A signal-like role for floral humidity in a nocturnal pollination system.

Previous studies have considered floral humidity to be an inadvertent consequence of nectar evaporation, which could be exploited as a cue by nectar-seeking pollinators. By contrast, our interdisciplinary study of a night-blooming flower, Datura wrightii, and its hawkmoth pollinator, Manduca sexta, reveals that floral relative humidity acts as a mutually beneficial signal in this system. The distinction between cue- and signal-based functions is illustrated by three experimental findings. First, floral humidity gradients in Datura are nearly ten-fold greater than those reported for other species, and result from active (stomatal conductance) rather than passive (nectar evaporation) processes. These humidity gradients are sustained in the face of wind and are reconstituted within seconds of moth visitation, implying substantial physiological costs to these desert plants. Second, the water balance costs in Datura are compensated through increased visitation by Manduca moths, with concomitant increases in pollen export. We show that moths are innately attracted to humid flowers, even when floral humidity and nectar rewards are experimentally decoupled. Moreover, moths can track minute changes in humidity via antennal hygrosensory sensilla but fail to do so when these sensilla are experimentally occluded. Third, their preference for humid flowers benefits hawkmoths by reducing the energetic costs of flower handling during nectar foraging. Taken together, these findings suggest that floral humidity may function as a signal mediating the final stages of floral choice by hawkmoths, complementing the attractive functions of visual and olfactory signals beyond the floral threshold in this nocturnal plant-pollinator system.

Multi-scale computational study of the Warburg effect, reverse Warburg effect and glutamine addiction in solid tumors.

Cancer metabolism has received renewed interest as a potential target for cancer therapy. In this study, we use a multi-scale modeling approach to interrogate the implications of three metabolic scenarios of potential clinical relevance: the Warburg effect, the reverse Warburg effect and glutamine addiction. At the intracellular level, we construct a network of central metabolism and perform flux balance analysis (FBA) to estimate metabolic fluxes; at the cellular level, we exploit this metabolic network to calculate parameters for a coarse-grained description of cellular growth kinetics; and at the multicellular level, we incorporate these kinetic schemes into the cellular automata of an agent-based model (ABM), iDynoMiCS. This ABM evaluates the reaction-diffusion of the metabolites, cellular division and motion over a simulation domain. Our multi-scale simulations suggest that the Warburg effect provides a growth advantage to the tumor cells under resource limitation. However, we identify a non-monotonic dependence of growth rate on the strength of glycolytic pathway. On the other hand, the reverse Warburg scenario provides an initial growth advantage in tumors that originate deeper in the tissue. The metabolic profile of stromal cells considered in this scenario allows more oxygen to reach the tumor cells in the deeper tissue and thus promotes tumor growth at earlier stages. Lastly, we suggest that glutamine addiction does not confer a selective advantage to tumor growth with glutamine acting as a carbon source in the tricarboxylic acid (TCA) cycle, any advantage of glutamine uptake must come through other pathways not included in our model (e.g., as a nitrogen donor). Our analysis illustrates the importance of accounting explicitly for spatial and temporal evolution of tumor microenvironment in the interpretation of metabolic scenarios and hence provides a basis for further studies, including evaluation of specific therapeutic strategies that target metabolism.

Adipose-derived stem cells increase angiogenesis through matrix metalloproteinase-dependent collagen remodeling.

Adipose-derived stem cells (ASCs) are key regulators of new blood vessel formation and widely investigated for their role in tissue regeneration and tumorigenesis. However, the cellular and molecular mechanisms through which ASCs regulate angiogenesis are not well understood. Here, it was our goal to test the functional contribution of ASC-mediated extracellular matrix (ECM) remodeling on endothelial cell invasion. To isolate the effect of ECM-remodeling, ASCs were embedded within 3-D collagen type I hydrogels and pre-cultured for 7 days; controls were not pre-cultured. A confluent monolayer of human umbilical vein endothelial cells (HUVECs) was seeded on top and its invasion into the underlying hydrogel was analyzed. Without pre-culture, ASCs inhibited vascular sprouting by stabilizing the endothelium. In contrast, 7 day pre-culture of ASCs drastically increased invasion by HUVECs. This effect was largely mediated by proteolytic ECM degradation by ASC-derived matrix metalloproteinases (MMPs) rather than vascular endothelial growth factor (VEGF), as our results indicated that blockade of MMPs, but not VEGF, inhibited endothelial sprouting. Collectively, these data suggest that the angiogenic capability of ASCs is modulated by their proteolytic remodeling of the ECM, opening new avenues for pro- and anti-angiogenic therapies.

