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In JoVE (2)
- Rat Mesentery Exteriorization: A Model for Investigating the Cellular Dynamics Involved in Angiogenesis
- Murine Spinotrapezius Model to Assess the Impact of Arteriolar Ligation on Microvascular Function and Remodeling
Other Publications (36)
- Annals of the New York Academy of Sciences
- Microcirculation (New York, N.Y. : 1994)
- American Journal of Physiology. Heart and Circulatory Physiology
- FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology
- Developmental Biology
- Microcirculation (New York, N.Y. : 1994)
- Microcirculation (New York, N.Y. : 1994)
- Annals of Biomedical Engineering
- Microcirculation (New York, N.Y. : 1994)
- Briefings in Bioinformatics
- BMC Systems Biology
- Birth Defects Research. Part C, Embryo Today : Reviews
- Annals of Plastic Surgery
- Annals of Plastic Surgery
- Stem Cells (Dayton, Ohio)
- Microcirculation (New York, N.Y. : 1994)
- Microcirculation (New York, N.Y. : 1994)
- Trends in Immunology
- PLoS Computational Biology
- Otolaryngology--head and Neck Surgery : Official Journal of American Academy of Otolaryngology-Head and Neck Surgery
- Tissue Engineering. Part A
- Tissue Engineering. Part A
- Microvascular Research
- Microcirculation (New York, N.Y. : 1994)
- Microcirculation (New York, N.Y. : 1994)
- Tissue Engineering. Part A
- Annals of Biomedical Engineering
- Microcirculation (New York, N.Y. : 1994)
- PloS One
- Frontiers in Physiology
- Annals of Biomedical Engineering
- American Journal of Physiology. Cell Physiology
- Frontiers in Physiology
- Current Opinion in Hematology
- Investigative Ophthalmology & Visual Science
- Microcirculation (New York, N.Y. : 1994)
Articles by Shayn M. Peirce in JoVE
JoVE General
Rat Mesentery Exteriorization: A Model for Investigating the Cellular Dynamics Involved in Angiogenesis
1Department of Biomedical Engineering, Tulane University, 2Department of Biomedical Engineering, University of Virginia, 3Center for Stem Cell Research and Regenerative Medicine, Tulane University
This article describes a simple model for stimulating angiogenesis in the rat mesentery. The model produces dramatic increases in capillary sprouting, vascular area and vascular density over a relatively short time course in a tissue that allows en face visualization of entire microvascular networks down to the single cell level.
JoVE Clinical and Translational Medicine
Murine Spinotrapezius Model to Assess the Impact of Arteriolar Ligation on Microvascular Function and Remodeling
1Department of Biomedical Engineering, University of Virginia, 2Department of Biomedical Engineering, California Polytechnic State University, 3Office of Animal Welfare, University of Virginia, 4Department of Biomedical Engineering & Institute for Computational Medicine, Johns Hopkins University
We demonstrate a novel arterial ligation model in murine spinotrapezius muscle, including a step-by-step procedure and description of required instrumentation. We describe the surgery and relevant outcome measurements relating to vascular network remodeling and functional vasodilation using intravital and confocal microscopy.
Other articles by Shayn M. Peirce on PubMed
Vascular Assembly in Natural and Engineered Tissues
With the advent of molecular embryology and exploitation of genetic models systems, many genes necessary for normal blood vessel formation during early development have been identified. These genes include soluble effectors and their receptors, as well as components of cell-cell junctions and mediators of cell-matrix interactions. In vitro model systems (2-D and 3-D) to study paracrine and autocrine interactions of vascular cells and their progenitors have also been created. These systems are being combined to study the behavior of genetically altered cells to dissect and define the cellular role(s) of specific genes and gene families in directing the migration, proliferation, and differentiation needed for blood vessel assembly. It is clear that a complex spatial and temporal interplay of signals, including both genetic and environmental, modulates the assembly process. The development of real-time imaging and image analysis will enable us to gain further insights into this process. Collaborative efforts among vascular biologists, biomedical engineers, mathematicians, and physicists will allow us to bridge the gap between understanding vessel assembly in vivo and assembling vessels ex vivo.
Microvascular Remodeling: a Complex Continuum Spanning Angiogenesis to Arteriogenesis
Angiogenesis, the arterialization of capillaries, and arteriogenesis are specific manifestations of the complex continuum of blood vessel-remodeling processes that are produced by environmental stimuli. Together, they determine the integrative control of vascular assembly and pattern formation. Vascular assembly and pattern formation are critical elements of therapeutic vascular collateralization of progressively ischemic organs and in the tissue engineering or organogenesis of various tissue substitutes. An integrative systems approach is useful to measure the dynamics of vascular assembly in vivo across time scales from the embryo to the adult, and spanning spatial scales from cells to whole networks, to understand the complex interplay of multiple interacting cells and signal molecules. This requires in vivo observations, multiscale computer simulations, and tools for the genetic regulation of cell interactions. The new view of vascular remodeling as a continuum that can be manipulated in various tissues and in different size blood vessels, using appropriately coordinated multisignal stimuli, should open new therapeutic avenues.
