Applying side effects of anti fibrotic compounds to promote neovascularization for tissue engineering

1. LITERATURE REVIEW 1.1. Neovascularization in Tissue Engineering Regenerative medicine encompasses a variety of research areas including cell therapy, biomaterials engineering, growth factors and transplantation science. Most of these efforts converge into tissue engineering, an exciting field which offers a means of developing biological substitutes for maintaining, restoring and improving tissue function1. One of the major challenges in tissue engineering today is the generation of large vascularized 3D-structures2,3. Cells require a constant supply of essential nutrients and oxygen as well as a constant removal of metabolic waste products. Vascularization is thus essential for maintaining the survival and function of tissue engineered constructs in vitro and vivo, as cellular viability decreases steeply beyond a few hundred μm from a blood supply4. One of the major challenges in tissue engineering is how accelerate neovascularization of an implanted tissue construct. Current technology limits the survival of implanted tissues as they depend initially on diffusion and later on neovascularization. Diffusion is insufficient for survival of the tissue and limits the thickness of the implants. Dependence on neovascularization can cause fibrovascular ingrowth and hence scarring. Presently, there are no established methods to prefabricate a capillary network in tissue constructs that could connect with the host vasculature after implantation5. Currently, strategies to improve vascularization prior to or after implantation involve the use of growth factors, endothelial cells and endothelial progenitor cells. They can be summarized into the following four approaches: i) Pre-vascularization of polymeric scaffolds by host. Prevascularization of polymeric scaffolds can be achieved by implanting the polymeric scaffold without the tissue construct at selected body sites first so that vascularization may occur before subsequent implantation of the tissue construct or cells into the vascularized matrix . This may be feasible in an animal model, but would require multiple surgeries for a human patient. Pre-vascularization using an animal host presents potential host immune reactions and risk of animal disease transmission. ii) Use of angiogenic growth factors. Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are potent inducers of angiogenesis. However, such angiogenic growth factors are inherently unstable in vivo, therefore bolus injections of the recombinant protein has limited use in neovascularization of implants. Sustained release of the exogenous growth factors may be achieved by integration or encapsulation within biodegradable polymers as microspheres7,8 or as part of a scaffold9. Another method of achieving sustained release of VEGF was developed using microsphere encapsulated cells transfected with VEGF cDNA, but this is dependent on cellular uptake and translation of the cDNA10. There are also issues with the safety of using transfected cells for gene therapy due to the transfection vector. Unfortunately, overproduction single angiogenic factors often results in deformed or non- functional blood vessels11. iii) Seeding endothelial cells on a biodegradable matrix together with VEGF transfected cells. This method ensures sustained release of VEGF to maintain the viability of endothelial cells after implantation2. Another group used embryonic fibroblasts to maintain VEGF levels in a tri-culture of endothelial cells and myoblasts to engineer vascularized skeletal muscle tissue 12 in vitro. Perfusion, vascularization and survival of the muscle tissue constructs after transplantation was improved. iv) Co-seeding scaffolds with progenitor cells. The use of stem cells is another avenue that shows promise. In the presence of angiogenic factors such as VEGF, they may be differentiated into endothelial progenitor cells13. Co-implantation of vascular endothelial cells with mesenchymal precursor cells on a three dimensional collagen gel resulted in differentiation of the precursor cells into mural cells. Implantation of this construct into mice produced capillary like tubes, some of which eventually became perfused and joined with the host vasculature, a process called inosculation14. Others have used bone marrow derived endothelial progenitor cells seeded together with other cell types, such as hematopoietic progenitor cells to produce a vascularized matrix15,16,17. 