Circulating Fibrocytes Stabilize Blood Vessels during Angiogenesis in a Paracrine Manner
2013; Elsevier BV; Volume: 184; Issue: 2 Linguagem: Inglês
10.1016/j.ajpath.2013.10.021
ISSN1525-2191
AutoresJinqing Li, Hong Tan, Xiaolin Wang, Yuejun Li, Lisa Samuelson, Xueyong Li, Cai-Bin Cui, David A. Gerber,
Tópico(s)Cell Adhesion Molecules Research
ResumoAccumulating evidence supports that circulating fibrocytes play important roles in angiogenesis. However, the specific role of fibrocytes in angiogenesis and the underlying mechanisms remain unclear. In this study, we found that fibrocytes stabilized newly formed blood vessels in a mouse wound-healing model by inhibiting angiogenesis during the proliferative phase and inhibiting blood vessel regression during the remodeling phase. Fibrocytes also inhibited angiogenesis in a Matrigel mouse model. In vitro study showed that fibrocytes inhibited both the apoptosis and proliferation of vascular endothelial cells (VECs) in a permeable support (Transwell) co-culture system. In a three-dimensional collagen gel, fibrocytes stabilized the VEC tubes by decreasing VEC tube density on stimulation with growth factors and preventing VEC tube regression on withdrawal of growth factors. Further mechanistic investigation revealed that fibrocytes expressed many prosurvival factors that are responsible for the prosurvival effect of fibrocytes on VECs and blood vessels. Fibrocytes also expressed angiogenesis inhibitors, including thrombospondin-1 (THBS1). THBS1 knockdown partially blocked the fibrocyte-induced inhibition of VEC proliferation in the Transwell co-culture system and recovered the fibrocyte-induced decrease of VEC tube density in collagen gel. Purified fibrocytes transfected with THBS1 siRNA partially recovered the fibrocyte-induced inhibition of angiogenesis in both the wound-healing and Matrigel models. In conclusion, our findings reveal that fibrocytes stabilize blood vessels via prosurvival factors and anti-angiogenic factors, including THBS1. Accumulating evidence supports that circulating fibrocytes play important roles in angiogenesis. However, the specific role of fibrocytes in angiogenesis and the underlying mechanisms remain unclear. In this study, we found that fibrocytes stabilized newly formed blood vessels in a mouse wound-healing model by inhibiting angiogenesis during the proliferative phase and inhibiting blood vessel regression during the remodeling phase. Fibrocytes also inhibited angiogenesis in a Matrigel mouse model. In vitro study showed that fibrocytes inhibited both the apoptosis and proliferation of vascular endothelial cells (VECs) in a permeable support (Transwell) co-culture system. In a three-dimensional collagen gel, fibrocytes stabilized the VEC tubes by decreasing VEC tube density on stimulation with growth factors and preventing VEC tube regression on withdrawal of growth factors. Further mechanistic investigation revealed that fibrocytes expressed many prosurvival factors that are responsible for the prosurvival effect of fibrocytes on VECs and blood vessels. Fibrocytes also expressed angiogenesis inhibitors, including thrombospondin-1 (THBS1). THBS1 knockdown partially blocked the fibrocyte-induced inhibition of VEC proliferation in the Transwell co-culture system and recovered the fibrocyte-induced decrease of VEC tube density in collagen gel. Purified fibrocytes transfected with THBS1 siRNA partially recovered the fibrocyte-induced inhibition of angiogenesis in both the wound-healing and Matrigel models. In conclusion, our findings reveal that fibrocytes stabilize blood vessels via prosurvival factors and anti-angiogenic factors, including THBS1. Angiogenesis is the growth of new blood vessels from pre-existing ones. It plays important roles during physiological processes, such as growth, development, wound healing, and the female reproductive cycle, and in many deadly and debilitating diseases, including cancer,1Gomes F.G. Nedel F. Alves A.M. Nor J.E. Tarquinio S.B. Tumor angiogenesis and lymphangiogenesis: tumor/endothelial crosstalk and cellular/microenvironmental signaling mechanisms.Life Sci. 2013; 92: 101-107Crossref PubMed Scopus (112) Google Scholar skin diseases, age-related blindness, diabetic ulcers, cardiovascular disease, and stroke.2Chung A.S. Ferrara N. Developmental and pathological angiogenesis.Annu Rev Cell Dev Biol. 2011; 27: 563-584Crossref PubMed Scopus (579) Google Scholar Thus, it is crucial to define the mechanisms underlying angiogenesis to combat angiogenesis-related diseases. Remarkable progress has led to the identification of specific factors that regulate angiogenesis.3Nguyen A. Hoang V. Laquer V. Kelly K.M. Angiogenesis in cutaneous disease: part I.J Am Acad Dermatol. 