Mary D. Frame

Ligand-Independent Activation of Vascular Endothelial Growth Factor Receptor 2 by Fluid Shear Stress Regulates Activation of Endothelial Nitric Oxide Synthase

Abstract— Fluid shear stress generated by blood flowing over the endothelium is a major determinant of arterial tone, vascular remodeling, and atherogenesis. Nitric oxide (NO) produced by endothelial NO synthase (eNOS) plays an essential role in regulation of vascular function and structure by blood flow, but the molecular mechanisms that transduce mechanical force to eNOS activation are not well understood. In this study, we found that laminar flow (shear stress=12 dyne/cm2) rapidly activates vascular endothelial growth factor receptor 2 (VEGFR2) in a ligand-independent manner and leads to eNOS activation in cultured endothelial cells. Flow-stimulated VEGFR2 recruits phosphoinositide 3-kinase and mediates activation of Akt and eNOS. Inhibiting VEGFR2 kinase with selective inhibitors blocks flow-induced activation of Akt and eNOS and production of NO. Decreasing VEGFR2 expression with antisense VEGFR2 oligonucleotides significantly attenuates activation of Akt and eNOS. Furthermore, Src kinases are involved in flow-stimulated VEGFR2 because inhibiting Src kinases by PP2, a selective inhibitor for Src kinases, abolishes flow-induced VEGFR2 tyrosine phosphorylation and downstream signaling. Finally, we show that inhibiting VEGFR2 kinase significantly reduces flow-mediated NO-dependent arteriolar dilation in vivo. These data identify VEGFR2 as a key mechanotransducer that activates eNOS in response to blood flow.

In Vitro Biocompatibility of Sheath–Core Cellulose-Acetate-Based Electrospun Scaffolds Towards Endothelial Cells and Platelets

Typically, tissue-engineered scaffolds mimic the topographical properties of the native extracellular matrix. However, other physical properties, such as the scaffold mechanical stiffness, are not imitated. The purpose of this study was to fabricate scaffolds with improved mechanical properties and investigate their biocompatibility towards endothelial cells and platelets. To enhance mechanical properties, an electrospinning apparatus was developed that fabricates fibers with sheath–core morphologies. Different combinations of cellulose acetate and chitosan were chosen to modulate the mechanical properties of the formed fibers. We hypothesized that mechanically stiffer scaffolds would improve endothelial cell growth and that all scaffolds would be compatible towards endothelial cells and platelets. Endothelial cell-culture conditions were quantified up to 5 days. Migration onto scaffolds was monitored for 10 days. Platelet aggregation, antagonized by thrombin receptor agonist peptide 6, was measured after scaffold incubation. A platelet activation time-course was assessed with the modified prothrombinase assay. As scaffold mechanical stiffness increased, endothelial cell growth within and adhesion to and migration throughout the scaffolds was promoted. Also, scaffolds did not induce platelet aggregation or activation. These results indicate that the mechanical stiffness of engineered scaffolds regulates endothelial cell-culture parameters and that these sheath–core electrospun scaffolds are compatible towards endothelial cells and platelets.

Bioassay Chamber for Angiogenesis with Perfused Explanted Arteries and Electrospun Scaffolding

Objective: The purpose of this study was to test the hypothesis that explanted perfused arteries can serve as the initial endothelial cell culture source to evaluate the onset of angiogenesis in a cellulose acetate electrospun scaffold.

Curcumin Mediates Both Dilation and Constriction of Peripheral Arterioles via Adrenergic Receptors

