Basic Fibroblast Growth Factor Induces Angiogenic Properties of Fibrocytes to Stimulate Vascular Formation during Wound Healing
2016; Elsevier BV; Volume: 186; Issue: 12 Linguagem: Inglês
10.1016/j.ajpath.2016.08.015
ISSN1525-2191
AutoresMiho Nakamichi, Yuri Akishima‐Fukasawa, Chie Fujisawa, Tetuo Mikami, Kiyoshi Onishi, Yoshikiyo Akasaka,
Tópico(s)Lymphatic System and Diseases
ResumoThe role of fibrocytes in wound angiogenesis remains unclear. We therefore demonstrated the specific changes in fibrocyte accumulation for angiogesis in basic fibroblast growth factor (bFGF)–treated wounds. bFGF-treated wounds exhibited marked formation of arterioles and inhibition of podoplanin+ lymph vessels that were lacking in vascular endothelial growth factor-A–treated wounds. Real-time PCR in bFGF-treated wounds manifested enhanced expression of CD34, CD31, and bFGF mRNA and reduced expression of podoplanin and collagen type I, III, and IV mRNA. Double immunofluorescence staining focusing on fibrocyte detection in bFGF-treated wounds showed increased formation of capillary-like structures composed of CD34+/procollagen I+ fibrocytes, with a lack of capillary-like structures formed by CD45+/procollagen I+ or CD11b+/procollagen I+ fibrocytes. However, vascular endothelial growth factor-A–treated wounds lacked capillary-like structures composed of CD34+/procollagen I+ fibrocytes, with increased numbers of CD34+/fetal liver kinase-1+ endothelial progenitor cells. Furthermore, fibroblast growth factor receptor 1 siRNA injection into wounds, followed by bFGF, inhibited the formation of capillary-like structures composed of CD34+/procollagen I+ fibrocytes, together with inhibited mRNA expression of CD34 and CD31 and enhanced mRNA expression of collagen type I, indicating the requirements of bFGF/fibroblast growth factor receptor 1 system for capillary structure formation. This study highlights the angiogenic properties of CD34+/procollagen I+ fibrocytes specifically induced by bFGF, providing new insight into the active contribution of fibrocytes for vascular formation during wound healing. The role of fibrocytes in wound angiogenesis remains unclear. We therefore demonstrated the specific changes in fibrocyte accumulation for angiogesis in basic fibroblast growth factor (bFGF)–treated wounds. bFGF-treated wounds exhibited marked formation of arterioles and inhibition of podoplanin+ lymph vessels that were lacking in vascular endothelial growth factor-A–treated wounds. Real-time PCR in bFGF-treated wounds manifested enhanced expression of CD34, CD31, and bFGF mRNA and reduced expression of podoplanin and collagen type I, III, and IV mRNA. Double immunofluorescence staining focusing on fibrocyte detection in bFGF-treated wounds showed increased formation of capillary-like structures composed of CD34+/procollagen I+ fibrocytes, with a lack of capillary-like structures formed by CD45+/procollagen I+ or CD11b+/procollagen I+ fibrocytes. However, vascular endothelial growth factor-A–treated wounds lacked capillary-like structures composed of CD34+/procollagen I+ fibrocytes, with increased numbers of CD34+/fetal liver kinase-1+ endothelial progenitor cells. Furthermore, fibroblast growth factor receptor 1 siRNA injection into wounds, followed by bFGF, inhibited the formation of capillary-like structures composed of CD34+/procollagen I+ fibrocytes, together with inhibited mRNA expression of CD34 and CD31 and enhanced mRNA expression of collagen type I, indicating the requirements of bFGF/fibroblast growth factor receptor 1 system for capillary structure formation. This study highlights the angiogenic properties of CD34+/procollagen I+ fibrocytes