Journal of Periodontology; Copyright 2015
DOI: 10.1902/jop.2015.140362
The Effect of Living Cellular Sheets on the Angiogenic Potential of Human Microvascular Endothelial Cells Cristina C. Villar, PhD*†, Xiang R. Zhao, MS*, Carolina B. Livi, PhD‡, David L. Cochran, PhD* *
Department of Periodontics, Dental School, University of Texas Health Science Center at San Antonio, Texas, USA.
† Division of Periodontics, Department of Stomatology, School of Dentistry, University of São Paulo, São Paulo, Brazil. ‡ Department of Molecular Medicine, Graduate School of Biomedical Sciences, University of Texas Health Science Center at San Antonio, Texas, USA. BACKGROUND: A fundamental issue limiting the efficacy of surgical approaches designed to correct periodontal mucogingival defects is that new tissues rely on limited sources of blood supply from the adjacent recipient bed. Accordingly, tissue-engineered-based therapies that leverage local self-healing potential may represent promising alternatives for the treatment of mucogingival defects by inducing local vascularization. The aim of this study was to evaluate the effect of commercially available living cellular sheets (LCS) on the angiogenic potential of human microvascular endothelial cells (HMVEC-dNeo). METHODS: The effect of LCS on HMVEC-dNeo proliferation, migration, capillary tube formation, gene expression and production of angiogenic factors was evaluated over time. RESULTS: LCS positively influenced HMVEC-dNeo proliferation and migration. Moreover, HMVEC-dNeo incubated with LCS showed transcriptional profiles different from untreated cells. While increased expression of angiogenic genes predominated early on in response to LCS, late-phase responses were characterized by up- and down-regulation of angiostatic and angiogenic genes. However, this trend was not confirmed at the protein level, as LCS induced increased production of most of the angiogenic factors tested (i.e. heparin-binding epidermal growth factor (EGF)-like growth factor, interleukin 6, angiopoietin, platelet-derived growth factor-BB, EGF, placental growth factor and vascular endothelial growth factor) throughout the investigational period. Finally, although LCS induced HMVEC-dNeo proliferation, migration and expression of angiogenic factors, additional factors/environmental pressures are likely to be required to promote the development of complex, mesh-like vascular structures. CONCLUSION: LCS favor initial mechanisms that govern angiogenesis but failed to enhance or accelerated HMVEC-dNeo morphological transition to complex vascular structures.
KEYWORDS: medical device, mucosal tissue, wound healing, endothelial cells, physiologic angiogenesis, growth factors.
Clinical application of tissue-engineered constructs was initially limited to implantation of threedimensional biodegradable scaffolds into surgical beds. 1 Accordingly, several types of three-dimensional biodegradable scaffolds were successfully engineered by adding a wide variety of human cells into polyglycolic acid, gelatin, alginate or collagen scaffolds. 1 However, deficient cell migration into scaffolds and prolonged inflammatory responses upon scaffold degradation limited the clinical success of these therapeutic devices. 1 To avoid these limitations, recent advances in tissue-engineering strategies led to the development of living cellular constructs that leverage local self-healing potential by positively modulating host cellular behavior. 2-6 These devices are engineered by layering cell sheets to construct functional tissues without artificial scaffolds. 2-6 One of the fundamental issues limiting the clinical efficacy of conventional surgical therapies directed to correct periodontal mucogingival defects is that new tissues must be formed directly onto avascular tooth root surfaces and rely on limited sources of blood supply from the adjacent surgically created wound bed. Accordingly, tissue-engineered-based therapies represent a promising alternative for the treatment of periodontal mucogingival defects by inducing and supporting adequate local vascularization during the 1
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DOI: 10.1902/jop.2015.140362
various phases of healing. Recently, commercially available allogeneic living cell-based tissue sheets were FDA-approved for intra-oral use. This living device consists of two layers, an upper cornified layer comprised of living human neonatal foreskin keratinocytes and a lower layer constructed of bovine-derived collagen, human extracellular matrix proteins, and living human neonatal foreskin fibroblasts. The hypothesis of this work is that living cellular sheets (LCS) stimulate the activation of an angiogenic program on endothelial cells. Based on this, the aim of this study was to evaluate the effect of commercially available LCS on the angiogenic potential of human microvascular endothelial cells (HMVEC-dNeo).
