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1.  Phthalimide neovascular factor 1 (PNF1) modulates MT1-MMP activity in human microvascular endothelial cells 
Biotechnology and bioengineering  2009;103(4):796-807.
We are creating synthetic pharmaceuticals with angiogenic activity and potential to promote vascular invasion. We previously demonstrated that one of these molecules, phthalimide neovascular factor 1 (PNF1), significantly expands microvascular networks in vivo following sustained release from poly(lactic-co-glycolic acid) (PLAGA) films. In addition, to probe PNF1 mode-of-action, we recently applied a novel pathway-based compendium analysis to a multi-timepoint, controlled microarray dataset of PNF1-treated (versus control) human microvascular endothelial cells (HMVECs), and we identified induction of tumor necrosis factor-alpha (TNF-α) and, subsequently, transforming growth factor-beta (TGF-β) signaling networks by PNF1. Here we validate this microarray data-set with quantitative real-time polymerase chain reaction (RT-PCR) analysis. Subsequently, we probe this dataset and identify three specific TGF-β-induced genes with regulation by PNF1 conserved over multiple timepoints—amyloid beta (A4) precursor protein (APP), early growth response 1 (EGR-1), and matrix metalloproteinase 14 (MMP14 or MT1-MMP)—that are also implicated in angiogenesis. We further focus on MMP14 given its unique role in angiogenesis, and we validate MT1-MMP modulation by PNF1 with an in vitro fluorescence assay that demonstrates the direct effects that PNF1 exerts on functional metalloproteinase activity. We also utilize endothelial cord formation in collagen gels to show that PNF1-induced stimulation of endothelial cord network formation in vitro is in some way MT1-MMP-dependent. Ultimately, this new network analysis of our transcriptional footprint characterizing PNF1 activity 1–48 h post-supplementation in HMVECs coupled with corresponding validating experiments suggests a key set of a few specific targets that are involved in PNF1 mode-of-action and important for successful promotion of the neovascularization that we have observed by the drug in vivo.
doi:10.1002/bit.22310
PMCID: PMC2711776  PMID: 19326468
Network analysis; transcriptional profiling; angiogenesis; matrix metalloproteinase; small molecule; drug discovery
2.  Expansion of microvascular networks in vivo by phthalimide neovascular factor 1 (PNF1) 
Biomaterials  2008;29(35):4698-4708.
Phthalimide neovascular factor (PNF1, formerly SC-3-149) is a potent stimulator of proangiogenic signaling pathways in endothelial cells. In this study, we evaluated the in vivo effects of sustained PNF1 release to promote ingrowth and expansion of microvascular networks surrounding biomaterial implants. The dorsal skinfold window chamber was used to evaluate the structural remodeling response of the local microvasculature. PNF1 was released from poly(lactic-co-glycolic acid) (PLAGA) films, and a transport model was utilized to predict PNF1 penetration into the surrounding tissue. PNF1 significantly expanded microvascular networks within a 2 mm radius from implants after 3 and 7 days by increasing microvessel length density and lumenal diameter of local arterioles and venules. Staining of histological sections with CD11b showed enhanced recruitment of circulating white blood cells, including monocytes, which are critical for the process of vessel enlargement through arteriogenesis. As PNF1 has been shown to modulate MT1-MMP, a facilitator of CCL2 dependent leukocyte transmigration, aspects of window chamber experiments were repeated in CCR2−/− (CCL2 receptor) mouse chimeras to more fully explore the critical nature of monocyte recruitment on the therapeutic benefits of PNF1 function in vivo.
doi:10.1016/j.biomaterials.2008.08.029
PMCID: PMC2885000  PMID: 18804278
Small molecule delivery; Controlled release; Microvascular remodeling; Monocyte recruitment
3.  Novel pathway compendium analysis elucidates mechanism of pro-angiogenic synthetic small molecule 
Bioinformatics  2008;24(20):2384-2390.
Motivation: Computational techniques have been applied to experimental datasets to identify drug mode-of-action. A shortcoming of existing approaches is the requirement of large reference databases of compound expression profiles. Here, we developed a new pathway-based compendium analysis that couples multi-timepoint, controlled microarray data for a single compound with systems-based network analysis to elucidate drug mechanism more efficiently.
Results: We applied this approach to a transcriptional regulatory footprint of phthalimide neovascular factor 1 (PNF1)—a novel synthetic small molecule that exhibits significant in vitro endothelial potency—spanning 1–48 h post-supplementation in human micro-vascular endothelial cells (HMVEC) to comprehensively interrogate PNF1 effects. We concluded that PNF1 first induces tumor necrosis factor-alpha (TNF-α) signaling pathway function which in turn affects transforming growth factor-beta (TGF-β) signaling. These results are consistent with our previous observations of PNF1-directed TGF-β signaling at 24 h, including differential regulation of TGF-β-induced matrix metalloproteinase 14 (MMP14/MT1-MMP) which is implicated in angiogenesis. Ultimately, we illustrate how our pathway-based compendium analysis more efficiently generates hypotheses for compound mechanism than existing techniques.
Availability: The microarray data generated as part of this study are available in the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/).
Contact: botchwey@virginia.edu; papin@virginia.edu
Supplementary information: Supplementary data are available at Bioinformatics online.
doi:10.1093/bioinformatics/btn451
PMCID: PMC2562016  PMID: 18718940
4.  Importance of interaction between nerve growth factor and α9β1 integrin in glial tumor angiogenesis 
Neuro-Oncology  2012;14(7):890-901.
