Basic Research—Biology
Hypoxia Enhances the Angiogenic Potential of Human Dental Pulp Cells Andreza M.F. Aranha, DDS, MS, PhD,*† Zhaocheng Zhang, MD, PhD,* Kathleen G. Neiva, DDS, MS, PhD,* Carlos A.S. Costa, DDS, MS, PhD,‡ Josimeri Hebling, DDS, MS, PhD,† and Jacques E. No¨r, DDS, MS, PhD*§k Abstract Introduction: Trauma can result in the severing of the dental pulp vessels, leading to hypoxia and ultimately to pulp necrosis. Improved understanding of mechanisms underlying the response of dental pulp cells to hypoxic conditions might lead to better therapeutic alternatives for patients with dental trauma. The purpose of this study was to evaluate the effect of hypoxia on the angiogenic response mediated by human dental pulp stem cells (DPSCs) and human dental pulp fibroblasts (HDPFs). Methods: DPSCs and HDPFs were exposed to experimental hypoxic conditions. Hypoxia-inducible transcription factor-1alpha (HIF-1alpha) was evaluated by Western blot and immunocytochemistry, whereas vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) expression was evaluated by enzyme-linked immunosorbent assay. YC-1, an inhibitor of HIF-1alpha, was used to evaluate the functional effect of this transcriptional factor on hypoxia-induced VEGF expression. Conditioned medium from hypoxic and normoxic pulp cells was used to stimulate human dermal microvascular endothelial cells (HDMECs). HDMEC proliferation was measured by WST-1 assay, and angiogenic potential was evaluated by a capillary sprouting assay in 3-dimensional collagen matrices. Results: Hypoxia enhanced HIF-1alpha and VEGF expression in DPSCs and HDPFs. In contrast, hypoxia did not induce bFGF expression in pulp cells. YC-1 partially inhibited hypoxia-induced HIF-1alpha and VEGF in these cells. The growth factor milieu of hypoxic HDPFs (but not hypoxic DPSCs) induced endothelial cell proliferation and sprouting as compared with medium from normoxic cells. Conclusions: Collectively, these data demonstrate that hypoxia induces complex and cell type–specific pro-angiogenic responses and suggest that VEGF (but not bFGF) participates in the revascularization of hypoxic dental pulps. (J Endod 2010;36:1633– 1637)
Key Words Angiogenesis, dental pulp, endodontics, tissue regeneration, trauma
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hildren and adolescents are a high-risk population for dental trauma. In severe traumatic injuries (eg, luxations and avulsions), a significant reduction in vascular blood supply to the dental pulp might occur as a result of the rupture of neurovascular bundle and periodontal ligament (1, 2). The ideal treatment for an avulsed permanent tooth is its immediate replantation into the socket. However, even when immediate replantation is achieved, complications such as ankylosis, root resorption, and pulp necrosis are frequently observed. Pulp tissue revascularization is one of the goals in the management of dental avulsion, but it occurs in only 30% of the immature permanent teeth that are replanted (1). Improvements in the successful outcome of replanted teeth will require better understanding of mechanisms underlying the response of dental pulps to hypoxic conditions. Angiogenesis is defined as the formation of new capillaries from preexisting blood vessels (3). The regulation of this process by hypoxia is an important mechanism for the maintenance of adequate influx of oxygen and nutrients required for the metabolic needs of tissues and organs (4). The regulation of the activity of hypoxia-inducible transcription factor-1 (HIF-1) is a very well-established mechanism for the maintenance of oxygen homeostasis (5). HIF-1 is a heterodimeric protein composed of an oxygensensitive subunit (HIF-1alpha) and a constitutive subunit (HIF-1beta). HIF-1alpha expression is rapidly increased under hypoxic conditions, and this protein subunit is degraded after reoxygenation (6, 7). HIF-1 plays a critical role in angiogenesis by activating the transcription of genes that encode pro-angiogenic growth factors, including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), angiopoietin 