Vascular Smooth Muscle Cell Stiffness and Adhesion to Collagen I Modified by Vasoactive Agonists

In vascular smooth muscle cells (VSMCs) integrin-mediated adhesion to extracellular matrix (ECM) proteins play important roles in sustaining vascular tone and resistance. The main goal of this study was to determine whether VSMCs adhesion to type I collagen (COL-I) was altered in parallel with the changes in the VSMCs contractile state induced by vasoconstrictors and vasodilators. VSMCs were isolated from rat cremaster skeletal muscle arterioles and maintained in primary culture without passage. Cell adhesion and cell E-modulus were assessed using atomic force microscopy (AFM) by repetitive nano-indentation of the AFM probe on the cell surface at 0.1 Hz sampling frequency and 3200 nm Z-piezo travelling distance (approach and retraction). AFM probes were tipped with a 5 μm diameter microbead functionalized with COL-I (1mg\ml). Results showed that the vasoconstrictor angiotensin II (ANG-II; 10−6 ) significantly increased (p<0.05) VSMC E-modulus and adhesion probability to COL-I by approximately 35% and 33%, respectively. In contrast, the vasodilator adenosine (ADO; 10−4 ) significantly decreased (p<0.05) VSMC E-modulus and adhesion probability by approximately −33% and −17%, respectively. Similarly, the NO donor (PANOate, 10−6 M), a potent vasodilator, also significantly decreased (p<0.05) the VSMC E-modulus and COL-I adhesion probability by −38% and −35%, respectively. These observations support the hypothesis that integrin-mediated VSMC adhesion to the ECM protein COL-I is dynamically regulated in parallel with VSMC contractile activation. These data suggest that the signal transduction pathways modulating VSMC contractile activation and relaxation, in addition to ECM adhesion, interact during regulation of contractile state

Effect of Longâ Term Oral Bisphosphonates on Implant Wound Healing: Literature Review and a Case Report

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141603/1/jper0584.pd

Acidic Extracellular pH Promotes Activation of Integrin αvβ3

Acidic extracellular pH is characteristic of the cell microenvironment in several important physiological and pathological contexts. Although it is well established that acidic extracellular pH can have profound effects on processes such as cell adhesion and migration, the underlying molecular mechanisms are largely unknown. Integrin receptors physically connect cells to the extracellular matrix, and are thus likely to modulate cell responses to extracellular conditions. Here, we examine the role of acidic extracellular pH in regulating activation of integrin [alpha]v[beta]3. Through computational molecular dynamics simulations, we find that acidic extracellular pH promotes opening of the [alpha]v[beta]3 headpiece, indicating that acidic pH can thereby facilitate integrin activation. This prediction is consistent with our flow cytometry and atomic force microscope-mediated force spectroscopy assays of integrin [alpha]v[beta]3 on live cells, which both demonstrate that acidic pH promotes activation at the intact cell surface. Finally, quantification of cell morphology and migration measurements shows that acidic extracellular pH affects cell behavior in a manner that is consistent with increased integrin activation. Taken together, these computational and experimental results suggest a new and complementary mechanism of integrin activation regulation, with associated implications for cell adhesion and migration in regions of altered pH that are relevant to wound healing and cancer.National Institute of Biomedical Imaging and Bioengineering (U.S.) (Award Number T32EB006348)Massachusetts Institute of Technology (Collamore-Rogers Fellowship)National Institutes of Health (U.S.) (NIH Cell Migration Consortium Grant U54-GM069668)National Science Foundation (U.S.) (CAREER Award)Singapore-MIT Alliance for Research and Technology (BioSystem and Micromechanics (BioSyM) Interdisciplinary Research Group

Angiopoietin-1 enhances neutrophil chemotaxis in vitro and migration in vivo through interaction with CD18 and release of CCL4

Angiopoietins are a family of growth factors that are ligands for the tyrosine kinase receptor, Tie2. Angiopoietin 1 (Ang-1) is agonistic for Tie2, plays a key role in blood vessel maturation and stability and has been shown to possess anti-inflammatory properties. However, Tie2 expression has been demonstrated on human neutrophils and the observation that neutrophils migrate in response to Ang-1 in vitro has confounded research into its exact role in inflammation as well as its potential use as a therapeutic agent. We used a mouse model of peritoneal neutrophilic inflammation to determine if Ang-1 could stimulate neutrophil migration in vivo. Tie2 expression was demonstrated on mouse neutrophils. In addition, recombinant human Ang-1 induced significant chemotaxis of isolated mouse neutrophils in a Tie2- and CD18-dependent manner. Subsequently, co-immunoprecipitation of Ang-1 and CD18 demonstrated their interaction. Intraperitoneal injection of an engineered angiopoietin-1, MAT.Ang-1, induced significant neutrophil migration into the peritoneum and a significant increase in the levels of CCL4 in peritoneal lavage fluid. Depletion of resident peritoneal macrophages prior to, or concomitant injections of an anti-CCL4 antibody with MAT.Ang-1 resulted in a significant reduction in neutrophil recruitment. These data indicate a pro-inflammatory role for Ang-1 with respect to neutrophil recruitment.British Heart Foundation Studentship FS/06/081/2172

Anti-Tumor Activity and Immunotherapeutic Potential of a Bisphosphonate Prodrug

Bisphosphonates have benefits in breast cancer and multiple myeloma patients and have been used with adoptive immunotherapy with γδ T cells expressing Vγ2?Vδ2 TCRs. Although treatment with γδ T cells is safe, it has shown limited efficacy. Present bisphosphonates stimulate γδ T cells but were designed to inhibit bone resorption rather than treating cancer and have limited oral absorption, tumor cell entry, and cause bone side effects. The development of phosphate and phosphonate nucleotide prodrugs has led to important drugs for hepatitis C and HIV. Using a similar approach, we synthesized bisphosphonate prodrugs and found that they efficiently limit tumor cell growth. Pivoxil bisphosphonate esters enter cells where esterases convert them to their active acids. The bisphosphonate esters stimulated γδ T cells to secrete TNF-α in response to a variety of tumor cells more efficiently than their corresponding acids. The most active compound, tetrakis-pivaloyloxymethyl 2-(thiazole-2-ylamino)ethylidene-1,1- bisphosphonate (7), specifically expanded γδ T cells and stimulated them to secrete interferon-γ and kill tumor cells. In preclinical studies, combination therapy with compound 7 and γδ T cells prolonged survival of mice inoculated with either human bladder cancer or fibrosarcoma cells. Therefore, bisphosphonate prodrugs could enhance the effectiveness of adoptive cancer immunotherapy with γδ T cells