specifically induced by bFGF, providing new insight into the active contribution of fibrocytes for vascular formation during wound healing. Wound healing is a complex process, and the mechanisms underlying granulation tissue formation remain obscure. Among the types of granulation tissue cells, fibroblasts have distinctive functions and have been postulated to partly originate from peripheral blood cells. Numerous studies have explored the pathways by which peripheral mononuclear cells undergo differentiation into fibroblasts. A distinct population of bone marrow–derived cells with fibroblast-like properties was described by Bucala et al1Bucala R. Spiegel L.A. Chesney J. Hogan M. Cerami A. Circulating fibrocytes define a new leukocyte subpopulation that mediates tissue repair.Mol Med. 1994; 1: 71-81Crossref PubMed Google Scholar that were termed fibrocytes and account for 0.1% to 0.5% of circulating leukocytes in the peripheral blood. Cultured fibrocytes display coexpression of a hematopoietic stem cell marker (CD34), leukocyte common antigen (CD45), monocyte lineage markers, such as CD11b, CD13, and major histocompatibility complex class II, and fibroblastic markers, such as collagens I, III, and IV, procollagen I, and fibronectin.1Bucala R. Spiegel L.A. Chesney J. Hogan M. Cerami A. Circulating fibrocytes define a new leukocyte subpopulation that mediates tissue repair.Mol Med. 1994; 1: 71-81Crossref PubMed Google Scholar, 2Abe R. Donnelly S.C. Peng T. Bucala R. Metz C.N. Peripheral blood fibrocytes: differentiation pathway and migration to wound sites.J Immunol. 2001; 166: 7556-7562Crossref PubMed Scopus (922) Google Scholar, 3Grieb G. Bucala R. Fibrocytes in fibrotic diseases and wound healing.Adv Wound Care (New Rochelle). 2012; 1: 36-40Crossref PubMed Google Scholar, 4Reilkoff R.A. Bucala R. Herzog E.L. Fibrocytes: emerging effector cells in chronic inflammation.Nat Rev Immunol. 2011; 11: 427-435Crossref PubMed Scopus (319) Google Scholar, 5Quan T.E. Cowper S. Wu S.P. Bockenstedt L.K. Bucala R. Circulating fibrocytes: collagen-secreting cells of the peripheral blood.Int J Biochem Cell Biol. 2004; 36: 598-606Crossref PubMed Scopus (488) Google Scholar These cells also secrete proinflammatory cytokines, such as tumor necrosis factor, matrix metalloproteinase-9, IL-6, IL-8, and IL-10,1Bucala R. Spiegel L.A. Chesney J. Hogan M. Cerami A. Circulating fibrocytes define a new leukocyte subpopulation that mediates tissue repair.Mol Med. 1994; 1: 71-81Crossref PubMed Google Scholar, 2Abe R. Donnelly S.C. Peng T. Bucala R. Metz C.N. Peripheral blood fibrocytes: differentiation pathway and migration to wound sites.J Immunol. 2001; 166: 7556-7562Crossref PubMed Scopus (922) Google Scholar, 3Grieb G. Bucala R. Fibrocytes in fibrotic diseases and wound healing.Adv Wound Care (New Rochelle). 2012; 1: 36-40Crossref PubMed Google Scholar, 4Reilkoff R.A. Bucala R. Herzog E.L. Fibrocytes: emerging effector cells in chronic inflammation.Nat Rev Immunol. 2011; 11: 427-435Crossref PubMed Scopus (319) Google Scholar, 5Quan T.E. Cowper S. Wu S.P. Bockenstedt L.K. Bucala R. Circulating fibrocytes: collagen-secreting cells of the peripheral blood.Int J Biochem Cell Biol. 2004; 36: 598-606Crossref PubMed Scopus (488) Google Scholar and express various chemokine receptors, such as CCR7 and chemokine receptor type 4, that regulate recruitment of inflammatory cells.6Mehrad B. Burdick M.D. Zisman D.A. Keane M.P. Belperio J.A. Strieter R.M. Circulating peripheral blood fibrocytes in human fibrotic interstitial lung disease.Biochem Biophys Res Commun. 