Blood vessel formation in the tissue-engineered bone with the constitutively active form of HIF-1α mediated BMSCs
Introduction
The possible risks of infection, rejection, the limited supply, and cost are serious limitations faced by autologous and allogeneic bone used to repair bone defects [1], [2]. The tissue-engineered bone offers new therapeutic strategies to repair tissue defects. When bone defects are repaired using tissue engineering technology, particularly larger bone defects, angiogenesis is a prerequisite step to achieve the successes of bone regeneration [3]. However, unlike organ transplants, where there is a pre-existing vascular supply, the tissue-engineered bone usually is devoid of pre-existing vasculature [4]. Therefore, how to promote the angiogenesis and vascularization of these bone constructs remains a big challenge in the clinic.
The importance of blood vessels in the formation of the skeleton and in bone repair was documented as early as the 1700s [5], [6]. The vasculature transports oxygen, nutrients, soluble factors, and numerous types of cells to the tissues of the body. Many of the factors that lead to the normal development of embryonic vasculature are recapitulated during neoangiogenesis in adults [7]. Factors involved in neoangiogenesis include VEGF, basic fibroblast growth factor (bFGF), various members of the transforming growth factor beta (TGF-β) family, Ang-1, placental growth factor (PLGF), angiopoietin 1 (ANGPT1), PDGF-BB, IGF-1, IGF-2, and SDF-1 [8]. Among these factors, VEGF, which is activated by hypoxia, plays a critical role in angiogenesis during the development of most tissues including bone [9]. It has been demonstrated that some growth factors can accelerate angiogenesis in the tissue-engineered bone, such as VEGF [10], [11]. However, the formation of blood vessels is a complex process that requires the coordination of multiple angiogenic factors, communication of cells with one another, and with their surrounding extracellular matrix (ECM) [12]. Besides VEGF, angiogenesis is also modulated by other growth factors, such as bFGF, PDGF, and SCF [13]. More recently, VEGF has been reported to be insufficient as a single agent to create the complex structures required for a functional vasculature. VEGF-induced vessels are often leaky and improperly connected to the existing vasculature [14]. To overcome these problems, several studies have used two angiogenic factors in combination to remodel and repair vasculature, such as Ang-1 and VEGF [15].
Hypoxia inducible factor-1 (HIF-1), which activates the transcription of hundreds of target genes in response to reduced O2 availability, is the master regulator of oxygen homeostasis in metazoans [16], [17], [18]. One of the main targets of HIF-1α-mediated gene induction is VEGF. As an upstream regulator, HIF-1α is known to activate the transcription of many other angiogenic genes, including TGF-β, Ang-1, PLGF, ANGPT1, PDGF-BB, stem cell factor (SCF), and SDF-1 [8], [19]. Therefore, HIF-1α should have more advantages in promoting angiogenesis compared with the above single agent gene therapies. Under normoxic conditions, HIF-1α is subject to degradation regulated by von Hippel-Lindau protein, which recruits an ubiquitin-protein ligase. A lack of oxygen, certain metabolic intermediates, or iron can inhibit prolyl-4-hydroxylase domain proteins (PHD) activity and stabilize HIF-1α [20], [21]. When stabilized, HIF-1α forms a heterodimer with HIF-1β and binds to the hypoxia response element to transactivate the expression of many downstream genes that are involved in angiogenesis.
We previously reported that a truncation mutant of HIF-1α (CA5), which contains a deletion (amino acids 392–520) and 2 substitutions (Pro567Thr and Pro658Gln), could effectively maintain the stability and activity of HIF-1α under normoxic conditions [22]. The expression of VEGF and SDF-1 in BMSCs was found up-regulated by HIF-1α overexpression [23]. However, whether HIF-1α could regulate angiogenesis in BMSCs through additional pathways and its in vivo role played in blood vessels formation in the tissue-engineered bone are largely unknown. In this study, we systematically explore the angiogenesis of HIF-1α mediated stem cells in vitro and evaluate the angiogenic function of CA5 gene inducing BMSCs in tissue-engineered bone in a rat model of critical-sized calvarial defects (CSD).