MATERIAL AND METHODS Endothelial Cells Neonatal Dermal Human Microvascular Endothelial Cells (HMVEC-dNeo)§ used include primary cells isolated from small vessels from neonatal foreskin. Cryopreserved HMVEC-dNeo were shipped in third passage and maintained in complete endothelial cell basal medium-2 (EBM-2)║ containing 5% fetal calf serum (FCS)¶ and EGM-2MV bullet kit# (consisting of hydrocortisone, gentamicin, amphotericin-B, epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF2), insulin-like growth factor 1 (IGF-1), and ascorbic acid). These cells were used between passages 4 and 6. Living Cellular Sheets Commercially available allogeneic living cell-based tissue sheets** were used in this study. LCS consist of two layers, an upper cornified layer comprised of living human neonatal foreskin keratinocytes and a lower layer constructed of bovine-derived type I collagen, human extracellular matrix proteins, and living human neonatal foreskin fibroblasts. Culture Systems Three experimental systems were used. Model A) To study HMVEC-dNeo proliferation, viability, gene and protein expression profiles, HMVEC-dNeo were seeded near confluence (3×105 cells/well and 1×105 cells/well, in 6- or 24-well polystyrene plates, respectively) and were incubated overnight. The following day, LCS (average diameter: 22.6 ± 1.4 mm or 6.2 ± 0.8 mm, for 6- or 24-well transwell tissue plates††, respectively) were loaded, with the cornified layer facing up, into transwell inserts with 8 µm pore size, and the inserts were transferred into wells containing HMVEC-dNeo in fresh medium. Model B) HMVEC-dNeo migration was evaluated using a modification of the collagen-sandwich experimental model described by Schor et al. (2006) 7. In brief, HMVEC-dNeo labeled with 5mM 5-(and6)-(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine (CMTMR) ‡‡ were seeded at 1×105 cells/well on type I collagen§§-coated 24-well polystyrene plates. The following day, 200 µl of a 1.2mg/ml solution of ready-to-use bovine type I collagen§§ was added to each well and was allowed to solidify for approximately 30 min. This resulted in a 1 mm thick layer of type I collagen covering HMVEC-dNeo. After collagen polymerization, fresh complete EBM-2 medium was added. LCS (average diameter: 6.3 ± 0.7 mm) were loaded into transwell inserts, and inserts were transferred to wells containing the experimental models. Model C) To study capillary formation, HMVEC-dNeo were seeded at subconfluency levels (7×104 cells/well) on basement membrane matrix║║-coated 24-well plates. The following day, LCS 2
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(average diameter: 6.3 ± 0.9 mm) were loaded into transwell inserts, and these were transferred to wells containing HMVEC-dNeo in fresh medium. Negative controls for all experiments included untreated HMVEC-dNeo and HMVEC-dNeo incubated in the presence of empty transwell inserts or transwell inserts containing acellular collagen constructs manufactured with the same type I bovine-derived collagen§§ as the one used for LCS manufacturing. LCS and acellular collagen constructs were maintained submerged in medium during the experimental period. All transwell inserts were removed prior to HMVEC-dNeo analysis. Altogether, the following groups were tested: -
Group LCS: HMVEC-dNeo incubated in the presence of transwell inserts containing LCS**
- Group collagen: HMVEC-dNeo incubated in the presence of transwell inserts containing acellular type I bovine-derived collagen gels§§ -
Group transwell: HMVEC-dNeo incubated in the presence of empty transwell inserts
-
Group untreated: HMVEC-dNeo alone
