The delivery of bone marrow-derived cells (BMDCs) has been widely used to stimulate angiogenesis and arteriogenesis. We identified a progenitor-enriched subpopulation of BMDCs that is able to augment venular remodeling, a generally unexplored area in microvascular research. Two populations of BMDCs, whole bone marrow (WBM) and Lin(-)/Sca-1(+) progenitor cells, were encapsulated in sodium alginate and delivered to a mouse dorsal skinfold chamber model. Upon observation that encapsulated Sca-1(+) progenitor cells enhance venular remodeling, the cells and tissue were analyzed on structural and molecular levels. Venule walls were thickened and contained more nuclei after Sca-1(+) progenitor cell delivery. In addition, progenitors expressed mRNA transcript levels of chemokine (C-X-C motif) ligand 2 (CXCL2) and interferon gamma (IFNγ) that are over 5-fold higher compared to WBM. Tissues that received progenitors expressed significantly higher protein levels of vascular endothelial growth factor (VEGF), monocyte chemotactic protein-1 (MCP-1), and platelet derived growth factor-BB (PDGF-BB) compared to tissues that received an alginate control construct. Nine days following cell delivery, tissue from progenitor recipients contained 39% more CD45(+) leukocytes, suggesting that these cells may enhance venular remodeling through the modulation of the local immune environment. Results show that different BMDC populations elicit different microvascular responses. In this model, Sca-1(+) progenitor cell-derived CXCL2 and IFNγ may mediate venule enlargement via modulation of the local inflammatory environment.

Early alterations in the skeletal muscle microvasculature may contribute to the onset and progression of type 2 diabetes (DM2) by limiting insulin and glucose availability to skeletal muscle. Microvascular alterations reported with DM2 are numerous and include impaired endothelium-mediated vasodilation, increased arteriole wall stiffness, and decreased capillary density. Most previous analyses of skeletal muscle microvascular architecture have been limited to skeletal muscle cross sections and thus have not presented an integrated, quantitative analysis of the relative significance of observed alterations to elevated microvascular network resistance and decreased blood flow. In this work, we tested the hypothesis that the onset of diabetes would influence microvascular architecture in a manner that would significantly increase capillary network resistance and reduce blood flow. In whole-mount spinotrapezius muscle capillary networks from Zucker diabetic fatty (ZDF) rats before and after the onset of DM2, we found a significant 37% decrease in microvascular branching and a 19% decrease in microvessel length density associated with the onset of the disease. This was previously indiscernible in skeletal muscle cross-section data. Hemodynamic computational analysis revealed that the changes in DM2 capillary network connectivity result in a significant 44% decrease in computed capillary network flow compared to controls. A hemodynamic sensitivity analysis showed that DM2 networks were predicted to be less robust in their ability to maintain perfused network surface area in the event of upstream terminal arteriole constriction. This study illustrates that capillary network connectivity is altered by DM2 and this negatively impacts microvascular hemodynamics. This work can serve as a basis for a more quantitative approach to evaluating DM2 microvascular networks and their potential use as an early diagnostic aid and/or method for identifying therapeutic targets.

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.

HASH(0x25a82300)

OBJECTIVE: Bone marrow-derived cells (BMCs) and inflammatory chemokine receptors regulate arteriogenesis and angiogenesis. Here, we tested whether arteriolar remodeling in response to an inflammatory stimulus is dependent on BMC-specific chemokine (C-C motif) receptor 2 (CCR2) expression and whether this response involves BMC transdifferentiation into smooth muscle. METHODS AND RESULTS: Dorsal skinfold window chambers were implanted into C57Bl/6 wild-type (WT) mice, as well as the following bone marrow chimeras (donor-host): WT-WT, CCR2(-/-)-WT, WT-CCR2(-/-), and EGFP(+)-WT. One day after implantation, tissue MCP-1 levels rose from "undetectable" to 463pg/mg, and the number of EGFP(+) cells increased more than 4-fold, indicating marked inflammation. A 66%(28 microm) increase in maximum arteriolar diameter was observed over 7 days in WT-WT mice. This arteriolar remodeling response was completely abolished in CCR2(-/-)-WT mice but largely rescued in WT-CCR2(-/-) mice. EGFP(+) BMCs were numerous throughout the tissue, but we found no evidence that EGFP(+) BMCs transdifferentiate into smooth muscle, based on examination of >800 arterioles and venules. CONCLUSIONS: BMC-specific CCR2 expression is required for injury/inflammation-associated arteriolar remodeling, but this response is not characterized by the differentiation of BMCs into smooth muscle.