NGF is a growth factor for which the role in the promotion of angiogenesis is still not completely understood. We found that NGF promotes the pathological neovascularization process in glioma through a direct interaction with α9β1 integrin, which is up-regulated on microvascular endothelial cells in cancer tissue. We propagated gHMVEC primary cells using a new method of immune-selection, and these cells demonstrated α9β1 integrin-dependent binding of NGF in a cell adhesion assay. Moreover, NGF induced gHMVEC proliferation and chemotaxis inhibited by specific blockers of α9β1 integrin, such as MLD-disintegrins and monoclonal antibody Y9A2. A Matrigel tube formation assay revealed that NGF significantly increased capillary-like growth from gHMVEC to a level comparable to treatment with VEGF. The snake venom disintegrin, VLO5, inhibited the agonistic effect of both growth factors, whereas the effect of Y9A2 was not statistically significant. Angiogenesis exogenously induced by NGF  was also α9β1-integrin dependent in an embryonic quail CAM system. However, angiogenesis pathologically induced by developing glioma in this system was only sensitive for inhibition with MLD-disintegrin, suggesting a more complex effect of cancer cells on the neovascularization process. The anti-angiogenic effect of MLD-disintegrins is probably related to their pro-apoptotic ability induced in activated tumoral endothelial cells. Therefore, the molecular basis of these disintegrins may be useful for developing new angiostatic pharmaceuticals for application in cancer therapy.
doi:10.1093/neuonc/nos119
PMCID: PMC3379805  PMID: 22611032
angiogenesis; disintegrins; glioma; integrins; nerve growth factor
5.  Resection of small plexiform neurofibromas in neurofibromatosis type 1 children 
Background
Plexiform neurofibromas (PNF) are benign tumors of the peripheral nerve which mostly develop in patients with neurofibromatosis type 1 (NF1). Surgical interventions are usually not applied to children with small tumors. These are rather restricted to debulking of larger tumors in adults that cause clinical complications or aesthetic disfigurement. In most cases, a total resection of PNF is not possible due to the network-like growth of the tumors.
Patients and methods
Early surgical intervention was carried out for 9 small PNFs in 7 NF1 children. Tumor resection was performed following the graphical delineation of the affected skin and according the MRI findings.
Results
Total resection was achieved for all 9 PNF without causing any neurological or organic deficit. Annual magnetic resonance tomography over a period of four years did not reveal any relapse of the tumors.
Conclusions
Early surgical intervention for small superficial PNFs in NF1 children have various advantages and may especially be considered a strategy to prevent progression.
doi:10.1186/1477-7819-3-6
PMCID: PMC549083  PMID: 15683544
6.  Regulation of shoot meristem integrity during Arabidopsis vegetative development 
Plant Signaling & Behavior  2011;6(8):1250-1252.
Shoot growth and development is mediated by the activity of the shoot meristem, which initiates leaves and axillary meristems. Meristem maintenance is achieved by a poorly understood process that functions to sustain the balance of stem cell perpetuation in the central zone (CZ) and organogenesis in the peripheral zone (PZ). A recent study showed that two related homeodomain transcription factors, PENNYWISE (PNY) and POUND-FOOLISH (PNF), regulate meristem maintenance by controlling the integrity of the CZ. The non-flower producing phenotype displayed by pny pnf plants can be rescued by genetically increasing the size of the shoot meristem. In this addendum, we show that augmenting the size of the central region of pny pnf shoot meristems partially rescues the meristem termination phenotype that occurs during early stages of vegetative development. Thus, regulation of CZ integrity by PNY and PNF is crucial for vegetative and reproductive development.
doi:10.4161/psb.6.8.16462
PMCID: PMC3260737  PMID: 21822063
development; shoot growth; meristem; homeodomain; stem cells
7.  Determinants of Microvascular Network Topologies in Implanted Neovasculatures 
Objectives
During neovascularization, the end result is a new functional microcirculation comprised of a network of mature microvessels with specific topologies. While much is known concerning the mechanisms underlying the initiation of angiogenesis, it remains unclear how the final architecture of microcirculatory beds is regulated. To begin to address this, we determined the impact of angiogenic neovessel pre-patterning on the final microvascular network topology using an implant model of implant neovascularization.
Methods and Results
To test this, we used 3-D direct-write bioprinting or physical constraints in a manner permitting post-angiogenesis vascular remodeling and adaptation to pattern angiogenic microvascular precursors (neovessels formed from isolated microvessel segments) in 3-dimensional collagen gels prior to implantation and subsequent network formation. Neovasculatures pre-patterned into parallel arrays formed functional networks following 4 weeks post-implantation, but lost the pre-patterned architecture. However, maintenance of uniaxial physical constraints during post-angiogenesis remodeling of the implanted neovasculatures produced networks with aligned microvessels as well as an altered proportional distribution of arterioles, capillaries and venules.