1 (Ang1) and 2 (Ang2), placental growth factor (PGF), and platelet-derived growth factor-B (PDGFB) (8–12). VEGF is a major regulator of both physiologic and pathologic angiogenesis (13, 14). It induces endothelial cell proliferation, migration, survival, and differentiation into angiogenic vessels (15, 16). Notably, we have recently reported that the application of recombinant VEGF induces angiogenesis in tooth slices implanted in immunodeficient mice (17). bFGF is also considered an important regulator of physiologic and pathologic angiogenesis. It is a particularly potent inducer of endothelial cell proliferation (18). Very little is known about molecular mechanisms underlying the response of dental pulp stem cells to hypoxia. Here we performed studies to investigate the effect
From the *Angiogenesis Research Laboratory, Department of Cariology, Restorative Sciences, and Endodontics, School of Dentistry, University of Michigan, Ann Arbor, Michigan; †Department of Orthodontics and Pediatric Dentistry, and ‡Laboratory of General Pathology and Biomaterials, Department of Physiology and Pathology, Sa˜o Paulo State University, Araraquara School of Dentistry, Araraquara, SP, Brazil; §Department of Otolaryngology, University of Michigan School of Medicine, and k Department of Biomedical Engineering, University of Michigan College of Engineering, Ann Arbor, Michigan. Address requests for reprints to Jacques E. No¨r, DDS, PhD, University of Michigan School of Dentistry, 1011 N University, Room 2309, Ann Arbor, MI 48109-1078. E-mail address:
[email protected]. 0099-2399/$0 - see front matter Copyright ª 2010 American Association of Endodontists. doi:10.1016/j.joen.2010.05.013
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Figure 1. Expression of HIF-1alpha, VEGF, and bFGF in human dental pulp cells. (A) Western blot assay for HIF-1alpha expression in DPSCs and in HDPFs. DPSCs and HDPFs were cultured under hypoxic conditions for 0–24 hours. Cells cultured in normoxia were used as a negative control, and cells treated with 1 mmol/L CoCl2 were used as positive control. (B) Immunocytochemistry for HIF-1alpha in DPSCs and HDPFs cultured for 4 hours under normoxia or hypoxia. (C, D) ELISA for evaluation of VEGF and bFGF expression in the 24-hour conditioned medium from DPSCs and HDPFs.
of hypoxia on the angiogenic potential of human dental pulp stem cells (DPSCs), with human dental pulp fibroblasts (HDPFs) as controls.
Materials and Methods Cell Culture HDPFs were cultured in Dulbecco modified Eagle medium (DMEM) (Sigma-Aldrich Co, St Louis, MO), and DPSCs (provided by Songtao Shi, University of Southern California, Los Angeles, CA) were cultured in low glucose DMEM. Both media were supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY), 100 U/mL penicillin, 100 mg/mL streptomycin, and 2 mmol/L glutamine (Gibco). Human dermal microvascular endothelial cells (HDMECs) were cultured in EGM2-MV medium (Cambrex, San Diego, CA). For normoxia, HDPFs and DPSCs were cultured at 37 C, 5% CO2, 21% O2, and 74% N2. Alternatively, HDPFs and DPSCs were cultured in a hypoxia chamber at 37 C (Modular Incubator Chamber; Billups-Rothenberg, San Diego, CA) containing 1% O2, 5% CO2, and 94% N2 (19). Western Blots DPSCs or HDPFs (60 104 cells) were cultured in normoxia or hypoxia for 0–24 hours. Cell lysates were resolved by sodium dodecylsulfate–polyacrylamide gel electrophoresis, and membranes were incu1634
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bated overnight with mouse anti-human HIF-1alpha (1:1000; BD Transduction Laboratories, San Jose, CA) at 4 C. Horseradish peroxidase–conjugated sheep anti-mouse antiserum immunoglobulin G (BD Laboratories Transduction) was used as a secondary antibody (1:10,000 dilution). The antigen-antibody complexes were visualized by chemiluminescence (Supersignal West Pico Chemiluminescent Substrate; Thermo Scientific, Rockford, IL). Cells cultured in normoxia were used as a negative control, and cells pretreated with 0.5 mmol/L cobalt chloride were used as a positive control (7).
