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1.  Macrophage recruitment and polarization during collateral vessel remodeling in murine adipose tissue 
During autologous flap transplantation for reconstructive surgeries, plastic surgeons use a surgical pre-treatment strategy called “flap delay”, which entails ligating a feeding artery into an adipose tissue flap 10–14 days prior to transfer. It is believed that this blood flow alteration leads to vascular remodeling in the flap, resulting in better flap survival following transfer; however, the structural changes in the microvascular network are poorly understood. Here, we evaluate microvascular adaptations within adipose tissue in a murine model of flap delay.
Methods and Results
We used a murine flap delay model in which we ligated an artery supplying the inguinal fat pad. Although the extent of angiogenesis appeared minimal, significant diameter expansion of pre-existing collateral arterioles was observed. There was a 5-fold increase in recruitment of CX3CR1+ monocytes to ligated tissue, a 3-fold increase in CD68+/CD206+ macrophages in ligated tissue, a 40% increase in collateral vessel diameters supplying ligated tissue, and a 6-fold increase in the number of proliferating cells in ligated tissue.
Our study describes microvascular adaptations in adipose in response to altered blood flow and underscores the importance of macrophages. Our data supports the development of therapies that target macrophages in order to enhance vascular remodeling in flaps.
PMCID: PMC4707992  PMID: 26638986
arteriogenesis; macrophage; adipose; collateral vessel
2.  Adipose-Derived Stem Cells From Diabetic Mice Show Impaired Vascular Stabilization in a Murine Model of Diabetic Retinopathy 
This study attempted to evaluate therapeutic efficacy of adipose-derived stem cells (ASCs) derived either from healthy, nondiabetic or from diabetic mice. ASCs from healthy mice were more effective than diabetic mouse ASCs in protecting the diabetic retina from further vascular dropout, supporting the utility of an allogeneic injection of ASCs versus autologous or conditioned media approaches in the treatment of diabetic retinopathy.
Diabetic retinopathy is characterized by progressive vascular dropout with subsequent vision loss. We have recently shown that an intravitreal injection of adipose-derived stem cells (ASCs) can stabilize the retinal microvasculature, enabling repair and regeneration of damaged capillary beds in vivo. Because an understanding of ASC status from healthy versus diseased donors will be important as autologous cellular therapies are developed for unmet clinical needs, we took advantage of the hyperglycemic Akimba mouse as a preclinical in vivo model of diabetic retinopathy in an effort aimed at evaluating therapeutic efficacy of adipose-derived stem cells (mASCs) derived either from healthy, nondiabetic or from diabetic mice. To these ends, Akimba mice received intravitreal injections of media conditioned by mASCs or mASCs themselves, subsequent to development of substantial retinal capillary dropout. mASCs from healthy mice were more effective than diabetic mASCs in protecting the diabetic retina from further vascular dropout. Engrafted ASCs were found to preferentially associate with the retinal vasculature. Conditioned medium was unable to recapitulate the vasoprotection seen with injected ASCs. In vitro diabetic ASCs showed decreased proliferation and increased apoptosis compared with healthy mASCs. Diabetic ASCs also secreted less vasoprotective factors than healthy mASCs, as determined by high-throughput enzyme-linked immunosorbent assay. Our findings suggest that diabetic ASCs are functionally impaired compared with healthy ASCs and support the utility of an allogeneic injection of ASCs versus autologous or conditioned media approaches in the treatment of diabetic retinopathy.
PMCID: PMC4414213  PMID: 25769654
Diabetic retinopathy; Stem cells; Adipose-derived stem cells; Pericyte; Diabetes
3.  Paradoxical Adipose Hyperplasia and Cellular Effects After Cryolipolysis: A Case Report 
Aesthetic Surgery Journal  2015;36(1):NP6-NP13.