Conclusions
Here we show that network topology resulting from implanted microvessel precursors is independent from pre-patterning of precursors but can be influenced by a patterning stimulus involving tissue deformation during post-angiogenesis remodeling and maturation.
doi:10.1161/ATVBAHA.111.238725
PMCID: PMC3256738  PMID: 22053070
microcirculation; regeneration; bioprinting; vascular engineering; neovascularization
8.  Motor Imagery Boosts Proprioceptive Neuromuscular Facilitation in the Attainment and Retention of Range-of -Motion at the Hip Joint 
This study examined the effect of proprioceptive neuromuscular facilitation (PNF) coupled with an internal mental imagery technique (PNFI) on both the attainment and retention of increased range-of-movement (ROM) at the hip joint. Twenty-four young adult subjects were randomly allocated to PNF, PNFI, and control treatments administered in fifteen sessions over a three-week period. ROM was assessed prior to training then at the completion of sessions 1 day, 3, 7, and 14 during training, then 28 days after program completion. Analysis-of-Variance with repeated measures showed both significant treatment (p < 0.01) and time effects (p < 0.05). Mean change of ROM values were always larger under the PNFI condition and significantly different (p < 0.05) at day 1 and 3 following training program completion. Thereafter, the diminution of ROM was comparable to the PNF condition. Mean ROM increment relative to baseline was 7.55 and 9.45 degrees for PNF and PNFI respectively receding to 5.86 and 6.5 degrees at twenty-eight days following treatment cessation. Motor imagery coupled with PNF to enhance and retain ROM yields superior results to physical training used alone and can benefit both athletes and those undergoing rehabilitation.
Key PointsA Proprioceptive Neuromuscular Facilitation (PNF) technique applied to enhance range-of-movement (ROM) at the hip joint was successful.The effect produced greater gains in participants who received and applied a motor imagery technique to supplement the regular PNF.Both effects receded by about 50% across a no-practice period of 21 days.Incorporation of motor imagery with physical therapy deemed worthwile.
PMCID: PMC3905298  PMID: 24482593
Mental and physical practice; flexibility training
9.  Combination oral antiangiogenic therapy with thalidomide and sulindac inhibits tumour growth in rabbits 
British Journal of Cancer  1999;79(1):114-118.
Neovascularization facilitates tumour growth and metastasis formation. In our laboratory, we attempt to identify clinically available oral efficacious drugs for antiangiogenic activity. Here, we report which non-steroidal anti-inflammatory drugs (NSAIDs) can inhibit corneal neovascularization, induced by basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF). This antiangiogenic activity may contribute to the known effects of NSAIDs on gastric ulcers, polyps and tumours. We found that sulindac was one of the most potent antiangiogenic NSAIDs, inhibiting bFGF-induced neovascularization by 50% and VEGF-induced neovascularization by 55%. Previously, we reported that thalidomide inhibited growth factor-induced corneal neovascularization. When we combined sulindac with thalidomide, we found a significantly increased inhibition of bFGF- or VEGF-induced corneal neovascularization (by 63% or 74% respectively) compared with either agent alone (P< 0.01). Because of this strong antiangiogenic effect, we tested the oral combination of thalidomide and sulindac for its ability to inhibit the growth of V2 carcinoma in rabbits. Oral treatment of thalidomide or sulindac alone inhibited tumour growth by 55% and 35% respectively. When given together, the growth of the V2 carcinoma was inhibited by 75%. Our results indicated that oral antiangiogenic combination therapy with thalidomide and sulindac may be a useful non-toxic treatment for cancer. © 1999 Cancer Research Campaign
doi:10.1038/sj.bjc.6690020
PMCID: PMC2362163  PMID: 10408702
basic fibroblast growth factor; vascular endothelial growth factor; corneal neovascularization; non-steroidal anti-inflammatory drug; cyclo-oxygenase
10.  Mindin is upregulated during colitis and may activate NF-κB in a TLR-9 mediated manner 
AIM: To investigate the regulation of mindin expression and the signaling pathway involved during inflammation.
METHODS: C57BL/6 mice were treated with 3% dextran sulfate sodium (DSS) in drinking water for 6 d to induce acute colitis, and then the colon was harvested for histological analysis or for RNA isolation. mRNA expression of mindin and nuclear factor (NF)-κB p65 was analyzed by quantitative real time polymerase chain reaction (RT-PCR) and mindin expression construct was confirmed by Western blotting. Mouse macrophage and intestinal epithelial lineage cells were stimulated with different cytokines and toll-like receptor (TLR) ligands, before pNF-κB-luciferase activity was assessed using the Dual-Luciferase reporter assay system.
RESULTS: mRNA expression of mindin was upregulated 4.7 ± 1.1 fold compared with the baseline during DSS-induced intestinal inflammation in the mice. Stimulation with CpG-ODN (a known TLR-9 ligand) induced 4.2 ± 0.3 fold upregulation of mindin expression in RAW 264.7 cells. Full-length of mindin was cloned from cDNA of mouse mesenteric lymph node, then the pCMV-Mindin-Flag expression vector was established and the protein expression level was confirmed. Transfection of the mindin construct and stimulation with CpG-ODN significantly increased the NF-κB-luciferase activity by 2.5 ± 0.3 and 4.5 ± 0.5 fold in RAW264.7 and CMT93 cells, respectively (P < 0.01).
CONCLUSION: Mindin expression is upregulated during intestinal inflammation and may induce NF-κB promoter activation in a TLR-9 mediated manner.
doi:10.3748/wjg.v16.i9.1070
PMCID: PMC2835782  PMID: 20205276
Mindin; Nuclear factor-κB promoter activity; Toll-like receptor-9
11.  Resistin Induces Expression of Pro-Inflammatory Cytokines and Chemokines in Human Articular Chondrocytes via Transcription and mRNA Stabilization 
Arthritis and rheumatism  2010;62(7):1993-2003.