1.2. HIF-1α: An Alternative Approach to Neovascularization Prolyl 4-hydroxylase inhibitors may be an alternative method of neovascularization by chemically stimulating angiogenesis via stabilization of HIF-1 and upregulation of HIF-1 target genes. As angiogenesis is a complex process involving many different factors, the use of exogenous angiogenic factors alone may not produce functional vascular networks. Instead of trying to optimize a recipe of cells and growth factors for vascularization to occur, this approach may achieve a more physiological response, similar to hypoxic stress, by using HIF-1 as a master switch. Targeting HIF-1 has the advantage of bypassing the downstream complexity of multiple cell signaling molecules and angiogenic factors involved in the process of angiogenesis. The cells are directed by HIF-1 to produce a number of resulting gene products endogenously, rather than attempting to recreate the series of events leading to angiogenesis by the use of exogenous growth factors and progenitor cell types. The amount and timing of these growth factors and signaling molecules are determined and controlled by the cells, therefore more closely resembling physiological stimulation of angiogenesis. This method would also remove the need of genetically manipulated cells. Alternatively, it may augment the rate of vascularization if used together with engineered tissues and scaffolds containing endothelial cells or endothelial progenitor cells. This proposed approach is novel for improving neovascularization in tissue engineering. 1.3. Regulation of HIF-1 Hypoxia inducible factor (HIF-1) is a key transcription factor that is upregulated in response to hypoxia. Hypoxia is a common physiological stress imposed by low oxygen conditions to cells or tissues. HIF-1 is a heterodimer consisting of two subunits, a hypoxia regulated HIF-1α and an oxygen insensitive subunit HIF-1β 18 , 19 . HIF-1α consists mainly of a basic-helix-loop-helix (bHLH) region, a Per/Arnt/Sim (PAS) region, a N-terminal transactivation domain (N-TAD) and a C-terminal transactivation domain (C-TAD) (Figure extracted from Kaelin 200519). Figure 1. Schematic representation of HIF-1α protein domain structures (Kaelin 200519) Under normoxia, posttranslational hydroxylation of HIF-1α occurs at conserved proline residues (Pro 402 and Pro 564) within a polypeptide segment known as the oxygen dependent domain (ODD) found at the N-terminal transactivating domain (N-TAD) region19,20,21. These hydroxylated prolyl sites interact with the von-Hippel Lindau tumor suppressor protein (pVHL), which is the recognition protein for the E3 Ubiquitin Ligase complex19,20,21, 22 , 23 . The ubiquitin ligase complex subsequently packages HIF-1 for proteosomal degradation via polyubiquitylation, therefore HIF-1 is highly unstable under normoxia19,20,22. In addition, an asparaginyl hydroxylase, Factor Inhibiting HIF-1 (FIH-1), acts on asparaginyl residue Asn 803, found on the C-terminal transactivating domain (CTAD) region of HIF-1α, blocking the site and preventing its interaction with the transcriptional activator p300 20,24,25 (Figure 2). Under hypoxic conditions, the oxygen dependent prolyl hydroxylase enzymes is inhibited, thus HIF-1 is not hydroxylated and accumulates in the cell. Hypoxia also prevents hydroxylation of the C-TAD region, allowing HIF-1α to interact with p300 and promote transcription of target genes. Figure 2. Regulation of the HIF-1 transcription factor (From Bruick & McKnight 200220). Under normoxic conditions, the ODD of HIF1α is modified by a HIF– prolyl hydroxylase, triggering HIF-1α recognition by pVHL and subsequent degradation by the proteasome. Similarly, an asparaginyl hydroxylase modifies the C-TAD of HIF1α, blocking its interaction with the transcriptional coactivator p300. Hypoxia blocks both prolyl hydroxylation and asparaginyl hydroxylation, allowing HIF-1α to accumulate and bind to p300, thereby promoting the transcription of downstream HIF-1 target genes, thus enabling cells and the whole organism to adapt to hypoxia. 1.4. HIF-1 Target Genes HIF-1 target genes (Figure 3) are expressed in hypoxic situations, with many of them found to be involved in cell metabolism, vascular remodeling and angiogenesis18, 26 . Specifically, vascular endothelial factor (VEGF) is well established as an angiogenesis promoting growth factor involved in endothelial cell proliferation, migration and differentiation27,28,29. It is often used as a positive control for stimulating angiogenesis. VEGF receptor-1 (VEGFR-1), also known as FLT-1, is a tyrosine kinase receptor involved in endothelial cell migration, autocrine signaling and induction of matrix metalloproteinase-9 (MMP-9)27,28,29. Others, such as erythropoietin (EPO), signal the requirement for production of red blood cells, which is important in hypoxia 30 , and ischemic diseases31. Gene product ABCG2 α1β-Adrenergic receptor Adrenomedullin Aldolase A (ALDA) Atrial natriuretic peptide Carbonic anhydrase CD18 Ceruloplasmin C-MET Connective tissue growth factor CYP3A6 CXCR4 DEC1 DEC2 Ecto-5’-nucleotidase (CD73) Endocrine