Michael J. Patrick

Interaction between alk1 and blood flow in the development of arteriovenous malformations.

Arteriovenous malformations (AVMs) are fragile direct connections between arteries and veins that arise during times of active angiogenesis. To understand the etiology of AVMs and the role of blood flow in their development, we analyzed AVM development in zebrafish embryos harboring a mutation in activin receptor-like kinase I (alk1), which encodes a TGFβ family type I receptor implicated in the human vascular disorder hereditary hemorrhagic telangiectasia type 2 (HHT2). Our analyses demonstrate that increases in arterial caliber, which stem in part from increased cell number and in part from decreased cell density, precede AVM development, and that AVMs represent enlargement and stabilization of normally transient arteriovenous connections. Whereas initial increases in endothelial cell number are independent of blood flow, later increases, as well as AVMs, are dependent on flow. Furthermore, we demonstrate that alk1 expression requires blood flow, and despite normal levels of shear stress, some flow-responsive genes are dysregulated in alk1 mutant arterial endothelial cells. Taken together, our results suggest that Alk1 plays a role in transducing hemodynamic forces into a biochemical signal required to limit nascent vessel caliber, and support a novel two-step model for HHT-associated AVM development in which pathological arterial enlargement and consequent altered blood flow precipitate a flow-dependent adaptive response involving retention of normally transient arteriovenous connections, thereby generating AVMs.

Intracellular pH measurements using perfluorocarbon nanoemulsions.

We report the synthesis and formulation of unique perfluorocarbon (PFC) nanoemulsions enabling intracellular pH measurements in living cells via fluorescent microscopy and flow cytometry. These nanoemulsions are formulated to readily enter cells upon coincubation and contain two cyanine-based fluorescent reporters covalently bound to the PFC molecules, specifically Cy3-PFC and CypHer5-PFC conjugates. The spectral and pH-sensing properties of the nanoemulsions were characterized in vitro and showed the unaltered spectral behavior of dyes after formulation. In rat 9L glioma cells loaded with nanoemulsion, the local pH of nanoemulsions was longitudinally quantified using optical microscopy and flow cytometry and displayed a steady decrease in pH to a level of 5.5 over 3 h, indicating rapid uptake of nanoemulsion to acidic compartments. Overall, these reagents enable real-time optical detection of intracellular pH in living cells in response to pharmacological manipulations. Moreover, recent approaches for in vivo cell tracking using magnetic resonance imaging (MRI) employ intracellular PFC nanoemulsion probes to track cells using (19)F MRI. However, the intracellular fate of these imaging probes is poorly understood. The pH-sensing nanoemulsions allow the study of the fate of the PFC tracer inside the labeled cell, which is important for understanding the PFC cell loading dynamics, nanoemulsion stability and cell viability over time.

Critical Transitions in Early Embryonic Aortic Arch Patterning and Hemodynamics

Transformation from the bilaterally symmetric embryonic aortic arches to the mature great vessels is a complex morphogenetic process, requiring both vasculogenic and angiogenic mechanisms. Early aortic arch development occurs simultaneously with rapid changes in pulsatile blood flow, ventricular function, and downstream impedance in both invertebrate and vertebrate species. These dynamic biomechanical environmental landscapes provide critical epigenetic cues for vascular growth and remodeling. In our previous work, we examined hemodynamic loading and aortic arch growth in the chick embryo at Hamburger-Hamilton stages 18 and 24. We provided the first quantitative correlation between wall shear stress (WSS) and aortic arch diameter in the developing embryo, and observed that these two stages contained different aortic arch patterns with no inter-embryo variation. In the present study, we investigate these biomechanical events in the intermediate stage 21 to determine insights into this critical transition. We performed fluorescent dye microinjections to identify aortic arch patterns and measured diameters using both injection recordings and high-resolution optical coherence tomography. Flow and WSS were quantified with 3D computational fluid dynamics (CFD). Dye injections revealed that the transition in aortic arch pattern is not a uniform process and multiple configurations were documented at stage 21. CFD analysis showed that WSS is substantially elevated compared to both the previous (stage 18) and subsequent (stage 24) developmental time-points. These results demonstrate that acute increases in WSS are followed by a period of vascular remodeling to restore normative hemodynamic loading. Fluctuations in blood flow are one possible mechanism that impacts the timing of events such as aortic arch regression and generation, leading to the variable configurations at stage 21. Aortic arch variations noted during normal rapid vascular remodeling at stage 21 identify a temporal window of increased vulnerability to aberrant aortic arch morphogenesis with the potential for profound effects on subsequent cardiovascular morphogenesis.