Cell Proliferation HMVEC-dNeo proliferation was measured by bromodeoxyuridine (BrdU) enzyme-linked immunosorbent assay (ELISA)†††, according to manufacturer’s instructions. Mean values were obtained by analysis of triplicate wells in four separate experiments. Viability HMVEC-dNeo viability was analyzed in the experimental model A at 24, 48 and 72 hours and in the collagen-sandwich migration model (model B) at days 2, 3, and 6, using a cytotoxicity assay‡‡‡, according to manufacturer’s instructions. Mean values were obtained by analysis of duplicate wells in three separate experiments. Cell Migration HMVEC-dNeo cell migration was examined using a confocal laser-scanning microscope (LSM)§§§. Zstack images of total thickness 200-500 µm were collected at intervals of 6.4 µm, as optimally computed by the LSM software║║║. Migrating cells were defined as cells moving at least the distance equivalent to their own length. Mean values were obtained by analysis of 20 view fields in triplicate wells in three separate experiments. Capillary Tube Formation Digital images obtained using brightfield inverted microscopy (at 40X) were scored blindly for the extent of angiogenesis according to a previously defined scoring system reported by Malinda et al. (1999). 8 Mean values were obtained by analysis of 15 view fields in triplicate wells in three separate experiments. Gene Expression Global gene expression was examined by BeadChips-based gene expression analysis¶¶¶. In brief, total ribonucleic acid (RNA) was isolated using a commercially available kit###. RNA yield was determined by measuring absorbance at 260 nm and RNA quality was analyzed using a Bioanalyzer****. Samples were labeled and hybridized onto expression BeadChips, following manufacture’s guidelines. Signal intensities were quantified using a data analysis software†††† and imported into GeneSpring GX ‡‡‡‡ for quantile 3
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normalization and analysis. The data presented show the average of three separate experiments, with single replicates. Comparative Quantitative Real Time Polymerase Chain Reaction (RT-PCR) RNA (1 μg) was reverse transcribed to complementary deoxyribonucleic acid (cDNA) using a reverse transcription Kit§§§§. RT-PCR was performed in a sequence detection system║║║║. Each reaction used cDNA (2 µl) mixed with DNA Master SYBR Green Reaction Mix (2 µl) ¶¶¶¶ and specific primers (1 pmol). Levels of messenger RNA (mRNA) expression were calculated and normalized to glyceraldehyde 3phosphate dehydrogenase (GAPDH) levels at each time point. The data presented show the average of four independent experiments with samples in duplicate Production and Storage of Angiogenic Factors Multiple angiogenic factors were simultaneously detected in cell lysates using multiplexed ELISA-based angiogenesis human angiogenesis protein array####. Results shown represent the mean value of three independent experiments, with samples in each experiment run in duplicate. Statistical Analysis Unless otherwise specified, results were expressed by mean ± SD and compared by analysis of variance (ANOVA) with post-hoc Tukey HSD (Honest Significant Differences) to asses differences between means. Linear regression was used to compare transcriptional profiles across biological replicates within the the groups. To compare transcriptional profiles across all 12 conditions tested (combinations of 4 treatments and 3 time points), hierarchical clustering was performed with a similarity measure using Pearson correlation coefficient and a complete linkage algorithm on genes that had differential expression levels with respect to untreated controls. Genes differentially expressed in response to treatment were identified using parametric Student’s t test with a multiple testing correction (Benjamini and Hochberg falsediscovery rate ≤0.05), and fold change ≥ 3.