Formation of microvascular networks in vitro.

This protocol describes how to form a 3D cell culture with explicit, endothelialized microvessels. The approach leads to fully enclosed, perfusable vessels in a bioremodelable hydrogel (type I collagen). The protocol uses microfabrication to enable user-defined geometries of the vascular network and microfluidic perfusion to control mass transfer and hemodynamic forces. These microvascular networks (µVNs) allow for multiweek cultures of endothelial cells or cocultures with parenchymal or tissue cells in the extra-lumen space. The platform enables real-time fluorescence imaging of living engineered tissues, in situ confocal fluorescence of fixed cultures and transmission electron microscopy (TEM) imaging of histological sections. This protocol enables studies of basic vascular and blood biology, provides a model for diseases such as tumor angiogenesis or thrombosis and serves as a starting point for constructing prevascularized tissues for regenerative medicine. After one-time microfabrication steps, the system can be assembled in less than 1 d and experiments can run for weeks.

Physicochemical regulation of endothelial sprouting in a 3D microfluidic angiogenesis model.

Both physiological and pathological tissue remodeling (e.g., during wound healing and cancer, respectively) require new blood vessel formation via angiogenesis, but the underlying microenvironmental mechanisms remain poorly defined due in part to the lack of biologically relevant in vitro models. Here, we present a biomaterials-based microfluidic 3D platform for analysis of endothelial sprouting in response to morphogen gradients. This system consists of three lithographically defined channels embedded in type I collagen hydrogels. A central channel is coated with endothelial cells, and two parallel side channels serve as a source and a sink for the steady-state generation of biochemical gradients. Gradients of vascular endothelial growth factor (VEGF) promoted sprouting, whereby endothelial cell responsiveness was markedly dependent on cell density and vessel geometry regardless of treatment conditions. These results point toward mechanical and/or autocrine mechanisms that may overwhelm pro-angiogenic paracrine signaling under certain conditions. To date, neither geometrical effects nor cell density have been considered critical determinants of angiogenesis in health and disease. This biomimetic vessel platform demonstrated utility for delineating hitherto underappreciated contributors of angiogenesis, and future studies may enable important new mechanistic insights that will inform anti-angiogenic cancer therapy.

Multiscale models of breast cancer progression.

Breast cancer initiation, invasion and metastasis span multiple length and time scales. Molecular events at short length scales lead to an initial tumorigenic population, which left unchecked by immune action, acts at increasingly longer length scales until eventually the cancer cells escape from the primary tumor site. This series of events is highly complex, involving multiple cell types interacting with (and shaping) the microenvironment. Multiscale mathematical models have emerged as a powerful tool to quantitatively integrate the convective-diffusion-reaction processes occurring on the systemic scale, with the molecular signaling processes occurring on the cellular and subcellular scales. In this study, we reviewed the current state of the art in cancer modeling across multiple length scales, with an emphasis on the integration of intracellular signal transduction models with pro-tumorigenic chemical and mechanical microenvironmental cues. First, we reviewed the underlying biomolecular origin of breast cancer, with a special emphasis on angiogenesis. Then, we summarized the development of tissue engineering platforms which could provide high-fidelity ex vivo experimental models to identify and validate multiscale simulations. Lastly, we reviewed top-down and bottom-up multiscale strategies that integrate subcellular networks with the microenvironment. We present models of a variety of cancers, in addition to breast cancer specific models. Taken together, we expect as the sophistication of the simulations increase, that multiscale modeling and bottom-up agent-based models in particular will become an increasingly important platform technology for basic scientific discovery, as well as the identification and validation of potentially novel therapeutic targets.

