arrhythmias. Knowledge of how myocyte and collagen micro-architecture changes affect the passive mechanical behavior of the border zone remains limited. Computational modeling provides us with an invaluable tool to identify and study the mechanisms driving the biomechanical remodeling of the myocardium post-MI. We utilized a rodent model of MI and an image-based approach to characterize the three-dimensional (3-D) myocyte and collagen micro-architecture at various timepoints post-MI. Left ventricular free wall (LVFW) samples were obtained from infarcted hearts at 1-week and 4-week post-MI (n = 1 each). Samples were labeled using immunoassays to identify the extracellular matrix (ECM) and myocytes. 3-D reconstructions of the infarct border zone were developed from confocal imaging and meshed to develop high-fidelity micro-anatomically accurate finite element models. We performed a parametric study using these models to investigate the influence of collagen undulation on the passive micromechanical behavior of the myocardium under a diastolic load. Our results suggest that although parametric increases in collagen undulation elevate the strain amount experienced by the ECM in both early- and late-stage MI, the sensitivity of myocytes to such increases is reduced from early to late-stage MI. Our 3-D micro-anatomical modeling holds promise in identifying mechanisms of border zone maladaptation post-MI.

Contractile Adaptation of the Left Ventricle Post-myocardial Infarction: Predictions by Rodent-Specific Computational Modeling.

Myocardial infarction (MI) results in cardiac myocyte death and the formation of a fibrotic scar in the left ventricular free wall (LVFW). Following an acute MI, LVFW remodeling takes place consisting of several alterations in the structure and properties of cellular and extracellular components with a heterogeneous pattern across the LVFW. The normal function of the heart is strongly influenced by the passive and active biomechanical behavior of the LVFW, and progressive myocardial structural remodeling can have a detrimental effect on both diastolic and systolic functions of the LV leading to heart failure. Despite important advances in understanding LVFW passive remodeling in the setting of MI, heterogeneous remodeling in the LVFW active properties and its relationship to organ-level LV function remain understudied. To address these gaps, we developed high-fidelity finite-element (FE) rodent computational cardiac models (RCCMs) of MI using extensive datasets from MI rat hearts representing the heart remodeling from one-week (1-wk) to four-week (4-wk) post-MI timepoints. The rat-specific models (n = 2 for each timepoint) integrate detailed imaging data of the heart geometry, myocardial fiber architecture, and infarct zone determined using late gadolinium enhancement prior to terminal measurements. The computational models predicted a significantly higher level of active tension in remote myocardium in early post-MI hearts (1-wk post-MI) followed by a return to near the control level in late-stage MI (3- and 4-wk post-MI). The late-stage MI rats showed smaller myofiber ranges in the remote region and in-silico experiments using RCCMs suggested that the smaller fiber helicity is consistent with lower contractile forces needed to meet the measured ejection fractions in late-stage MI. In contrast, in-silico experiments predicted that collagen fiber transmural orientation in the infarct region has little influence on organ-level function. In addition, our MI RCCMs indicated that reduced and potentially positive circumferential strains in the infarct region at end-systole can be used to infer information about the time-varying properties of the infarct region. The detailed description of regional passive and active remodeling patterns can complement and enhance the traditional measures of LV anatomy and function that often lead to a gross and limited assessment of cardiac performance. The translation and implementation of our model in patient-specific organ-level simulations offer to advance the investigation of individualized prognosis and intervention for MI.

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.

A machine learning model to estimate myocardial stiffness from EDPVR.

In-vivo estimation of mechanical properties of the myocardium is essential for patient-specific diagnosis and prognosis of cardiac disease involving myocardial remodeling, including myocardial infarction and heart failure with preserved ejection fraction. Current approaches use time-consuming finite-element (FE) inverse methods that involve reconstructing and meshing the heart geometry, imposing measured loading, and conducting computationally expensive iterative FE simulations. In this paper, we propose a machine learning (ML) model that feasibly and accurately predicts passive myocardial properties directly from select geometric, architectural, and hemodynamic measures, thus bypassing exhaustive steps commonly required in cardiac FE inverse problems. Geometric and fiber-orientation features were chosen to be readily obtainable from standard cardiac imaging protocols. The end-diastolic pressure-volume relationship (EDPVR), which can be obtained using a single-point pressure-volume measurement, was used as a hemodynamic (loading) feature. A comprehensive ML training dataset in the geometry-architecture-loading space was generated, including a wide variety of partially synthesized rodent heart geometry and myofiber helicity possibilities, and a broad range of EDPVRs obtained using forward FE simulations. Latin hypercube sampling was used to create 2500 examples for training, validation, and testing. A multi-layer feed-forward neural network (MFNN) was used as a deep learning agent to train the ML model. The model showed excellent performance in predicting stiffness parameters [Formula: see text] and [Formula: see text] associated with fiber direction ([Formula: see text] and [Formula: see text]). After conducting permutation feature importance analysis, the ML performance further improved for [Formula: see text] ([Formula: see text]), and the left ventricular volume and endocardial area were found to be the most critical geometric features for accurate predictions. The ML model predictions were evaluated further in two cases: (i) rat-specific stiffness data measured using ex-vivo mechanical testing, and (ii) patient-specific estimation using FE inverse modeling. Excellent agreements with ML predictions were found for both cases. The trained ML model offers a feasible technology to estimate patient-specific myocardial properties, thus, bridging the gap between EDPVR, as a confounded organ-level metric for tissue stiffness, and intrinsic tissue-level properties. These properties provide incremental information relative to traditional organ-level indices for cardiac function, improving the clinical assessment and prognosis of cardiac diseases.