Spatial and Temporal Control of Angiogenesis and Arterialization Using Focal Applications of VEGF164 and Ang-1
Microvascular networks undergo patterning changes that determine and reflect functional adaptations during tissue remodeling. Alterations in network architectures are a result of complex and integrated signaling events. To understand how two growth factor signals interact to stimulate angiogenesis and arterialization, we engineered spatially directed microvascular pattern changes in vivo by using combinations of focally delivered exogenous growth factors. We implanted microdelivery beads containing recombinant vascular endothelial growth factor-164 (VEGF(164)) and recombinant angiopoietin-1* (Ang-1*) into the dorsal subcutaneous tissue of fully anesthetized male Fischer 344 rats implanted with backpack window chambers, and we quantified vascular patterning changes by using intravital microscopy, a combination of architectural metrics, and immunohistochemistry. Focal delivery of VEGF(164) caused spatially directed increases in both the total number and the density of vessels with diameters <25 microm 7 days after microbead implantation. Increases were maintained out to 14 days but were reduced to control values by day 21. The addition of Ang-1* on day 7 maintained these increases out to day 21, induced vessel order ratios comparable to control levels, and was accompanied by increases in the length density of smooth muscle alpha-actin-positive vessels. We achieved spatial control of patterning changes in vivo by using multisignal stimulation via focal delivery of exogenous growth factor combinations and conclude that Ang-1* administered subsequent to VEGF(164) stimulation induces vascular growth while maintaining a network pattern consistent with native patterns that persist in the presence of vehicle control stimulation.
Multicellular Simulation Predicts Microvascular Patterning and in Silico Tissue Assembly
Remodeling of microvascular networks in mammals is critical for physiological adaptations and therapeutic revascularization. Cellular behaviors such as proliferation, differentiation, and migration are coordinated in these remodeling events via combinations of biochemical and biomechanical signals. We developed a cellular automata (CA) computational simulation that integrates epigenetic stimuli, molecular signals, and cellular behaviors to predict microvascular network patterning events. Over 50 rules obtained from published experimental data govern independent behaviors (including proliferation, differentiation, and migration) of thousands of interacting cells and diffusible growth factors in their tissue environment. From initial network patterns of in vivo blood vessel networks, the model predicts emergent patterning responses to two stimuli: 1) network-wide changes in hemodynamic mechanical stresses, and 2) exogenous focal delivery of an angiogenic growth factor. The CA model predicts comparable increases in vascular density (370+/-29 mm/mm3) 14 days after treatment with exogenous growth factor to that in vivo (480+/-41 mm/mm3) and approximately a twofold increase in contractile vessel lengths 5-10 days after 10% increase in circumferential wall strain, consistent with in vivo results. The CA simulation was thus able to identify a functional patterning module capable of quantitatively predicting vessel network remodeling in response to two important epigenetic stimuli.
Multicellular Computer Simulation of Morphogenesis: Blastocoel Roof Thinning and Matrix Assembly in Xenopus Laevis
In the blastocoel roof (BCR) of the Xenopus laevis embryo, epibolic movements are driven by the radial intercalation of deep cell layers and the coordinate spreading of the overlying superficial cell layer. Thinning of the lateral margins of the BCR by radial intercalation requires fibronectin (FN), which is produced and assembled into fibrils by the inner deep cell layer of the BCR. A cellular automata (CA) computer model was developed to analyze the spatial and temporal movements of BCR cells during epiboly. Simulation parameters were defined based on published data and independent results detailing initial tissue geometry, cell numbers, cell intercalation rates, and migration rates. Hypotheses regarding differential cell adhesion and FN assembly were also considered in setting system parameters. A 2-dimensional model simulation was developed that predicts BCR thinning time of 4.8 h, which closely approximates the time required for the completion of gastrulation in vivo. Additionally, the model predicts a temporal increase in FN matrix assembly that parallels fibrillogenesis in the embryo. The model is capable of independent predictions of cell rearrangements during epiboly, and here was used to predict successfully the lateral dispersion of a patch of cells implanted in the BCR, and increased assembly of FN matrix following inhibition of radial intercalation by N-cadherin over-expression.
Differential Arterial/venous Expression of NG2 Proteoglycan in Perivascular Cells Along Microvessels: Identifying a Venule-specific Phenotype
Similar to other vascular pericyte markers, including smooth muscle (SM) alpha-actin, desmin, and PDGF-beta-receptor, NG2 proteoglycan is not pericyte specific. Therefore, the use of NG2 as a pericyte marker, especially in cell lineage studies, in comparison to other nonspecific pericyte markers requires an understanding of how its expression varies spatially within a microvascular network. The objective of this study was to characterize NG2 expression along vessels within rat microvascular networks and compare this to SM alpha-actin expression.