Phosphorescent nanoparticles for quantitative measurements of oxygen profiles in vitro and in vivo.

We present the development and characterization of nanoparticles loaded with a custom phosphor; we exploit these nanoparticles to perform quantitative measurements of the concentration of oxygen within three-dimensional (3-D) tissue cultures in vitro and blood vessels in vivo. We synthesized a customized ruthenium (Ru)-phosphor and incorporated it into polymeric nanoparticles via self-assembly. We demonstrate that the encapsulated phosphor is non-toxic with and without illumination. We evaluated two distinct modes of employing the phosphorescent nanoparticles for the measurement of concentrations of oxygen: 1) in vitro, in a 3-D microfluidic tumor model via ratiometric measurements of intensity with an oxygen-insensitive fluorophore as a reference, and 2) in vivo, in mouse vasculature using measurements of phosphorescence lifetime. With both methods, we demonstrated micrometer-scale resolution and absolute calibration to the dissolved oxygen concentration. Based on the ease and customizability of the synthesis of the nanoparticles and the flexibility of their application, these oxygen-sensing polymeric nanoparticles will find a natural home in a range of biological applications, benefiting studies of physiological as well as pathological processes in which oxygen availability and concentration play a critical role.

Mathematical modeling and frequency gradient analysis of cellular and vascular invasion into integra and strattice: toward optimal design of tissue regeneration scaffolds.

BACKGROUND: Rapid, effective host cell invasion and vascularization is essential for durable incorporation of avascular tissue-replacement scaffolds. In this study, the authors sought to qualitatively and quantitatively determine which of two commercially available products (i.e., Strattice and Integra) facilitates more rapid cellular and vascular invasion in a murine model of graft incorporation. METHODS: Integra and Strattice were implanted subcutaneously into the dorsa of C57BL/6 mice; harvested after 3, 7, or 14 days; and stained with hematoxylin and eosin, 4',6-diamidino-2-phenylindole, and immunohistochemical stains for CD31 and α-smooth muscle actin. Exponential decay equations describing cellular invasion through each layer were fit to each material/time point. Mean cell density and cell frequency maps were created denoting extent of invasion by location within the scaffold. RESULTS: Qualitative analysis demonstrated extensive cellular infiltration into Integra by 3 days and increasing over the remaining 14 days. Invasion of Strattice was sparse, even after 14 days. α-Smooth muscle actin immunohistochemistry revealed blood vessel formation within Integra by 14 days but no analogous neovascularization in Strattice. Mean decay equations for Integra and Strattice were y = 76.3(0.59) and y = 75.5(0.33), respectively. Both cell density measurements and frequency mapping demonstrated that, at all time points, Integra manifested a greater density/depth of cellular invasion when compared with Strattice. CONCLUSIONS: These data confirm empiric clinical observations that Integra is more rapidly invaded than Strattice when placed in a suitable host bed. A remnant microvasculature template is not sufficient for effective cellular ingrowth into an artificial tissue construct. These findings provide insight into means for improving future dermal replacement products.

Microstructured templates for directed growth and vascularization of soft tissue in vivo.

Tissue templates for reconstruction and regeneration in vivo have achieved clinical successes for homogeneous tissues in well vascularized regions. Outstanding challenges exist for applications in poorly vascularized regions and for spatially differentiated tissues. Here, we present a strategy to control the spatial patterns of cell and vascular ingrowth throughout the volume of a bioremodelable and resorbable matrix via well-defined micropores and networks of microchannels. Our presentation of this approach includes: a description of a lithographic technique to form deterministic microstructures within a matrix of native collagen; elucidation of multistep process by which microstructures drive rapid invasion and vascularization; and demonstration of long range guidance of invasion through the full thickness of patterned templates. Experiments were performed in a murine wound model. These microstructured tissue templates (MTTs) could improve outcomes in reconstructive surgery and open paths to directed regeneration of spatially heterogeneous tissues or organs.

Dense type I collagen matrices that support cellular remodeling and microfabrication for studies of tumor angiogenesis and vasculogenesis in vitro.