gland-derived VEGF Endoglin Endothelin-1 Enolase ENOS Erythropoietin ETS-1 Glucose transporter (GLUT1) Glyceraldehyde-3-phosphate dehydrogenase Glucose regulated protein, 94-kDa (GRP94) Heme oxygenase-1 HIF-1α prolyl hydroxylase PHD3 (EGLN3) HIF-1α prolyl hydroxylase PHD2 (EGLN1) HGTD-P ID2 Integrin β2 Intestinal trefoil factor Lactate dehydrogenase A (LDHA) Lactase Leptin Membrane type-1 matrix metalloproteinase Multi-drug resistance (ABCB1) Myeloid cell factor (MNL1) Nitric oxide synthase NIP3 NUR77 p35srj (CITED2) Phosphoglycerate kinase 6-Phosphofructo-2-kinase/fructose-2,6bisphosphatase-3 (PFKFB3) 6-Phosphofructo-2-kinase/fructose-2,6bisphosphatase-4 (PFKFB4) Plasminogen activator inhibitor Procollagen prolyl-4-hydroxylase α(I) RORα Stromal-derived factor (SDF-1) Telomerase (TERT) Transferrin Transferrin receptor Transforming growth factor β3 Vascular endothelial growth factor (VEGF) VEGF receptor-1 (Flt-1) Figure 3. Genes that are directly regulated by HIF-118,32. Many of these gene products of HIF-1 transcription are related to angiogenesis, such as erythropoietin for the production of red blood cells, insulin like growth factor and its binding proteins, VEGF and its receptor FLT-1. 1.5. HIF-1α Promotes Angiogenesis There is a considerable amount of evidence published on the involvement of HIF-1α in promoting angiogenesis. This has been reviewed extensively18,26,32, therefore only a brief summary will be given here. Ischemia results in cellular exposure to hypoxic microenvironments. By stabilization of HIF-1, the cell automatically signals the requirement for growth of blood vessels to the ischemic site by upregulating the local production of angiogenic factors. This is also often the case in cancers, where HIF is activated by physiological hypoxia within a rapidly growing tumour cell mass32,33. Overexpression of HIF-1α has been found with an associated increase in microvessel density and VEGF expression in several cancers, such as bladder, breast, colon, ovarian and pancreatic cancers32. HIF-1 is also critical in angiogenesis and embryogenesis. It was reported that HIF-1 deficient mouse embryonic cells produce major vascular defects in the yolk sac and developing embryo, associated with severe hypoxia due to lack of perfusion34. Injection of these HIF-1 deficient mouse embryonic stem cells into immunocompromised mice resulted in smaller and less vascularized tumors compared to normal embryonic stem cells. Transgenic mice expressing constitutively active HIF-1α in the epidermis exhibited an increase capillary density in the dermis and marked increase in VEGF 35 . Despite hypervascularization, there was no edema, inflammation or vascular leakage, compared to transgenic mice overexpressing VEGF cDNA in the skin35,11. Additionally, administration of HIF-1α/VP16 recombinant plasmids constructed by fusion of HIF-1α to the transactivation domain of herpes simplex virus VP16 has improved recovery of local blood flow and vascularization in a rabbit hindlimb ischemic model36. Similar results were obtained using an acute myocardial infarction rat model 37 . These evidences demonstrate the involvement of HIF-1 in angiogenesis. Consequently, several approaches have been undertaken to target HIF-1 for cancer therapy and treatment for ischemic diseases38,39. 1.6. HIF Prolyl 4-Hydroxylases There are three identified human cytoplasmic prolyl 4-hydroxylase isoenzymes that hydroxylate HIF-1α 40 , 41 . Like collagen prolyl 4-hydroxylases, they are dioxygenases dependent on Fe2+, oxygen, ascorbate and 2-oxoglytarate. 1.7. Prolyl 4-Hydroxylase Inhibitors (PHi) Promote Angiogenesis By Stabilizing HIF-1α Available literature on promotion of angiogenesis using prolyl 4-hydroxylase inhibitors (PHi) is scarce at the moment. Gleadle et al (1995)42 was one of the first to look at the effect of prolyl hydroxylase inhibitors on HIF-1. Cobalt ions and iron chelators such as desferrioxamine (also known as deferioxamine, or DFO), upregulated mRNA expression of some HIF-1 target genes, namely VEGF, platelet derived growth factor A and B (PDGF-A, PDGF-B), erythropoietin (EPO) and transforming growth factor β-1 (TGFβ-1). The mRNA expression levels were compared to that induced by hypoxia in human tumour cell lines like Hep3B, HepG2 (hepatoma) and HT1080 (fibrosarcoma). Stabilization of HIF-1α has subsequently been reported using cobalt chloride 43 , 2oxoglutarate analogues such as N-oxalylglycine and dimethyloxalyglycine (DMOG)22,43,44, nitric oxide (S-nitroglutathione)45, proprietary substances developed by Fibrogen, Inc 46,47,48 , Proteosome inhibitors such as MG-132 may also induce nuclear accumulation of HIF-1α44,43 by preventing its degradation. In addition, aliphatic and aromatic compounds such as pyridine-2,4-dicarboxylate and 3,4-dihydroxybenzoic acid were found to be