Perivascular Cells Along Venules Upregulate NG2 Expression During Microvascular Remodeling
Recently the authors have shown that neuron-glial antigen 2 (NG2) is expressed by perivascular cells along arterioles and capillaries, but not along venules in quiescent rat mesenteric microvascular networks. To investigate how the spatial distribution of this proteoglycan changes during microvascular remodeling, the objective of this study was to characterize the expression of NG2 in adult rat mesenteric microvascular networks undergoing active remodeling.
Multi-cell Agent-based Simulation of the Microvasculature to Study the Dynamics of Circulating Inflammatory Cell Trafficking
Leukocyte trafficking through the microcirculation and into tissues is central in angiogenesis, inflammation, and the immune response. Although the literature is rich with mechanistic detail describing molecular mediators of these processes, integration of signaling events and cell behaviors within a unified spatial and temporal framework at the multi-cell tissue-level is needed to achieve a fuller understanding. We have developed a novel computational framework that combines agent-based modeling (ABM) with a network flow analysis to study monocyte homing. A microvascular network architecture derived from mouse muscle was incorporated into the ABM. Each individual cell was represented by an individual agent in the simulation. The network flow model calculates hemodynamic parameters (blood flow rates, fluid shear stress, and hydrostatic pressures) throughout the simulated microvascular network. These are incorporated into the ABM to affect monocyte transit through the network and chemokine/cytokine concentrations. In turn, simulated monocytes respond to their local mechanical and biochemical environments and make behavioral decisions based on a rule set derived from independent literature. Simulated cell behaviors give rise to emergent leukocyte rolling, adhesion, and extravasation. Molecular knockout simulations were performed to validate the model, and predictions of monocyte adhesion, rolling, and extravasation show good agreement with the independently published corresponding mouse studies.
EphB4 Expression Along Adult Rat Microvascular Networks: EphB4 is More Than a Venous Specific Marker
EphrinB2 and EphB4 are considered to be markers of arterial/venous identity during embryonic development. However, the expression patterns of these arterial/venous-specific markers in adult microvascular networks remain unclear. The objective of this study was to characterize the cellular distribution of EphB4 expression along the hierarchy of unstimulated and remodeling adult rat mesenteric microvascular networks.
Combining Experiments with Multi-cell Agent-based Modeling to Study Biological Tissue Patterning
Agent-based modeling (ABM), also termed 'Individual-based modeling (IBM)', is a computational approach that simulates the interactions of autonomous entities (agents, or individual cells) with each other and their local environment to predict higher level emergent patterns. A literature-derived rule set governs the actions of each individual agent. While this technique has been widely used in the ecological and social sciences, it has only recently been applied in biomedical research. The purpose of this review is to provide an introduction to ABM as it has been used to study complex multi-cell biological phenomena, underscore the importance of coupling models with experimental work, and outline future challenges for the ABM field and its application to biomedical research. We highlight a number of published examples of ABM, focusing on work that has combined experimental with ABM analyses and how this pairing produces new understanding. We conclude with suggestions for moving forward with this parallel approach.
Multiscale Computational Analysis of Xenopus Laevis Morphogenesis Reveals Key Insights of Systems-level Behavior
Tissue morphogenesis is a complex process whereby tissue structures self-assemble by the aggregate behaviors of independently acting cells responding to both intracellular and extracellular cues in their environment. During embryonic development, morphogenesis is particularly important for organizing cells into tissues, and although key regulatory events of this process are well studied in isolation, a number of important systems-level questions remain unanswered. This is due, in part, to a lack of integrative tools that enable the coupling of biological phenomena across spatial and temporal scales. Here, we present a new computational framework that integrates intracellular signaling information with multi-cell behaviors in the context of a spatially heterogeneous tissue environment.
Agent-based Modeling of Multicell Morphogenic Processes During Development
A central challenge in the field of developmental biology is to understand how mechanisms at one level of biological scale (i.e., cell-level) interact to produce higher-level (i.e., tissue-level) phenomena. This challenge is particularly relevant to the study of tissue morphogenesis, the process that generates newly formed, remodeled, or regenerated tissue structures. Morphogenesis arises from the spatially- and temporally-dynamic interactions of individual cells with each other and their local environment. Computational models have been combined with experimental efforts to accelerate the discovery processes. Agent-based modeling (ABM) is a computational technique that can be used to model collections of individual biological cells and compute their interactions, which generate emergent tissue-level results. Recently, ABM has been applied to the study of various developmental morphogenic processes, and the purpose of this review is to summarize these studies in order to demonstrate the types of advances that can be expected from pursuing a multicell ABM approach. We also highlight some challenges associated with ABM and suggest strategies for overcoming them. While ABM's application to the study of ecology, epidemiology, and social sciences has a much longer history, we suggest that the application of ABM to the study of morphogenesis has great utility, and when paired with benchtop experimentation, ABM can provide new insights and direct future experimentation.
