The role of the cytoskeleton in the formation and properties of membrane tethers

Abstract only availableMembrane tethers play a critical role in cell adhesion and cell motility. This can be observed in the arrest of neutrophils on the endothelial wall of blood vessels during inflammatory response. Tethers are employed to slow down neutrophils when they are attracted to the periphery of the vessels due to chemotactic gradients set up by cytokines. As a result of slowing down, the neutrophil has time to form specific bonds with endothelial cells and start extravasating from the circulatory system into the surrounding tissue. Metastasizing cancer cells use a similar mechanism. A wide array of factors affects the mechanical characteristics of tethers. A major contribution is provided by the interaction between the cytoskeleton and the membrane. Interbilayer slip and the interaction between the membrane and the glycocalix are additional determinants of tether properties. Previous studies have shown a strong dependence of force needed to extract and pull a tether on the interaction between the membrane and the cytoskeleton. It has also been shown that disrupting the integrity of the cytoskeleton significantly reduces the tether force. The focus of this study was to further elucidate the contribution of the cytoskeleton-lipid bilayer interaction to the tether force, in particular how it affects cell membrane surface viscosity. Atomic Force Microscopy based force spectroscopy was used to determine the tether force and surface viscosity of the membrane prior and after the actin microfilament system had been depolyermerized by latrunculin-A. Two cells lines, Chinese Hamster Ovary (CHO) and Human Brain tumor (HB) cells were investigated. The tether force was determined when the membrane was stretched by a cantilever moving at a constant velocity over a range 3 to 21 micron/s. Surface viscosity was obtained from the slope of the linear force-speed curve. Quantitative information on tether forces and membrane surface viscosities allow for a better understanding of the mechanism responsible for the arrest of neutrophils during their attachment to the endothelial wall.NSF-REU Biosystems Modelin

Angiopoietin-like protein 2 regulates endothelial colony forming cell vasculogenesis

Angiopoietin-like 2 (ANGPTL2) has been reported to induce sprouting angiogenesis; however, its role in vasculogenesis, the de novo lumenization of endothelial cells (EC), remains unexplored. We sought to investigate the potential role of ANGPTL2 in regulating human cord blood derived endothelial colony forming cell (ECFC) vasculogenesis through siRNA mediated inhibition of ANGPTL2 gene expression. We found that ECFCs in which ANGPTL2 was diminished displayed a threefold decrease in in vitro lumenal area whereas addition of exogenous ANGPTL2 protein domains to ECFCs lead to increased lumen formation within a 3 dimensional (3D) collagen assay of vasculogenesis. ECFC migration was attenuated by 36 % via ANGPTL2 knockdown (KD) although proliferation and apoptosis were not affected. We subsequently found that c-Jun NH2-terminal kinase (JNK), but not ERK1/2, phosphorylation was decreased upon ANGPTL2 KD, and expression of membrane type 1 matrix metalloproteinase (MT1-MMP), known to be regulated by JNK and a critical regulator of EC migration and 3D lumen formation, was decreased in lumenized structures in vitro derived from ANGPTL2 silenced ECFCs. Treatment of ECFCs in 3D collagen matrices with either a JNK inhibitor or exogenous rhTIMP-3 (an inhibitor of MT1-MMP activity) resulted in a similar phenotype of decreased vascular lumen formation as observed with ANGPTL2 KD, whereas stimulation of JNK activity increased vasculogenesis. Based on gene silencing, pharmacologic, cellular, and biochemical approaches, we conclude that ANGPTL2 positively regulates ECFC vascular lumen formation likely through its effects on migration and in part by activating JNK and increasing MT1-MMP expression

Notch ligand Delta-like 1 promotes in vivo vasculogenesis in human cord blood-derived endothelial colony forming cells

BACKGROUND AIMS: Human cord blood (CB) is enriched in circulating endothelial colony forming cells (ECFCs) that display high proliferative potential and in vivo vessel forming ability. Because Notch signaling is critical for embryonic blood vessel formation in utero, we hypothesized that Notch pathway activation may enhance cultured ECFC vasculogenic properties in vivo. METHODS: In vitro ECFC stimulation with an immobilized chimeric Notch ligand (Delta-like1(ext-IgG)) led to significant increases in the mRNA and protein levels of Notch regulated Hey2 and EphrinB2 that were blocked by treatment with γ-secretase inhibitor addition. However, Notch stimulated preconditioning in vitro failed to enhance ECFC vasculogenesis in vivo. In contrast, in vivo co-implantation of ECFCs with OP9-Delta-like 1 stromal cells that constitutively expressed the Notch ligand delta-like 1 resulted in enhanced Notch activated ECFC-derived increased vessel density and enlarged vessel area in vivo, an effect not induced by OP9 control stromal implantation. RESULTS: This Notch activation was associated with diminished apoptosis in the exposed ECFC. CONCLUSIONS: We conclude that Notch pathway activation in ECFC in vivo via co-implanted stromal cells expressing delta-like 1 promotes vasculogenesis and augments blood vessel formation via diminishing apoptosis of the implanted ECFC