RESULTS A) Effect of LCS on HMVEC-dNeo Proliferation, Viability, Migration and Capillary Tube Formation HMVEC-dNeo viability was not affected by the conditions tested (not shown). HMVEC-dNeo proliferation was sustained at high levels for up to 72 h in cultures treated with LCS (Figure 1a). In sharp contrast, HMVEC-dNeo proliferation was significantly reduced at the latest time point in response to acellular collagen gels (Figure 1a). As expected, empty transwell inserts had no effect on HMVEC-dNeo proliferation rate (Figure 1a). Number of migrating HMVEC-dNeo increased 37.45 fold after 2 days of treatment with LCS (Figure 1b). LCS also stimulated HMVEC-dNeo migration at days 3 and 6, albeit in a less pronounced manner (Figure 1b). Lastly, empty transwell inserts and transwell inserts containing acellular collagen gels had no effects on HMVEC-dNeo migration (Figure 1b). In initial experiments using complete EBM-2 (containing FCS and EGM-2MV bullet kit]), HMVECdNeo produced capillary tubes at 3 hours and closed polygons within 6 hours. HMVEC-dNeo morphological transition was not enhanced or accelerated in response to LCS or acellular collagen gels (Figure 1c). Angiogenic factors present in complete EBM-2 may provide strong inducing signals that favor HMVEC-dNeo capillary tube formation and may obscure the stimulatory effect of LCS. Therefore, in additional experiments, complete EBM-2 was replaced by EBM-2 supplemented with 5% FCS only (EMB4
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2/serum) or EBM-2 alone (EBM-2/alone). HMVEC-dNeo formed tube structures and sprouts within 3 h following incubation in EMB-2/serum, and longer incubation did not allow for organization of these cells into closed polygons and mesh-like structures (Figure 1c). These results were also not affected by treatment with LCS or acellular collagen gels (Figure 1c). In contrast, HMVEC-dNeo maintained in EBM2/alone showed changes suggestive of loss of viability (not shown). B) Gene Expression Responses Prior to differential gene expression analysis, quality control diagnostic measurements were performed. To ensure that the microarray data were consistent among biological replicates, scatterplots were constructed. Replicates in each of the groups displayed a very high correlation as indicated by the regression line and r2 values (0.99–1). In contrast, when expression values for untreated HMVEC-dNeo cells were compared to LCS treated cells, a broad scatter was observed, indicating numerous gene expression changes. To compare transcriptional profiles across all 12 conditions tested (combinations of 4 treatments and 3 time points), hierarchical clustering was performed and followed by a complete linkage algorithm on 352 genes that had differential expression levels with respect to untreated controls (Figure 2). Expression profiles of HMVECdNeo incubated with empty transwell inserts and transwell inserts containing acellular collagen gels clustered together with those of untreated cells (Figure 2), indicating the similarity of the transcriptome among these conditions. In contrast, HMVEC-dNeo treated with LCS contained a high number of differentially expressed transcripts and different transcriptional profiles (Figure 2). A total of 8,238 genes were detected in HMVEC-dNeo. Out of this, 71 genes were up-regulated and 15 genes were down-regulated after 24 h of LCS treatment. Kinetic experiments demonstrated that the number of genes regulated by LCS increased over time (not shown). Along with this line, at 72 h, 203 genes were up-regulated and 210 genes were down-regulated in response to LCS. In contrast, acellular collagen gels regulated the expression of a limited number of genes in HMVEC-dNeo. Moreover, empty transwell inserts did not affect HMVEC-dNeo gene expression. C) Expression of Angiogenic Genes The effect of LCS on the angiogenic potential of HMVEC-dNeo cells was indirectly evaluated by investigating its effect on the expression levels of angiogenesis-related genes. After 24 h of incubation in the presence of LCS, HMVEC-dNeo had significantly altered expression of 13 angiogenic genes (Table 1). Of the genes identified from this analysis, 12 encode for factors that positively stimulates angiogenic responses. Among these, 11 were up-regulated and only 01 was down-regulated (Table 1). ANGPT2 was the only negative regulator of angiogenesis, which expression was affected by LCS at 24 h (Table 1). A similar trend was observed at 48 h. At this time point, 17 angiogenic genes (16 positive regulators and 1 negative regulator of angiogenesis) were differentially regulated in response to LCS (Table 1). Among the differentially expressed genes encoding for positive regulators of angiogenesis, 12 were upregulated and 4 were down-regulated (Table 1). RECK was the only negative regulator of angiogenesis that showed altered expression at 48 h (Table 1). At 72 h, LCS induced increased expression of 25 genes encoding for positive regulators of angiogenesis, but inhibited the expression of other 29 pro-angiogenic genes (Table 1). Finally, 3 genes encoding for negative regulators of angiogenesis showed were affected by LCS. Among them, THBS1 and RECK showed decreased expression and ANGPT2 increased expression in response to LCS (Table 1). Regulation of key genes identified by gene arrays was confirmed by qRT-PCR. LCS induced increased expression of 5 out of the 8 tested genes encoding for positive regulators of angiogenesis (ADM2, VEGF, TYMP, IL-6 and IL-8) (Figure 3). The remaining genes encoding for positive regulators of angiogenesis 5