Functional Binding of Human Adipose-derived Stromal Cells: Effects of Extraction Method and Hypoxia Pretreatment
Human adipose-derived stromal cells (hASCs) were evaluated in vitro for their ability to bind vascular adhesion and extracellular matrix proteins to arrest (firmly adhere) under physiological flow conditions. hASCs were flowed through a parallel plate flow chamber containing substrates presenting immobilized type I collagen, fibronectin, E-selectin, L-selectin, P-selectin, vascular cell adhesion molecule-1 (VCAM-1), or intercellular adhesion molecule-1 (ICAM-1) under static and laminar flow conditions (wall shear stress = 1 dyn/cm). hASCs were able to firmly adhere to type I collagen, fibronectin, VCAM-1, and ICAM-1 substrates, but not to any of the selectins. Pretreatment with hypoxia increased the ability of hASCs isolated by liposuction to adhere to VCAM-1 and ICAM-1, but this effect was not seen in cells isolated by tissue excision. These results indicate that hASCs possess the ability to adhere key adhesion proteins, illustrate the importance of hASC harvest procedure, and suggest mechanisms for homing in a setting where interaction with inflamed or injured tissue is necessary.
Topical Poloxamer-188 Improves Blood Flow Following Thermal Injury in Rat Mesenteric Microvasculature
Microvascular changes of sludging and stasis are indications of thermal injury in tissue. This study investigates whether microvascular thermal injury can be decreased via topical application of poloxamer-188. Rat mesenteric microvessels were thermally injured and topically suffused with either Ringer's solution (control) or 5% poloxamer-188 in Ringer's solution (experiment). Blood flow was characterized in microvessels as normal or abnormal (ie, sludging and stasis). Topical treatment with poloxamer-188 reduced the percentage of capillaries with abnormal blood flow from 62% to 23%. In venules, this treatment resulted in a decrease from 54% to 34%. These results demonstrated that topically applied poloxamer-188 dramatically reduces the microvascular changes of sludging and stasis because of thermal injury in rat mesenteric microvessels.
IFATS Collection: The Role of Human Adipose-derived Stromal Cells in Inflammatory Microvascular Remodeling and Evidence of a Perivascular Phenotype
A growing body of literature suggests that human adipose-derived stromal cells (hASCs) possess developmental plasticity both in vitro and in vivo, and might represent a viable cell source for therapeutic angiogenesis and tissue engineering. We investigate their phenotypic similarity to perivascular cell types, ability to contribute to in vivo microvascular remodeling, and ability to modulate vascular stability. We evaluated hASC surface expression of vascular and stem/progenitor cell markers in vitro, as well as any effects of platelet-derived growth factor B chain (PDGF-BB) and vascular endothelial growth factor 165 on in vitro hASC migration. To ascertain in vivo behavior of hASCs in an angiogenic environment, hASCs were isolated, expanded in culture, labeled with a fluorescent marker, and injected into adult nude rat mesenteries that were stimulated to undergo microvascular remodeling. Ten, 30, and 60 days after injection, tissues from anesthetized animals were harvested and processed with immunohistochemical techniques to determine hASC quantity, positional fate in relation to microvessels, and expression of endothelial and perivascular cell markers. After 60 days, 29% of hASCs exhibited perivascular morphologies compared with 11% of injected human lung fibroblasts. hASCs exhibiting perivascular morphologies also expressed markers characteristic of vascular pericytes: smooth muscle alpha-actin (10%) and neuron-glia antigen 2 (8%). In tissues treated with hASCs, vascular density was significantly increased over age-matched controls lacking hASCs. This study demonstrates that hASCs express pericyte lineage markers in vivo and in vitro, exhibit increased migration in response to PDGF-BB in vitro, exhibit perivascular morphology when injected in vivo, and contribute to increases in microvascular density during angiogenesis by migrating toward vessels. Disclosure of potential conflicts of interest is found at the end of this article.
Arteriolar Remodeling Following Ischemic Injury Extends from Capillary to Large Arteriole in the Microcirculation
Skeletal muscle vasculature undergoes arteriogenesis to restore tissue perfusion and function following loss of blood flow. This process has been shown to occur in large vessels following ischemia, although recent studies suggest this may occur in the microcirculation as well. We tested the hypothesis that ischemia induces microvascular remodeling in the skeletal muscle microcirculation on the scale of capillary to sub-35 mum diameter arterioles.
Computational and Mathematical Modeling of Angiogenesis
Over the past two decades, a number of mathematical and computational models have been developed to study different aspects of angiogenesis that span the spatial and temporal scales encompassed by this complex process. For example, models have been built to investigate how growth factors and receptors signal endothelial cell proliferation, how groups of endothelial cells assemble into individual vessels, and how tumors recruit the ingrowth of whole microvascular networks. A prudent question to pose is: "what have we learned from these models?" This review aims to answer this question as it pertains to angiogenesis in the context of normal physiological growth, tumorigenesis, wound healing, tissue engineering, and the design of therapeutic strategies. We also provide a framework for parsing angiogenesis models into categories, according to the type of modeling approach used, the spatial and temporal scales simulated, and the overarching question being posed to the model. Finally, this review introduces some of the simplification strategies and assumptions used in model building, discusses model validation, and makes recommendations for application of modeling approaches to unresolved questions in the field.
