Lino F. Miele

Angiogenesis in Wounds Treated by Microdeformational Wound Therapy

BACKGROUND Mechanical forces play an important role in tissue neovascularization and are a constituent part of modern wound therapies. The mechanisms by which vacuum assisted closure (VAC) modulates wound angiogenesis are still largely unknown. OBJECTIVE To investigate how VAC treatment affects wound hypoxia and related profiles of angiogenic factors as well as to identify the anatomical characteristics of the resultant, newly formed vessels. METHODS Wound neovascularization was evaluated by morphometric analysis of CD31-stained wound cross-sections as well as by corrosion casting analysis. Wound hypoxia and mRNA expression of HIF-1α and associated angiogenic factors were evaluated by pimonidazole hydrochloride staining and quantitative reverse transcription-polymerase chain reaction (RT-PCR), respectively. Vascular endothelial growth factor (VEGF) protein levels were determined by western blot analysis. RESULTS VAC-treated wounds were characterized by the formation of elongated vessels aligned in parallel and consistent with physiological function, compared to occlusive dressing control wounds that showed formation of tortuous, disoriented vessels. Moreover, VAC-treated wounds displayed a well-oxygenated wound bed, with hypoxia limited to the direct proximity of the VAC-foam interface, where higher VEGF levels were found. By contrast, occlusive dressing control wounds showed generalized hypoxia, with associated accumulation of HIF-1α and related angiogenic factors. CONCLUSIONS The combination of established gradients of hypoxia and VEGF expression along with mechanical forces exerted by VAC therapy was associated with the formation of more physiological blood vessels compared to occlusive dressing control wounds. These morphological changes are likely a necessary condition for better wound healing.

Dynamic determination of oxygenation and lung compliance in murine pneumonectomy

ABSTRACT Thoracic surgical procedures in mice have been applied to a wide range of investigations, but little is known about the murine physiologic response to pulmonary surgery. Using continuous arterial oximetry monitoring and the FlexiVent murine ventilator, the authors investigated the effect of anesthesia and pneumonectomy on mouse oxygen saturation and lung mechanics. Sedation resulted in a dose-dependent decline of oxygen saturation that ranged from 55% to 82%. Oxygen saturation was restored by mechanical ventilation with increased rate and tidal volumes. In the mouse strain studied, optimal ventilatory rates were a rate of 200/minute and a tidal volume of 10 mL/kg. Sustained inflation pressures, referred to as a “recruitment maneuver,” improved lung volumes, lung compliance, and arterial oxygenation. In contrast, positive end-expiratory pressure (PEEP) had a detrimental effect on oxygenation; an effect that was ameliorated after pneumonectomy. These results confirm that lung volumes in the mouse are dynamically determined and suggest a threshold level of mechanical ventilation to maintain perioperative oxygen saturation.

Cross-circulation and cell distribution kinetics in parabiotic mice.

Blood‐borne nucleated cells participate not only in inflammation, but in tissue repair and regeneration. Because progenitor and stem cell populations have a low concentration in the blood, the circulation kinetics and tissue distribution of these cells is largely unknown. An important approach to tracking cell lineage is the use of fluorescent tracers and parabiotic models of cross‐circulation. Here, we investigated the cross‐circulation and cell distribution kinetics of C57/B6 GFP+/wild‐type parabionts. Flow cytometry analysis of the peripheral blood after parabiosis demonstrated no evidence for a “parabiotic barrier” based on cell size or surface characterstics; all peripheral blood cell subpopulations in this study reached equilibrium within 14 days. Whole blood fluorescence analysis indicated that the mean exchange flow rate was 16 µl/h or 0.66% of the circulating blood volume per hour. Studies of peripheral lymphoid organs indicated differential cell distribution kinetics. Some subpopulations, such as CD8+ and CD11c+, equilibrated in both lymph nodes and spleen indicating a residence time <28 days; in contrast, other lymphocyte subpopulations, such as B220+ and CD4+ cells, had not yet reached equilibrium at 28 days. We conclude that parabiosis can provide important insights into defining tissue distribution, residence times, and recirculating pools using fluorochrome markers of cell lineage. J. Cell. Physiol. 227: 821–828, 2012.