Objective
To elucidate the effect of resistin on human articular chondrocytes, and generate a picture of their regulation at the transcriptional and post-transcriptional levels.
Methods
Human articular chondrocytes were cultured with resistin. Changes in gene expression were analyzed at various doses and times. Cells were also treated with the transcriptional inhibitor actinomycin D after resistin treatment, or the nuclear factor kappa B (NF-κB) inhibitor IKK-NBD before resistin treatment. Gene expression was tested by quantitative real-time polymerase chain reaction. Computational analysis for transcription factor binding motifs was performed on the promoter regions of differentially expressed genes. TC28 chondrocytes were transfected with CCL3 and CCL4 promoter constructs, pNF-κB reporter, and NF-κB and CCAAT/enhancer-binding protein β (C/EBPβ) expression vectors with or without resistin.
Results
Resistin-treated human articular chondrocytes increased the expression of cytokines and chemokines. The mRNAs for MMP-1, MMP-13 and ADAMTS-4 also increased while COL2A1 and aggrecan were down-regulated. Cytokine and chemokine genes could be categorized into three groups according to the pattern of mRNA expression in a 24 h time course. One pattern suggested rapid regulation by mRNA stability. The second and third patterns were consistent with transcriptional regulation. Computational analysis suggested the transcription factors NF-κB and C/EBPβ were involved in the resistin-induced up-regulation. This prediction was confirmed by the co-transfection of NF-κB and C/EBPβ, and IKK-NBD inhibition.
Conclusion
Resistin has diverse effects on gene expression in human chondrocytes affecting chemokines, cytokines and matrix genes. mRNA stabilization and transcriptional up-regulation are involved in resistin-induced gene expression in human chondrocytes.
doi:10.1002/art.27473
PMCID: PMC3979471  PMID: 20506172
12.  Evaluation of Strength and Irradiated Movement Pattern Resulting from Trunk Motions of the Proprioceptive Neuromuscular Facilitation 
Introduction. The proprioceptive neuromuscular facilitation (PNF) is a physiotherapeutic concept based on muscle and joint proprioceptive stimulation. Among its principles, the irradiation is the reaction of the distinct regional muscle contractions to the position of the application of the motions. Objective. To investigate the presence of irradiated dorsiflexion and plantar flexion and the existing strength generated by them during application of PNF trunk motions. Methods. The study was conducted with 30 sedentary and female volunteers, the PNF motions of trunk flexion, and extension with the foot (right and left) positioned in a developed equipment coupled to the load cell, which measured the strength irradiated in Newton. Results. Most of the volunteers irradiated dorsal flexion in the performance of the flexion and plantar flexion during the extension motion, both presenting an average force of 8.942 N and 10.193 N, respectively. Conclusion. The distal irradiation in lower limbs became evident, reinforcing the therapeutic actions to the PNF indirect muscular activation.
doi:10.1155/2012/281937
PMCID: PMC3472517  PMID: 23094160
13.  Thrombospondin-1 as a Paradigm for the Development of Antiangiogenic Agents Endowed with Multiple Mechanisms of Action 
Pharmaceuticals  2010;3(4):1241-1278.
Uncontrolled neovascularization occurs in several angiogenesis-dependent diseases, including cancer. Neovascularization is tightly controlled by the balance between angiogenic growth factors and antiangiogenic agents. The various natural angiogenesis inhibitors identified so far affect neovascularization by different mechanisms of action. Thrombospondin-1 (TSP-1) is a matricellular modular glycoprotein that acts as a powerful endogenous inhibitor of angiogenesis. It acts both indirectly, by sequestering angiogenic growth factors and effectors in the extracellular environment, and directly, by inducing an antiangiogenic program in endothelial cells following engagement of specific receptors including CD36, CD47, integrins and proteoglycans (all involved in angiogenesis ). In view of its central, multifaceted role in angiogenesis, TSP-1 has served as a source of antiangiogenic tools, including TSP-1 fragments, synthetic peptides and peptidomimetics, gene therapy strategies, and agents that up-regulate TSP-1 expression. This review discusses TSP-1-based inhibitors of angiogenesis, their mechanisms of action and therapeutic potential, drawing our experience with angiogenic growth factor-interacting TSP-1 peptides, and the possibility of exploiting them to design novel antiangiogenic agents.
doi:10.3390/ph3041241
PMCID: PMC4034032
angiogenesis; tumor; integrins; interactions; thrombospondin-1
14.  Oxaliplatin-induced loss of phosphorylated heavy neurofilament subunit neuronal immunoreactivity in rat DRG tissue 
Molecular Pain  2009;5:66.
Background
Oxaliplatin and related chemotherapeutic drugs cause painful chronic peripheral neuropathies in cancer patients. We investigated changes in neuronal size profiles and neurofilament immunoreactivity in L5 dorsal root ganglion (DRG) tissue of adult female Wistar rats after multiple-dose treatment with oxaliplatin, cisplatin, carboplatin or paclitaxel.