2007; 353: 104-108Crossref PubMed Scopus (218) Google Scholar, 7Sakai N. Wada T. Yokoyama H. Lipp M. Ueha S. Matsushima K. Kaneko S. Secondary lymphoid tissue chemokine (SLC/CCL21)/CCR7 signaling regulates fibrocytes in renal fibrosis.Proc Natl Acad Sci U S A. 2006; 103: 14098-14103Crossref PubMed Scopus (220) Google Scholar, 8Phillips R.J. Burdick M.D. Hong K. Lutz M.A. Murray L.A. Xue Y.Y. Belperio J.A. Keane M.P. Strieter R.M. Circulating fibrocytes traffic to the lungs in response to CXCL12 and mediate fibrosis.J Clin Invest. 2004; 114: 438-446Crossref PubMed Scopus (920) Google Scholar, 9Keeley E.C. Mehrad B. Strieter R.M. The role of fibrocytes in fibrotic diseases of the lungs and heart.Fibrogenesis Tissue Repair. 2011; 4: 2Crossref PubMed Scopus (60) Google Scholar Based on these findings, fibrocytes have been assumed to play a pivotal role in both wound healing10Yang L. Scott P.G. Dodd C. Medina A. Jiao H. Shankowsky H.A. Ghahary A. Tredget E.E. Identification of fibrocytes in postburn hypertrophic scar.Wound Repair Regen. 2005; 13: 398-404Crossref PubMed Scopus (147) Google Scholar, 11Wang J.F. Jiao H. 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Bone marrow-derived progenitor cells in pulmonary fibrosis.J Clin Invest. 2004; 113: 243-252Crossref PubMed Scopus (650) Google Scholar, 15Wang C.H. Huang C.D. Lin H.C. Lee K.Y. Lin S.M. Liu C.Y. Huang K.H. Ko Y.S. Chung K.F. Kuo H.P. Increased circulating fibrocytes in asthma with chronic airflow obstruction.Am J Respir Crit Care Med. 2008; 178: 583-591Crossref PubMed Scopus (148) Google Scholar, 16Schmidt M. Sun G. Stacey M.A. Mori L. Mattoli S. Identification of circulating fibrocytes as precursors of bronchial myofibroblasts in asthma.J Immunol. 2003; 171: 380-389Crossref PubMed Scopus (563) Google Scholar, 17Andersson-Sjoland A. de Alba C.G. Nihlberg K. Becerril C. Ramirez R. Pardo A. Westergren-Thorsson G. Selman M. Fibrocytes are a potential source of lung fibroblasts in idiopathic pulmonary fibrosis.Int J Biochem Cell Biol. 2008; 40: 2129-2140Crossref PubMed Scopus (291) Google Scholar During the process of tissue fibrosis, fibrocytes have been speculated to infiltrate the extravascular tissues and differentiate into myofibroblasts, leading to collagen production and subsequent fibrosis.2Abe R. Donnelly S.C. Peng T. Bucala R. Metz C.N. Peripheral blood fibrocytes: differentiation pathway and migration to wound sites.J Immunol. 2001; 166: 7556-7562Crossref PubMed Scopus (922) Google Scholar, 18Mori L. Bellini A. Stacey M.A. Schmidt M. Mattoli S. Fibrocytes contribute to the myofibroblast population in wounded skin and originate from the bone marrow.Exp Cell Res. 2005; 304: 81-90Crossref PubMed Scopus (318) Google Scholar However, the mechanisms underlying granulation tissue formation mediated by fibrocytes in wounds, especially those underlying the role of fibrocytes in angiogenesis, remain obscure. Recent accumulating evidence suggested that fibrocytes could play an important role in angiogenesis, because in vitro evidence indicated that fibrocytes express high levels of angiogenetic growth factors, such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and platelet-derived growth factor.5Quan T.E. Cowper S. Wu S.P. Bockenstedt L.K. Bucala R. Circulating fibrocytes: collagen-secreting cells of the peripheral blood.Int J Biochem Cell Biol. 2004; 36: 598-606Crossref PubMed Scopus (488) Google Scholar Further investigations provided supporting evidence for an angiogenic role of fibrocytes, by demonstrating that the culture medium of fibrocytes promoted angiogenesis in vitro and in vivo.19Hartlapp I. Abe R. Saeed R.W. Peng T. Voelter W. Bucala R. Metz C.N. Fibrocytes induce an angiogenic phenotype in cultured endothelial cells and promote