Blood flow shapes intravascular pillar geometry in the chick chorioallantoic membrane

The relative contribution of blood flow to vessel structure remains a fundamental question in biology. To define the influence of intravascular flow fields, we studied tissue islands--here defined as intravascular pillars--in the chick chorioallantoic membrane. Pillars comprised 0.02 to 0.5% of the vascular system in 2-dimensional projection and were predominantly observed at vessel bifurcations. The bifurcation angle was generally inversely related to the length of the pillar (R = -0.47, P < .001). The pillar orientation closely mirrored the axis of the dominant vessel with an average variance of 5.62 ± 6.96 degrees (p = .02). In contrast, the variance of pillar orientation relative to nondominant vessels was 36.78 ± 21.33 degrees (p > .05). 3-dimensional computational flow simulations indicated that the intravascular pillars were located in regions of low shear stress. Both wide-angle and acute-angle models mapped the pillars to regions with shear less than 1 dyn/cm2. Further, flow modeling indicated that the pillars were spatially constrained by regions of higher wall shear stress. Finally, the shear maps indicated that the development of new pillars was limited to regions of low shear stress. We conclude that mechanical forces produced by blood flow have both a limiting and permissive influence on pillar development in the chick chorioallantoic membrane.

A morphometric study of mechanotransductively induced dermal neovascularization.

Background: Mechanical stretch has been shown to induce vascular remodeling and increase vessel density, but the pathophysiologic mechanisms and the morphologic changes induced by tensile forces to dermal vessels are poorly understood. Methods: A custom computer-controlled stretch device was designed and applied to the backs of C57BL/6 mice (n = 38). Dermal and vascular remodeling was studied over a 7-day period. Corrosion casting and three-dimensional scanning electron microscopy and CD31 staining were performed to analyze microvessel morphology. Hypoxia was assessed by immunohistochemistry. Western blot analysis of vascular endothelial growth factor (VEGF) and mRNA expression of VEGF receptors was performed. Results: Skin stretching was associated with increased angiogenesis as demonstrated by CD31 staining and vessel corrosion casting where intervascular distance and vessel diameter were decreased (p < 0.01). Immediately after stretching, VEGF dimers were increased. Messenger RNA expression of VEGF receptor 1, VEGF receptor 2, neuropilin 1, and neuropilin 2 was increased starting as early as 2 hours after stretching. Highly proliferating epidermal cells induced epidermal hypoxia starting at day 3 (p < 0.01). Conclusions: Identification of significant hypoxic cells occurred after identification of neovessels, suggesting an alternative mechanism. Increased expression of angiogenic receptors and stabilization of VEGF dimers may be involved in a mechanotransductive, prehypoxic induction of neovascularization.

Spatial calibration of structured illumination fluorescence microscopy using capillary tissue phantoms.

Quantitative assessment of microvascular structure is relevant to the investigations of ischemic injury, reparative angiogenesis and tumor revascularization. In light microscopy applications, thick tissue specimens are necessary to characterize microvascular networks; however, thick tissue leads to image distortions due to out‐of‐focus light. Structured illumination confocal microscopy is an optical sectioning technique that improves contrast and resolution by using a grid pattern to identify the plane‐of‐focus within the specimen. Because structured illumination can be applied to wide‐field (nonscanning) microscopes, the microcirculation can be studied by sequential intravital and confocal microscopy. To assess the application of structured illumination confocal microscopy to microvessel imaging, we studied cell‐sized microspheres and fused silica microcapillary tissue phantoms. As expected, structured illumination produced highly accurate images in the lateral (X‐Y) plane, but demonstrated a loss of resolution in the Z‐Y plane. Because the magnitude of Z‐axis distortion was variable in complex tissues, the silica microcapillaries were used as spatial calibration standards. Morphometric parameters, such as shape factor, were used to empirically optimize Z‐axis software compression. We conclude that the silica microcapillaries provide a useful tissue phantom for in vitro studies as well as spatial calibration standard for in vivo morphometry of the microcirculation. Microsc. Res. Tech., 2009.