Immunocytochemistry DPSCs or HDPFs (2.5 104 cells) were cultured in chamber slides (Lab-Tek Brand Products, Naperville, IL) and then fixed in 10% phosphate-buffered formalin (Fisher Scientific, Pittsburgh, PA). An immunoperoxidase detection system (4 Plus Universal HRP-DAB Sample Kit; Biocare Medical, Concord, CA) was used according to the manufacturer’s instructions. Anti–HIF-1alpha antibody was used as a primary antibody (mouse antiserum; 1:100; BD Transduction Laboratories). After DAB chromogen staining (Biocare Medical), slides were counterstained with hematoxylin (Sigma). WST-1 Assay To evaluate the proliferation of endothelial cells, HDMECs were seeded in 96-well plates and cultured in EGM2-MV supplemented JOE — Volume 36, Number 10, October 2010
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Figure 2. Effect of HIF-1alpha on VEGF expression in human dental pulp cells. (A) WST-1 assay to evaluate the relative cell density of DPSCs and HDPFs treated with a HIF-1alpha inhibitor (YC-1) for 24 hours. (B) Western blot to evaluate the effect of YC-1 on HIF-1alpha expression induced by cobalt chloride. (C) ELISA for evaluation of VEGF expression in normoxic or hypoxic DPSCs and HDPFs treated with 0 or 40 mmol/L YC-1 for 24 hours. Different lowercase letters indicate statistically significant difference (P < .05). Data were derived from 4 microscopic fields/well, 3 wells/experimental condition.
with conditioned medium collected from normoxic or hypoxic DPSCs or HDPFs. Cell proliferation was evaluated for 24–72 hours by WST-1 assay (Roche Applied Science, Penzberg, Germany) (20).
Determination of YC-1 Concentration for Functional Studies YC-1 (AG Scientific Inc, San Diego, CA) is an inhibitor of HIF1alpha (21). To determine the YC-1 concentration required for HIF1alpha inhibition, DPSCs and HDPFs (60 104 cells/60-mm dish) were treated with 0–80 mmol/L YC-1 in presence of 0.5 mmol/L cobalt chloride at 37 C for 4 hours. Western blots were performed to evaluate HIF-1alpha expression. To ensure that the YC-1 concentration capable to inhibit HIF-1alpha expression would not affect cell viability, the metabolic activity of the pulp cells was assessed by WST-1 assay, as described above. Enzyme-linked Immunosorbent Assay VEGF and bFGF expression was measured by enzyme-linked immunosorbent assay (ELISA) (Quantikine; R&D Systems Inc, Minneapolis, MN). DPSCs and HDPFs (3 104 cells/well) were cultured in triplicate wells per condition in 12-well plates and incubated at 37 C in normoxia or in hypoxia. The supernatants were collected, centrifuged, and evaluated. The optical density obtained from the samples was measured at 450 nm in a spectrophotometer (GENious; Tecan, JOE — Volume 36, Number 10, October 2010
Ma¨nnedorf, Switzerland). Data were normalized by the number of cells per well.