Characterizing Emergent Properties of Immunological Systems with Multi-cellular Rule-based Computational Modeling
The immune system is comprised of numerous components that interact with one another to give rise to phenotypic behaviors that are sometimes unexpected. Agent-based modeling (ABM) and cellular automata (CA) belong to a class of discrete mathematical approaches in which autonomous entities detect local information and act over time according to logical rules. The power of this approach lies in the emergence of behavior that arises from interactions between agents, which would otherwise be impossible to know a priori. Recent work exploring the immune system with ABM and CA has revealed novel insights into immunological processes. Here, we summarize these applications to immunology and, particularly, how ABM can help formulate hypotheses that might drive further experimental investigations of disease mechanisms.
Agent-based Model of Therapeutic Adipose-derived Stromal Cell Trafficking During Ischemia Predicts Ability to Roll on P-selectin
Intravenous delivery of human adipose-derived stromal cells (hASCs) is a promising option for the treatment of ischemia. After delivery, hASCs that reside and persist in the injured extravascular space have been shown to aid recovery of tissue perfusion and function, although low rates of incorporation currently limit the safety and efficacy of these therapies. We submit that a better understanding of the trafficking of therapeutic hASCs through the microcirculation is needed to address this and that selective control over their homing (organ- and injury-specific) may be possible by targeting bottlenecks in the homing process. This process, however, is incredibly complex, which merited the use of computational techniques to speed the rate of discovery. We developed a multicell agent-based model (ABM) of hASC trafficking during acute skeletal muscle ischemia, based on over 150 literature-based rules instituted in Netlogo and MatLab software programs. In silico, trafficking phenomena within cell populations emerged as a result of the dynamic interactions between adhesion molecule expression, chemokine secretion, integrin affinity states, hemodynamics and microvascular network architectures. As verification, the model reasonably reproduced key aspects of ischemia and trafficking behavior including increases in wall shear stress, upregulation of key cellular adhesion molecules expressed on injured endothelium, increased secretion of inflammatory chemokines and cytokines, quantified levels of monocyte extravasation in selectin knockouts, and circulating monocyte rolling distances. Successful ABM verification prompted us to conduct a series of systematic knockouts in silico aimed at identifying the most critical parameters mediating hASC trafficking. Simulations predicted the necessity of an unknown selectin-binding molecule to achieve hASC extravasation, in addition to any rolling behavior mediated by hASC surface expression of CD15s, CD34, CD62e, CD62p, or CD65. In vitro experiments confirmed this prediction; a subpopulation of hASCs slowly rolled on immobilized P-selectin at speeds as low as 2 microm/s. Thus, our work led to a fundamentally new understanding of hASC biology, which may have important therapeutic implications.
Construct Validity of a Simulator for Myringotomy with Ventilation Tube Insertion
To establish construct validity of an anatomic model as a simulator for myringotomy with ventilation tube insertion and to assess its subjective appeal.
FTY720 Promotes Local Microvascular Network Formation and Regeneration of Cranial Bone Defects
The calvarial bone microenvironment contains a unique progenitor niche that should be considered for therapeutic manipulation when designing regeneration strategies. Recently, our group demonstrated that cells isolated from the dura are multipotent and exhibit expansion potential and robust mineralization on biodegradable constructs in vitro. In this study, we evaluate the effectiveness of healing critical-sized cranial bone defects by enhancing microvascular network growth and host dura progenitor trafficking to the defect space pharmacologically by delivering drugs targeted to sphingosine 1-phosphate (S1P) receptors. We demonstrate that delivery of pharmacological agonists to (S1P) receptors S1P(1) and S1P(3) significantly increase bone ingrowth, total microvessel density, and smooth muscle cell investment on nascent microvessels within the defect space. Further, in vitro proliferation and migration studies suggest that selective activation of S1P(3) promotes recruitment and growth of osteoblastic progenitors from the meningeal dura mater.
Human Adipose-derived Stromal Cells Accelerate Diabetic Wound Healing: Impact of Cell Formulation and Delivery
Human adipose-derived stromal cells (ASCs) have been shown to possess therapeutic potential in a variety of settings, including cutaneous wound healing; however, it is unknown whether the regenerative properties of this cell type can be applied to diabetic ulcers. ASCs collected from elective surgical procedures were used to treat full-thickness dermal wounds in leptin receptor-deficient (db/db) mice. Cells were delivered either as multicellular aggregates or as cell suspensions to determine the impact of cell formulation and delivery methods on biological activity and in vivo therapeutic effect. After treatment with ASCs that were formulated as multicellular aggregates, diabetic wounds experienced a significant increase in the rate of wound closure compared to wounds treated with an equal number of ASCs delivered in suspension. Analysis of culture supernatant and gene arrays indicated that ASCs formulated as three-dimensional aggregates produce significantly more extracellular matrix proteins (e.g., tenascin C, collagen VI alpha3, and fibronectin) and secreted soluble factors (e.g., hepatocyte growth factor, matrix metalloproteinase-2, and matrix metalloproteinase-14) compared to monolayer culture. From these results, it is clear that cell culture, formulation, and delivery method have a large impact on the in vitro and in vivo biology of ASCs.