Curcumin has wound healing attributes mediated through a plethora of biological activities that in general are not ascribed to specific receptors. Recently, we have demonstrated that intravenous administration of curcumin limits burn injury progression in a rat model. As decreased microvascular perfusion is a central element of burn injury progression, we hypothesized that curcumin may induce vasodilation in peripheral arterioles, to improve perfusion. Using mucosal microcirculation as an in situ assay, cheek pouch tissue was exteriorized in anesthetized (phentobarbital 70 mg kg(-1) intraperitoneal) male hamsters (N=60) to observe the terminal feed arterioles (∼8 μm diameter) and the immediately upstream arcade arterioles (∼20 μm). Curcumin (10(-12)-10(-4) mol l(-1)) was applied dose-wise (micropipette, 60 seconds). Subnanomolar curcumin dilated, whereas micromolar doses constricted, the arterioles. For the terminal arteriole: vasodilation logEC(50) -10.3±0.2, peak dilation +39±1%; vasconstriction logEC(50) -8.0±0.4, peak constriction -14±2%. Simultaneous atropine (muscarinic antagonist) or PD142893 (endothelin antagonist) had no effect. Propranolol (β-adrenergic receptor (β-Ad) antagonist) enhanced constriction by removing the vasodilation response to curcumin. Phentolamine (α-adrenergic receptor (α-Ad) antagonist) enhanced dilation to curcumin by removing the vasoconstriction response. Thus, the curcumin vasomotor activity on microcirculation was α-Ad and β-Ad receptor-dependent and its net vasoactive effect was concentration- and time-dependent.

Terminal Arteriolar Network Structure/Function and Plasma Cytokine Levels in db/db and ob/ob Mouse Skeletal Muscle

Please cite this paper as: Georgi, Vigilance, Dewar, and Frame (2011). Terminal Arteriolar Network Structure/Function and Plasma Cytokine Levels in db/db and ob/ob Mouse Skeletal Muscle. Microcirculation 18(3), 238–251.

In vivo validation of biological responses of bFGF released from microspheres formulated by blending poly-lactide-co-glycolide and poly(ethylene glycol)-grafted-chitosan in hamster cheek pouch microcirculatory models.

Microspheres formulated from blending poly(lactide-co-glycolide) (PLGA) and poly(ethylene glycol)-grafted-chitosan (PEG-g-CHN), using a modified in-emulsion-solvent-evaporation method, were investigated for the delivery of protein. A model protein, bovine serum albumin (BSA), was incorporated into the PLGA/PEG-g-CHN microspheres and both initial burst and release kinetics could be modulated by varying the PEG-g-CHN content. Basic fibroblast growth factor (bFGF) was formulated into the microspheres containing 5% PEG-g-CHN and the bFGF contents in the releasates were determined by a receptor-based ELISA with their in vitro bioactivities validated by fibroblast cell culture. The in vivo effect of the bFGF microspheres formulation was evaluated in a hamster cheek pouch model using a 7 day exposure (e.g., before significant vascular remodeling was expected). Using intravital microscopy, the tissue showed no evidence of inflammation with any formulation; deliberate activation of a preconditioning response linked to inflammation was attenuated by BSA microspheres alone. Vasoactive responses (receptor-dependant and independent constriction and dilation) linked to nitric oxide were attenuated, and constriction to endothelin was enhanced in bFGF and not BSA containing microspheres. PLGA/PEG-g-CHN blended microspheres were also demonstrated to be non-inflammatory and non-thrombogenic in vivo by observing the vascular changes in the cheek pouch. In conclusion, the addition of PEG-g-CHN to PLGA microspheres can serve as a sustained delivery vehicle for bFGF and the released protein provides vasoactive changes consistent with chronic bFGF exposure.

Remote Microvascular Preconditioning Alters Specific Vasoactive Responses

Objective: The mechanism by which remote microvascular preconditioning (RMP) response is initiated was recently reported (Am J Physiol 290:H264, 2006). The goal of this study was to further characterize RMP and to investigate the extent to which RMP altered local vasoactive responses.

Vasoactive effects of stable aqueous suspensions of single walled carbon nanotubes in hamsters and mice