Capillary Tube Assays DPSCs and HDPFs were cultured in DMEM and maintained at 37 C in normoxia or in hypoxia. After 24 hours, conditioned medium was collected, centrifuged, concentrated 10, and frozen at –80 C. EGM2-MV and EGM2-MV supplemented with 50 ng/mL rhVEGF were used as negative and positive controls, respectively (13, 15). To evaluate angiogenesis in vitro (13, 14), 6-well plates were coated with 1.5 mL/well type I collagen (Vitrogen 100; Cohesion Technologies, Palo Alto, CA). HDMECs (5 104 cells) were cultured on the collagen gel in EGM-2MV containing conditioned medium from DPSCs (CM-DPSC) or HDPFs (CM-HDPF) collected under hypoxic or normoxic conditions. The number of sprouts was counted daily in a phase contrast microscope at 100 magnification, as described (14). Data were obtained from triplicate wells per condition and time point. Statistical Analysis The Kruskal-Wallis nonparametric test was performed to compare groups, and it was complemented by Mann-Whitney U or Wilcoxon-2-related tests. HIF-1alpha expression was analyzed descriptively. Hypoxia and Pulp Angiogenesis
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Figure 3. Effect of the growth factor milieu secreted by DPSCs and HDPFs on the angiogenic potential of endothelial cells. (A) WST-1 assay to evaluate the effect of conditioned medium from normoxic or hypoxic DPSCs and HDPFs on endothelial cell proliferation. EGM2-MV and EGM2-MV supplemented with 50 ng/mL VEGF were used as negative and positive controls, respectively. (B) Photomicrographs (original magnification, 100) of representative fields of endothelial cells cultured in 3-dimensional collagen matrices and treated with conditioned medium from normoxic or hypoxic cells. Arrows point to capillary sprouts. (C) Graph showing the quantification of capillary sprouts observed in the experiment depicted in (B). Values for (A) and (C) are medians (interquartile interval, P25/P75). Data were derived from 4 microscopic fields/well, 3 wells/experimental condition.
Results Hypoxia Induces HIF-1alpha and VEGF in Pulp Cells Western blots (Fig. 1A) and immunocytochemistry (Fig. 1B) showed that HIF-1alpha is induced by hypoxia in both DPSCs and HDPFs. HIF-1alpha expression peaked at 4 hours of hypoxia, and then it decreased gradually through 24 hours (Fig. 1A). Immunocytochemistry revealed that HIF-1alpha was localized primarily in the cell nuclei under hypoxic conditions. Correlated with the nuclear expression of HIF-1alpha, we observed a significant increase in VEGF expression in both cell types (Fig. 1C). VEGF expression in hypoxia was approximately 8-fold higher in DPSCs and 3-fold higher in HDPFs when compared with normoxic conditions. However, HDPFs expressed higher VEGF levels than DPSCs in both normoxic and hypoxic conditions (Fig. 1C). In contrast, bFGF expression was low in DPSCs and HDPFs, and hypoxia did not stimulate it beyond baseline levels (Fig. 1D). Effect of HIF-1alpha on VEGF in Dental Pulp Cells When used in concentrations up to 40 mmol/L, the HIF-1alpha inhibitor YC-1 showed relatively mild toxicities to DPSCs or HDPFs (Fig. 2A). We used cobalt chloride to mimic the effect of hypoxia and to help with the determination of the concentration of YC-1 necessary to block HIF-1alpha expression in pulp cells (Fig. 2B). For DPSCs, 40 mmol/L YC-1 was sufficient to abrogate HIF-1alpha expression induced by cobalt chloride (Fig. 2B). In contrast, HIF-1alpha expres1636
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sion was only partially inhibited in HDPFs at this concentration of YC1. On the basis of these results, 40 mmol/L YC-1 was selected for subsequent experiments. We observed only partial inhibition of VEGF expression when pulp cells treated with YC-1 were exposed to hypoxic conditions (Fig. 2C).