Cryolipolysis is a noninvasive technique for the reduction of subcutaneous adipose tissue by controlled, localized cooling, causing adipocyte apoptosis, reportedly without affecting surrounding tissue. Although cryolipolysis has a low incidence of adverse side effects 33 cases of paradoxical adipose hyperplasia (PAH) have been reported and the precise pathogenesis of PAH is poorly understood. This present case study of PAH aims to characterize the pathological changes in the adipose tissue of PAH on a cellular level by using multiple different assays [hematoxy lin and eosin staining, LIVE/DEAD staining, BODIPY® 558/568 C12 (4,4-Difluoro-5-(2-Thienyl)-4-Bora-3a,4a-Diaza-s-Indacene-3-dodecanoic acid) staining]. to identify the underlying mechanism of PAH and reduce the prevalence of PAH in the future. Tissue with PAH had fewer viable cells, significantly decreased quantities of interstitial cells (p = 0.04), and fewer vessels per adipose tissue area when compared to the control tissue. Adipocytes from the PAH tissue were on average slightly smaller than the control adipocytes. Adipocytes of PAH tissue had irregularly contoured edges when compared to the smooth, round edges of the control tissue. These findings from a neutral third party are contrary to prior reports from the inventors of this technique regarding effects of cryolipolysis on both the microvasculature and interstitial cells in adipose tissue. Our use of different assays to compare cryolipolysis-treated PAH tissue with untreated adipose tissue in the same patient showed adipose tissue that developed PAH was hypocellular and hypovascular. Contrary to prior reports from the inventors, cryolipolysis may cause vessel loss, which could lead to ischemia and/or hypoxia that further contributes to adipocyte death.
Level of Evidence: 5
PMCID: PMC4678995  PMID: 26590197
4.  Integration of Experimental and Computational Approaches to Sprouting Angiogenesis 
Current opinion in hematology  2012;19(3):184-191.
Purpose of review
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.
Recent findings
Experimental data extends beyond the tip cell vs. stalk cell paradigm, and involves numerous molecular inputs such as VEGF and Notch. This data is being used to generate and validate computational models, which can then be used to predict the results of hypothetical experiments that are difficult to perform in the laboratory, and to generate new hypotheses that account for system-wide interactions. As a result of this integration, descriptions of critical gradients of growth factor-receptor complexes have been generated, and new modulators of cell behavior have been described.
We suggest that the recent emphasis on the different stages of sprouting angiogenesis, and integration of experimental and computational approaches, should provide a way to manage the complexity of this process and help identify new regulatory paradigms and therapeutic targets.
PMCID: PMC4132663  PMID: 22406822
Sprouting angiogenesis; experimental models; computational models; stages of angiogenesis
5.  Multiscale Computational Models of Complex Biological Systems 
Integration of data across spatial, temporal, and functional scales is a primary focus of biomedical engineering efforts. The advent of powerful computing platforms, coupled with quantitative data from high-throughput experimental platforms, has allowed multiscale modeling to expand as a means to more comprehensively investigate biological phenomena in experimentally relevant ways. This review aims to highlight recently published multiscale models of biological systems while using their successes to propose the best practices for future model development. We demonstrate that coupling continuous and discrete systems best captures biological information across spatial scales by selecting modeling techniques that are suited to the task. Further, we suggest how to best leverage these multiscale models to gain insight into biological systems using quantitative, biomedical engineering methods to analyze data in non-intuitive ways. These topics are discussed with a focus on the future of the field, the current challenges encountered, and opportunities yet to be realized.
