Ron Jankowski

Microbubbles Targeted to Intercellular Adhesion Molecule-1 Bind to Activated Coronary Artery Endothelial Cells

BACKGROUND Preclinical atherosclerosis is associated with increased endothelial cell (EC) expression of leukocyte adhesion molecules (LAMs), which mediate monocyte adhesion during atherogenesis. Identification of cell-surface LAMs may uniquely allow assessment of endothelial function, but there are no in vivo methods for detecting LAMs. We tested a new microbubble designed to bind to and allow specific ultrasound detection of intercellular adhesion molecule-1 (ICAM-1). METHODS AND RESULTS A perfluorobutane gas-filled lipid-derived microsphere with monoclonal antibody to ICAM-1 covalently bound to the bubble shell was synthesized. Bubbles with either nonspecific IgG or no protein on the shell were synthesized as controls. Coverslips of cultured human coronary artery ECs were placed in a parallel-plate perfusion chamber and exposed to 1 of the 3 microbubble species, followed by perfusion with culture medium. Experiments were performed with either normal or interleukin-1beta-activated ECs overexpressing ICAM-1, and bubble adherence was quantified with epifluorescent videomicroscopy. There was limited adherence of control bubbles to normal or activated ECs, whereas a 40-fold increase in adhesion occurred when anti-ICAM-1-conjugated bubbles were exposed to activated ECs compared with normal ECs (8.1+/-3.5 versus 0.21+/-0.09 bubbles per cell, respectively, P<0.001). Although diminished, this difference persisted even after perfusion at higher wall shear rates. CONCLUSIONS A gas-filled microbubble with anti-ICAM-1 antibody on its shell specifically binds to activated ECs overexpressing ICAM-1. Diagnostic ultrasound in conjunction with targeted contrast agents has the unique potential to characterize cell phenotype in vivo.

Muscle-derived stem cells

The existence of cells with stem cell-like abilities derived from various tissues can now be extended to include the skeletal muscle compartment. Although researchers have focused on the utilization of these cells with regard to their myogenic capacity, initially exploring more efficient cellular therapy treatments for muscular dystrophy, it is becoming increasingly apparent that such cells may one day be used in the treatment of non-myogenic disorders. Evidence regarding the existence and differentiation capacity of muscle-derived stem cells is discussed, along with current theories regarding their proposed position within the myogenic hierarchy.

Enhancement of Bone Healing Based on Ex Vivo Gene Therapy Using Human Muscle-Derived Cells Expressing Bone Morphogenetic Protein 2

Molecular biological advances have allowed the use of gene therapy in a clinical setting. In addition, numerous reports have indicated the existence of inducible osteoprogenitor cells in skeletal muscle. Because of this, we hypothesized that skeletal muscle cells might be ideal vehicles for delivery of bone-inductive factors. Using ex vivo gene transfer methods, we genetically engineered freshly isolated human skeletal muscle cells with adenovirus and retrovirus to express human bone morphogenetic protein 2 (BMP-2). These cells were then implanted into nonhealing bone defects (skull defects) in severe combined immune deficiency (SCID) mice. The closure of the defect was monitored grossly and histologically. Mice that received BMP-2-producing human muscle-derived cells experienced a full closure of the defect by 4 to 8 weeks posttransplantation. Remodeling of the newly formed bone was evident histologically during the 4- to 8-week period. When analyzed by fluorescence in situ hybridization, a small fraction of the transplanted human muscle-derived cells was found within the newly formed bone, where osteocytes normally reside. These results indicate that genetically engineered human muscle-derived cells enhance bone healing primarily by delivering BMP-2, while a small fraction of the cells seems to differentiate into osteogenic cells.