2009; 61 (quiz 43-44): 921-942Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar These molecules are divided into two categories, based on their functions in angiogenesis3Nguyen A. Hoang V. Laquer V. Kelly K.M. Angiogenesis in cutaneous disease: part I.J Am Acad Dermatol. 2009; 61 (quiz 43-44): 921-942Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar: angiogenic growth factors, which promote angiogenesis, including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (FGF2), platelet-derived growth factor (PDGF), and angiogenin (ANG); and angiogenesis inhibitors, which inhibit angiogenesis, including thrombospondin (THBS), angioarrestin, and angiostatin. Only a few studies have focused on the roles of different cells in angiogenesis, even though angiogenesis is orchestrated by multiple cell types. Circulating fibrocytes (alias fibrocytes or peripheral blood fibrocytes) are bone marrow-derived mesenchymal progenitors that co-express hematopoietic stem cell markers (eg, CD34), monocyte lineage markers (eg, CD11b+, CD13, and CD86), the leukocyte common antigen (CD45), and fibroblast proteins (eg, collagens I, III, and IV, procollagen I, and lamin B).4Abe 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 (947) Google Scholar, 5Bucala 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, 6Chesney J. Bacher M. Bender A. Bucala R. The peripheral blood fibrocyte is a potent antigen-presenting cell capable of priming naive T cells in situ.Proc Natl Acad Sci U S A. 1997; 94: 6307-6312Crossref PubMed Scopus (315) Google Scholar, 7Pilling D. Vakil V. Gomer R.H. Improved serum-free culture conditions for the differentiation of human and murine fibrocytes.J Immunol Methods. 2009; 351: 62-70Crossref PubMed Scopus (53) Google Scholar Fibrocytes comprise 0.1% to 1% of the nucleated cells in the peripheral blood. Fibrocytes play an important role in granulation tissue formation during wound healing and in some fibrotic diseases, such as airway remodeling in asthma,8Kaur D. Saunders R. Berger P. Siddiqui S. Woodman L. Wardlaw A. Bradding P. Brightling C.E. Airway smooth muscle and mast cell-derived CC chemokine ligand 19 mediate airway smooth muscle migration in asthma.Am J Respir Crit Care Med. 2006; 174: 1179-1188Crossref PubMed Scopus (127) Google Scholar interstitial pulmonary fibrosis, cardiac fibrosis,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 (61) Google Scholar chronic pancreatitis and cystitis,10Barth P.J. Ebrahimsade S. Hellinger A. Moll R. Ramaswamy A. CD34+ fibrocytes in neoplastic and inflammatory pancreatic lesions.Virchows Arch. 2002; 440: 128-133Crossref PubMed Scopus (92) Google Scholar and nephrogenic systemic fibrosis.11Galan A. Cowper S.E. Bucala R. Nephrogenic systemic fibrosis (nephrogenic fibrosing dermopathy).Curr Opin Rheumatol. 2006; 18: 614-617Crossref PubMed Scopus (139) Google Scholar In these diseases, fibrocytes infiltrate the diseased tissue and differentiate into myofibroblasts that deposit extracellular matrix (ECM) and subsequently induce fibrosis. The hypothesis that fibrocytes regulate angiogenesis was based on the observation that fibrocytes secrete angiogenic growth factors, including VEGF, PDGF, and FGF2.12Metz C.N. Fibrocytes: a unique cell population implicated in wound healing.Cell Mol Life Sci. 2003; 60: 1342-1350Crossref PubMed Scopus (206) Google Scholar Further investigations from different groups found that conditioned medium from the fibrocytes promoted angiogenesis both in vitro and in vivo,13Hartlapp 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 (243) Google Scholar and injection of the circulating fibrocytes enhanced angiogenesis during wound healing in diabetic mice.14Kao 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 (96) Google Scholar However, these findings only provided indirect evidence because fibrocytes were not co-cultured with vascular endothelial cells (VECs) and had not been injected into the peripheral blood of healthy mice when using angiogenesis models. Thus, the role of fibrocytes in angiogenesis remains unclear. The current study was designed to investigate the roles and underlying mechanisms of fibrocytes in angiogenesis. In this study, we co-cultured fibrocytes and VECs in a permeable support (ie, Transwell) co-culture system and three-dimensional (3D) collagen gels. In addition, we injected fibrocytes into the peripheral blood of healthy mice with a wound-healing model or a Matrigel model. In addition to the widely accepted pro-angiogenic role of fibrocytes, we demonstrate that fibrocytes also stabilized blood vessels during angiogenesis both in vitro and in vivo. Our mechanistic study showed that fibrocytes expressed many factors that are responsible for the prosurvival role of fibrocytes in angiogenesis and some anti-angiogenic factors, including THBS1, which partially mediated the fibrocyte-induced inhibition of angiogenesis. Balb-C and C57BL/6J mice, aged 8 to 10 weeks, were purchased from the Animal Center of the Fourth Military Medical University (Xi'an, China). Green fluorescent protein (GFP) transgenic mice on a C57BL/6J mouse background (Hr-GFPTg/+) were purchased from Cyagen Biosciences Inc. (Guangzhou, China). All animal procedures were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Fourth Military Medical University's Institutional Animal Care and Use Committee. Human and mouse circulating fibrocytes were isolated and purified, as described previously,15Xueyong L. Shaozong C. Wangzhou L. Yuejun L. Xiaoxing L. Jing L. Yanli W. Jinqing L. Differentiation of the pericyte in wound healing: the precursor, the process, and the role of the vascular endothelial cell.Wound Repair Regen. 2008; 16: 346-355Crossref PubMed Scopus (19) Google Scholar with some modifications. Briefly, human circulating fibrocytes (CFs) were isolated from leukapheresis packs (kindly provided by the Xijing Hospital Blood Center, Xi'an, China) using a Percoll density centrifugation. Then, the cells were purified by negative immunomagnetic selection, which removed contaminating T cells, B cells, and monocytes using pan-T, anti-CD2, pan-B, anti-CD19, and anti-CD14 Dynabeads (Dynal Inc., Lake Success, NY). Mouse fibrocytes were isolated from Balb-C and C57BL/6J mouse blood (heparinized) collected via the inferior vena cava. Mouse fibrocytes were isolated using Percoll density centrifugation and purified by immunomagnetic removal of contaminating T cells, B cells, and monocytes using pan-T Dynabeads (anti-CD90), pan-B Dynabeads (anti-B220), and anti-mouse CD14 attached to Dynabeads, respectively. The resultant enriched human and mouse fibrocyte populations were >95% pure based on collagen I and CD13 staining. Human dermal microvascular endothelial cells (HDMECs; catalog number 2000) were purchased from ScienCell Research Laboratories (Minneapolis, MN). HDMECs were cultured according to the manufacturer's protocol. Briefly, HDMECs were cultured in ECM supplemented with 5% fetal bovine serum (FBS; ScienCell Research Laboratories), 4 mmol/L l-glutamine, 100 U/mL penicillin-G, 100 U/mL streptomycin, and 1% endothelial cell growth supplement (v/v; ScienCell Research Laboratories). HDMECs were used from passage 4 to 5. Balb-C or C57BL6/J mice were weighed and anesthetized by 100 μL/20 g body weight i.p. injection of 1% sodium pentobarbital. After the hair on the dorsum was shaved and then removed by Nair hair-removal lotion (Church & Dwight Co, Inc., Princeton, NJ), the dorsal skin was disinfected with 70% ethanol, and one full-thickness wound was generated aseptically with a biopsy punch (7 mm in diameter in Balb-C mice and 6 mm in diameter in C57BL/6J mice). Fifty-nine wounds were generated on Balb-C mice, and the