PMCID: PMC3970111  PMID: 23642247
data integration; model validation; systems biology; bioinformatics; biochemical networks; model design
6.  Murine Spinotrapezius Model to Assess the Impact of Arteriolar Ligation on Microvascular Function and Remodeling 
The murine spinotrapezius is a thin, superficial skeletal support muscle that extends from T3 to L4, and is easily accessible via dorsal skin incision. Its unique anatomy makes the spinotrapezius useful for investigation of ischemic injury and subsequent microvascular remodeling. Here, we demonstrate an arteriolar ligation model in the murine spinotrapezius muscle that was developed by our research team and previously published1-3. For certain vulnerable mouse strains, such as the Balb/c mouse, this ligation surgery reliably creates skeletal muscle ischemia and serves as a platform for investigating therapies that stimulate revascularization. Methods of assessment are also demonstrated, including the use of intravital and confocal microscopy. The spinotrapezius is well suited to such imaging studies due to its accessibility (superficial dorsal anatomy) and relative thinness (60-200 μm). The spinotrapezius muscle can be mounted en face, facilitating imaging of whole-muscle microvascular networks without histological sectioning. We describe the use of intravital microscopy to acquire metrics following a functional vasodilation procedure; specifically, the increase in arterilar diameter as a result of muscle contraction. We also demonstrate the procedures for harvesting and fixing the tissues, a necessary precursor to immunostaining studies and the use of confocal microscopy.
PMCID: PMC3622090  PMID: 23486360
Biomedical Engineering; Issue 73; Medicine; Anatomy; Physiology; Surgery; Immunology; Hematology; Microvessels; Capillaries; Arterioles; Venules; Vascular Diseases; Ischemia; spinotrapezius; peripheral vascular disease; functional vasodilation; arteriolar ligation; vessels; circulation; confocal microscopy; animal model
7.  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.
Thrombin or vehicle was locally applied to both ligated and non-operated Balb/c spinotrapezius muscles which were harvested after three or seven days, imaged using confocal microscopy, and analyzed.
Thrombin treatment resulted in accelerated arterialization of collateral capillaries and accelerated tissue reperfusion in ischemic muscles. Uninjured muscle treated with thrombin displayed increased vascular cell adhesion molecule 1 expression on arteriole and venule endothelium, increased expression of smooth muscle α-actin on capillary-sized vessels, increased infiltration by CD11b+ leukocytes, and mast cell infiltration and degranulation.
Exogenous delivery of thrombin enhances microvascular collateral development in response to ischemic insult and accelerates tissue reperfusion. Elicited responses from multiple cell types likely contribute to these effects.
PMCID: PMC3262124  PMID: 21954923
thrombin; arteriogenesis; collateral vessel; ischemia
8.  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.
Genetically manipulated mice with attenuated ephrinB2 reverse signaling (ephrinB2lacZ/+), along with wild-type (WT) controls, were exposed to oxygen-induced retinopathy (OIR), a postnatal model of proliferative retinopathy. At peak disease (postnatal day 18), microvascular networks were analyzed to examine intraretinal revascularization, capillary sprouting, and pathologic neovascularization responses. EphB4 and phosphorylated ephrinB protein expression patterns along retinal microvessels were also assessed.
EphrinB2lacZ/+ mice exhibited reduced hypoxia-induced revascularization (P ≤ 0.04) and reduced formation of neovascular tufts (P < 0.001), as compared with WT controls. Corresponding to the observed inhibition of retinal angiogenesis, ephrinB2lacZ/+ retinas displayed an increased number of blind-ended capillary sprout tips (P < 0.02) and endothelial filopodial processes (P = 0.001). In WT and ephrinB2lacZ/+ OIR-exposed retinas, ephrinB was confined to endothelial cells, with expression detected along angiogenic vascular processes including neovascular tufts and blind-ended capillary sprouts.
EphrinB2 reverse signaling is a regulator of key processes during retinal vascularization and controls pathologic retinal angiogenesis through direct effects on capillary sprouting and endothelial filopodia formation.
We have uncovered a novel role for ephrinB2 reverse signaling in modulating pathologic (hyperoxia induced) retinal angiogenesis using genetically manipulated mice with attenuated ephrinB2 reverse signaling.
PMCID: PMC3948501  PMID: 22789927
9.  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.
PMCID: PMC3185289  PMID: 22007175
angiogenesis; vascular development; computational model; mathematical model; sFlt-1; VEGF; capillary sprouting
10.  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.
Canonical Wnt inhibitor DKK-1, Wnt inhibitor sFRP-1, BSA or saline was superfused onto the exteriorized mesenteric windows of 300g adult female Sprague-Dawley rats for 20 minutes. Three days following surgery, mesenteric windows were imaged intravitally and harvested for immunofluorescence staining with smooth muscle alpha-actin and BRDU.