External application of static magnetic fields, utilized specifically for the treatment of inflammatory conditions such as soft tissue injuries, has recently become popular as a complementary and/or alternative therapy with minimal investigation into efficacy or mechanism. Localized inflammation was induced via injection of inflammatory agents lambda-carrageenan (CA) or histamine into rat hindpaws, alone or in conjunction with pharmacologic agents, resulting in a spatially and temporally defined inflammatory reaction. Application of a 10mT or 70mT, but not a 400mT SMF for 15 or 30 minutes immediately following histamine-induced edema resulted in a significant, 20-50% reduction in edema formation. Additionally, a 2 hour, 70mT field application to CA-induced edema also resulted in significant (33-37%) edema reduction. Field application before injection or at the time of maximal edema did not influence edema formation or resolution, respectively. Together these results suggest the existence of a therapeutic threshold of SMF strength (below 400mT) and a temporal dependence of efficacy. Administration of pharmacologic agents directed at NO signaling and L-type Ca(2+) channel dynamics in conjunction with SMF treatment and histamine-induced edema revealed that the potential mechanism of SMF action may be via modulation of vascular tone through effects on L-type Ca(2+) channels in vascular smooth muscle cells. Key words: Static Magnetic Field, Edema, Microvascular Tone.

Magnetic field therapy has recently become a widely used complementary/alternative medicine for the treatment of vascular as well as other musculoskeletal pathologies including soft tissue injuries. Recent studies in our laboratory and others have suggested that acute static magnetic field (SMF) exposure can have a modulatory influence on the microvasculature, acting to normalize vascular function; however, the effect of chronic SMF exposure has not been investigated. This study aimed to measure, for the first time, the adaptive microvascular response to a chronic, 7-day continuous magnetic field exposure. Murine dorsal skinfold chambers were applied on day 0 and neodymium static magnets (or size and weight matched shams) were affixed to the chambers at day 0, where they remained until day 7. Separate analysis of arteriolar and venular diameters revealed that chronic SMF application significantly abrogated the luminal diameter expansion observed in sham treated networks. Magnet treated venular diameters were significantly reduced at day 4 and day 7 (34.3% and 54.4%, respectively) as compared to sham treated vessels. Arteriolar diameters were also significantly reduced by magnet treatment at day 7 (50%) but not significantly at day 4 (31.6%), although the same trend was evident. Venular functional length density was also significantly reduced by chronic field application (60%). These results suggest that chronic SMF exposure can alter the adaptive microvascular remodeling response to mechanical injury, thus supporting the further study of chronic application of static magnetic fields for the therapeutic treatment of vascular pathologies involving the dysregulation of microvascular structure. Key words: static magnetic field, microvascular, remodeling.

Objective: 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.Methods: The distribution of NG2 expression was evaluated in adult rat mesenteric microvascular networks. Tissues were harvested from 250 g, female, Sprague-Dawley rats at 1, 3, and 5 days poststimulation and double immunolabeled for NG2 and CD31 (endothelial cell marker).Results: After 1 day, NG2 expression was observed along 27 +/- 11% of network draining venules (14-55 microm) and after 3 days, 59 +/- 10% of draining venules (13-59 microm) stained positive for the proteoglycan. By 5 days poststimulation, the percentage of network draining venules (18-59 microm) staining positive for NG2 returned to 18 +/- 7%, indicating a downregulation of the proteoglycan toward quiescent levels along larger-sized venules.Conclusions: The results suggest that NG2 proteoglycan expression is transiently upregulated along venules during microvascular remodeling, implicating NG2 as a marker of activated venules.

Bone marrow-derived cells (BMCs) have been implicated as a modifiers of vascular growth either directly by transdifferentiation into endothelial cells (ECs) or indirectly through growth factor release. To examine these possibilities under physiological conditions, we developed a model of hypoxia-mediated angiogenesis in the mouse spinotrapezius muscle. This allows whole-mount analysis; therefore, the morphology and location of BMCs within the vascular network may be observed along with differentiation markers. We exposed bone marrow transplant chimeric mice to hypoxia and treated a subset with granulocyte macrophage colony-stimulating factor. Exposure to hypoxia caused an 13% increase in capillary density relative to control. Hypoxia did not increase the overall number of muscle-resident BMCs, but did increase the number of rounded BMCs by 25%. There was no discernable BMC contribution to the endothelium, although some BMCs assumed a pericyte morphology around capillaries. Granulocyte macrophage colony-stimulating factor treatment further increased the number of round BMCs within the muscle and caused a 23% increase in angiogenesis. The results of this study suggest a potentially beneficial action of BMCs during hypoxia through paracrine release of growth factors but not transdifferentiation into ECs.

OBJECTIVE: 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. METHODS: Mesenteric tissue, subcutaneous tissue, spinotrapezius muscle, and gracilis muscle were harvested from 250-g, female, Sprague-Dawley rats and stained for NG2 and SM alpha-actin. The distribution of NG2 expression was evaluated in mesenteric networks (n = 28) with complementary observations in subcutaneous tissue and skeletal muscle. RESULTS: Perivascular cells, including mature smooth muscle cells (SMCs), immature SMCs, and pericytes, expressed NG2. Most importantly, NG2 expression was primarily confined to perivascular cells along arterioles and capillaries, and continuous expression was not observed along venules beyond the immediate postcapillary vessels. The differential expression of NG2 along the arteriolar side of microvascular networks was also observed in rat subcutaneous and skeletal muscle. CONCLUSIONS: The results indicate that NG2 is expressed by all perivascular cells along arterioles, and its absence denotes a venule-specific phenotype. These results identify for the first time a marker that differentiates venous smooth muscle and pericytes from other capillary- and arteriole-associated perivascular cells.