Results
After treatment with oxaliplatin, phosphorylated neurofilament heavy subunit (pNF-H) immunoreactivity was reduced in neuronal cell bodies, but unchanged in nerve fibres, of the L5 DRG. Morphometric analysis confirmed significant changes in the number (-75%; P < 0.0002) and size (-45%; P < 0.0001) of pNF-H-immunoreactive neurons after oxaliplatin treatment. pNF-H-immunoreactive neurons had overlapping size profiles and co-localisation with neurons displaying cell body immunoreactivity for parvalbumin, non-phospho-specific neurofilament medium subunit (NF-M) and non-phospho-specific neurofilament heavy subunit (NF-H), in control DRG. However, there were no significant changes in the numbers of neurons with immunoreactivity for parvalbumin (4.6%, P = 0.82), NF-M (-1%, P = 0.96) or NF-H (0%; P = 0.93) after oxaliplatin treatment, although the sizes of parvalbumin (-29%, P = 0.047), NF-M (-11%, P = 0.038) and NF-H (-28%; P = 0.0033) immunoreactive neurons were reduced. In an independent comparison of different chemotherapeutic agents, the number of pNF-H-immunoreactive neurons was significantly altered by oxaliplatin (-77.2%; P < 0.0001) and cisplatin (-35.2%; P = 0.03) but not by carboplatin or paclitaxel, and their mean cell body area was significantly changed by oxaliplatin (-31.1%; P = 0.008) but not by cisplatin, carboplatin or paclitaxel.
Conclusion
This study has demonstrated a specific pattern of loss of pNF-H immunoreactivity in rat DRG tissue that corresponds with the relative neurotoxicity of oxaliplatin, cisplatin and carboplatin. Loss of pNF-H may be mechanistically linked to oxaliplatin-induced neuronal atrophy, and serves as a readily measureable endpoint of its neurotoxicity in the rat model.
doi:10.1186/1744-8069-5-66
PMCID: PMC2785764  PMID: 19922644
15.  The Effects of Abdominal Hollowing in Lower-limb PNF Pattern Training on the Activation of Contralateral Muscles 
Journal of Physical Therapy Science  2013;25(10):1335-1338.
[Purpose] The purpose of this study was to determine the effects of abdominal hollowing during lower-limb proprioceptive neuromuscular facilitation (PNF) training on the activation of contralateral muscles. [Subjects] Twenty male college students without symptoms or signs of muscular or nervous disease participated in this experiment after signing a consent form. [Methods] All the subjects were measured with electromyography (EMG) in a muscle activation test before and after abdominal hollowing. In the PNF program, the lower-limb pattern of PNF training, was maintained for 5 seconds, followed by a 2-minute break. This was repeated three times. The resting time between sets was 30 minutes. Surface EMG (Keypoint, Medtronic Inc., USA) was used for the measurements, and the highest value of three measurements was used in the analysis. [Result] The results revealed a significant change in the muscular activation of the opposite-side lower limbs. The muscular activations of the vastus lateralis, tibialis anterior, semitendinosus and gastrocnemius were increased significantly after the abdominal hollowing. [Conclusion] The findings suggest that abdominal hollowing in PNF pattern training can be effective at promoting muscular activation of the contralateral muscles. To promote muscular activation of the opposite side in lower-limb PNF pattern training, abdominal hollowing should be considered to improve the effect of PNF pattern training.
doi:10.1589/jpts.25.1335
PMCID: PMC3820202  PMID: 24259788
PNF pattern; Abdominal hollowing; Contralateral muscle activation
16.  Angiogenic properties of dehydrated human amnion/chorion allografts: therapeutic potential for soft tissue repair and regeneration 
Vascular Cell  2014;6:10.
Background
Chronic wounds are associated with a number of deficiencies in critical wound healing processes, including growth factor signaling and neovascularization. Human-derived placental tissues are rich in regenerative cytokines and have been shown in randomized clinical trials to be effective for healing chronic wounds. In this study, PURION® Processed (MiMedx Group, Marietta, GA) dehydrated human amnion/chorion membrane tissue allografts (dHACM, EpiFix®, MiMedx) were evaluated for properties to support wound angiogenesis.
Methods
Angiogenic growth factors were identified in dHACM tissues using enzyme-linked immunosorbent assays (ELISAs), and the effects of dHACM extract on human microvascular endothelial cell (HMVEC) proliferation and production of angiogenic growth factors was determined in vitro. Chemotactic migration of human umbilical vein endothelial cells (HUVECs) toward pieces of dHACM tissue was determined using a standard in vitro transwell assay. Neovascularization of dHACM in vivo was determined utilizing a murine subcutaneous implant model.
Results
Quantifiable levels of the angiogenic cytokines angiogenin, angiopoietin-2 (ANG-2), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), heparin binding epidermal growth factor (HB-EGF), hepatocyte growth factor (HGF), platelet derived growth factor BB (PDGF-BB), placental growth factor (PlGF), and vascular endothelial growth factor (VEGF) were measured in dHACM. Soluble cues promoted HMVEC proliferation in vitro and increased endogenous production of over 30 angiogenic factors by HMVECs, including granulocyte macrophage colony-stimulating factor (GM-CSF), angiogenin, transforming growth factor β3 (TGF-β3), and HB-EGF. 6.0 mm disks of dHACM tissue were also found to recruit migration of HUVECs in vitro. Moreover, subcutaneous dHACM implants displayed a steady increase in microvessels over a period of 4 weeks, indicative of a dynamic intra-implant neovascular process.