competitive inhibitors of HIF-prolyl 4-hydroxylase isoenzymes in comparison to 2-oxoglutarate41. Another study made use of recombinant polypeptide constructs bearing either of the two prolyl residues on HIF-1α that are targeted for hydroxylation to induce nuclear accumulation and stabilization of HIF-1α in transfected cell 49 . Stimulation of angiogenesis was shown by formation of tubules by microvascular endothelial cells cocultured with these transfected cells and increased vascularization of subcutaneously implanted polyurethane sponges injected with the fusion protein in a murine model49. There are three reports on stimulation of angiogenesis using pharmaceutically induced prolyl hydroxylase inhibition to stabilize HIF-1α, all published shortly before this research study was begun50,51,52. Warnecke et al (2003) showed accumulation of HIF-1α in western blots of lysates from human HT1080 fibrosarcoma cells, rat PC12W 10 It was observed that the HUVECs were found mainly on the edges of the scaffolds, similar to what was observed in tissue culture plates, where the HUVECs were found primarily near the edges of the wells rather than the center (data not shown). While it is plausible that the cells have better access to nutrients near the edges of the scaffold compared to the center, this does not explain why a similar trend was found in tissue culture plate wells. However, it should be noted that the DAPI staining in the center of the scaffolds, although present, were weaker, suggesting that either the staining solutions did not penetrate the cells, or fewer cells were found in the center. It is possible that in cutting the PLLA felt scaffolds using the biopsy punch, the PLLA fibers that were severed at the edges of the scaffold produce more open pores and spaces for fluids to enter. Dynamic culture using a bioreactor or spinner flasks may improve cell distribution, but this was not investigated as the angiogenic effects of CPX could still be assessed within the limits of static scaffold culture. The results obtained show that CPX was effective in stimulating angiogenesis, evidenced by the formation of more numerous and thinner CLS compared to untreated controls (Figure 37). These CLS more greatly resembled a capillary network with multiple interconnections compared to those formed in two dimensional cultures. They were also formed within cell sheets of fibroblasts and did not follow the pattern of PLLA scaffold fibers, which were found to coincide with dark regions devoid of cells (Figure 38). This was important as it demonstrated that the endothelial cells were not merely growing 104 along the fibers of the scaffold to form a CLS, but were actively migrating between cells to align and form CLS within fibroblast cell sheets. It was difficult to quantify the extent of angiogenesis in three dimensional scaffolds as the capillary like structures were different in thickness within any captured image and the nodes present at the junctions of interconnections were difficult to completely include or exclude from the quantification. The outcome of such measurements would not be an accurate quantification, but merely a very rough indication that may be observer biased by the lack of measurement criteria. Nevertheless, the confocal microscopy images provide sufficient information on the general angiogenic stimulatory effects of CPX and VEGF compared to untreated controls. 4.9. Advantages of Using PHi In Tissue Engineering The use of prolyl hydroxylase inhibitors has the advantage of acting via the transcription factor HIF-1α, a master switch for angiogenesis. Instead of delivery of one or two specific angiogenic peptides such as VEGF and bFGF at non physiological doses7,8,9,10, this method allows several genes involved in angiogenesis to be upregulated at the same time. This allows the cells to regulate the timing and amount of cytokines and signaling molecules produced. For example, we found that the levels of VEGF production by fibroblasts were 300-2000 pg/ml compared to the bolus dose of 10 ng/ml VEGF used as a positive control, indicating that cells produced more physiological levels of VEGF and these lower levels were able to induce angiogenesis, probably due to the presence of other factors and signaling molecules. 