Analysis of early embryonic great-vessel microcirculation in zebrafish using high-speed confocal μPIV.

In the developing cardiovascular system, hemodynamic vascular loading is critical for angiogenesis and cardiovascular adaptation. Normal zebrafish embryos with transgenically-labeled endothelial and red blood cells provide an excellent in vivo model for studying the fluid-flow induced vascular loading. To characterize the developmental hemodynamics of early embryonic great-vessel microcirculation in the zebrafish embryo, two complementary studies (experimental and numerical) are presented. Quantitative comparison of the wall shear stress (WSS) at the first aortic arch (AA1) of wild-type zebrafish embryos during two consecutive developmental stages is presented, using time-resolved confocal micro-particle image velocimetry (μPIV). Analysis showed that there was significant WSS difference between 32 and 48 h post-fertilization (hpf) wild-type embryos, which correlates with normal arch morphogenesis. The vascular distensibility of the arch wall at systole and the acceleration/deceleration rates of time-lapse phase-averaged streamwise blood flow curves were also analyzed. To estimate the influence of a novel intermittent red-blood cell (RBC) loading on the endothelium, a numerical two-phase, volume of fluid (VOF) flow model was further developed with realistic in vivo conditions. These studies showed that near-wall effects and cell clustering increased WSS augmentation at a minimum of 15% when the distance of RBC from arch vessel wall was less than 3 μm or when RBC cell-to-cell distance was less than 3 μm. When compared to a smooth wall, the WSS augmentation increased by a factor of ~1.4 due to the roughness of the wall created by the endothelial cell profile. These results quantitatively highlight the contribution of individual RBC flow patterns on endothelial WSS in great-vessel microcirculation and will benefit the quantitative understanding of mechanotransduction in embryonic great vessel biology, including arteriovenous malformations (AVM).

Two-color fluorescent (near-infrared and visible) triphasic perfluorocarbon nanoemulsions

Abstract. Design and development of a new formulation as a unique assembly of distinct fluorescent reporters with nonoverlapping fluorescence spectra and a F19 magnetic resonance imaging agent into colloidally and optically stable triphasic nanoemulsion are reported. Specifically, a cyanine dye-perfluorocarbon (PFC) conjugate was introduced into the PFC phase of the nanoemulsion and a near-infrared dye was introduced into the hydrocarbon (HC) layer. To the best of our knowledge, this is the first report of a triphasic nanoemulsion system where each oil phase, HC, and PFC are fluorescently labeled and formulated into an optically and colloidally stable nanosystem. Having, each oil phase separately labeled by a fluorescent dye allows for improved correlation between in vivo imaging and histological data. Further, dual fluorescent labeling can improve intracellular tracking of the nanodroplets and help assess the fate of the nanoemulsion in biologically relevant media. The nanoemulsions were produced by high shear processing (microfluidization) and stabilized with biocompatible nonionic surfactants resulting in mono-modal size distribution with average droplet size less than 200 nm. Nanoemulsions demonstrate excellent colloidal stability and only moderate changes in the fluorescence signal for both dyes. Confocal fluorescence microscopy of macrophages exposed to nanoemulsions shows the presence of both fluorescence agents in the cytoplasm.

Enhanced aqueous solubility of long wavelength voltage-sensitive dyes by covalent attachment of polyethylene glycol

Long wavelength voltage-sensitive dyes (VSDs) called Pittsburgh (PGH) dyes were recently synthesized by coupling various heterocyclic groups to a styryl-thiophene intermediate forming extended, partially rigid chromophores. Unlike most styryl VSDs, dyes with a sulfonic acid anchor directly attached to the chromophore showed no solvatochromic absorption shifts. The limited water solubility of many long wavelength VSDs requires the use of surfactants to transport the dye through physiological saline solutions and effectively label biological membranes. Here, we tested the chemical substitution of the sulfonic acid moiety with polyethyleneglycol (PEG) chains, ranging from MW 750 to 5000, to overcome the poor solubility of VSDs while retaining their properties as VSDs. The chemical synthesis of PGH dyes and their PEG derivatives are described. The PEG derivatives were soluble in aqueous solutions (>1 mM) and still reported membrane potential changes. In frog and mouse hearts, the voltage sensitivity (DeltaF/F per action potential) and spectral properties of PEG dyes were the same as the sulfonated analogues. Thus, the solubility of VSDs can be considerably improved with small polyethyleneglycol chains and can provide an effective approach to improve staining of excitable tissues and optical recordings of membrane potential.