We observed prominent differences in the response of the mesenteric microvasculature amongst the various treatment groups. Significant increases in hemorrhage area, vascular density, and draining vessel diameter were observed in windows treated with Wnt inhibitors as compared to control-treated windows. Additionally, confocal imaging analysis showed significant increases in proliferating cells as well as evidence of proliferating smooth muscle cells along venules.
Together, our results suggest that canonical Wnt inhibition plays an important role in microvascular remodeling, specifically venular remodeling.
PMCID: PMC2904644  PMID: 20618692
Wnt; Dickkopf-1 (DKK-1); secreted frizzled-related protein 1 (sFRP-1); venular remodeling; hemorrhage
11.  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.
PMCID: PMC3125711  PMID: 18720228
angiogenesis; computational modeling; mathematical modeling; validation; multi-scale; validation; systems biology
12.  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.
PMCID: PMC3118494  PMID: 21720536
agent-based modeling; constrained mixture modeling; hypertension; vascular remodeling; multi-scale modeling
13.  Chronic Whole-body Hypoxia Induces Intussusceptive Angiogenesis and Microvascular Remodeling in the Mouse Retina 
Microvascular research  2010;79(2):93-101.
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-wk old female C57Bl/6 mice were exposed to 10% O2 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 α-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 ≤ 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.
PMCID: PMC2828864  PMID: 20080108
angiogenesis; hypoxia; intussusceptive microvascular growth; microvascular remodeling; mouse; retina
14.  Functional Binding of Human Adipose-Derived Stromal Cells: Effects of Extraction Method & Hypoxia Pretreatment 
Annals of plastic surgery  2008;60(4):437-444.
Human adipose-derived stromal cells (hASCs) were evaluated in vitro for their ability to bind vascular adhesion and extracellular matrix proteins in order 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/cm2). 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.
PMCID: PMC2829884  PMID: 18362576
Adipose-derived stromal cells; hypoxia; liposuction; parallel plate flow chamber; adhesion cascade
15.  IFATS Series: The Role of Human Adipose-Derived Stromal Cells in Inflammatory Microvascular Remodeling and Evidence of a Perivascular Phenotype 
Stem cells (Dayton, Ohio)  2008;26(10):2682-2690.
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 PDGF-BB and VEGF165 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. 10, 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 to 11% of injected human lung fibroblasts. hASCs exhibiting perivascular morphologies also expressed markers characteristic of vascular pericytes: smooth muscle α-actin (SMA) (10%) and NG2 (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.
PMCID: PMC2672107  PMID: 18436860
adipose-derived stromal cells; microcirculation; pericyte; angiogenesis
16.  Computational Network Model Prediction of Hemodynamic Alterations Due to Arteriolar Rarefaction and Estimation of Skeletal Muscle Perfusion in Peripheral Arterial Disease 
To estimate the relative influence of input pressure and arteriole rarefaction on gastrocnemius muscle perfusion in patients with peripheral arterial disease (PAD) after exercise and/or percutaneous interventions.
A computational network model of the gastrocnemius muscle microcirculation was adapted to reflect rarefaction based on arteriolar density measurements from PAD patients, with and without exercise. A normalized input pressure was applied at the feeder artery to simulate both reduced and restored ankle-brachial index (ABI) in the PAD condition.
In simulations of arteriolar rarefaction, resistance increased non-linearly with rarefaction, leading to a disproportionally large drop in perfusion. Additionally, perfusion was less sensitive to changes in input pressure as the degree of rarefaction increased. Reduced arteriolar density was observed in PAD patients and improved 33.8% after 3 months of exercise. In model simulations of PAD, ABI restoration yielded perfusion recovery to only 66% of baseline. When exercise training was simulated by reducing rarefaction, ABI restoration increased perfusion to 80% of baseline.