105 One potential application of prolyl hydroxylase inhibitors for improving neovascularization is to accelerate angiogenesis in pre-vascularized tissue constructs which are cultured in vitro before implantation. This proof-of-concept has been demonstrated here using three dimensional cultures in PLLA scaffolds and has the potential to be applied to other tissue engineering systems that aim to form tissue constructs to regenerate tissues at sites of critical defects, such as the heart, liver, bone or skin. Acceleration of angiogenesis using PHi would lower costs of tissue culture. In addition, pre-vascularized tissue engineered skin has been shown to improve angiogenesis by inosculation with the host vasculature after grafting within days, compared to 15 days when grafts without vascular analogs were used97,98. This suggests that the survival of implanted cells could be improved by pre-vascularizing the tissue construct prior to implantation. An alternative means of using PHi in tissue engineering applications is to deliver the substances to the site of implantation together with the tissue construct. This may be explored through the use of implantation studies in animal models. CPX and HDZ are both FDA approved drugs and hence have the potential to be employed in this manner. The substances could be encapsulated or coated on the biomaterial used to make the tissue scaffold and delivered locally together with the implanted cells by controlled release or degradation of the biomaterial. As discussed earlier, the main advantage of using PHi is that these compounds would evoke a physiological broadly based angiogenic response centered around the HIF-1 transcription factor, compared to what could be 106 achieved by the delivery of one or two down-stream acting angiogenic growth factors. As PHi also inhibit collagen secretion, they could exert a dual angiogenic and anti-fibrotic effect, which could improve the viability and survival of an implanted tissue engineered construct. Targeted local delivery of these substances is important to ensure that the angiogenic stimulatory and collagen inhibitory effects are specific to the implantation site to induce neovascularization into the implanted tissue construct and prevent periimplantational fibrosis. 107 5. CONCLUSIONS The in vitro and in vivo angiogenic and anti-fibrotic effects of HDZ, CPX and PDCA investigated in this research study are summarized in Table 1. Of the three substances, HDZ appears to be the most promising candidate for tissue engineering applications, as it is a FDA (Food Drug Administration) approved hypertensive drug and promoted angiogenesis in vitro and in vivo. The drug may be used to pre-vascularize tissue constructs in static culture or bioreactors prior to implantation and could also be incorporated into scaffolds for local release after implantation. Concentration PDCA CPX HDZ HIF-1α (WB) VEGF ELISA mM 10 mM 15 mM 20 mM 30 mM µM + +(+) 3.5 fold fold µM µM 16 µM ++++ +++++ 25 µM +(+) CLS formation sequential co-culture (+) + 2.3 fold Inhibition - fold fold 10 fold fold fold Inhibition No increase fold fold Inhibition Inhibition Ectopic SIV in zebrafish embryos In vitro reduction in collagen I secretion Collagen reduction in zebrafish embryos 72% fold 42% fold Toxic at 0.5 µM 71% 95% 50 µM 100 µM fold 32% 200 µM (+) 1.7 fold 400 µM + 5.3 fold 96% 500 µM fold 1000 µM fold VEGF 10 ng/ml fold 100 ng/ml fold + depicts one arbitrary unit and (+) depicts approximately 0.5 arbitrary units. 6% 30% 35% Table 1. Summary of the in vitro and in vivo angiogenic and anti-fibrotic effects of PDCA, CPX, HDZ and VEGF. 108 CPX is another promising candidate as it is also FDA approved as a topical anti-mycotic on skin and nails. It had the most potent effect in inducing HIF-1α stabilization, VEGF increase and formation of CLS in co-cultures and was shown to induce formation of numerous and well interconnected CLS in PLLA felt scaffolds. Although CPX interfered with the early development of zebrafish embryos it showed very low toxicity in coculture and up to 33 µM CPX has been used in rat kidney perfusion experiments53, therefore further work will be needed to establish its potential use in implantation studies. PDCA was the least potent in inducing angiogenesis in co-cultures, but had the most potent effect on zebrafish angiogenesis. While it is not an FDA approved substance, several analogues have been produced for clinical purposes, particularly by the company Fibrogen Inc (South San Francisco), which has purchased patents of the former Hoechst AG and is pursuing preclinical and phase I clinical trials using PH inhibitors for systemic application to treat renal anaemia. The successful induction of CLS in co-cultures using HDZ and CPX demonstrates their potential applications in larger scale co-cultures of fibroblasts and endothelial cells involving scaffolds or bioreactors. Pre-vascularized tissue engineered skin can speed up neovascularization by inosculation with the host vasculature after grafting compared to wound healing angiogenesis alone 97 , 98 . The use of prolyl hydroxylase inhibitors to simulate neovascularization may stimulate endothelial cells to form vascular analogs within engineered tissue constructs in vitro, thus saving cost and time. If locally delivered to the site of implantation, they may also further improve the surgical outcome by 109 speeding up angiogenesis and inosculation time after implantation of the tissue construct, at the same time exerting an anti-fibrotic effect to prevent peri-implantational fibrosis. In conclusion, we provide evidence that HDZ, CPX and PDCA induce angiogenesis in vitro and in vivo by stabilization of HIF-1α and upregulation of VEGF. Combined with the advantage of their anti-fibrotic effects, these substances offer a promising strategy for promoting neovascularization in engineered tissues and improving the survival and function of such implanted tissue. 