angiogenesis in vivo.FASEB J. 2001; 15: 2215-2224Crossref PubMed Scopus (240) Google Scholar, 20Kao H.K. Chen B. Murphy G.F. Li Q. Orgill D.P. Guo L. Peripheral blood fibrocytes: enhancement of wound healing by cell proliferation, re-epithelialization, contraction, and angiogenesis.Ann Surg. 2011; 254: 1066-1074Crossref PubMed Scopus (89) Google Scholar These combined findings indicate that fibrocytes can act as a positive regulator of angiogenesis. However, a recent investigation demonstrated that fibrocytes can also play a role as a negative regulator of angiogenesis, because certain types of fibrocytes secrete angiogenesis inhibitors.21Li J. Tan H. Wang X. Li Y. Samuelson L. Li X. Cui C. Gerber D.A. Circulating fibrocytes stabilize blood vessels during angiogenesis in a paracrine manner.Am J Pathol. 2014; 184: 556-571Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar Therefore, the angiogenetic properties of fibrocytes and the role of specific subtypes of fibrocytes in angiogenesis during wound healing need to be clarified. The growth factor bFGF plays a role as a potent inducer of angiogenesis because it can enhance mitogenesis of endothelial cells. bFGF is also a positive regulator of angiogenesis by recruitment of inflammatory cells, such as monocytes, macrophages, and neutrophils, as a result of its up-regulation of the expression of various chemokines, such as chemokine (C-C motif) ligands 2 and 7 and CXCL1.22Presta M. Andres G. Leali D. Dell'Era P. Ronca R. Inflammatory cells and chemokines sustain FGF2-induced angiogenesis.Eur Cytokine Netw. 2009; 20: 39-50PubMed Google Scholar VEGF-A is an important growth factor for promotion of the early events of angiogenesis, particularly for the promotion of endothelial cell migration.23Barrientos S. Stojadinovic O. Golinko M.S. Brem H. Tomic-Canic M. Growth factors and cytokines in wound healing.Wound Repair Regen. 2008; 16: 585-601Crossref PubMed Scopus (2392) Google Scholar Therefore, the contribution of bFGF to fibrocyte recruitment during the process of angiogenesis needs to be investigated. Our aim was to examine the role of fibrocytes in angiogenesis in wound healing and then to clarify the mechanisms by which bFGF promotes angiogenesis in wounds and how such mechanisms might involve angiogenic properties of fibrocytes. All experimental studies were approved by Toho University Laboratory Animal Research Committee (authorization number 15-33-239), complied with the Guidelines on Animal Experiments of Toho University in accordance with the Japanese Government Animal Protection and Management Law (number 105), and conformed to the European Union Directive 2010/63/EU for Animal Experiments. Male Sprague-Dawley rats were purchased from CLEA Japan, Inc. (Tokyo, Japan) and were acclimatized in our laboratory for ≥7 days before use. Sprague-Dawley rats were anesthetized with isoflurane (WAKO Pure Chemical Industries, Ltd, Osaka, Japan) at a concentration of 3% in oxygen, using a precision vaporizer (TK-7; Bio Machinery Co Ltd, Chiba, Japan). The following four treatment groups were established: a control group and a bFGF group in which phosphate-buffered saline (PBS) and 10 ng of bFGF (100 ng/mL; Kaken Pharmaceutical Co Ltd, Tokyo, Japan), respectively, were applied onto full-thickness wounds. The last two groups were a control group and a VEGF-A (Peprotech, Rocky Hill, NJ) group, in which PBS and 1 μg of VEGF-A, respectively, were applied onto full-thickness wounds. A total of 20 male Sprague-Dawley rats (body weight, 390 to 400 g) were used for this study. The rats were randomly assigned to one of the four groups before surgery, and the groups were evaluated