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1.  Arteriolar Remodeling Following Ischemic Injury Extends from Capillary to Large Arteriole in the Microcirculation 
Objective
During ischemia, vascular beds in skeletal muscle have been shown to undergo arteriolar remodeling, or arteriogenesis, in order to restore tissue perfusion and function. This process has traditionally been thought to occur predominately in large vessels (diameters > 50 μm), although recent studies showing arteriogenesis in the microcirculation (diameters < 35 μm) following ultrasound-induced microbubble destruction suggest this may occur during skeletal muscle ischemia, as well. Using a surgical model of ischemia that allows en face visualization of microvasculature, we tested the hypothesis that ischemic injury induces arteriolar remodeling in the skeletal muscle microcirculation on the scale of capillary to sub-35 μm diameter arterioles.
Methods
Surgical ligations of the main feeding arteriole (70 μm in diameter) to the caudal-half of the spinotrapezius muscle were performed on age- and weight-matched C57BL/6 mice. The microvascular remodeling response to the ischemic insult, including enlargement and formation of new arterioles, as well as degree of vascular branching and collateral formation, were then quantified and compared to contralateral control muscles using intravital and whole-mount confocal microscopy. Immunohistochemical techniques were used to verify the presence of inflammatory cells (monocytes and tissue-resident macrophages; MOMA-2+ or CD11b+), as well as the absence of chronic hypoxia (Hypoxyprobe-1+ kit; Chemicon International).
Results
Five days post-arteriole ligation, ischemic tissue underwent reproducible and localized microvascular remodeling characteristic of arteriogenesis. Using intravital microscopy and examining functional vessels (arterioles and venules) with diameters 15–35 μm, we observed significant increases in vascular density (38%), branching (90%) and collateral development (36.5%). The formation of new arterioles (diameters 6–35 μm) was quantified and significantly increased, as evidenced by expanded smooth-muscle α-actin (24.3%) arteriolar-coverage. However, arcade arteriole (AA) length densities did not significantly increase following ligation. The absence of chronic hypoxia and pronounced vessel tortuosity was also consistently observed.
Conclusions
Ischemic ligations induce arteriolar remodeling responses in the microcirculation (vessel diameters < 35 μm) of the spinotrapezius muscle in the C57BL/6 mouse. Furthermore, the surgical model that allowed this quantification enabled en face analysis of skeletal muscle microvascular network adaptations with single-cell resolution and has the capability to provide investigators with functional and morphometric data on a microscale difficult to achieve using other animal models.
doi:10.1080/10739680701708436
PMCID: PMC3129987  PMID: 18574742
spinotrapezius; hindlimb; animal model; ischemia; arteriogenesis; arterialization; collateral; vascular remodeling; microcirculation; microvessel; hypoxia; arteriole; monocyte; arcade; transverse
2.  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.
doi:10.3791/50218
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
3.  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.
doi:10.1097/SAP.0b013e318095a771
PMCID: PMC2829884  PMID: 18362576
Adipose-derived stromal cells; hypoxia; liposuction; parallel plate flow chamber; adhesion cascade
4.  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.
doi:10.1634/stemcells.2008-0030
PMCID: PMC2672107  PMID: 18436860
adipose-derived stromal cells; microcirculation; pericyte; angiogenesis
5.  Agent-Based Model of Therapeutic Adipose-Derived Stromal Cell Trafficking during Ischemia Predicts Ability To Roll on P-Selectin 
PLoS Computational Biology  2009;5(2):e1000294.
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 µm/s. Thus, our work led to a fundamentally new understanding of hASC biology, which may have important therapeutic implications.
Author Summary
Ischemic pathologies, such as acute myocardial infarction and peripheral vascular disease, continue to be associated with high morbidities and mortalities. Recently, therapies wherein adult stem cells are injected into the circulation have been shown to increase blood flow and help to restore tissue function following injury. Pre-clinical animal models and human trials have shown successes utilizing this approach, but variable trafficking efficiencies and low incorporation of cells into the injured tissue severely limit effectiveness and may preclude clinical adoption. To address this, we sought to study the complex process of how injected stem cells traffic through the microcirculation and home to sites of injury, in an effort to identify bottlenecks in this process that could be manipulated for therapeutic gain. We developed an agent-based computer model to speed the rate of discovery, and we identified a key cell–cell adhesion interaction that could be targeted to enhance stem cell homing efficiencies during injectable stem cell therapies.
doi:10.1371/journal.pcbi.1000294
PMCID: PMC2636895  PMID: 19247427

Results 1-5 (5)