Chronic Whole-body Hypoxia Induces Intussusceptive Angiogenesis and Microvascular Remodeling in the Mouse Retina
Currently, little is known about the response of the adult retinal microvasculature to hypoxia. To test the hypothesis that chronic systemic hypoxia induces angiogenesis and microvascular remodeling in the adult mouse retina, adult 10-week old female C57Bl/6 mice were exposed to 10% O(2) for 2 or 3 weeks. After hypoxia exposure, retinas were harvested, whole-mounted, and processed for immunohistochemistry. Retinas were stained with lectin, anti-smooth muscle alpha-actin antibody, and anti-NG2 antibody to visualize microvascular networks and their cellular components. Confocal microscopy was used to obtain images of superficial retinal networks. Images were analyzed to assess vessel diameter, vascular length density, branch point density, and the presence of vascular loops, a hallmark of intussusceptive angiogenesis. Both 2 and 3 weeks of hypoxia exposure resulted in a significant increase in the diameters of arterioles and post-arteriole capillaries (p<0.003). After 3 weeks of hypoxia, vascular length density and branch point density were significantly increased in retinas exposed to hypoxia as compared to normoxic controls (p<0.001). The number of vascular loops in the superficial retinal networks was significantly greater in hypoxia-exposed retinas (p < or = 0.001). Our results demonstrate, for the first time, intussusceptive angiogenesis as a tissue-level mechanism of vascular adaptation to chronic systemic hypoxia in the adult mouse retina and contribute to our understanding of hypoxia-induced angiogenesis and microvascular remodeling in the adult animal.
Collateral Capillary Arterialization Following Arteriolar Ligation in Murine Skeletal Muscle
Chronic and acute ischemic diseases-peripheral artery disease, coronary artery disease, stroke-result in tissue damage unless blood flow is maintained or restored in a timely manner. Mice of different strains recover from arteriolar ligation (by increasing collateral blood flow) at different speeds. We quantify the spatio-temporal patterns of microvascular network remodeling following arteriolar ligation in different mouse strains to better understand inter-individual variability.
Inhibition of Canonical Wnt Signaling Increases Microvascular Hemorrhaging and Venular Remodeling in Adult Rats
The canonical Wnt signaling pathway, heavily studied in development and cancer, has recently been implicated in microvascular growth with the use of developmental and in vitro models. To date, however, no study exists showing the effects of perturbing the canonical Wnt pathway in a complete microvascular network undergoing physiological remodeling in vivo. Our objective was to investigate the effects of canonical Wnt inhibition on the microvascular remodeling of adult rats.
Selective Activation of Sphingosine 1-phosphate Receptors 1 and 3 Promotes Local Microvascular Network Growth
Proper spatial and temporal regulation of microvascular remodeling is critical to the formation of functional vascular networks, spanning the various arterial, venous, capillary, and collateral vessel systems. Recently, our group has demonstrated that sustained release of sphingosine 1-phosphate (S1P) from biodegradable polymers promotes microvascular network growth and arteriolar expansion. In this study, we employed S1P receptor-specific compounds to activate and antagonize different combinations of S1P receptors to elucidate those receptors most critical for promotion of pharmacologically induced microvascular network growth. We show that S1P(1) and S1P(3) receptors act synergistically to enhance functional network formation via increased functional length density, arteriolar diameter expansion, and increased vascular branching in the dorsal skinfold window chamber model. FTY720, a potent activator of S1P(1) and S1P(3), promoted a 107% and 153% increase in length density 3 and 7 days after implantation, respectively. It also increased arteriolar diameters by 60% and 85% 3 and 7 days after implantation. FTY720-stimulated branching in venules significantly more than unloaded poly(D, L-lactic-co-glycolic acid). When implanted on the mouse spinotrapezius muscle, FTY720 stimulated an arteriogenic response characterized by increased tortuosity and collateralization of branching microvascular networks. Our results demonstrate the effectiveness of S1P(1) and S1P(3) receptor-selective agonists (such as FTY720) in promoting microvascular growth for tissue engineering applications.
Systems Analysis of Small Signaling Modules Relevant to Eight Human Diseases
Using eight newly generated models relevant to addiction, Alzheimer's disease, cancer, diabetes, HIV, heart disease, malaria, and tuberculosis, we show that systems analysis of small (4-25 species), bounded protein signaling modules rapidly generates new quantitative knowledge from published experimental research. For example, our models show that tumor sclerosis complex (TSC) inhibitors may be more effective than the rapamycin (mTOR) inhibitors currently used to treat cancer, that HIV infection could be more effectively blocked by increasing production of the human innate immune response protein APOBEC3G, rather than targeting HIV's viral infectivity factor (Vif), and how peroxisome proliferator-activated receptor alpha (PPARα) agonists used to treat dyslipidemia would most effectively stimulate PPARα signaling if drug design were to increase agonist nucleoplasmic concentration, as opposed to increasing agonist binding affinity for PPARα. Comparative analysis of system-level properties for all eight modules showed that a significantly higher proportion of concentration parameters fall in the top 15th percentile sensitivity ranking than binding affinity parameters. In infectious disease modules, host networks were significantly more sensitive to virulence factor concentration parameters compared to all other concentration parameters. This work supports the future use of this approach for informing the next generation of experimental roadmaps for known diseases.