mice were randomly divided into the control group (n = 17), the peripheral blood mononuclear cell (PBMC) group (n = 16), and the fibrocyte group (n = 26). Immediately after the wounds were generated, 100 μL of 0.85% sterile sodium chloride was injected into the control group mice via the tail vein; 4 × 104 PBMCs/10 g body weight were injected into the mice in the PBMC group via the tail vein; and fibrocytes isolated and purified from 65 Balb-C mice were injected into the mice in the fibrocyte group via the tail vein (4 × 104 cells/10 g body weight). The wounds were harvested at 3, 6, and 10 days after wounding (six, six, and five mice, respectively, in the control group; six, five, and five mice, respectively, in the PBMC group; and eight, eight, and five mice, respectively, in the fibrocyte group). Parts of the specimens were fixed in 4% paraformaldehyde and embedded in paraffin for immunohistochemistry; parts of the specimens were stored in liquid nitrogen for division into frozen sections or myeloperoxidase (MPO) activity determination; and parts of the specimens were processed for transmission electron microscopy (TEM). Five wounds in the fibrocyte group were allowed to heal undisturbed to observe the wound area and wound closure time. Skin specimens were harvested from five mice from each of the control, PBMC, and fibrocyte groups and were processed to observe capillary vessel density, fibrocyte infiltration, and MPO activity at baseline for each group. Six additional wounds were generated in Balb-C mice. A total of 4 × 104 fibrocytes per 10 g body weight were injected into these mice via the tail vein 6 days after wounding. Then, the wounds were harvested 4 days after injection (10 days after wounding) and processed for immunofluorescent staining. Twenty-one wounds (diameter, 6 mm) were generated in the C57BL/6J mice to investigate the role of THBS1 in fibrocyte-regulated angiogenesis. The mice were randomly divided into the following groups: the control group (n = 5), in which 100 μL of 0.85% sterile sodium chloride was injected into the mice via the tail vein; the fibrocyte group (n = 8), in which 4 × 104 fibrocytes per 10 g body weight isolated from Hr-GFPTg+ mice and transfected with a control siRNA were injected into the recipient mice via the tail vein; and the fibrocyte/THBS1 siRNA group (n = 8), in which 4 × 104 fibrocytes per 10 g body weight isolated from Hr-GFPTg+ mice and transfected with THBS1 siRNA were injected into the recipient mice via the tail vein. The wounds were harvested 6 days after wounding, and the specimens were stored in liquid nitrogen for further measurements. An in vivo Matrigel angiogenesis model was generated as described previously.16Woodman S.E. Ashton A.W. Schubert W. Lee H. Williams T.M. Medina F.A. Wyckoff J.B. Combs T.P. Lisanti M.P. Caveolin-1 knockout mice show an impaired angiogenic response to exogenous stimuli.Am J Pathol. 2003; 162: 2059-2068Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 17Zhu K. Amin M.A. Zha Y. Harlow L.A. Koch A.E. Mechanism by which H-2g, a glucose analog of blood group H antigen, mediates angiogenesis.Blood. 2005; 105: 2343-2349Crossref PubMed Scopus (23) Google Scholar Briefly, Matrigel (catalog number 354248; BD Bioscience, Franklin Lakes, NJ) was diluted with serum-free Dulbecco's modified Eagle's medium to 10 mg/mL and mixed with FGF2 (catalog number GF003-AF; Millipore Corp, Billerica, MA) at a final concentration of 0.2 μg/mL and heparin at a final concentration of 30 μg/mL. Balb-C mice were anesthetized as previously described. Matrigel (400 μL) supplemented with FGF2 and heparin was injected s.c. in the back of the mice. Twenty-one mice were injected with Matrigel supplemented with FGF2 and heparin and randomly divided into three groups: the control group (n = 5), in which 100 μL of 0.85% sterile sodium chloride was injected into the mice; the fibrocyte group (n = 8), in which Hr-GFPTg/+ 4 × 104 fibrocytes per10 g body weight transfected with a control siRNA were injected into the mice via the tail vein; and the fibrocyte/THBS1 siRNA group (n = 8), in which Hr-GFPTg/+ 4 × 104 fibrocytes per 10 g body