Proliferation and Capillary Tube–like Formation by Endothelial Cells To evaluate the angiogenic potential of the growth factor milieu secreted by pulp cells, we performed proliferation and capillary sprouting assays by using the conditioned medium of DPSCs or HDPFs cultured under normoxic or hypoxic conditions. The effect of pulp cell conditioned medium on endothelial cell proliferation was time-dependent (Fig. 3A). We did not observe statistically significant differences during the 24-hour period. During the 48-hour period, endothelial cells treated with conditioned medium collected from HDPFs and DPSCs showed an increase in proliferation when compared with untreated controls, but no differences were observed with hypoxic versus normoxic conditioned medium. During the 72-hour period, endothelial cell proliferation was stimulated by conditioned medium from hypoxic HDPFs (but not hypoxic DPSCs), as compared with conditioned medium from normoxic cells (Fig. 3A). Interestingly, capillary sprouting was enhanced in endothelial cells treated with conditioned medium from hypoxic HDPFs when compared with normoxic HDPFs (Fig. 3B, C). JOE — Volume 36, Number 10, October 2010
Basic Research—Biology Discussion The present study was designed to investigate the responses of dental pulp cells exposed to low oxygen levels in vitro. This was done as an attempt to simulate the conditions of dental pulp hypoxia developed by the rupture of the neurovascular bundle in intrusive luxations and avulsions. We focused on the events mediated by HIF-1alpha because this transcriptional factor is known to play an important role on angiogenic responses to hypoxic conditions (8–12, 22, 23). We observed an induction of HIF-1alpha in both DPSCs and HDPFs exposed to hypoxia. It has been demonstrated in other cell types that HIF-1alpha DNA binding activity and protein levels increase exponentially as cells are subjected to decreasing oxygen concentrations, with a half maximal response between 1.5% and 2% oxygen and maximal response at 0.5% oxygen (24). Therefore, we elected to perform most studies with cells cultured in a hypoxia chamber at 1% oxygen. Under these conditions, HIF-1alpha expression peaked at 4 hours. Notably, both DPSCs and HDPFs presented an increase in VEGF expression as compared with cells cultured in normoxia. Collectively, these data showed that DPSCs and HDPFs are capable of organizing a rapid response to the changes in oxygen tension induced experimentally. HIF-1alpha activity was inhibited with YC-1 to investigate its functional role in the regulation of VEGF expression in DPSCs and HDPFs. We observed that inhibition of HIF-1alpha partially inhibited VEGF expression in pulp cells. Although there are not many studies with DPSCs under hypoxic conditions, a recent study demonstrated a significant increase in VEGF expression associated with an increase in HIF1alpha activity in hypoxic bone marrow–derived stem cells (BMSCs) (25). By inhibiting HIF-1alpha, the authors observed a partial inhibition of VEGF expression in BMSCs. It is important to point out, however, that YC-1 treatment was not capable of reducing VEGF expression down to baseline levels in any of the conditions examined here, suggesting that other transcriptional factors might be involved in hypoxia-induced VEGF in these cells. We observed that VEGF expression in HDPFs was 12-fold greater than that of DPSCs in normoxia and 5-fold greater than that of DPSCs in hypoxia. Furthermore, conditioned medium from hypoxic HDPFs increased endothelial cell proliferation and capillary sprouting, whereas DPSC-secreted factors did not. We speculate that these responses might be related to the fact that HDPFs are differentiated cells required for tissue homeostasis and response to injury. In contrast, DPSCs are undifferentiated cells that provide a cellular source for tissue regeneration but might not be so involved in the recovery of the vascular supply of dental pulps exposed to hypoxic conditions. Interestingly, there was no difference in bFGF expression in normoxia or hypoxia for both cell types. Such pattern has also been observed in rat chondrosarcoma cells (26). Together, these data suggest that dental pulp fibroblasts are highly responsive to changes in oxygen tension, and that VEGF (not bFGF) is the factor that mediates the pro-angiogenic responses triggered by these cells. In summary, this work unveils complex and cell type–specific signaling events triggered by hypoxia in human dental pulp stem cells and pulp fibroblasts. The understanding of mechanisms involved in the response of dental pulp cells to hypoxia is critical for the development of new therapeutic strategies for the management of trauma that leads to damage in the dental pulp vascular networks.
Acknowledgments The Department of Cariology, Restorative Sciences, and Endodontics at the University of Michigan School of Dentistry (J.E.N.) and CAPES (A.M.F.A.) supported this work. This work was based on a thesis submitted to the graduate faculty, Sa˜o Paulo State
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University, in partial fulfillment of the requirements for a doctoral degree (A.M.F.A.).
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