Conclusions
Taken together, these results demonstrate that dHACM grafts: 1) contain angiogenic growth factors retaining biological activity; 2) promote amplification of angiogenic cues by inducing endothelial cell proliferation and migration and by upregulating production of endogenous angiogenic growth factors by endothelial cells; and 3) support the formation of blood vessels in vivo. dHACM grafts are a promising wound care therapy with the potential to promote revascularization and tissue healing within poorly vascularized, non-healing wounds.
doi:10.1186/2045-824X-6-10
PMCID: PMC4016655  PMID: 24817999
Amnion; Chorion; Amnion/chorion grafts; dHACM; Angiogenesis; Growth factors; VEGF; Endothelial cells; Soft tissue regeneration; Wound healing; Chronic wounds
17.  Acute Effects of Three Different Stretching Protocols on the Wingate Test Performance 
The purpose of this study was to examine the acute effects of different stretching exercises on the performance of the traditional Wingate test (WT). Fifteen male participants performed five WT; one for familiarization (FT), and the remaining four after no stretching (NS), static stretching (SS), dynamic stretching (DS), and proprioceptive neuromuscular facilitation (PNF). Stretches were targeted for the hamstrings, quadriceps, and calf muscles. Peak power (PP), mean power (MP), and the time to reach PP (TP) were calculated. The MP was significantly lower when comparing the DS (7.7 ± 0.9 W/kg) to the PNF (7.3 ± 0.9 W/kg) condition (p < 0.05). For PP, significant differences were observed between more comparisons, with PNF stretching providing the lowest result. A consistent increase of TP was observed after all stretching exercises when compared to NS. The results suggest the type of stretching, or no stretching, should be considered by those who seek higher performance and practice sports that use maximal anaerobic power.
Key points
The mean power was significantly lower when comparing dynamic stretching.to proprioceptive neuromuscular facilitation.
For peak power, significant differences were observed between more comparisons, with proprioceptive neuromuscular facilitation stretching providing the lowest result.
A consistent increase of time to reach the peak was observed after all stretching exercises when compared to non-stretching.
The type of stretching, or no stretching, should be considered by those who seek higher performance and practice sports that use maximal anaerobic power.
PMCID: PMC3737835  PMID: 24149116
Static stretching; proprioceptive neuromuscular facilitation; dynamic stretching; anaerobic power
18.  Harnessing Systems Biology Approaches to Engineer Functional Microvascular Networks 
Microvascular remodeling is a complex process that includes many cell types and molecular signals. Despite a continued growth in the understanding of signaling pathways involved in the formation and maturation of new blood vessels, approximately half of all compounds entering clinical trials will fail, resulting in the loss of much time, money, and resources. Most pro-angiogenic clinical trials to date have focused on increasing neovascularization via the delivery of a single growth factor or gene. Alternatively, a focus on the concerted regulation of whole networks of genes may lead to greater insight into the underlying physiology since the coordinated response is greater than the sum of its parts. Systems biology offers a comprehensive network view of the processes of angiogenesis and arteriogenesis that might enable the prediction of drug targets and whether or not activation of the targets elicits the desired outcome. Systems biology integrates complex biological data from a variety of experimental sources (-omics) and analyzes how the interactions of the system components can give rise to the function and behavior of that system. This review focuses on how systems biology approaches have been applied to microvascular growth and remodeling, and how network analysis tools can be utilized to aid novel pro-angiogenic drug discovery.
doi:10.1089/ten.teb.2009.0611
PMCID: PMC2946904  PMID: 20121415
19.  Proprioceptive Neuromuscular Facilitation Techniques in Sports Medicine: A Reassessment 
Journal of Athletic Training  1997;32(1):34-39.
Objective:
The purpose of this survey was for comparison with a similar 1981 survey to determine if proprioceptive neuromuscular facilitation (PNF) techniques are being implemented in the same manner today.
Design and Setting:
The survey was made available at the 1993 NATA Clinical Symposium.
Subjects:
The subjects were 131 athletic trainers representing all major national athletic conferences who attended the 1993 NATA Clinical Symposium and who stated that they used PNF exercise in their practice.
Measurements:
The survey consisted of 15 questions dealing with academic preparation, years of practice, scope and method of preparation in PNF, application of nine PNF techniques to various joints and regions of the body, and the most successful use of PNF techniques.
Results:
PNF techniques are most frequently applied during rehabilitation of the knee, shoulder, and hip, similar to 1981 except that the use of these techniques during ankle rehabilitation has increased. In both studies, the most frequently used techniques were contract-relax and hold-relax. Two techniques not surveyed in 1981, contract-relax-contract and hold-relax-contract, are becoming techniques of choice for elbow, wrist, hip, and knee rehabilitation. The use of PNF techniques in the muscle re-education phase of rehabilitation is an application identified in this survey not cited by athletic trainers in the 1981 survey.
Conclusions:
Proprioceptive and kinesthetic deficits are known to occur after certain types of injuries, and the use of PNF techniques to correct these problems is a natural application A contemporary trend in exercise rehabilitation is multiplanar exercises, which are typified by PNF techniques.
PMCID: PMC1319233  PMID: 16558430
proprioception; neuromuscular; facilitation; injuries
20.  Neovascularization in Biodegradable Inverse Opal Scaffolds with Uniform and Precisely Controlled Pore Sizes** 
Advanced healthcare materials  2012;2(1):145-154.