A New Method for in Vivo Visualization of Vessel Remodeling Using a Near-infrared Dye
Vascular obstructive events can be partially compensated for by remodeling processes that increase vessel diameter and collateral tortuosity. However, methods for visualizing remodeling events in vivo and with temporal comparisons from the same animal remain elusive.
Rapid Analysis of Vessel Elements (RAVE): a Tool for Studying Physiologic, Pathologic and Tumor Angiogenesis
Quantification of microvascular network structure is important in a myriad of emerging research fields including microvessel remodeling in response to ischemia and drug therapy, tumor angiogenesis, and retinopathy. To mitigate analyst-specific variation in measurements and to ensure that measurements represent actual changes in vessel network structure and morphology, a reliable and automatic tool for quantifying microvascular network architecture is needed. Moreover, an analysis tool capable of acquiring and processing large data sets will facilitate advanced computational analysis and simulation of microvascular growth and remodeling processes and enable more high throughput discovery. To this end, we have produced an automatic and rapid vessel detection and quantification system using a MATLAB graphical user interface (GUI) that vastly reduces time spent on analysis and greatly increases repeatability. Analysis yields numerical measures of vessel volume fraction, vessel length density, fractal dimension (a measure of tortuosity), and radii of murine vascular networks. Because our GUI is open sourced to all, it can be easily modified to measure parameters such as percent coverage of non-endothelial cells, number of loops in a vascular bed, amount of perfusion and two-dimensional branch angle. Importantly, the GUI is compatible with standard fluorescent staining and imaging protocols, but also has utility analyzing brightfield vascular images, obtained, for example, in dorsal skinfold chambers. A manually measured image can be typically completed in 20 minutes to 1 hour. In stark comparison, using our GUI, image analysis time is reduced to around 1 minute. This drastic reduction in analysis time coupled with increased repeatability makes this tool valuable for all vessel research especially those requiring rapid and reproducible results, such as anti-angiogenic drug screening.
Toward a Multi-scale Computational Model of Arterial Adaptation in Hypertension: Verification of a Multi-cell Agent Based Model
Agent-based models (ABMs) represent a novel approach to study and simulate complex mechano chemo-biological responses at the cellular level. Such models have been used to simulate a variety of emergent responses in the vasculature, including angiogenesis and vasculogenesis. Although not used previously to study large vessel adaptations, we submit that ABMs will prove equally useful in such studies when combined with well-established continuum models to form multi-scale models of tissue-level phenomena. In order to couple agent-based and continuum models, however, there is a need to ensure that each model faithfully represents the best data available at the relevant scale and that there is consistency between models under baseline conditions. Toward this end, we describe the development and verification of an ABM of endothelial and smooth muscle cell responses to mechanical stimuli in a large artery. A refined rule-set is proposed based on a broad literature search, a new scoring system for assigning confidence in the rules, and a parameter sensitivity study. To illustrate the utility of these new methods for rule selection, as well as the consistency achieved with continuum-level models, we simulate the behavior of a mouse aorta during homeostasis and in response to both transient and sustained increases in pressure. The simulated responses depend on the altered cellular production of seven key mitogenic, synthetic, and proteolytic biomolecules, which in turn control the turnover of intramural cells and extracellular matrix. These events are responsible for gross changes in vessel wall morphology. This new ABM is shown to be appropriately stable under homeostatic conditions, insensitive to transient elevations in blood pressure, and responsive to increased intramural wall stress in hypertension.
Ensuring Congruency in Multiscale Modeling: Towards Linking Agent Based and Continuum Biomechanical Models of Arterial Adaptation
There is a need to develop multiscale models of vascular adaptations to understand tissue-level manifestations of cellular level mechanisms. Continuum-based biomechanical models are well suited for relating blood pressures and flows to stress-mediated changes in geometry and properties, but less so for describing underlying mechanobiological processes. Discrete stochastic agent-based models are well suited for representing biological processes at a cellular level, but not for describing tissue-level mechanical changes. We present here a conceptually new approach to facilitate the coupling of continuum and agent-based models. Because of ubiquitous limitations in both the tissue- and cell-level data from which one derives constitutive relations for continuum models and rule-sets for agent-based models, we suggest that model verification should enforce congruency across scales. That is, multiscale model parameters initially determined from data sets representing different scales should be refined, when possible, to ensure that common outputs are consistent. Potential advantages of this approach are illustrated by comparing simulated aortic responses to a sustained increase in blood pressure predicted by continuum and agent-based models both before and after instituting a genetic algorithm to refine 16 objectively bounded model parameters. We show that congruency-based parameter refinement not only yielded increased consistency across scales, it also yielded predictions that are closer to in vivo observations.