Microvascular resistance increases non-linearly with increasing arteriole rarefaction. Therefore, muscle perfusion becomes disproportionally less sensitive to ABI restoration as arteriole rarefaction increases. These results highlight the importance of restoring both microvascular structure and upstream input pressure in PAD therapy.
PMCID: PMC4506703  PMID: 25866235
rarefaction; microcirculation; network model; peripheral artery disease
17.  Multiscale models of skeletal muscle reveal the complex effects of muscular dystrophy on tissue mechanics and damage susceptibility 
Interface Focus  2015;5(2):20140080.
Computational models have been increasingly used to study the tissue-level constitutive properties of muscle microstructure; however, these models were not created to study or incorporate the influence of disease-associated modifications in muscle. The purpose of this paper was to develop a novel multiscale muscle modelling framework to elucidate the relationship between microstructural disease adaptations and modifications in both mechanical properties of muscle and strain in the cell membrane. We used an agent-based model to randomly generate new muscle fibre geometries and mapped them into a finite-element model representing a cross section of a muscle fascicle. The framework enabled us to explore variability in the shape and arrangement of fibres, as well as to incorporate disease-related changes. We applied this method to reveal the trade-offs between mechanical properties and damage susceptibility in Duchenne muscular dystrophy (DMD). DMD is a fatal genetic disease caused by a lack of the transmembrane protein dystrophin, leading to muscle wasting and death due to cardiac or pulmonary complications. The most prevalent microstructural variations in DMD include: lack of transmembrane proteins, fibrosis, fatty infiltration and variation in fibre cross-sectional area. A parameter analysis of these variations and case study of DMD revealed that the nature of fibrosis and density of transmembrane proteins strongly affected the stiffness of the muscle and susceptibility to membrane damage.
PMCID: PMC4342948  PMID: 25844152
multiscale; muscular dystrophy; agent-based; micromechanical; fibrosis; muscle
18.  Applications of computational models to better understand microvascular remodelling: a focus on biomechanical integration across scales 
Interface Focus  2015;5(2):20140077.
Microvascular network remodelling is a common denominator for multiple pathologies and involves both angiogenesis, defined as the sprouting of new capillaries, and network patterning associated with the organization and connectivity of existing vessels. Much of what we know about microvascular remodelling at the network, cellular and molecular scales has been derived from reductionist biological experiments, yet what happens when the experiments provide incomplete (or only qualitative) information? This review will emphasize the value of applying computational approaches to advance our understanding of the underlying mechanisms and effects of microvascular remodelling. Examples of individual computational models applied to each of the scales will highlight the potential of answering specific questions that cannot be answered using typical biological experimentation alone. Looking into the future, we will also identify the needs and challenges associated with integrating computational models across scales.
PMCID: PMC4342945  PMID: 25844149
microcirculation; angiogenesis; agent-based model; computational fluid dynamics; hypertension; multiscale computational model
19.  Monocytes are Recruited from Venules during Arteriogenesis in the Murine Spinotrapezius Ligation Model 
Chronic arterial occlusion results in arteriogenesis of collateral blood vessels. This process has been shown to be dependent upon the recruitment of growth-promoting macrophages to remodeling collaterals. However, the potential role of venules in monocyte recruitment during microvascular arteriogenesis is not well demonstrated. First, we aim to document that arteriogenesis occurs in the mouse spinotrapezius ligation model. Then, we investigate the temporal and spatial distribution, as well as proliferation, of monocytes/macrophages recruited to collateral arterioles in response to elevated fluid shear stress.
Approach and Results
Laser speckle flowmetry confirmed a post-ligation increase in blood velocity within collateral arterioles but not venules. After 72 hours post-ligation, collateral arteriole diameters were increased, proliferating cells were identified in vessel walls of shear-activated collaterals, and perivascular CD206+ macrophages demonstrated proliferation. An EdU assay identified proliferation. CD68+CD206+ cells around collaterals were increased 96%, while CX3CR1(+/GFP ) cells were increased 126% in ligated versus sham groups after 72 hours. CX3CR1(+/GFP ) cells were predominately venule-associated at 6 hours post-ligation; and CX3CR1(+/GFP hi) cells shifted from venule-associated to arteriole-associated between 6 and 72 hours post-surgery exclusively in ligated muscle. We report accumulation and extravasation of adhered CX3CR1(+/GFP) cells in and from venules, but not arterioles, following ligation.