Section snippets
Culture of rat BMSCs
Male F344 rats were obtained from the Ninth People’s Hospital Animal Center (Shanghai, China), and all procedures were approved by the Animal Research Committee of the Shanghai Ninth People’s Hospital. Rat bone marrow was extracted from the femurs and tibias of 4-week-old male F344 rats, as described previously [22], and the BMSCs were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco BRL, Grand Island, NY, USA) containing 10% FBS and 1% penicillin/streptomycin at 37 °C in 5% CO2 for
Cell culture and gene transduction
F344 rat BMSCs had characteristics of stem cells, including high expression of CD90 and CD105. Our previous data have showed that CD90 and CD105 were overexpression, whereas CD31 and CD34 are rarely detected in BMSCs of F344 rat [22]. After preliminary experiments to test for the correct dose of lentivirus, an MOI of 15 plaque forming units (pfu)/cell was determined to have the optimal effect on transfer efficiency in vitro. Four days after transduction with 15 pfu/cell Lenti-GFP, Lenti-WT, or
Discussion
The development of new therapeutic approaches that can enhance vascularized tissue growth has become one of the most active areas of tissue engineering. Due to the close association between angiogenesis and osteogenesis, neovascularization is considered as an important element in the repair of bone defects [26], [27]. This study investigated the angiogenesis of HIF-1α-transduced BMSCs in tissue-engineered bone that was used to repair a CSD. Our results indicate that overexpression of HIF-1α may
Conclusions
In summary, this study demonstrates that HIF-1α overexpression in BMSCs by gene therapy can increase angiogenesis in tissue-engineered bone, thereby promoting bone regeneration in a CSD in vivo. At the molecular level, the mRNA and protein expression of angiogenic factors in BMSCs were significantly up-regulated by CA5 in vitro. These results provide preclinical data for the potential applications of BMSCs engineered with CA5 to promote vascularization, vascular remodeling, and bone defect
Acknowledgments
The authors thank Xiuli Zhang, Xiaochen Zhang, Jun Zhao, and Shuhong Wang for their help with the animal studies and data collection. This work was supported by the National Natural Science Foundation of China (81070806, 81070864, 81100788, 31140007, 30973342), NCET-08-0353, the Science and Technology Commission of Shanghai Municipality (09411954800, 10JC1413300, 10430710900, 10dz2211600), and the Science and Technology Commission of Anhui Municipality (11040606M173, 11040606M204) and is a Key
References (44)
- et al.
Future potentials for using osteogenic stem cells and biomaterials in orthopedics
Bone
(1999) - et al.
Improved tissue-engineered bone regeneration by endothelial cell mediated vascularization
Biomaterials
(2009) - et al.
Genome-wide association of hypoxia-inducible factor (HIF)-1alpha and HIF-2alpha DNA binding with expression profiling of hypoxia-inducible transcripts
J Biol Chem
(2009) - et al.
Activation of hypoxia-inducible factor-1; definition of regulatory domains within the alpha subunit
J Biol Chem
(1997) - et al.
Repairing critical-sized calvarial defects with BMSCs modified by a constitutively active form of hypoxia-inducible factor-1alpha and a phosphate cement scaffold
Biomaterials
(2011) - et al.
Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method
Methods
(2001) - et al.
Development of in vivo muCT evaluation of neovascularisation in tissue engineered bone constructs
Bone
(2008) - et al.
Transcriptional regulation of vascular endothelial cell responses to hypoxia by HIF-1
Blood
(2005) - et al.
SCF and G-CSF lead to the synergistic induction of proliferation and gene expression through complementary signaling pathways
Blood
(2000) Effect of decalcified freeze-dried bone allograft on the healing of jaw defects after cyst enucleation
J Oral Maxillofac Surg
(1996)
Oxygen sensing and osteogenesis
Ann N Y Acad Sci
Bone graft materials. An overview of the basic science
Clin Orthop Relat Res
Vascularization in bone tissue engineering: physiology, current strategies, major hurdles and future challenges
Macromol Biosci
Treatise on the blood, inflammation and gunshot wounds
Angiogenesis in health and disease
Nat Med
Therapeutic angiogenesis and vasculogenesis for tissue regeneration
Exp Physiol
Role of hypoxia-inducible factor-1alpha in angiogenic-osteogenic coupling
J Mol Med (Berl)
Bone marrow stromal cells with a combined expression of BMP-2 and VEGF-165 enhanced bone regeneration
Biomed Mater
Angiogenesis and osteogenesis enhanced by bFGF ex vivo gene therapy for bone tissue engineering in reconstruction of calvarial defects
J Biomed Mater Res A
The FAKs about blood vessel assembly
Circ Res
In vitro models for the evaluation of angiogenic potential in bone engineering
Acta Pharmacol Sin
Cited by (125)
HIF-1α and periodontitis: Novel insights linking host-environment interplay to periodontal phenotypes
2023, Progress in Biophysics and Molecular BiologyHIF signaling: A new propellant in bone regeneration
2022, Biomaterials AdvancesThe role of rare earth elements in bone tissue engineering scaffolds - A review
2022, Composites Part B: Engineering