110 6. REFERENCES Langer, R., Vacanti, J.P. 1993. Tissue engineering. Science. 260, 920–6. Nomi, M., Atala, A., De Coppi, P. and Soker, S. 2003. Principals of neovascularization for tissue engineering. Molecular Aspects of Medicine. 23, 463-483. Cassell, O.C.S., Hofer, S.O.P.,Morrison, W.A. and Knight, K.R. 2002. Vascularisation of tissue-engineered grafts: the regulation of angiogenesis in reconstructive surgery and in disease states. British Journal of Plastic Surgery. 55, 603-610 Vogel, V., Barneyx G. 2003. The tissue engineering puzzle: A molecular prespective. Annual Reviews in Biomedical Engineering. 5, 441-463. Kannan, R.Y., Salacinski, H.J., Sales, K., Butler, P., Seifalian, A.M. 2005. The roles of tissue engineering and vascularization in the development of micro-vascular networks: a review. Biomaterials. 26, 1857-1875. Mikos, A.G., Sarakinos, G., Lyman, M.D., Ingber, D.E., Vacanti, J.P., Langer, R. 1993. Prevascularization of porous biodegradable polymers. Biotechnology and Bioengineering. 42:716-723. Richardson, T.O., Peters, M.C., Ennett, A.B., Mooney, D.J. 2001. Polymeric system for dual growth factor delivery. Nature Biotechnology. 19:1029-1034. Perets, A., Baruch, Y., Weisbuch, F., Shoshany, G., Neufeld, G. and Cohen, S. 2003. Enhancing the vascularization of three-dimensional porous alginate scaffolds by incorporating controlled release basic fibroblast growth factor microspheres. Journal of Biomedical Materials Research. 65A: 485-497. Murphy, W.L., Peters, M.C., Kohn, D.H., Mooney, D.J. 2000. Sustained release of vascular endothelial growth factor from mineralized poly(lactide-co-glycolide) scaffolds for tissue engineering. Biomaterials. 21:2521-2527. 10 Soker, S., Machado, M., Atala, A. 2000. Systems for therapeutic angiogenesis in tissue engineering. World Journal of Urology. 18:10-18. Larcher, F., Murillas, R., Bolontrade, M., Conti, C.J., Jorcano, J.L. 1998. VEGF/VPF overexpression in skin of transgenic mice induces angiogenesis, vascular hyperpermeability and accelerated tumour development. Oncogene. 17:303-311. 11 12 Levenberg, S., Rouwkema, J., Macdonald, M., Garfein, E.S., Kohane, D.S., Darland, D.C., Marini, R., Blitterswijk, C.,A, Mulligan, R.C., D’Amore, P.A. and Langer, R. 2005. Engineering vascularized skeletal muscle tissue. Nature Biotechnology. 23:879-884. 111 13 Zwaginga, J.J. and Doevendans, P. 2003. Stem cell-derived angiogenic/ vasculogenic cells: possible therapies for tissue repair and tissue engineering. Clinical and Experimental Pharmacology and Physiology. 30:900-908. 14 Koike, N., Fukumura, D., Gralla, O., Au, P., Schechner, J.S., Jain, R.K. 2004. Creation of long-lasting blood vessels. Nature. 428:138-139. 15 Yamashita, J., Itoh, H., Hirashima, M., Ogawa, M., Nishikawa, S., Yurugi, T., Naito, M., Nakao, J., Nishikawa, S. 2000. Flk1-positive cells derived from embryonic stem cells serve as vascular progenitors. Nature. 408:92-96. 16 Mertsching, H., Walles, T., Hofmann, M., Schanz, J., Knapp, W.H. 2005. Engineering of a vascularized scaffold for artificial tissue and organ generation. Biomaterials. 26:6610-6617. 17 Rafii, S. and Lyden, D. 2003. Therapeutic stem and progenitor cell transplantation for organ vascularization and regeneration. Nature Medicine. 9:702-712. Semenza, G.L. 2000. HIF-1: mediator of physiological and pathophysiological responses to hypoxia. Journal of Applied Physiology. 88:1474-1480. 18 19 Kaelin, W.G. 2005. Proline hydroxylation and gene expression. Annual Reviews Biochemistry. 74:115-28. 20 Bruick R.K. and McKnight, S.L. 2002 Oxygen sensing gets a second wind. Science 295: 807-808. 21 Mazure, N.M., Brahimi-Horn, C., Berta, M.A., Benizri, E., Bilton, R.L., Dayan, F., Ginouves, A., Berra, E., Pouyssegur, J. 2004. HIF-1: master and commander of the hypoxic world. A pharmacological approach to its regulation by siRNAs. Biochemical Pharmacology. 68:971-980. 22 Jaakkola, P., Mole, D.R., Tian, Y.M., Wilson, M.I., Gielbert, J., Gaskell, S.J., von Kriegsheim, A., Hebestreit, H.F., Mukherji, M., Schofield, C.J., Maxwell, P.H., Pugh, C.W., Ratcliffe, P.J. Targeting of HIF-α to the von Hippel-Lindau ubiquitylation complex by O2- regulated prolyl hydroxylation. Science.2001;292:468-472. 