Our results demonstrate that arteriogenesis occurs in the murine spinotrapezius ligation model and implicate post-capillary venules as the site of tissue entry for circulating monocytes. Local proliferation of macrophages also is documented. These data open up questions concerning the role of arteriole-venule communication during monocyte recruitment.
PMCID: PMC4373588  PMID: 24969773
Vascular biology; vascular remodeling; flow measurement; macrophage; monocyte
20.  Targeting Perciytes for Angiogenic Therapies 
In pathological scenarios, such as tumor growth and diabetic retinopathy, blocking angiogenesis would be beneficial. In others, such as myocardial infarction and hypertension, promoting angiogenesis might be desirable. Due to their putative influence on endothelial cells, vascular pericytes have become a topic of growing interest and are increasingly being evaluated as a potential target for angioregulatory therapies. For example, the strategy of manipulating pericyte recruitment to capillaries could result in anti- or pro-angiogenic effects. However, our current understanding of pericytes is limited by knowledge gaps regarding pericyte identity and lineage. To use a music analogy, this review is a “mash-up” that attempts to integrate what we know about pericyte functionality and expression with what is beginning to be elucidated regarding their regenerative potential. We explore the lingering questions regarding pericyte phenotypic identity and lineage. The expression of different pericyte markers (e.g., SMA, Desmin, NG2 and PDGFR-β) varies for different subpopulations and tissues. Previous use of these markers to identify pericytes has suggested potential phenotypic overlaps and plasticity toward other cell phenotypes. Our review chronicles the state of the literature, identifies critical unanswered questions, and motivates future research aimed at understanding this intriguing cell type and harnessing its therapeutic potential.
PMCID: PMC4079092  PMID: 24267154
pericytes; stem cell; angiogenesis; therapy
21.  Human adipose-derived stromal/stem cells demonstrate short-lived persistence after implantation in both an immunocompetent and an immunocompromised murine model 
Mesenchymal cells are emerging as a promising cell platform for regenerative therapies. However, the fate of cells after transplantation in many different disease settings and tissue beds remains unclear.
In this study, human adipose-derived stromal/stem (ASCs) cells were fluorescently labeled with a membrane dye and injected into both immunocompetent and immunocompromised mouse strains. Cells were injected either as single cell suspensions, or as self-assembling spheroids. In parallel, cells were purposefully devitalized prior to injection and then implanted in the opposite side in a randomized fashion. These ‘control’ groups were included to determine whether the fluorescent membrane dye would remain localized at the injection site despite the use of nonviable cells. Cell implants and the surrounding tissues were harvested on days 3, 10 and 21 after in vivo delivery and evaluated in a blinded manner. Injection sites were analyzed by fluorescent microscopy, and human cell numbers were quantified using PCR detection of a human-specific endogenous retrovirus (ERV-3). Host response was evaluated by immunofluorescent staining of macrophages.
ERV-3 quantification showed that 95% of the human cells that were viable when they were injected were undetectable at the three-week time-point. Although fluorescent signal persisted for the entire study period, further analysis revealed that much of this signal was located within host macrophages.
These results suggest that human ASCs survive for less than three weeks after injection into even immunocompromised mice, and call into question the notion that human ASCs are immuno-privileged and capable of surviving for extended periods in xenogeneic and/or allogeneic models.
PMCID: PMC4445497  PMID: 25523792
23.  Pericytes Derived from Adipose-Derived Stem Cells Protect against Retinal Vasculopathy 
PLoS ONE  2013;8(5):e65691.