Existence, functional impairment, and lung repair potential of endothelial colony-forming cells in oxygen-induced arrested alveolar growth

BACKGROUND: Bronchopulmonary dysplasia and emphysema are life-threatening diseases resulting from impaired alveolar development or alveolar destruction. Both conditions lack effective therapies. Angiogenic growth factors promote alveolar growth and contribute to alveolar maintenance. Endothelial colony-forming cells (ECFCs) represent a subset of circulating and resident endothelial cells capable of self-renewal and de novo vessel formation. We hypothesized that resident ECFCs exist in the developing lung, that they are impaired during arrested alveolar growth in experimental bronchopulmonary dysplasia, and that exogenous ECFCs restore disrupted alveolar growth. METHODS AND RESULTS: Human fetal and neonatal rat lungs contain ECFCs with robust proliferative potential, secondary colony formation on replating, and de novo blood vessel formation in vivo when transplanted into immunodeficient mice. In contrast, human fetal lung ECFCs exposed to hyperoxia in vitro and neonatal rat ECFCs isolated from hyperoxic alveolar growth-arrested rat lungs mimicking bronchopulmonary dysplasia proliferated less, showed decreased clonogenic capacity, and formed fewer capillary-like networks. Intrajugular administration of human cord blood-derived ECFCs after established arrested alveolar growth restored lung function, alveolar and lung vascular growth, and attenuated pulmonary hypertension. Lung ECFC colony- and capillary-like network-forming capabilities were also restored. Low ECFC engraftment and the protective effect of cell-free ECFC-derived conditioned media suggest a paracrine effect. Long-term (10 months) assessment of ECFC therapy showed no adverse effects with persistent improvement in lung structure, exercise capacity, and pulmonary hypertension. CONCLUSIONS: Impaired ECFC function may contribute to arrested alveolar growth. Cord blood-derived ECFC therapy may offer new therapeutic options for lung diseases characterized by alveolar damage

Balloon Atrial Septostomy as Initial Therapy in Pediatric Pulmonary Hypertension

Balloon atrial septostomy is a palliative procedure currently used to bridge medically refractory pulmonary hypertension patients to lung transplantation. In the current report, we present balloon atrial septostomy as an initial therapy for high-risk pediatric pulmonary hypertension patients at our institution. Nineteen patients with median age of 4.3 years (range 0.1-14.3 years) underwent balloon atrial septostomy during initial admission for pulmonary hypertension. There were no procedural complications or deaths within 24 h of balloon atrial septostomy. Patients were followed for a median of 2.6 years (interquartile range 1.0-4.8 years). Three (16%) patients died, 3 (16%) underwent lung transplantation, and 1 (5%) underwent reverse Potts shunt. Transplant-free survival at 30 days, 1 year, and 3 years was 84%, 76%, and 67% respectively. This single-center experience suggests early-BAS in addition to pharmacotherapy is safe and warrants consideration in high-risk pediatric pulmonary hypertension patients

Matrix mechanical properties modulate ECFC vascular network formation

Development of a functional vascular network is a major problem limiting current tissue engineering strategies targeting repair and regeneration of damaged or diseased tissue. Recently endothelial colony forming cells (ECFCs) have been shown to vascularize a type I collagen scaffold in vivo. ECFCs are the only cells that have been shown to possess direct in vivo vessel forming ability upon transplantation. This has generated much interest in the use of ECFCs for tissue engineering strategies. However, there is still a great need for refinement of a defined microenvironment to locally deliver ECFCs and guide vessel formation in vivo. By regulating cross link formation, a novel mechanism to alter the matrix microenvironment and influence endothelial cell behavior, type I collagen scaffolds can potentially be engineered to support the formation of long lasting ECFC derived vessels. We investigated how collagen matrix microenvironment and physical parameters (matrix stiffness, fibril density, and collagen cross link composition) affected vascular network formation by ECFCs in vitro. Further we have characterized how collagen matrix design parameters modulate a matrix-integrin-cytoskeleton signaling axis known to regulate endothelial cell lumen formation. The results from this study will provide critical information for the development of vascularized tissue constructs that can be controllably delivered to ischemic areas and improve the efficacy of human umbilical cord blood derived ECFC therapies for human subjects