on days 2, 4, 6, 7, and 14 postoperatively. Before incision, the back was clipped and disinfected with Hibitane (5% chlorhexidine gluconate; AstraZeneca Pharmaceutical Co, Osaka, Japan) containing alcohol. For each rat, four full-thickness wounds (10 mm in diameter) composed of two bFGF-treated wounds and two PBS-treated wounds or two VEGF-A–treated wounds and two PBS-treated wounds were generated on the dorsal skin using a biopsy punch (Kai Industries Co, Ltd, Gifu, Japan). After the generation of full-thickness wounds at 2-cm intervals, a bFGF- or VEGF-A–treated artificial dermis (Pelnac; Gunze Corp., Osaka, Japan) was prepared by dropping 0.1 mL of PBS containing bFGF or VEGF-A onto the artificial dermis (Figure 1A). The full-thickness wounds were then treated with the artificial dermis that had been immersed in bFGF or VEGF-A. To protect against dehydration and infection, wounds were covered with occlusive polyurethane foam dressings (Hydrosite plus; Smith & Nephew Wound Management KK, Tokyo, Japan) that were fixed using nylon sutures (A18-40N3; Natsume Seisakusho, Tokyo, Japan). Each treatment was assigned to a different position on the dorsal skin to eliminate any effect of wound placement. Groups of rats received in the animal-euthanizing chambers were euthanized by using carbon dioxide fed from a gas feeder (KN-750-2-A; Natsume Seisakusho) on days 2, 4, 6, 7, and 14 postoperatively, with a total of 16 wounds (four bFGF-treated wounds and four PBS-treated wounds or four VEGF-treated wounds and four PBS-treated wounds) obtained on each day. For the following examination, the wound and surrounding tissue, including the entire wound bed, were excised. Excised tissue samples were then cut into two pieces. One piece was fixed in buffered 4% paraformaldehyde for histologic examination and embedded in paraffin, according to standard protocols (Figure 1, B and C). Another piece was excised for RNA extraction and was stored at −20°C in a deep freezer. Histologic analysis was performed according to methods described previously.24Ishiguro S. Akasaka Y. Kiguchi H. Suzuki T. Imaizumi R. Ishikawa Y. Ito K. Ishii T. Basic fibroblast growth factor induces down-regulation of alpha-smooth muscle actin and reduction of myofibroblast areas in open skin wounds.Wound Repair Regen. 2009; 17: 617-625Crossref PubMed Scopus (51) Google Scholar Briefly, for identification of four different histologic types of vessels (capillaries, arterioles, arteries, and lymph vessels), horizontal sections of the wound granulation tissue stained by the triple immunohistochemical staining method described below were scanned using ImageJ software version 1.41 (NIH, Bethesda, MD; http://imagej.nih.gov/ij), and the digital images were analyzed. Three unbiased observers (Y.A.-F., T.M., and K.O.) randomly selected 10 fields in granulation tissues per tissue section. In each field, they counted the number of four types of vessels (capillaries, arterioles, arteries, and lymph vessels) at ×80 magnification, and the average number of each type of vessel per field was finally calculated as the density of each type of vessel. For counting of fibrocytes, the three observers also assessed the number of double-positive fibrocytes. Ten to twelve randomly selected fields in granulation tissues per tissue section were examined, and the numbers of double-positive fibrocytes were counted and calculated as the mean number of positively stained cells per total number of DAPI-positive granulation tissue cells at ×80 magnification. We conducted immunohistochemical staining for counting the numbers of vessels using von Willebrand factor (vWF) antibody, as described previously.25Gartland A. Mason-Savas A. Yang M. MacKay C.A. Birnbaum M.J. Odgren P.R. Septoclast deficiency accompanies postnatal growth plate chondrodysplasia in the toothless (tl) osteopetrotic, colony-stimulating factor-1 (CSF-1)-deficient rat and is partially responsive to CSF-1 injections.Am J Pathol. 