Retinal vasculopathies, including diabetic retinopathy (DR), threaten the vision of over 100 million people. Retinal pericytes are critical for microvascular control, supporting retinal endothelial cells via direct contact and paracrine mechanisms. With pericyte death or loss, endothelial dysfunction ensues, resulting in hypoxic insult, pathologic angiogenesis, and ultimately blindness. Adipose-derived stem cells (ASCs) differentiate into pericytes, suggesting they may be useful as a protective and regenerative cellular therapy for retinal vascular disease. In this study, we examine the ability of ASCs to differentiate into pericytes that can stabilize retinal vessels in multiple pre-clinical models of retinal vasculopathy.
Methodology/Principal Findings
We found that ASCs express pericyte-specific markers in vitro. When injected intravitreally into the murine eye subjected to oxygen-induced retinopathy (OIR), ASCs were capable of migrating to and integrating with the retinal vasculature. Integrated ASCs maintained marker expression and pericyte-like morphology in vivo for at least 2 months. ASCs injected after OIR vessel destabilization and ablation enhanced vessel regrowth (16% reduction in avascular area). ASCs injected intravitreally before OIR vessel destabilization prevented retinal capillary dropout (53% reduction). Treatment of ASCs with transforming growth factor beta (TGF-β1) enhanced hASC pericyte function, in a manner similar to native retinal pericytes, with increased marker expression of smooth muscle actin, cellular contractility, endothelial stabilization, and microvascular protection in OIR. Finally, injected ASCs prevented capillary loss in the diabetic retinopathic Akimba mouse (79% reduction 2 months after injection).
ASC-derived pericytes can integrate with retinal vasculature, adopting both pericyte morphology and marker expression, and provide functional vascular protection in multiple murine models of retinal vasculopathy. The pericyte phenotype demonstrated by ASCs is enhanced with TGF-β1 treatment, as seen with native retinal pericytes. ASCs may represent an innovative cellular therapy for protection against and repair of DR and other retinal vascular diseases.
PMCID: PMC3669216  PMID: 23741506
24.  Ensuring Congruency in Multiscale Modeling: Towards Linking Agent Based and Continuum Biomechanical Models of Arterial Adaptation 
Annals of biomedical engineering  2011;39(11):2669-2682.
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.
PMCID: PMC3207323  PMID: 21809144
Agent Based Model (ABM); Constrained Mixture Model (CMM); Growth and Remodeling; Model Verification
25.  Rat Mesentery Exteriorization: A Model for Investigating the Cellular Dynamics Involved in Angiogenesis 
Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease conditions1, 2. Network growth is commonly attributed to angiogenesis, defined as the growth of new vessels from pre-existing vessels. The angiogenic process is also directly linked to arteriogenesis, defined as the capillary acquisition of a perivascular cell coating and vessel enlargement. Needless to say, angiogenesis is complex and involves multiple players at the cellular and molecular level3. Understanding how a microvascular network grows requires identifying the spatial and temporal dynamics along the hierarchy of a network over the time course of angiogenesis. This information is critical for the development of therapies aimed at manipulating vessel growth.
The exteriorization model described in this article represents a simple, reproducible model for stimulating angiogenesis in the rat mesentery. It was adapted from wound-healing models in the rat mesentery4-7, and is an alternative to stimulate angiogenesis in the mesentery via i.p. injections of pro-angiogenic agents8, 9. The exteriorization model is attractive because it requires minimal surgical intervention and produces dramatic, reproducible increases in capillary sprouts, vascular area and vascular density over a relatively short time course in a tissue that allows for the two-dimensional visualization of entire microvascular networks down to single cell level. The stimulated growth reflects natural angiogenic responses in a physiological environment without interference of foreign angiogenic molecules. Using immunohistochemical labeling methods, this model has been proven extremely useful in identifying novel cellular events involved in angiogenesis. Investigators can readily correlate the angiogenic metrics during the time course of remodeling with time specific dynamics, such as cellular phenotypic changes or cellular interactions4, 5, 7, 10, 11.
PMCID: PMC3466932  PMID: 22643964
Cellular Biology;  Issue 63;  mesentery;  rat;  angiogenesis;  microcirculation;  microvascular;  remodeling

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