23 Marxsen, J.H., Stengel, P., Doege, K., Heikkinen, P., Jokilehto, T., Wagner, T., Jelkmann, W., Jaakkola, P and Metzen, E. 2004. Biochemistry Journal 381:761-767. 24 Lando, D., Peet, D.J., Whelan, D.A., Gorman, J.J., Whitelaw, M.L. 2002. Asparagine hydroxylation of the HIF transactivation domain: A hypoxic switch. Science. 295:858861. 112 25 Lando, D., Peet, D.J., Gorman, J.J., Whelan, D.A., Whitelaw, M.L., Bruick, R.K. 2002. FIH-1 is an asparaginyl hydroxylase enzme that regulates the transcriptional activity of hypoxia-inducible factor. Genes & Development. 16:1466-1471. Pugh, C.W., Ratcliffe, P.J. 2003. Regulation of angiogenesis by hypoxia: role of the HIF system. Nature Medicine. 9:677-684. 26 27 Neufeld, G., Cohen, T., Gengrinovitch, S., Poltorak, Z. 1999. Vascular endothelial growth factor (VEGF) and its receptors. FASEB Journal. 13:9-22. 28 Ferrara, N. Role of vascular endothelial growth factor in regulation of physiological angiogenesis. Am J Physiol Cell Physiol. 2001;280:C1358-1366. 29 Ferrara, N. Vascular endothelial growth factor: Basic science and clinical progress. Endocrine Reviews. 2004;25:581-611. 30 Wanner, R.M., Spielmann, P., Stroka, D.M., Camenisch, G., Camenisch, I., Scheid, A., Houck, D.R., Bauer, C., Gassmann, M., Wenger, R.H. 2000. Epolones induce erythropoietin expression via hypoxia-indicible factor-1α activation. Blood. 96:15581565. 31 Prass, K., Scharff, A., Ruscher, K., Lowl, D., Muselmann, C., Victorov, H., Kapinya, K., Dirnagl, U., Meisel, A. 2003. Hypoxia-induced stroke tolerance in the mouse is mediated by erythropoietin. Stroke. 34:1981-1986. 32 Hirota, K., Semenza, G.L. 2006. Regulation of angiogenesis by hypoxia-inducible factor 1. Critical Reviews in Oncology/Hematology. 59:15-26. 33 Choi, K.S., Bae, M.K., Jeong, J.W., Moon, H.E., Kim, K.W. 2003. Hypoxia induced angiogenesis during carcinogenesis. Journal of Biochemistry & Molecular Biology. 36:120-127. 34 Ryan, H.E., Lo, J., Johnson, R.S. 1998. HIF-1α is required for solid tumor formation and embryonic vascularization. The EMBO Journal. 17:3005-3015. 35 Elson, D.A., Thurston, G., Huang, L.E., Ginzinger, D.G., McDonald, D.M., Johnson, R.S., Arbeit, J. 2001. Induction of hypervascularity without leakage or inflammation in transgenic mice overexpressing hypoxia-inducible factor-1α. Genes & Development. 15:2520-2532. 36 Vincent, K.A., Shyu, K.G., Luo, Y., Magner, M., Tio, R.A., Jiang, C., Goldberg, M.A., Akita, G.Y., Gregory, R.J., Isner, J.M. 2000. Angiogenesis is induced in a rabbit model of hindlimb ischemia by naked DNA encoding an HIF-1α/VP16 hybrid transcription factor. Circulation. 102:2255-2261. 113 37 Shyu, K.G., Wang, M.T., Wang, B.W., Chang, C.C., Leu, J.G., Kuan, P., Chang, H. 2002. Intramyocardial injection of naked DNA encoding HIF-1α/VP16 hybrid to enhance angiognesis in an acute myocardial infarction model in the rat. Cardiovascular Research. 54:576-583. 38 Semenza, G.L. 2003. Targetting HIF-1 for cancer therapy. Nature Reviews. 3:721-732. 39 Giaccia, A., Siim, B.G., Johnson, R.S. 2003. HIF-1 as a target for drug development. Nature Reviews. 2:1-9. 40 Epstein, A.C.R., Gleadle, J.M., McNeill, L.A., Hewitson, K.S., O’Rourke, J., Mole, D.R., Mukherji, M., Meetzen, E., Wilson, M.I., Dhanda, A., Tian, Y.M., Masson, N., Hamilton, D.L., Jaakkola, P., Barstead, R., Hodgkin, J., Maxwell, P.H., Pugh, C.W., Schofield, C.J., Ratcliffe, P.J. 2001. C. elegans EGL-9 and mammalian homologs define a family of dioxygenases that regulate HIF by prolyl hydroxylation. Cell. 107:43-54. 41 Hirsila, M., Koivunen, P., Gunzler, V., Kiviikko, K.I., Myllyharju, J. 2003. Characterization of the human prolyl 4-hydroxylases that modify the hypoxia-inducible factor. The Journal of Biological Chemistry. 278:30772-30780. 42 Gleadle, J.M., Ebert, B.L., Firth, J.D., Ratcliffe, P.J. 1995. Regulation of angiogenic growth factor expression by hypoxia, transition metals, and chelating agents. American Journal of Physiology (Cell Physiology) 268:C1362-1368. 43 Chan, D.A., Sutphin, P.D., Denko, N.C., Giaccia, A.J. 2002. Role of prolyl hydroxylation in oncogenically stabilized hypoxia-inducible factor-1α. Journal of Biological Chemistry. 277:40112-40117. 44 Hagg, M., Wennstrom, S. 2005. Activation of hypoxia0induced transcription in normoxia. Experimental Cell Research. 306:180-191. 45 Metzen, E., Zhou, J., Jelkmann, W., Fandrey, J., Brune, B. 2003. Nitric oxide impairs normoxic degradation of HIF-1α by inhibition of prolyl hydroxylases. Molecular Biology of the Cell. 14:3470-3481. 46 Ivan, M., Haberberger, T., Gervasi, D.C., Michelson, K.S., Gunzler, V., Kondo, K., Yang, H., Sorokina, I., Conaway, R., Conaway, J.W., Kaelin, W.G. 2002. Biochemical purification and pharmacological inhibition of a mammalian prolyl hydroxylase acting on hypoxia-inducible factor. PNAS. 99:13459-113464. 