Effect of intraluminal pillars on particle motion in bifurcated microchannels

A central feature of intussusceptive angiogenesis is the development of an intravascular pillar that bridges the opposing sides of the microvessel lumen. In this report, we created polydimethyl siloxane (PDMS) microchannels with geometric proportions based on corrosion casts of the colon microcirculation. The structure of the PDMS microchannels was a bifurcated channel with an intraluminal pillar in the geometric center of the bifurcation. The effect of the intraluminal pillar on particle flow paths was investigated using an in vitro perfusion system. The microchannels were perfused with fluorescent particles, and the particle movements were recorded using fluorescence videomicroscopy. We found that the presence of an intravascular pillar significantly decreased particle velocity in the bifurcation system (p < 0.05). In addition, the pillar altered the trajectory of particles in the center line of the flow stream. The particle trajectory resulted in prolonged pillar contact as well as increased residence time within the bifurcation system (p < 0.001). Our results suggest that the intravascular pillar not only provides a mechanism of increasing resistance to blood flow but may also participate in spatial redistribution of cells within the flow stream.

Vascular Microarchitecture of Murine Colitis-Associated Lymphoid Angiogenesis

In permissive tissues, such as the gut and synovium, chronic inflammation can result in the ectopic development of anatomic structures that resemble lymph nodes. These inflammation‐induced structures, termed lymphoid neogenesis or tertiary lymphoid organs, may reflect differential stromal responsiveness to the process of lymphoid neogenesis. To investigate the structural reorganization of the microcirculation involved in colonic lymphoid neogenesis, we studied a murine model of dextran sodium sulfate (DSS)‐induced colitis. Standard 2‐dimensional histology demonstrated both submucosal and intramucosal lymphoid structures in DSS‐induced colitis. A spatial frequency analysis of serial histologic sections suggested that most intramucosal lymphoid aggregates developed de novo. Intravital microscopy of intravascular tracers confirmed that the developing intramucosal aggregates were supplied by capillaries arising from the quasi‐polygonal mucosal plexus. Confocal optical sections and whole mount morphometry demonstrated capillary networks (185 ± 46 μm diameter) involving six to ten capillaries with a luminal diameter of 6.8 ± 1.1 μm. Microdissection and angiogenesis PCR array analysis demonstrated enhanced expression of multiple angiogenic genes including CCL2, CXCL2, CXCL5, Il‐1b, MMP9, and TNF within the mucosal plexus. Intravital microscopy of tracer particle flow velocities demonstrated a marked decrease in flow velocity from 808 ± 901 μm/sec within the feeding mucosal plexus to 491 ± 155 μm/sec within the capillary structures. We conclude that the development of ectopic lymphoid tissue requires significant structural remodeling of the stromal microcirculation. A feature of permissive tissues may be the capacity for lymphoid angiogenesis. Anat Rec, 292:621–632, 2009.

Abstract 121: Evaluation of Otology Outcomes After Surgical Treatment of Symptomatic Pierre Robin Sequence: A Cohort Comparison Study Between Furlow Palatoplasty vs. Radical Intravelar Veloplasty.

morbidity or SSO in breast cancer patients undergoing mastectomy with or without immediate reconstruction. This results suggest that NRT is not a contraindication to immediate breast reconstruction, and provide a strong basis for future prospective studies to assess long-term morbidity and survival associated with NRT. 121 evaluation of otology outcomes after Surgical treatment of Symptomatic Pierre robin Sequence: a cohort comparison Study Between Furlow Palatoplasty vs. radical intravelar Veloplasty

160A: MECHANOTRANSDUCTIVE INDUCED RECEPTOR MODULATION AS A PREHYPOXIC MECHANISM OF NEOVASCULARIZATION

Methods: A custom computer controlled stretch device was designed and applied to the backs of C57BL/6 mice (n=38). Mice were stretched continuously with a 50 g force. Seven days after stretching, corrosion casting and three-dimensional (3D) scanning electron microscopy (SEM) were performed to analyze microvessel morphology. Hypoxia was assessed by immunhistochemistry using pimonidazole hydrochloride as an in vivo marker. Western blot analysis of VEGF and mRNA expression of vascular endothelial growth factor receptor 1 (VEGFR1) and 2 (VEGFR2), neuropilin receptor 1 (NP1) and 2 (NP2) was performed.