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were suppressed in response to LCS (FGF2, TGFB and TGFA) (Figure 3). Lastly, ANGPT2 expression was significantly increased at 48 h, in response to LCS (Figure 3). D) Production of Angiogenic Factors Production of multiple angiogenic factors were measured in cell lysates (Figure 4). Angiogenic factors heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF), EGF, placental growth factor (PIGF), ANGPT2, VEGF, Platelet-derived growth factor-BB (PDGF-BB), and FGF2 were synthesized and stored intracellularly in untreated HMVEC-dNeo (Figure 4). Intracellular levels of ANGPT2 and VEGF increased over time, whereas HB-EGF, EGF, PIGF levels tended to decrease from 24 to 72 h (Figure 4). Contrarily, PDGF-BB and FGF2 levels remained unchanged throughout the observational period (Figure 4). HMVEC-dNeo cells responded to LCS with marked increases and qualitative differences in the synthesis of angiogenic factors. In brief, HMVEC-dNeo produced and stored higher levels of HB-EGF, EGF, PIGF, VEGF, and PDGF-BB in response to LCS (Figure 4). Moreover, intracellular levels of IL-6 and ANG were only detected in HMVEC-dNeo incubated in the presence of LCS (Figure 4). In contrast, production of angiogenic factors by HMVEC-dNeo cells was minimally affected by empty transwell inserts and tranwell inserts containing acellular collagen gels (Figure 4).
DISCUSSION The commercially available LCS used in this study are bilayer living devices, constructed of type I bovine collagen seeded with viable allogenic human fibroblasts and keratinocytes. In vivo studies demonstrated that these LCS survive for up to 6 weeks in human wounds and exhibits no signs of engraftment. 9 Although the precise mechanism by which these LCS stimulate wound healing remains unclear, it is postulated that LCS living fibroblasts and keratinocytes stimulate angiogenic and inflammatory pathways through the release of a wide variety of cytokines and growth factors. 9,10 Moreover, it is also suggested that cells in LCS physically interact with host cells to alter their behavior during wound. 11 Angiogenesis involves a coordinated sequence of events initiated by selective degradation of vascular basement membranes and surrounding extracellular matrix with migration of endothelial cells, which is followed by proliferation and differentiation of migrated cells into tubular structures. In the present study, we demonstrated for the first time that commercially available LCS successfully favor the initial mechanisms that govern angiogenesis, including endothelial cell migration and proliferation. Although a wide variety of growth factors and chemokines are involved in the regulation of cell migration, VEGF, FGF, and angiopoietins are the major drivers of endothelial cell migration during angiogenesis. 12 Endothelial cell proliferation occurs through the tightly regulated and coordinated action of VEGF, FGF, and PDGF. 13, 14 In accordance to this, here we presented evidence that HMVEC-dNeo respond to LCS with marked increases in the synthesis of VEGF and PDGF-BB, which were maintained for the entire investigational period (72 h). In agreement with our results, a clinical trial has reported previously that PDGF-BB and VEGF levels are up-regulated in gingival recession-type defects treated with the same LCS. 15
We demonstrated that while increased expression of angiogenic genes predominates early on in response to LCS, late-phase responses are characterized by up-regulation of more than 20 angiogenic genes, but also by the down-regulation of almost 30 other angiogenic genes. However, this trend was not confirmed at the protein level, as LCS induced increased production of most of the angiogenic factors tested (i.e. HB-EGF, IL-6, ANG, PDGF-BB, EGF, PIGF and VEGF) throughout the investigational period. It is plausible that reductions in mRNA levels detected at 72 h would be followed by a drop in protein levels at later time points. Thus, it is speculated that levels of angiogenic proteins would fall if experiments 6