Type I collagen is a favorable substrate for cell adhesion and growth and is remodelable by many tissue cells; these characteristics make it an attractive material for the study of dynamic cellular processes. Low mass fraction (1.0-3.0 mg/ml), hydrated collagen matrices used for three-dimensional cell culture permit cellular movement and remodeling, but their microstructure and mechanics fail to mimic characteristics of many extracellular matrices in vivo and limit the definition of fine-scale geometrical features (<1 mm) within scaffolds. In this study, we worked with hydrated type I collagen at mass fractions between 3.0 and 20 mg/ml to define the range of densities over which the matrices support both microfabrication and cellular remodeling. We present pore and fiber dimensions based on confocal microscopy and longitudinal modulus and hydraulic permeability based on confined compression. We demonstrate faithful reproduction of simple pores of 50 µm-diameter over the entire range and formation of functional microfluidic networks for mass fractions of at least 10.0 mg/ml. We present quantitative characterization of the rate and extent of cellular remodelability using human umbilical vein endothelial cells. Finally, we present a co-culture with tumor cells and discuss the implications of integrating microfluidic control within scaffolds as a tool to study spatial and temporal signaling during tumor angiogenesis and vascularization of tissue engineered constructs.

Microfluidic culture models of tumor angiogenesis.

Blood vessels control all stages of tumor development and therapy by defining the physicochemical and cellular state of the tumor microenvironment. However, no pathologically relevant culture systems currently exist that recapitulate the associated cellular and convective mass transfer processes that are implicated in tumor angiogenesis. By integrating tissue engineering and microfluidic technologies, it will be possible to develop tumor-mimetic culture environments with embedded microvascular structures. Utilization of these microfluidic tumor models will help reveal the importance of the transport of chemical and cellular factors in tumor angiogenesis, and provide a test bed that may ultimately improve current strategies to antiangiogenic therapy.

Oxygen-controlled three-dimensional cultures to analyze tumor angiogenesis.

Tumor angiogenesis is controlled by the integrated action of physicochemical and biological cues; however, the individual contributions of these cues are not well understood. We have designed alginate-based microscale tumor models to define the distinct importance of oxygen concentration, culture dimensionality, and cell-extracellular matrix interactions on the angiogenic capability of oral squamous cell carcinoma, and have verified the relevance of our findings with U87 glioblastoma cells. Our results revealed qualitative differences in the microenvironmental regulation of vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) secretion in three-dimensional (3D) culture. Specifically, IL-8 secretion was highest under ambient conditions, whereas VEGF secretion was highest in hypoxic cultures. Additionally, 3D integrin engagement by RGD-modified alginate matrices increased IL-8 secretion independently of oxygen, whereas VEGF secretion was only moderately affected by cell-extracellular matrix interactions. Using two-dimensional migration assays and a new 3D tumor angiogenesis model, we demonstrated that the resulting angiogenic signaling promotes tumor angiogenesis by increasing endothelial cell migration and invasion. Collectively, tissue-engineered tumor models improve our understanding of tumor angiogenesis, which may ultimately advance anticancer therapies.

Protein translocation through a tunnel induces changes in folding kinetics: a lattice model study.

Compaction of a nascent polypeptide chain inside the ribosomal exit tunnel, before it leaves the ribosome, has been proposed to accelerate the folding of newly synthesized proteins following their release from the ribosome. Thus, we used Kinetic Monte Carlo simulations of a minimalist on-lattice model to explore the effect that polypeptide translocation through a variety of channels has on protein folding kinetics. Our results demonstrate that tunnel confinement promotes faster folding of a well-designed protein relative to its folding in free space by displacing the unfolded state towards more compact structures that are closer to the transition state. Since the tunnel only forbids rarely visited, extended configurations, it has little effect on a "poorly designed" protein whose unfolded state is largely composed of low-energy, compact, misfolded configurations. The beneficial effect of the tunnel depends on its width; for example, a too-narrow tunnel enforces unfolded states with limited or no access to the transition state, while a too-wide tunnel has no effect on the unfolded state entropy. We speculate that such effects are likely to play an important role in the folding of some proteins or protein domains in the cellular environment and might dictate whether a protein folds co-translationally or post-translationally.