Abstract Single-walled carbon nanotubes synthesized with iron (Fe-SWCNT) or gadolinium (Gd-SWCNT) show promise as high performance multimodal contrast and drug-delivery agents. Our purpose was to evaluate potential vasoactive effects of SWCNT. Stable aqueous solutions of Fe-SWCNTs or Gd-SWCNTs were made using the biocompatible amphiphilic polymer N-(carbonyl-methoxypolyethyleneglycol 2000)-1,2-distearoylsn-glycero-3- phosphoethanolamine (PEG-DSPE). Both aggregated and non-aggregated (sonicated) formulations were tested. The initial vasoactivity of the formulations and their potential for inducing pro-inflammatory endothelial dysfunction were investigated in the hamster cheek pouch and murine cremaster muscle intravital microscopy models. These models provide an assay to test several formulations/dosages in a paired fashion. Abluminal exposure to small arterioles exposes both endothelial and vascular smooth muscle cells. Using abluminal exposures of dosages that would approximate the first pass of an i.v. bolus injection, both Fe-SWCNTs and Gd-SWCNTs were immediately vasoactive. Aggregated formulations induced dilation and non-aggregated formulations induced constriction in both hamsters and mice. Endothelial dysfunction was evident after exposure to either aggregated or non-aggregated forms. General loss of dilator capability was seen after exposure to non-aggregated but not aggregated forms. Thus concentrations mimicking bolus dosing of PEG-DSPE coated SWCNT induce both acute and chronic vascular responses.

Downstream exposure to growth factors causes elevated velocity and dilation in arteriolar networks.

Our goal was to characterize changes in flow and diameter with vascular endothelial cell growth factor A (VEGF-A) and fibroblast growth factor 2 (FGF2). Observations were made in arteriolar networks of the cheek pouch tissue in anesthetized hamsters (pentobarbital 70 mg/kg, i.p., n = 45). Local and remote dilation responses to micropipette-applied VEGF or FGF2 yielded similar EC50 values. The role of gap junctions in the remote response was tested by applying sucrose, halothane or 18αGA to the feed arteriole midway between the remote stimulation and upstream observation sites; all remote dilation to FGF2 was prevented, while only the early dilation to VEGF was blocked. The remote dilation to VEGF displayed a second rheologic mechanism. The second mechanism involved an abrupt increase in upstream velocity and shear rate, followed by nitro-arginine sensitive dilation. To test whether the abrupt increase in shear could be caused by other agents known to cause edema, remote responses to histamine and thrombin were tested. Each caused an abrupt increase in velocity followed by nitro-arginine-sensitive dilation. This study shows that VEGF or agents that increase permeability can initiate an upstream velocity increase with dilation that recruits flow to the network; this is in addition to simultaneous gap junction-mediated dilation.

A novel nanofiber scaffold by electrospinning and its utility in microvascular tissue engineering

AbstractCellulose acetate (CA) thin, porous membranes were produced by electrospinningprecursor polymer solutions in acetone at room temperature. During this process, CAnanofibers were produced when a high electric field of 12 kV was applied to theprecursor solution. The diameters of fibers obtained varied from 100 nm to 1.2 P mwhile the average diameter was approximately 500 nm. The electrospinningparameters used to control the morphology of the fibers and their membranes are flowrate, the distance between the syringe needle that ejects fluid and the collector, and thevoltage applied. These membranes were used as scaffolds for microvascular cellsgrowth. The structure of the membranes that were produced mimic the topographyand porosity of extracellular matrix (ECM) in two key ways. The fiber diametermimics extracellular protein fiber diameter, thus enabling cellular attachment andfacilitating cellular migration. The porosity mimics that of extracellular matrix suchthat microvascular capillary tube formation is enhanced. The non-woven fiber matswere examined by means of electron microscopy and the nanofibers were seen to beoriented randomly. The issue of strengthening the CA scaffold is currently studied byadding ceramic nano-structured component (carbon nanotubes) in the polymermembranes.Keywords: electrospinning, cellulose acetate, microvascular, tissue engineering1. IntroductionIn 1934, Formhals [I] patented a novel process for producing polymer fibers by usingelectrostatic force, which is called electrospinning. In this process, two electrodes areattached to the capillary of a syringe which contains the polymer solution and thecollector respectively. Under the applied electric field, a polymer jet is formed anddeposited on the collector, usually metal screen [2]. In this experiment, dry depositionconditions were used, only solutes can be finally deposited on the collector due to theevaporation of the solvent during the ejection process. By means of controlling thevoltage of the electric field, the flow rate of the polymer solution, the distance*Corresponding author.E-mail address: donhan@notes.cc.sunysb.edu (Dong Han)