2009; 175: 2668-2675Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 26Sen V. Guzel A. Sen H.S. Ece A. Uluca U. Soker S. Dogan E. Kaplan I. Deveci E. Preventive effects of dexmedetomidine on the liver in a rat model of acid-induced acute lung injury.Biomed Res Int. 2014; 2014: 621827Google Scholar The primary antibody was a rabbit anti-vWF antibody at a dilution of 1:100 that had been purchased from Abcam, Inc. (ab6994; Cambridge, MA). The secondary antibody/reagent was Envision+ mouse peroxidase detection system that had been purchased from Dako (K4001; Glostrup, Denmark). We conducted triple immunohistochemical staining for identification of four different types of vessels (capillaries, arterioles, arteries, and lymph vessels) using anti-CD34, anti–α-smooth muscle actin (α-SMA), and anti-podoplanin antibodies. Sections were heated to 95°C for 5 minutes in a jar with an antigen activation solution at pH 9 (Nichirei Corp., Tokyo, Japan) and were then incubated with rabbit anti-CD34 (ab150060; Abcam, Inc.) and mouse anti-podoplanin antibodies (D2-40) (NB120-11842; Novus Biologicals, Littleton, CO) at dilutions both of 1:100. The sections were washed with Tris-buffered saline containing 0.05% Tween 20, incubated with anti-rabbit alkaline phosphatase–conjugated secondary antibodies (Nichirei Corp.) at a dilution of 1:100, and further incubated with the New Fuchsin substrate solution (Nichirei Corp.). After washing with Tris-buffered saline containing 0.05% Tween 20, the sections were incubated with biotin-conjugated secondary antibodies directed against mouse (BA2000; Vector Laboratories, Burlingame, CA) and with streptavidin conjugated to horseradish peroxidase (Nichirei Corp.) at dilutions both of 1:100 and were further incubated with the Deep Space Black Chromogen Kit (Biocare Medical, Concord, CA). After washing with Tris-buffered saline containing 0.05% Tween 20, the sections were incubated with a mouse anti–α-SMA antibody, followed by a streptavidin-biotin-peroxidase complex (U7033; Dako) at dilutions both of 1:100 and staining with 3,3′-diaminobenzidine (Dako). We conducted double immunofluorescence (IF) staining for identification of fibrocytes, as described previously.10Yang L. Scott P.G. Dodd C. Medina A. Jiao H. Shankowsky H.A. Ghahary A. Tredget E.E. Identification of fibrocytes in postburn hypertrophic scar.Wound Repair Regen. 2005; 13: 398-404Crossref PubMed Scopus (147) Google Scholar, 20Kao H.K. Chen B. Murphy G.F. Li Q. Orgill D.P. Guo L. Peripheral blood fibrocytes: enhancement of wound healing by cell proliferation, re-epithelialization, contraction, and angiogenesis.Ann Surg. 2011; 254: 1066-1074Crossref PubMed Scopus (89) Google Scholar The primary antibodies were as follows: a rabbit anti-CD34 antibody (ab150060) at a dilution of 1:100, a rabbit anti-CD45 antibody (ab10558) at a dilution of 1:100, a rabbit anti-CD11b antibody (ab75476) at a dilution of 1:200, and a mouse anti-fibroblast growth factor receptor 1 (FGFR1) antibody (ab829) at a dilution of 1:100, which were all purchased from Abcam, Inc. A goat anti-procollagen I antibody (sc25973) at a dilution of 1:100 was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX). The secondary antibodies used were as follows: biotin-conjugated secondary antibodies directed against goat (BA9500), biotin-conjugated secondary antibodies directed against mouse (BA2000), Texas Red avidin D (A2006), Texas Red anti-goat IgG antibody (TI-5000), and fluorescein avidin D (A2001), which had been all purchased from Vector Laboratories. Fluorescein isothiocyanate–conjugated anti-rabbit immunoglobulins (F0205) were purchased from Dako. Negative controls were generated by incubating tissue sections (bFGF-treated wounds on day 6) with Ig from the same species [goat Ig (I-5000; Vector Laboratories) and rabbit Ig (I-1000; Vector Laboratories)] at the same final concentration, but with no primary antibody. Unstained controls were generated by incubating tissue sections (bFGF-treated wounds on day 6) with PBS, but with no primary antibody. For identification of endothelial progenitor cells, double IF staining was performed as described previously.27Yao W. Firth A.L. Sacks R.S. Ogawa A. Auger W.R. Fedullo P.F. Madani M.M. Lin G.Y. Sakakibara N. Thistlethwaite P.A. Jamieson S.W. Rubin L.J. Yuan J.X. Identification of putative endothelial progenitor cells (CD34+CD133+Flk-1+) in endarterectomized tissue of patients with chronic thromboembolic pulmonary hypertension.Am J Physiol Lung Cell Mol Physiol. 2009; 296: L870-L878Crossref PubMed Scopus (69) Google Scholar The primary antibodies were as follows: a rabbit anti-CD34 antibody (ab150060; Abcam, Inc.) at a dilution of 1:100 and a mouse anti-fetal liver kinase (Flk)-1 antibody (sc393163; Santa Cruz Biotechnology, Inc.) at a dilution of 1:100. The secondary antibodies used were as follows: biotin-conjugated secondary antibodies directed against mouse (BA2000) were purchased from Vector Laboratories. Fluorescein isothiocyanate–conjugated swine anti-rabbit immunoglobulins (F0205) and Texas Red avidin D (A2006) were purchased from Dako and Vector Laboratories, respectively. Cellular colocalization was evaluated using Graphic Converter Universal Binary software version 6.1.2J (Lemke Software GmbH, Peine, Germany). siRNA targeting the human and rat FGFR1 mRNA (http://www.ncbi.nlm.nih.gov/nuccore/D12498.1; accession number D12498.1) was designed and synthesized by Nippon Gene Co, Ltd (Toyama, Japan). The siRNA sense strand sequence was as follows: CCAAUGAGCUGUACAUGAUdTdT. As a negative control, control siRNA (scrambled siRNA; sc37007) was purchased from Santa Cruz Biotechnology, Inc. The synthesized siRNAs were dissolved in TransIT-QR hydrodynamic delivery solution (Mirus Bio LCC, Madison, WI), which also served as control, and were injected into rat skin. The rats were dosed with a single intradermal injection of 20 μg of siRNA (siFGFR1 group) or control siRNA (siCON group) per rat, three times at intervals of 2 days, with two rats in each group. bFGF treatment after siRNA injection was performed as follows. At 24 hours after the last injection, full-thickness wounds 10 mm in diameter were generated on the siRNA-injected sites using a biopsy punch (Kai Industries Co, Ltd). After the generation of full-thickness wounds that had been injected with siRNA (siFGFR1 group) or control siRNA (siCON group), the full-thickness wounds were treated with the bFGF-treated artificial dermis (Pelnac), as described above. At 2, 4, 6, 7, and 14 days after bFGF treatment, skin tissue samples were excised from the wounds and were cut into two pieces, one for immunohistochemistry and one for PCR analysis. Tissue samples excised from wounds were homogenized in FastPrep-24 (MP Biomedicals, Solon, OH), and the RNA was isolated using NucleoSpin RNA (Macherey-Nagel, Düren, Germany) and then reverse transcribed using the PrimeScript RT Reagent Kit (Takara Bio Inc., Kyoto, Japan). Target gene expression was estimated using TaqMan gene expression assays specific for CD34 (Rn03416140), CD31 (Rn01467262), podoplanin (Rn01637526), bFGF (Rn00570809), FGFR1 (Rn01478647), and β-actin (Rn01759928) (Applied Biosystems, Foster, CA).28Kinoshita T. Ishikawa Y. Arita M. Akishima-Fukasawa Y. Fujita K. Inomata N. Suzuki T. Namiki A. Mikami T. Ikeda T. Yamazaki J. Ishii T. Akasaka Y. Antifibrotic response of cardiac fibroblasts in hypertensive hearts through enhanced TIMP-1 expression by basic fibroblast growth factor.Cardiovasc Pathol. 