The formation of a stable vascular network in a scaffold is one of the most challenging tasks in tissue engineering and regenerative medicine. Despite the common use of porous scaffolds in these applications, little is known about the effect of pore size on the neovascularization in these scaffolds. Here we fabricated poly(D, L-lactide-co-glycolide) inverse opal scaffolds with uniform pore sizes of 79, 147, 224, and 312 μm in diameter and then used them to systematically study neovascularization in vivo. Histology analyses revealed that scaffolds with small pores (<200 μm) favored the formation of vascular networks with small vessels at high densities and poor penetration depth. By contrast, scaffolds with large pores (>200 μm) favored the formation of vascular networks with large blood vessels at low densities and deep penetration depth. Based on the different patterns of vessel ingrowth as regulated by the pore size, we proposed a model to describe vascularization in a three-dimensional porous scaffold, which can potentially serve as a guideline for future design of porous scaffolds.
doi:10.1002/adhm.201200106
PMCID: PMC3541475  PMID: 23184495
inverse opal scaffolds; pore dimension; blood vessels; tissue engineering; regenerative medicine
21.  Hypoxia down-regulates sFlt-1 (sVEGFR-1) expression in human microvascular endothelial cells by a mechanism involving mRNA alternative processing 
Biochemical Journal  2011;436(Pt 2):399-407.
sFlt-1 (soluble Flt-1) potently inhibits angiogenesis by binding extracellularly to VEGF (vascular endothelial growth factor). In the present paper, we report that hypoxia down-regulates sFlt-1 expression in HMVECs (human microvascular endothelial cells), a constituent of microvessels where angiogenesis occurs. Hypoxia (5–1% O2) increased VEGF expression in HMVECs. In contrast, the levels of sFlt-1 mRNA and protein in HMVECs decreased significantly as the O2 concentration fell, whereas mFlt-1 (membrane-bound Flt-1) mRNA and protein remained unchanged. This suggested that hypoxia selectively regulates alternative 3′-end processing of sFlt-1 pre-mRNA. We have also demonstrated that sFlt-1 overexpression in lentiviral-construct-infected HMVECs counteracted VEGF-induced endothelial cell growth. We next identified cis-elements involved in sFlt-1 mRNA processing in HMVECs using a human Flt-1 minigene and found that two non-contiguous AUUAAA sequences function as the poly(A) signal. Furthermore, we identified a cis-element in intron 13 that regulates sFlt-1 mRNA processing. Mutagenesis of the U-rich region in intron 13 caused a significant decrease in the soluble-form/membrane-form RNA ratio in the minigene-transfected HMVECs. These results suggest that decreased sFlt-1 expression due to hypoxia contributes to hypoxia-induced angiogenesis and reveals a novel mechanism regulating angiogenesis by alternative mRNA 3′-end processing.
doi:10.1042/BJ20101490
PMCID: PMC3133880  PMID: 21382012
angiogenesis; hypoxia; mRNA 3′-end processing; soluble Flt-1; vascular endothelial cell; vascular endothelial growth factor (VEGF); ARE, A+U-rich element; bFGF, basic fibroblast growth factor; CFIM25, 25-kDa subunit of the cleavage factor Im; CstF, cleavage stimulation factor; Cst F64, 64-kDa subunit of CstF; DMOG, dimethyloxaloylglycine; EC, endothelial cell; FBS, fetal bovine serum; HEK-293TN cell, HEK (human embryonic kidney)-293 cell expressing the large T-antigen of SV40 (simian virus 40) and the neomycin-resistance gene; HIF, hypoxia-inducible factor; hnRNP, heterogeneous nuclear ribonucleoprotein; HUVEC, human umbilical vein EC; KDR, kinase insert domain-containing receptor; mFlt-1, membrane-bound Flt-1; MVEC, microvascular EC; HMVEC, human MVEC; PHD, prolyl hydroxylase; RT, reverse transcription; sFlt-1, soluble Flt-1; VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor; sVEGFR, soluble VEGFR
22.  Influence of the oxygen microenvironment on the proangiogenic potential of human endothelial colony forming cells 
Angiogenesis  2009;12(4):303-311.
Therapeutic angiogenesis is a promising strategy to promote the formation of new or collateral vessels for tissue regeneration and repair. Since changes in tissue oxygen concentrations are known to stimulate numerous cell functions, these studies have focused on the oxygen microenvironment and its role on the angiogenic potential of endothelial cells. We analyzed the proangiogenic potential of human endothelial colony-forming cells (hECFCs), a highly proliferative population of circulating endothelial progenitor cells, and compared outcomes to human dermal microvascular cells (HMVECs) under oxygen tensions ranging from 1% to 21% O2, representative of ischemic or healthy tissues and standard culture conditions. Compared to HMVECs, hECFCs (1) exhibited significantly greater proliferation in both ischemic conditions and ambient air; (2) demonstrated increased migration compared to HMVECs when exposed to chemotactic gradients in reduced oxygen; and (3) exhibited comparable or superior proangiogenic potential in reduced oxygen conditions when assessed using a vessel-forming assay. These data demonstrate that the angiogenic potential of both endothelial populations is influenced by the local oxygen microenvironment. However, hECFCs exhibit a robust angiogenic potential in oxygen conditions representative of physiologic, ischemic, or ambient air conditions, and these findings suggest that hECFCs may be a superior cell source for use in cell-based approaches for the neovascularization of ischemic or engineered tissues.
doi:10.1007/s10456-009-9152-6
PMCID: PMC2778716  PMID: 19544080
Angiogenesis; Endothelial cells; Endothelial progenitor cells; Neovascularization
23.  Sprouting and intussusceptive angiogenesis in postpneumonectomy lung growth: mechanisms of alveolar neovascularization 
Angiogenesis  2013;17:541-551.