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were extended over longer periods of time. Along with this line and in accordance to our results of increased protein levels at 72 h, in vivo clinical data has demonstrated that angiogenic factors are increased during the first week after LCS transplantation into mucogingival defects and their levels progressively decrease from the first to the fourth week of healing. 15 Altogether, these results indicate that expression of angiogenic factors is stimulated early on in response to LCS and is followed by a dynamic equilibrium between positive and negative regulators of angiogenesis to avoid excessive new blood vessel formation. Our data is in accordance with results from a clinical study that demonstrated elevated levels of VEGF, ANG e PDGF-BB at the crevicular gingival fluid from sites treated with LCS. 15 Nonetheless, we were unable to demonstrate increased levels of FGF2 in response to LCS, as has been reported in vivo. 15 There are a few possible explanations for this discrepancy. First, while our study measured FGF2 levels in wholecell lysates, the clinical study by Morelli et al. evaluated FGF2 protein levels at gingival crevicular fluid. 15 FGF2 lacks a consensus signal peptide for secretion, 16 but can be released from producing cells after cell damage. 17 Therefore, increased levels of FGF2 at gingival crevicular fluid of sites treated with LCS might be related to surgical trauma. Second, while we measured FGF2 exclusively produced by endothelial cells, FGF2 levels in crevicular fluid reflects the synthesis and/or release of FGF2 by a wide variety of cell sources, mainly fibroblasts, macrophages, endothelial and epithelial cells. 18, 19 Currently, it is not known whether these cells differ in their responses to LCS. Moreover, it remains to be established how co-cultures of mixed cell populations behave in response to LCS. Studies evaluating the early events of oral mucosal wound healing indicate that the presence of angiogenic factors correlates positively to increased blood vessel formation. 20 In contrast to these findings, we noted that although LCS induced production of pro-angiogenic factors, it failed to accelerate or enhance the formation of complex, mesh-like vascular structures. Until recently, little was known about the molecular mechanisms underlying vascular morphogenesis. However, novel findings revealed that proangiogenic factors VEGF, FGF2 and angiopoietins only prime endothelial cells for vascular morphogenesis and that hematopoietic stem cell cytokines (stem cell factor, IL-3, and CXCL12) are required for pericyte-induced tube maturation. 21 Analysis of HMVEC-dNeo expression profiles at 72 h revealed that CXCL12 levels decrease in response to LCS. Moreover, stem cell factor and IL-3 expression levels are not affected by LCS (not shown). Thus, providing a plausible biological explanation for the inability of LCS to induce accelerated or enhanced vascular network formation. This is corroborated by findings from a clinical study that showed that increases in VEGF, angiogenin, IL-8, FGF2, PDGF-BB, TIMP-1 and TIMP-2 levels at LCS-treated sites did not translate to angiogenesis occurring in vivo. 15 The first phase of wound healing is characterized by production of proinflammatory cytokines 22. LCS promoted increased HMVEC-dNeo expression and/or secretion of proinflammatory mediators (i.e. IL-6, IL-8, TNFIP2, TNFIP3, TNFRSF12A , CCL2, CCL3, CX3CL1, ANGPTL2, ANGPT2, LOX, CBS, JAG1) throughout the 72 h observational period. However, LCS also stimulated increased expression of genes associated with anti-inflammatory responses (HMOX1, SERPINE1, S100P, ADORA2B). Future studies should explore the effect of LCS on the regulation of proinflammatory and anti-inflammatory mediators over longer periods of time, as prolonged inflammation can limit the clinical success of reconstructive therapies. 1
CONCLUSION In the light of the findings presented, this study demonstrates that LCS boost the initial steps of the angiogenic program (i.e. endothelial cell migration and proliferation), but failed to enhance or accelerated HMVEC-dNeo morphological transition to complex vascular structures.
7
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ACKNOWLEDGEMENTS This work was an Investigator Initiated Study sponsored by Organogenesis Inc., Canton, MA, USA. We would like to thank Jack K. Spitznagel, Medical Director, for his assistance in our research relationship with Organogenesis and Mandy Rolando, from UTHSCSA Genomics Core Shared Resource, for her assistance with the array data. Dr. David Cochran appeared before the Food and Drug Administration panel that reviewed the biologics licensing application for the product and was compensated for his efforts before the panel. The remaining authors have reported no conflicts of interest.
DISCLAIMERS: This work was an Investigator Initiated Study sponsored by Organogenesis Inc. Dr. David Cochran appeared before the Food and Drug Administration panel that reviewed the biologics licensing application for the product and was compensated for his efforts before the panel. The remaining authors have reported no conflicts of interest.