Suppressing inflammation from inside out with novel NIR visible perfluorocarbon nanotheranostics

Highly innovative multimodal perfluorocarbon (PFC) nanoemulsions are presented. They serve simultaneously as dual-mode imaging reagents (NIR and 19F MRI), and drug delivery vehicles for water insoluble cyclooxgenase-2 (COX-2) inhibitors. These features qualify them as theranostic. Cancer progression and metastasis are highly influenced by tumor microenvironment and inflammation. Infiltration of primary tumors with inflammation-promoting cells (e.g. macrophages) is a negative prognostic factor for cancer patient survival. We postulate that the suppression of COX-2 enzyme in macrophages by theranostic PFC nanoemulsions will result in changes in macrophage levels of accumulation in tumors and/or their phenotype, which can suppress tumor- promoting activity. The presented theranostic nanoemulsions are designed to label immune cells such as macrophages, and deliver celecoxib, a COX-2 inhibitor. The designed theranostic incorporates two fluorescent reporters: a near-infrared (NIR) fluorescent dye for improved optical in vivo imaging, and a distinct fluorescent dye for histological analysis of excised tissues. A high content of PFC in the theranostic allows 19F MRI to quantitatively assess the distribution of the injected nanomedicine in the peritumoral area, and measure tumor-associated inflammation, while 1H MRI provides anatomical context. NIR imaging is used as a complementary in vivo technique to assess biodistribution of the theranostic. We report preparation and characterization of the nanoemulsions’ colloidal and optical stability, in vitro toxicity, and imaging capabilities. This theranostic offers flexibility for in vitro and in vivo inflammation imaging and histological analysis using three different imaging functionalities (fluorescence, NIR and 19F MRI), advancing the monitoring and modulating of tumor-infiltrating immune cells in vivo.

Hemodynamic Investigation of Normal Developing Aortic Arch in the Chick Embryo

Governed by genetic and epigenetic feedback [1], during embryonic cardiac development, the anatomy of aortic arches demonstrates drastic three dimensional (3D) changes that interact with the function of cardiovascular system. Six major pairs of aortic arches appear at different embryonic periods and eventually form the two brachiocephalic arteries (left and right third), an aortic arch (left fourth) and pulmonary arteries and ductus arteriosus (left and right sixth) [2–4], Fig 1. Flow-driven hemodynamic loading plays a major role in this dynamic process. Morphological studies on the embryonic aortic arches began over 100 years ago while the recent remarkable developments include understanding genetic determinants such as the effects of neural crest cells [5,6]. However the relationship between hemodynamic factors and the dynamic 3D geometry changes is still limited requiring an interdisciplinary research effort [7,8].Copyright

In VIVO hemodynamic performance of wild-type vs. Mutant zebrafish embryos using high-speed confocal micro-PIV

In developing cardiovascular systems, definite performance comparison between disease and healthy hemodynamics requires quantitative tools to support advanced microscopy. Mutations in the activin receptor-like kinase 1 (ALK1) gene are responsible for the autosomal dominant vascular disease, hereditary hemorrhagic telangiectasia type 2 (HHT2), characterized by high flow arteriovenous malformations (AVMs) [1]. Recent studies show that the zebrafish mutant violet beauregrade (vbg), which harbors a mutation in alk1, develops an abnormal circulation with dilated cranial vessels and AVMs [2]. Quantitative understanding of mechanical influences on the alk1 mutant phenotype will aid treatment of HHT2 patients. Inspired by earlier studies that demonstrate the capability of using confocal micro-PIV technique to quantify biofluid dynamics in vivo [3], primarily in major vessels (dorsal aorta, vitelline veins), the present study focused on secondary branching great vessels of zebrafish embryos where microcirculation flow regimes are different. Furthermore, confocal microscopy, essentially being an imaging modality, requires rigorous validation efforts with respect to the gold standard measurement protocols (such as PIV) and synthetic scan data. Another objective of this work was to document the intra-species differences of wall shear stress (WSS) and flow physics during embryonic development in aortic arch systems of zebrafish [4].Copyright