weight transfected with the THBS1 siRNA were injected into the mice via the tail vein. For each group, Matrigel mixed with heparin only was injected into five mice as a control. The Matrigel plugs were dissected and imaged 7 days after the Matrigel injection. The plugs were stored in liquid nitrogen for division into frozen sections. The wound area was quantified by computerized planimetry, as described previously.18Li J. Topaz M. Xun W. Li W. Wang X. Liu H. Yuan Y. Chen S. Li Y. Li X. New swine model of infected soft tissue blast injury.J Trauma Acute Care Surg. 2012; 73: 908-913Crossref PubMed Scopus (6) Google Scholar, 19Mayrovitz H.N. Soontupe L.B. Wound areas by computerized planimetry of digital images: accuracy and reliability.Adv Skin Wound Care. 2009; 22: 222-229Crossref PubMed Google Scholar, 20Li J. Topaz M. Tan H. Li Y. Li W. Xun W. Yuan Y. Chen S. Li X. Treatment of infected soft tissue blast injury in swine by regulated negative pressure wound therapy.Ann Surg. 2013; 257: 335-344Crossref PubMed Scopus (23) Google Scholar Briefly, the wounds were imaged with a scale adjacent to the wound, and the wound area was calculated with Image-Pro Plus version 6.0 (Media Cybernetics, Inc., Warrendale, PA). A rabbit anti-platelet endothelial cell adhesion molecule (PECAM) 1 (cell marker of vascular endothelial cells) polyclonal antibody and a rabbit anti-von Willebrand factor (VWF) were purchased from Abcam Inc. (Cambridge, MA; catalog numbers ab28364 and ab6994, respectively). A mouse anti-CD13 monoclonal antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA; catalog number sc-13536). A rabbit anti-collagen I polyclonal antibody was purchased from Boster Biotechnology (Wuhan, China; catalog number BA0325). A biotin-conjugated goat anti-mouse antibody and a biotin-conjugated goat anti-rabbit antibody were purchased from Santa Cruz Biotechnology, Inc. (catalog numbers sc-2039 and sc-2040, respectively). Streptavidin-conjugated Alexa Fluor 488 and streptavidin-conjugated Alexa Fluor 594 were purchased from Life Technologies/Invitrogen (Grand Island, NY; catalog numbers S32354 and S32356, respectively). Immunofluorescent staining was performed as described previously.15Xueyong L. Shaozong C. Wangzhou L. Yuejun L. Xiaoxing L. Jing L. Yanli W. Jinqing L. Differentiation of the pericyte in wound healing: the precursor, the process, and the role of the vascular endothelial cell.Wound Repair Regen. 2008; 16: 346-355Crossref PubMed Scopus (19) Google Scholar, 21Martin D. Galisteo R. Gutkind J.S. CXCL8/IL8 stimulates vascular endothelial growth factor (VEGF) expression and the autocrine activation of VEGFR2 in endothelial cells by activating NFkappaB through the CBM (Carma3/Bcl10/Malt1) complex.J Biol Chem. 2009; 284: 6038-6042Crossref PubMed Scopus (317) Google Scholar Toluidine blue staining of the vasculature in the collagen gel was performed as described previously.22Koh W. Stratman A.N. Sacharidou A. Davis G.E. In vitro three dimensional collagen matrix models of endothelial lumen formation during vasculogenesis and angiogenesis.Methods Enzymol. 2008; 443: 83-101Crossref PubMed Scopus (159) Google Scholar Blood vessel density in the granulation tissue and the Matrigel plugs was measured as described previously.20Li J. Topaz M. Tan H. Li Y. Li W. Xun W. Yuan Y. Chen S. Li X. Treatment of infected soft tissue blast injury in swine by regulated negative pressure wound therapy.Ann Surg. 2013; 257: 335-344Crossref PubMed Scopus (23) Google Scholar Briefly, the specimens were labeled with VEC markers, CD34, VWF, or platelet/endothelial cell adhesion molecule 1 (PECAM1 or CD31) by immunofluorescent staining and then observed under an Olympus FV1000 MPE SIM Laser Scanning Confocal Microscope (Olympus Corp, Center Valley, PA). Because CD34, VWF, and PECAM1 are not exclusive cell markers of VECs and are expressed by other cell types, only lumens formed by CD34−, VWF−, or PECAM1+ cells were counted as blood vessels. For each wound or Matrigel specimen, four sections were processed, and eight fields of view (two per section) were imaged using a 30× objective lens. The area of the field (A) was calculated