In most rodents and some other mammals, the removal of one lung results in compensatory growth associated with dramatic angiogenesis and complete restoration of lung capacity. One pivotal mechanism in neoalveolarization is neovascularization, because without angiogenesis new alveoli can not be formed. The aim of this study is to image and analyze three-dimensionally the different patterns of neovascularization seen following pneumonectomy in mice on a sub-micron-scale. C57/BL6 mice underwent a left-sided pneumonectomy. Lungs were harvested at various timepoints after pneumonectomy. Volume analysis by microCT revealed a striking increase of 143 percent in the cardiac lobe 14 days after pneumonectomy. Analysis of microvascular corrosion casting demonstrated spatially heterogenous vascular densitities which were in line with the perivascular and subpleural compensatory growth pattern observed in anti-PCNA-stained lung sections. Within these regions an expansion of the vascular plexus with increased pillar formations and sprouting angiogenesis, originating both from pre-existing bronchial and pulmonary vessels was observed. Also, type II pneumocytes and alveolar macrophages were seen to participate actively in alveolar neo-angiogenesis after pneumonectomy. 3D-visualizations obtained by high-resolution synchrotron radiation X-ray tomographic microscopy showed the appearance of double-layered vessels and bud-like alveolar baskets as have already been described in normal lung development. Scanning electron microscopy data of microvascular architecture also revealed a replication of perialveolar vessel networks through septum formation as already seen in developmental alveolarization. In addition, the appearance of pillar formations and duplications on alveolar entrance ring vessels in mature alveoli are indicative of vascular remodeling. These findings indicate that sprouting and intussusceptive angiogenesis are pivotal mechanisms in adult lung alveolarization after pneumonectomy. Various forms of developmental neoalveolarization may also be considered to contribute in compensatory lung regeneration.
Electronic supplementary material
The online version of this article (doi:10.1007/s10456-013-9399-9) contains supplementary material, which is available to authorized users.
doi:10.1007/s10456-013-9399-9
PMCID: PMC4061467  PMID: 24150281
Intussusceptive angiogenesis; Pneumonectomy; Septal alveolarization; Corrosion cast; Synchrotron radiation tomographic microscopy; Lung surgery
24.  Mecamylamine Suppresses Basal and Nicotine-Stimulated Choroidal Neovascularization 
Purpose
Nicotinic acetylcholine receptors (nAChR) are best known for their role in neurotransmission, but they have recently been demonstrated on vascular endothelial cells. Acetylcholine is their endogenous ligand, but they are also stimulated by nicotine. By stimulating nAChR, nicotine promotes tumor angiogenesis as well as atherosclerotic plaque neovascularization. In this study, we investigated the role of nAChR in the pathogenesis of choroidal neovascularization (CNV).
Methods
The effect of the nAChR antagonist mecamylamine was tested on human retinal and choroidal endothelial cells in vitro and in a murine model of CNV.
Results
Several nAChR isoforms were identified in retinal and choroidal microvascular endothelial cells and the ability of these cells to form tubules when grown in Matrigel and VEGF was suppressed by the nAChR antagonist mecamylamine. Supplementation of the drinking water of mice with nicotine increased the size of CNV lesions at Bruch’s membrane rupture sites, an effect that was blocked by infusion of mecamylamine by osmotic pump. In the absence of nicotine, CNV formation was suppressed by mecamylamine given by osmotic pump or by topical application to the cornea.
Conclusions
These data suggest that endogenous activation of nAChR promotes CNV and activation of nAChR by nicotine may contribute to the increased incidence of CNV seen in smokers with age-related macular degeneration (AMD). Topically administered mecamylamine could provide an appealing new treatment approach for CNV.
doi:10.1167/iovs.07-0089
PMCID: PMC2636721  PMID: 18385094
25.  Implanted Microvessels Progress through Distinct Neovascularization Phenotypes 
Microvascular research  2009;79(1):10.
We have previously demonstrated that implanted microvessels form a new microcirculation with minimal host-derived vessel investment. Our objective was to define the vascular phenotypes present during neovascularization in these implants and identify post-angiogenesis events. Morphological, functional and transcriptional assessments identified three distinct vascular phenotypes in the implants: sprouting angiogenesis, neovascular remodeling, and network maturation. A sprouting angiogenic phenotype appeared first, characterized by high proliferation and low mural cell coverage. This was followed by a neovascular remodeling phenotype characterized by a perfused, poorly organized neovascular network, reduced proliferation, and re-associated mural cells. The last phenotype included a vascular network organized into a stereotypical tree structure containing vessels with normal perivascular cell associations. In addition, proliferation was low and was restricted to the walls of larger microvessels. The transition from angiogenesis to neovascular remodeling coincided with the appearance of blood flow in the implant neovasculature. Analysis of vascular-specific and global gene expression indicates that the intermediate, neovascular remodeling phenotype is transcriptionally distinct from the other two phenotypes. Therefore, this vascular phenotype likely is not simply a transitional phenotype but a distinct vascular phenotype involving unique cellular and vascular processes. Furthermore, this neovascular remodeling phase may be a normal aspect of the general neovascularization process. Given that this phenotype is arguably dysfunctional, many of the microvasculatures present within compromised or diseased tissues may not represent a failure to progress appropriately through a normally occurring neovascularization phenotype.
doi:10.1016/j.mvr.2009.10.001
PMCID: PMC2813398  PMID: 19833141
neovascularization; neovessel; sprouting; angiogenesis; microvascular remodeling; microcirculation; structural adaptation; gene expression

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