Flow Cytometric Characterization of Myogenic Cell Populations Obtained via the Preplate Technique: Potential for Rapid Isolation of Muscle-Derived Stem Cells

Myoblast transplantation has been investigated as a therapy for muscle-related diseases and as a gene delivery vehicle for therapeutic recombinant proteins. Clinical successes involving muscle cell transplantation have been limited, in part because of poor donor cell survival, and the heterogeneous nature of myogenic donor cells has largely been ignored. We have previously reported an isolation technique, preplating, that results in purified myogenic cells that are capable of significantly higher rates of donor cell survival leading to enhanced gene transfer to skeletal muscle. Characterization of these purified cells revealed that they display markers common to stem cells and are capable of multilineage differentiation. This study was performed to phenotypically characterize, by flow cytometry, muscle-derived cell populations obtained by the preplate technique for the purpose of eventually developing a method to quickly identify and isolate viable muscle cells best suited for transplantation. Muscle cell cultures were analyzed for expression of the surface proteins Sca-1, c-Kit, and CD34. We found that the preplate technique purifies distinct myogenic cell subpopulations expressing CD34 alone (Sca-1 negative) and Sca-1 alone (CD34 negative), but that this expression is subject to change with time in culture. Isolation and transplantation of phenotypically pure Sca-1-positive myogenic cells, obtained by magnetic cell sorting, demonstrates the ability to quickly select viable myogenic cells capable of regenerating skeletal muscle and restoring dystrophin expression within dystrophic host skeletal muscle. Flow cytometric described phenotypes will aid in the rapid isolation of specific donor cell populations for muscle cell transplants and muscle cell-mediated gene therapies, thereby enhancing their future success.

The role of CD34 expression and cellular fusion in the regeneration capacity of myogenic progenitor cells

Characterization of myogenic subpopulations has traditionally been performed independently of their functional performance following transplantation. Using the preplate technique, which separates cells based on their variable adhesion characteristics, we investigated the use of cell surface proteins to potentially identify progenitors with enhanced regeneration capabilities. Based on previous studies, we used cell sorting to investigate stem cell antigen-1 (Sca-1) and CD34 expression on myogenic populations with late adhesion characteristics. We compared the regeneration efficiency of these sorted progenitors, as well as those displaying early adhesion characteristics, by quantifying their ability to regenerate skeletal muscle and restore dystrophin following transplantation into allogenic dystrophic host muscle. Identification and utilization of late adhering populations based on CD34 expression led to differential regeneration, with CD34-positive populations exhibiting significant improvements in dystrophin restoration compared with both their CD34-negative counterparts and early adhering cell populations. Regenerative capacity was found to correspond to the level of myogenic commitment, defined by myogenic regulatory factor expression, and the rate and degree of induced cell differentiation and fusion. These results demonstrate the ability to separate definable subpopulations of myogenic progenitors based on CD34 expression and reveal the potential implications of defining myogenic cell behavioral and phenotypic characteristics in relation to their regenerative capacity in vivo.

Albumin microbubble adherence to human coronary endothelium: implications for assessment of endothelial function using myocardial contrast echocardiography.

OBJECTIVES We hypothesized that sonicated 5% human albumin microbubbles (Albunex) adhere to disrupted vascular endothelium and that this interaction is a marker of endothelial integrity. This study sought to identify sites and determinants of Albunex-endothelial cell (EC) attachment. BACKGROUND Under normal conditions, Albunex microbubbles used in myocardial contrast echocardiography (MCE) pass unimpeded through the coronary microcirculation. During pathophysiologic states associated with endothelial dysfunction, however, microbubbles linger in the myocardium despite normal flow. The sites and conditions regulating microbubble adhesion are unknown. METHODS Coverslips with cultured human coronary artery ECs were mounted in a parallel plate perfusion system and perfused with a suspension of fluorescein-labeled Albunex in culture medium, followed by a bubble-free wash at a wall shear rate of 100 s-1. To create inflammatory ECs, phorbol myristate acetate was added 4.5 h before perfusion, and flow cytometry was used to confirm an inflammatory response. Perfusions were performed under normal and inflammatory conditions using surfaces of confluent and subconfluent ECs and isolated extracellular matrix. Bubble adherence was quantified in 20 random fields per cover-slip using epifluorescent video microscopy. RESULTS No microbubbles adhered to normal confluent ECs, although small numbers adhered to inflamed ECs (0.03 +/- 0.01 bubbles/cell, p < 0.01 vs. normal cells). Fever microbubbles attached to normal versus inflamed matrix of both partially exposed (1,800 +/- 520 vs. 4,100 +/- 1,000 bubbles/mm2, p = 0.05) and completely denuded (2,700 +/- 1,300 vs. 7,200 +/- 1,100 bubbles/ mm2, p < 0.03) endothelium. CONCLUSIONS Albunex microbubbles preferentially adhere to inflammatory endothelial extracellular matrix. These data suggest that MCE can be used to noninvasively study endothelial integrity and may have implications for the assessment of preclinical atherosclerotic heart disease.