using Image Pro Plus version 6.0 (Media Cybernetics, Inc.). The number of the blood vessels (N) in the eight fields was counted, and the mean of the blood vessel density (D) was calculated using the following equation: D = N/A. A total of 1 × 105 purified human fibrocytes per well were cultured in 6-well plates (catalog number 3516; Corning Life Science, Lowell, MA). The HDMECs (2 × 104 per insert) were cultured in the Transwell insert (0.4-μm pore size; catalog number 3491; Corning Life Science). The fibrocytes and HDMECs were cultured separately for 2 days before initiating the co-culture experiments. In some groups, the fibrocytes were transfected with control siRNA or THBS1 siRNA before seeding. Then, the fibrocytes and HDMECs were co-cultured in starvation medium (ECM supplemented with 0.5% FCS and 0.1% endothelial cell growth supplement, v/v) for 24 hours, followed by 20 ng/mL FGF2 or 20 ng/mL VEGF, with or without 40 ng/mL exogenous THBS1 stimulation, for 48 hours. Some HDMECs and fibrocytes were cultured separately in the inserts and culture plates, respectively, in starvation medium, with or without FGF2 or VEGF stimulation. Cells were lysed to isolate mRNA or protein for real-time PCR or Western blot analysis, respectively. The [3H]-thymidine incorporation assay was performed as described previously.23Li J. Ichikawa T. Villacorta L. Janicki J.S. Brower G.L. Yamamoto M. Cui T. Nrf2 protects against maladaptive cardiac responses to hemodynamic stress.Arterioscler Thromb Vasc Biol. 2009; 29: 1843-1850Crossref PubMed Scopus (205) Google Scholar Briefly, 5 × 104 HDMECs per well and 1 × 104 fibrocytes per well were cultured separately for 24 hours in 24-well plates (catalog number CLS3524; Corning Life Science) and inserts (0.4-μm pore size; catalog number 3495; Corning Life Science), respectively. The fibrocytes were transfected with THBS1 siRNA or control siRNA before seeding. Then, the cells were treated with FGF2, VEGF, and exogenous THBS1, as described in Transwell Co-Culture of Fibrocytes and VECs. [3H]-thymidine (catalog number 2403095; MP Biomedicals, Solon, OH) was added to the medium to a final concentration of 1 μCi/mL 6 hours after VEGF or FGF2 treatment. After washing twice with ice-cold PBS, the cells were precipitated with 5% ice-cold trichloroacetic acid (model T9159; Sigma-Aldrich, St. Louis, MO) for 20 minutes. Then, the cells were washed twice with 5% ice-cold trichloroacetic acid, washed twice with ice-cold PBS, and then lysed with 0.2 mL of 0.5 mol/L NaOH for 30 minutes at 37°C. [3H]-thymidine radioactivity was measured with a Beckman LS6000 scintillation counter (Beckman Coulter, Inc., Fullerton, CA). The 3D collagen gel was prepared as described previously.24Baxter S.C. Morales M.O. Goldsmith E.C. Adaptive changes in cardiac fibroblast morphology and collagen organization as a result of mechanical environment.Cell Biochem Biophys. 2008; 51: 33-44Crossref PubMed Scopus (32) Google Scholar Briefly, the collagen gel was prepared by mixing collagen I solution (3.0 mg/mL collagen I; Advanced BioMatrix, Inc., San Diego, CA), 10× M199 solution (Sigma-Aldrich), and 0.2 N HEPES (pH 9.0; Sigma-Aldrich) at a ratio of 8:1:1. All solutions were kept on ice to prevent gel formation. HMDECs and fibrocytes were diluted to a 6 × 106/mL cell suspension in M199 supplemented with 1% FBS, 40 ng/mL VEGF, 40 ng/mL FGF2, and 100 U/mL penicillin and streptomycin. In the VEC-only group, the HMDEC cell solution was mixed with the collagen gel solution and ECM medium at a ratio of 1:8:1. In the VEC and CF group, the HMDEC cell solution, fibrocyte solution, ECM medium, and collagen gel solution were mixed at the following ratio:1:X:(1−X):8(1) where X is determined by the ratio of VECs/CFs in the indicated groups. The gel was placed in a 37°C incubator without CO2 for 30 minutes, and M199 supplemented with 1% FBS, 40 ng/mL VEGF, 40 ng/mL FGF2, and 100 U/mL penicillin and streptomycin was carefully added on top of the gel. To assess cell-cell interactions in 3D collagen gel culture, VECs were labeled with Vybrant Dil cell tracer (Invitrogen Corp.) and CFs were labeled with Vybrant