Hypoxic Culture and in Vivo Inflammatory Environments Affect the Assumption of Pericyte Characteristics by Human Adipose and Bone Marrow Progenitor Cells
Previous studies have shown that exposure to a hypoxic in vitro environment increases the secretion of pro-angiogenic growth factors by human adipose-derived stromal cells (hASCs) [Cao Y, et al., Biochem Biophys Res Commun 332: 370-379, 2005; Kokai LE, et al., Plast Reconstr Surg 116: 1453-1460, 2005; Park BS, et al., Biomed Res (Tokyo) 31: 27-34, 2010; Rasmussen JG, et al., Cytotherapy 13: 318-328, 2010; Rehman J, et al., Circulation 109: 1292-1298, 2004]. Previously, it has been demonstrated that hASCs can differentiate into pericytes and promote microvascular stability and maintenance during angiogenesis in vivo (Amos PJ, et al., Stem Cells 26: 2682-2690, 2008; Traktuev DO, et al., Circ Res 102: 77-85, 2008). In this study, we tested the hypotheses that angiogenic induction can be increased and pericyte differentiation decreased by pretreatment of hASCs with hypoxic culture and that hASCs are similar to human bone marrow-derived stromal cells (hBMSCs) in these regards. Our data confirms previous studies showing that hASCs: 1) secrete pro-angiogenic proteins, which are upregulated following culture in hypoxia, and 2) migrate up gradients of PDGF-BB in vitro, while showing for the first time that a rat mesenteric model of angiogenesis induced by 48/80 increases the propensity of both hASCs and hBMSCs to assume perivascular phenotypes following injection. Moreover, culture of both cell types in hypoxia before injection results in a biphasic vascular length density response in this model of inflammation-induced angiogenesis. The effects of hypoxia and inflammation on the phenotype of adult progenitor cells impacts both the therapeutic and the basic science applications of the cell types, as hypoxia and inflammation are common features of natural and pathological vascular compartments in vivo.
Computational Modeling of Interacting VEGF and Soluble VEGF Receptor Concentration Gradients
Experimental data indicates that soluble vascular endothelial growth factor (VEGF) receptor 1 (sFlt-1) modulates the guidance cues provided to sprouting blood vessels by VEGF-A. To better delineate the role of sFlt-1 in VEGF signaling, we have developed an experimentally based computational model. This model describes dynamic spatial transport of VEGF, and its binding to receptors Flt-1 and Flk-1, in a mouse embryonic stem cell model of vessel morphogenesis. The model represents the local environment of a single blood vessel. Our simulations predict that blood vessel secretion of sFlt-1 and increased local sFlt-1 sequestration of VEGF results in decreased VEGF-Flk-1 levels on the sprout surface. In addition, the model predicts that sFlt-1 secretion increases the relative gradient of VEGF-Flk-1 along the sprout surface, which could alter endothelial cell perception of directionality cues. We also show that the proximity of neighboring sprouts may alter VEGF gradients, VEGF receptor binding, and the directionality of sprout growth. As sprout distances decrease, the probability that the sprouts will move in divergent directions increases. This model is a useful tool for determining how local sFlt-1 and VEGF gradients contribute to the spatial distribution of VEGF receptor binding, and can be used in conjunction with experimental data to explore how multi-cellular interactions and relationships between local growth factor gradients drive angiogenesis.
Integration of Experimental and Computational Approaches to Sprouting Angiogenesis
We summarize recent experimental and computational studies that investigate molecular and cellular mechanisms of sprouting angiogenesis. We discuss how experimental tools have unveiled new opportunities for computational modeling by providing detailed phenomenological descriptions and conceptual models of cell-level behaviors underpinned by high-quality molecular data. Using recent examples, we show how new understanding results from bridging computational and experimental approaches.
Attenuation of EphrinB2 Reverse Signaling Decreases Vascularized Area and Preretinal Vascular Tuft Formation in the Murine Model of Oxygen-induced Retinopathy
EphB4 and ephrinB2 are known key regulators of retinal vascular development, but due to their capacity for bidirectional signaling, delineation of their individual roles in this process remains unclear. To better dissect out individual contributions, a model of proliferative retinopathy in mice with attenuated ephrinB2 reverse signaling was studied. It was hypothesized that endothelial ephrinB2 reverse signaling regulates hypoxia-induced capillary sprouting, as well as the pathologic formation of neovascular tufts in postnatal retinal microvascular networks.
Exogenous Thrombin Delivery Promotes Collateral Capillary Arterialization and Tissue Reperfusion in the Murine Spinotrapezius Muscle Ischemia Model
We examined the effects of exogenously delivered thrombin on cell recruitment in skeletal muscle and the formation of new collateral arterioles in the microvasculature in response to ligation-induced ischemia.