Targeting and ultrasound imaging of microbubble-based contrast agents.

Preparation and characterization of targeted microbubbles (ultrasound contrast agents) is described. Specific ligands were attached to the microbubble shell, and ligand-coated microbubbles were selectively attached to various targets, using either an avidin-biotin model system or an antigen-antibody system for targeting to live activated endothelial cells. Firm attachment of microbubbles to the target was achieved. Forces necessary to detach microbubbles from the target were estimated to exceed dozens of pN. Microbubbles were bound to the target even in the rapidly moving stream of the aqueous medium. Down to 20 ng of the ultrasound contrast material on the target surface could be detected by the ultrasound imaging with a commercial medical imaging system. At high bubble density on the target surface, strong ultrasound image attenuation was observed.

Human muscle-derived cell injection in a rat model of stress urinary incontinence

We investigated the use of human muscle‐derived cells (hMDCs) for the treatment of stress urinary incontinence (SUI) in a nude rat model. hMDCs were isolated from adult skeletal muscle. Three groups of six animals consisting of controls, animals undergoing sciatic nerve transection (SNT) with periurethral sham‐injection, and SNT with hMDCs (1 × 106 cells/20 μl saline) were utilized. Leak point pressure (LPP) was measured 4 weeks following injection. Bilateral SNT resulted in a significantly lower LPP that was significantly higher following hMDCs than sham injection. The results demonstrate the efficacy of human muscle cell therapy alone in improving physiologic outcomes in an animal model of SUI. Muscle Nerve, 2007

Human Skeletal Muscle Cells With a Slow Adhesion Rate After Isolation and an Enhanced Stress Resistance Improve Function of Ischemic Hearts

Identification of cells that are endowed with maximum potency could be critical for the clinical success of cell-based therapies. We investigated whether cells with an enhanced efficacy for cardiac cell therapy could be enriched from adult human skeletal muscle on the basis of their adhesion properties to tissue culture flasks following tissue dissociation. Cells that adhered slowly displayed greater myogenic purity and more readily differentiated into myotubes in vitro than rapidly adhering cells (RACs). The slowly adhering cell (SAC) population also survived better than the RAC population in kinetic in vitro assays that simulate conditions of oxidative and inflammatory stress. When evaluated for the treatment of a myocardial infarction (MI), intramyocardial injection of the SACs more effectively improved echocardiographic indexes of left ventricular (LV) remodeling and contractility than the transplantation of the RACs. Immunohistological analysis revealed that hearts injected with SACs displayed a reduction in myocardial fibrosis and an increase in infarct vascularization, donor cell proliferation, and endogenous cardiomyocyte survival and proliferation in comparison with the RAC-treated hearts. In conclusion, these results suggest that adult human skeletal muscle-derived cells are inherently heterogeneous with regard to their efficacy for enhancing cardiac function after cardiac implantation, with SACs outperforming RACs.

The potential of muscle-derived stem cells for stress urinary incontinence

The suburethral sling procedures, such as transvaginal tape (TVT), have recently gained popularity for the treatment of stress urinary incontinence (SUI). This TVT procedure can reinforce the weakness of pelvic floor muscles but urethral sphincter deficiency remains. Adult stem cell injection therapy for SUI has recently been at the forefront of the repair of deficient urethral function. Muscle-derived stem cells and adipose-derived stem cells are regarded as candidates for the treatment of SUI because these stem cells can be easily obtained in large quantities under local anesthesia, they have the potential to undergo long-term proliferation, self-renewal and multipotent differentiation, and can serve as a vehicle of releasing neurotrophins, such as nerve growth factor, to repair the deficient urethra.