2014; 23: 92-100Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar Relative quantification and statistics were estimated as the mean of three replicate assays calculated by the 7500 FAST sequence detection software version 2.3 (Applied Biosystems). Results are presented as means ± SEM. Statistical significance for two-group comparisons in quantitative RT-PCR analysis was determined using unpaired t-tests using GraphPad QuickCalcs (GraphPad Software, Inc., La Jolla, CA; http://www.graphpad.com/quickcalcs/ttest2). Comparisons of the numbers of immunohistochemical-positive vessels and IF-positive fibrocytes between two groups (bFGF versus control or VEGF versus control) were statistically analyzed using one-way analysis of variance, followed by Bonferroni's post hoc test. Differences with P < 0.05 were considered statistically significant. We first assessed the number of vWF-positive (vWF+) vessels, including capillaries, arterioles, arteries, and lymph vessels, in bFGF-treated or control wounds on days 2, 4, 6, 7, and 14 after operation. The average number of vWF+ vessels per field was significantly greater in the bFGF-treated wounds than in the control wounds on day 6 (P < 0.05) (Figure 2A). In addition, the average number of vWF+ vessels per field was significantly greater in the VEGF-A–treated wounds than in the control wounds observed on days 2 and 4 (P < 0.05) (Figure 2B). We further confirmed the positive effects of bFGF on angiogenesis by assessing the mRNA expression levels of the two endothelial cell markers, CD34 and CD31, using quantitative RT-PCR (real-time PCR). Compared to the control, bFGF-treated wounds showed a significant increase in CD34 mRNA expression on days 4, 6, and 14 (P < 0.0001) (Figure 2C). CD31 mRNA expression was also significantly higher in bFGF-treated wounds than in control wounds on days 4 and 14 (P = 0.0011 and P = 0.0001, respectively) (Figure 2D). These combined data further confirmed a positive effect of bFGF on angiogenesis. Regarding collagen expression in wounds, bFGF treatment for 6 days significantly decreased expression of collagen type I, III, and IV mRNA compared to control wounds (P < 0.0001) (Figure 2, E–G). bFGF treatment for 14 days also significantly decreased expression of collagen type I, III, and IV mRNA expression compared to control wounds (P < 0.0001, P = 0.0002, and P < 0.0001, respectively) (Figure 2, E–G). No significant difference in the three types of collagen mRNA expression was evident between bFGF-treated and control wounds on day 4 (Figure 2, E–G). On the other hand, bFGF mRNA expression in bFGF-treated wounds was significantly higher than that in control wounds on all three days (P < 0.0001) (Figure 2H). This suggests that the persistent induction of bFGF mRNA after bFGF treatment contributes to enhanced angiogenesis and inhibited collagen mRNA expression in bFGF-treated wounds. We next assessed the vessel densities in four different types of vessels in bFGF- or VEGF-A–treated wounds on days 2, 4, 6, 7, and 14 after operation. We considered immunohistochemically stained CD34+/α-SMA– vessels as capillaries, CD34+/α-SMA+ vessels in the thin smooth
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