carboxyfluorescein diacetate, succinimidyl ester (CFDA SE) cell tracer (Invitrogen Corp.). Images were obtained using laser scanning confocal microscopy, and 3D rendering was performed using Volocity software version 6.0 (PerkinElmer Inc., Waltham, MA). The wounds and the collagen gels were harvested and fixed in 2.5% glutaraldehyde/0.1 mol/L cacodylate buffer. The wounds and part of the collagen gels were processed for TEM using a standard protocol, and part of the collagen gels was processed for scanning electron microscopy (SEM) following a standard protocol and using critical point drying to preserve the structure of the collagen gel. Cell lysates were subjected to Western blot analysis using an anti-THBS1 antibody (catalog number sc-59887; Santa Cruz Biotechnology, Inc.), an anti-chemokine (C-X-C motif) ligand 12 (CXCL12) antibody (catalog number ab9797; Abcam Inc.), an anti-C-X-C chemokine receptor type 4 (CXCR4) antibody (catalog number ab2074; Abcam Inc.), and an anti-activated caspase 3 antibody (catalog number 9661; Cell Signaling Technology, Inc., Danvers, MA), as described previously.21Martin D. Galisteo R. Gutkind J.S. CXCL8/IL8 stimulates vascular endothelial growth factor (VEGF) expression and the autocrine activation of VEGFR2 in endothelial cells by activating NFkappaB through the CBM (Carma3/Bcl10/Malt1) complex.J Biol Chem. 2009; 284: 6038-6042Crossref PubMed Scopus (317) Google Scholar Quantitative real-time PCR (qPCR) was performed as described previously.23Li J. Ichikawa T. Villacorta L. Janicki J.S. Brower G.L. Yamamoto M. Cui T. Nrf2 protects against maladaptive cardiac responses to hemodynamic stress.Arterioscler Thromb Vasc Biol. 2009; 29: 1843-1850Crossref PubMed Scopus (205) Google Scholar Total RNA was extracted from the cells using the RNeasy Mini kit (catalog number 74104; Qiagen Inc., Valencia, CA), and reverse transcription reactions were performed with 0.5 μg of DNase I (Qiagen)-treated RNA using the SuperScript III First-Strand Synthesis System (catalog number 18080-051; Invitrogen Corp, Carlsbad, CA). PCR and qPCR were performed using a Mastercycler EP Realplex (Eppendorf, Westbury, NY). The expression levels of target genes were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression, which was measured concurrently, as described by Pfaffl.25Pfaffl M.W. A new mathematical model for relative quantification in real-time RT-PCR.Nucleic Acids Res. 2001; 29: e45Crossref PubMed Scopus (26452) Google Scholar THBS1 siRNA was transfected into human or mouse circulating fibrocytes using Nucleofector 2b (Lonza Walkersville, Inc., Walkersville, MD), according to the manufacturer's protocol. The optimal concentration of THBS1 siRNA was determined by transfecting 1 × 106 293 cells with THBS1 siRNA at a different concentration and 5 μg of pcDNA3 hTHBS1 using Nucleofector 2b. The cells were cultured for 48 hours; then, the expression of THBS1 protein was determined by using Western blot analysis. A concentration of 50 nmol/L of THBS1 siRNA was chosen based on the results shown in Supplemental Figure S1. A live/dead cell viability assay was performed according to the manufacturer's protocol. Briefly, cells were washed once with HBSS and then incubated with live green and dead red solutions (Live/Dead Cell Imaging Kit, catalog number R37601; Invitrogen Corp) for 15 minutes at room temperature. The cells were then imaged using SimplePCI Image Analysis Software (Compix Inc., Sewickley, PA) with phase-contrast, red fluorescence, and green fluorescence channels. The data are presented as the means ± SD. The means were compared by analysis of variance, followed by a t-test with Bonferroni correction for multiple comparisons. We expected that the injection of fibrocytes into healthy recipient mice would accelerate wound healing based on previous reports.13Hartlapp 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 (243) Google Scholar, 14Kao 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 (96) Google Scholar We generated skin wounds on the backs of Balb-C mice and then injected purified circulating fibrocytes into the mice via the tail vein. Surprisingl
Referência(s)