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18. Duraisamy Y, Slevin M, Smith N, et al. Effect of glycation on basic fibroblast growth factor induced angiogenesis and activation of associated signal transduction pathways in vascular endothelial cells: possible relevance to wound healing in diabetes. Angiogenesis 2001;4:277-288. 19. Akimoto S, Ishikawa O, Iijima C, Miyachi Y. Expression of basic fibroblast growth factor and its receptor by fibroblast, macrophages and mast cells in hypertrophic scar. Eur J Dermatol 1999;9:357-362. 20. Kumar I, Staton CA, Cross SS, Reed MW, Brown NJ. Angiogenesis, vascular endothelial growth factor and its receptors in human surgical wounds. Br J Surg 2009;96:1484-1491 21. Stratman AN, Davis MJ, Davis GE. VEGF and FGF prime vascular tube morphogenesis and sprouting directed by hematopoietic stem cell cytokines. Blood 2011;117:3709-3719. 22. Eming SA, Krieg T, Davidson JM. Inflammation in wound repair: molecular and cellular mechanisms. J Invest Dermatol 2007; 127:514-525.
Corresponding author for proof and reprints: Cristina C. Villar, Division of Periodontics, Department of Stomatology, School of Dentistry, University of São Paulo, Avenida Prof. Lineu Prestes, 2227, São Paulo – São Paulo, 05508-000, Brazil, + 55 (11) 2648-8055 (phone), + 55 (11) 3091-7833 (fax, can be published),
[email protected] (e-mail, can be published) Submitted June 16, 2014; accepted for publication November 14, 2014. Figure 1. Effect of LCS on HMVEC-dNeo morphology, proliferation, migration and capillary tube formation. A) HMVEC-dNeo proliferation was examined using BrdU ELISA assay. Mean values were obtained by analysis of triplicate wells in four separate experiments, and error bars indicate one SD of the mean. An asterisk indicates that p value is ≤0.0005 for a comparison with HMVEC-dNeo alone. B) HMVEC-dNeo migration was examined using a confocal LSM. Z-stack images of total thickness 200500 µm were collected at intervals of 6.4 µm. Migrating cells were defined as cells moving at least the distance equivalent to their own length. Mean values were obtained by analysis of 20 view fields in triplicate wells in three separate experiments. Error bars indicate one SD of the mean. An asterisk indicates that p value is ≤0.05 for a comparison with HMVEC-dNeo alone and HMVEC-dNeo incubated in the presence of empty tranwell inserts or tranwell inserts with collagen gels. C) Pattern recognition methods were used to quantify the degree of tube formation by HMVEC-dNeo. In this system score 0 represents individual endothelial cells, well separated; score 1 represents migration and alignment of endothelial cells; score 2 represents capillary tube formation with no sprouting; score 3 represents sprouting on new capillary tubes; score 4 represents formation of closed polygons; and score 5 represents development of complex, mesh-like structures. Mean values were obtained by analysis of 15 view fields in triplicate wells in three separate experiments. Error bars indicate one SD of the mean. Figure 2. Hierarchical clustering of expression profiles for 352 genes differentially expressed across different conditions tested. Global gene expression was examined at 24, 48 and 72 h using expression BeadChip platforms. Each colored line in the heat map represents the expression level of one gene that was found to be differentially expressed in at least of one the conditions tested. Individual gene expression profiles are plotted horizontally against vertical columns representing the 12 conditions evaluated (combinations of 4 treatments and 3 time points). For each condition, profiles for the three biological replicates derived from three separate experiments are displayed. The heat map shows strongly expressed and weakly expressed genes with respect to the common median, represented in red and blue, respectively. Figure 3. Confirmation of the expression level of differentially expressed angiogenesis-related genes. Levels of mRNA expression were calculated and normalized to the level of GAPDH mRNA at each time point. Results are shown as average fold-change values ± SD, of four independent experiments with samples in duplicate. Data is presented in two panels, one with an axis that shows all the data, and the other with an axis that focuses on the small values. A color gradient confined to the very top of the bar in the bottom panel indicate that the bar have been truncated and extend higher than their neighbors. Asterisks indicate statistically significant differences (p