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.

Identification of Inhibitors of Integrin Cytoplasmic Domain Interactions With Syk.

Leukocyte inflammatory responses require integrin cell-adhesion molecule signaling through spleen tyrosine kinase (Syk), a non-receptor kinase that binds directly to integrin ß-chain cytoplasmic domains. Here, we developed a high-throughput screen to identify small molecule inhibitors of the Syk-integrin cytoplasmic domain interactions. Screening small molecule compound libraries identified the ß-lactam antibiotics cefsulodin and ceftazidime, which inhibited integrin ß-subunit cytoplasmic domain binding to the tandem SH2 domains of Syk (IC50 range, 1.02-4.9 µM). Modeling suggested antagonist binding to Syk outside the pITAM binding site. Ceftazidime inhibited integrin signaling via Syk, including inhibition of adhesion-dependent upregulation of interleukin-1ß and monocyte chemoattractant protein-1, but did not inhibit ITAM-dependent phosphorylation of Syk mediated by FcγRI signaling. Our results demonstrate a novel means to target Syk independent of its kinase and pITAM binding sites such that integrin signaling via this kinase is abrogated but ITAM-dependent signaling remains intact. As integrin signaling through Syk is essential for leukocyte activation, this may represent a novel approach to target inflammation.

Interactions Between Structural Remodeling and Hypertrophy in the Right Ventricle in Response to Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) exerts substantial pressure overload on the right ventricle (RV), inducing RV remodeling and myocardial tissue adaptation often leading to right heart failure. The associated RV free wall (RVFW) adaptation involves myocardial hypertrophy, augmented intrinsic contractility, collagen fibrosis, and structural remodeling in an attempt to cope with pressure overload. If RVFW adaptation cannot maintain the RV stroke volume (SV), RV dilation will prevail as an exit mechanism, which usually decompensates RV function, leading to RV failure. Our knowledge of the factors determining the transition from the upper limit of RVFW adaptation to RV decompensation and the role of fiber remodeling events such as extracellular fibrosis and fiber reorientation in this transition remains very limited. Computational heart models that connect the growth and remodeling (G&R) events at the fiber and tissue levels with alterations in the organ-level function are essential to predict the temporal order and the compensatory level of the underlying mechanisms. In this work, building upon our recently developed rodent heart models (RHM) of PAH, we integrated mathematical models that describe volumetric growth of the RV and structural remodeling of the RVFW. The time-evolution of RV remodeling from control and post-PAH time points was simulated. The results suggest that the augmentation of the intrinsic contractility of myofibers, accompanied by an increase in passive stiffness of RVFW, is among the first remodeling events through which the RV strives to maintain the cardiac output. Interestingly, we found that the observed reorientation of the myofibers toward the longitudinal (apex-to-base) direction was a maladaptive mechanism that impaired the RVFW contractile pattern and advanced along with RV dilation at later stages of PAH. In fact, although individual fibers were more contractile post-PAH, the disruption in the optimal transmural fiber architecture compromised the effective contractile function of the RVFW, contributing to the depressed ejection fraction (EF) of the RV. Our findings clearly demonstrate the critical need for developing multiscale approaches that can model and delineate relationships between pathological alterations in cardiac function and underlying remodeling events across fiber, cellular, and molecular levels.

A Computational Cardiac Model for the Adaptation to Pulmonary Arterial Hypertension in the Rat.

Pulmonary arterial hypertension (PAH) imposes pressure overload on the right ventricle (RV), leading to RV enlargement via the growth of cardiac myocytes and remodeling of the collagen fiber architecture. The effects of these alterations on the functional behavior of the right ventricular free wall (RVFW) and organ-level cardiac function remain largely unexplored. Computational heart models in the rat (RHMs) of the normal and hypertensive states can be quite valuable in simulating the effects of PAH on cardiac function to gain insights into the pathophysiology of underlying myocardium remodeling. We thus developed high-fidelity biventricular finite element RHMs for the normal and post-PAH hypertensive states using extensive experimental data collected from rat hearts. We then applied the RHM to investigate the transmural nature of RVFW remodeling and its connection to wall stress elevation under PAH. We found a strong correlation between the longitudinally-dominated fiber-level adaptation of the RVFW and the transmural alterations of relevant wall stress components. We further conducted several numerical experiments to gain new insights on how the RV responds both normally and in the post-PAH state. We found that the effect of pressure overload alone on the increased contractility of the RV is comparable to the effects of changes in the RV geometry and stiffness. Furthermore, our RHMs provided fresh perspectives on long-standing questions of the functional role of the interventricular septum in RV function. Specifically, we demonstrated that an inaccurate identification of the mechanical adaptation of the septum can lead to a significant underestimation of RVFW contractility in the post-PAH state. These findings show how integrated experimental-computational models can facilitate a more comprehensive understanding of the cardiac remodeling events during PAH.

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.

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.

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 γ-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.

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.

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.

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.

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.

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 beta1 integrin function. This was apparent through increased resistance to shear flow-dependent detachment of T cells adherent to the alpha4beta1 and alpha5beta1 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 beta1 integrins and to activate adhesion-strengthening processes.

Cell adhesion antagonists: therapeutic potential in asthma and chronic obstructive pulmonary disease.

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 beta1 and beta2 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 alphaIIbbeta3 (e.g. abciximab), integrin alphaLbeta2 (efalizumab), and integrin alpha4beta1 (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 alpha4beta1 antagonist (valategrast [R411]), have generated enthusiastic anticipation that novel strategies to treat asthma and COPD may be forthcoming.

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 alpha4 and beta2 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.