47 Siddiq, A., Ayoub, I.A., Chavez, J.C., Aminova, L., Shah, S., LaManna, J.C., Patton, S.M., Connor, J.R., Cherny, R.A., Volitakis, I., Bush, A.I., Langsetmo, I., Seeley, T., Gunzler, V., Ratan, R.R. 2005. Hypoxia-inducible factor prolyl 4-hydroxylase inhibition. A target for neuroprotection in the central nervous system. Journal of Biological Chemistry. 280:41732-41743. 114 48 Asikainen, T.M., Ahmad, A., Schneider, B.K., Ho, W.B., Arend, M., Brenner, M., Gunzler, V., White, C.W. 2005. Stimulation of HIF-1α, HIF-2α, and VEGF by prolyl 4hydroxylase inhibition in human lung endothelial and epithelial cells. Free Radical Biology & Medicine. 38:1002-1013. 49 Willam, C., Masson, N., Tian, Y.M., Mahmood, S.A., Wilson, M.I., Bicknell, R., Eckardt, K.U., Maxwell, P.H., Ratcliffe, P.J., Pugh, C.W. 2002. Peptide blockade of HIF1α degradation modulates cellular metabolism and angiogenesis. PNAS. 99:1042310428. 50 Warnecke, C., Griethe, W., Weidemann, A., Jurgensen, J.S., Willam, C., Bachmann, S., Ivashchenko, Y., Wagner, Y., Frei, U., Wiesener, M. and Eckardt, K.U. 2003. Activation of the hypoxia-inducible factor pathway and stimulation of angiogenesis by application of prolyl hydroxylase inhibitors. FASEB Journal. 17:1186-8. 51 Linden, T., Katschinski, D.M., Eckhardt, K., Scheid, A., Pagel H. and Wenger, R.H. 2003. The antimycotic ciclopirox olamine induces HIF-1α stability, VEGF expression, and angiogenesis. FASEB Journal. 17:761-3. 52 Knowles, H.J., Tian Y.M., Mole, D.R., Harris, A.L. Novel mechanism of action for hydralazine. 2004. Induction of hypoxia-inducible factor-1 α, vascular endothelial growth factor, and angiogenesis by inhibition of prolyl hydroxylases. Circulation Research. 95:162-169. 53 Clement, P.M.J., Hanauske-Abel, H.M., Wolff, E.C., Kleinman, H.K., Park, M.H. 2002. The antifungal drug ciclopirox inhibits deoxyhypusine and proline hydroxylation, endothelial cell growth and angiogenesis in vitro. International Journal of Cancer 100:491-498. 54 Kleinman, H.K., Martin, G.R. 2005. Matrigel: Basement membrane matrix with biological activity. Seminars in Cancer Biology. 15:378-386. 55 Staton, C.A., Stribbling, S.M., Tazzyan, S., Hughes, R., Browth, N.J., Lewis, C.E. 2004. Current methods for assaying angiogenesis in vitro and in vivo. Int J Exp Path. 85:233-248. 56 Bishop, E.T., Bell, G.T., Bloor, S., Broom, I.J., Hendry, N.F.K., Wheatley, D.N. 1999. An in vitro model of angiogenesis: basic features. Angiogenesis. 3:335-344. 57 Friis, T., Sorensen, B.K., Engel, A.M., Rygaard, J., Houen, G. 2003. A quantitative ELISA based co-culture angiogenesis and cell proliferation assay. APMIS. 111:658-668. 58 Langheinrich, U. 2003. Zebrafish: a new model on the pharmaceutical catwalk. BioEssays. 25:904-912. 115 59 Kidd, K.R., Weinstein, B.M. 2003. Fishing for novel angiogenic therapies. British Journal of Pharmacology. 140:585-594. 60 Parng, C., Seng, W.L., Semino, C., McGrath, P. 2002. Zebrafish: A preclinical model for drug screening. Assay and Drug Development Technologies. 1:41-48. 61 Lawson, N.D., Weinstein, B.M. 2002. In vivo imaging of embryonic vascular development using transgenic zebrafish. Developmental Biology. 258:307-318. 62 Childs, S., Chen, J., Garrity, D.M., Fishman, M.C. 2002. Patterning of angiogenesis in the zebrafish embryo. Development. 129:9773-982. 63 Cross, L.M., Cook, M.A., Lin, S., Chen, J.N. and Rubinstein, A.L. 2003. Rapid analysis of angiogenesis drugs in a live fluorescent zebrafish assay. Arterioscler Thromb Vasc Biol 23, 911-2 64 Seng, W.L., Eng, K., Lee, J., McGrath, P. 2004. Use of a monoclonal antibody specific for activated endothelial cells to quantitate angiogenesis in vivo in zebrafish after drug treatment. Angiogenesis. 7:243-253. 65 Serbedzija, G.N., Flynn, E., Willett, C.E. 1999. Zebrafish angiogenesis: A new model for drug screening. Angiogenesis. 3:353-359. 66 Habeck, H., Odenthal, J., Walderich, B., Maischein, H., Schulte-Merker, S., Tubingen 2000 screen consortium. 2002. Analysis of a zebrafish VEGF receptor mutant reveals specific disuption of angiognesis. Current Biology. 12:1405-1412. 67 Babensee, J.E., Anderson, J.M., McIntire, L.V., Mikos, A.G. 1998. Host response to tissue engineered devices. Advanced Drug Delivery Reviews. 33:111-139. 68 Phillips, S.J. 2000. Physiology of wound heaing and surgical wound care. ASAIO Journal. 46:S2-S5. 69 Martin, P., Leibovich, S.J. 2005. Inflammatory cells during wound repair: the good, the bad and the ugly. Trends in Cell Biology. 15:599-607. 70 Ratner, B.D. 2002. Reducing capsular thickness and enhancing angiogenesis around implant drug release systems. Journal of Controlled Release. 78:211-218. 71 Gelse, K., Poschl, E., Aigner, T. 2003. Collagens – structure, function and biosynthesis. Advances Drug Delivery Reviews. 55:1531-1546. 72 Kar, K., Amin, P., Bryan, M.A., Persikov, A.V., Mohs, A., Wang, Y.H., Brodsky, B. 2006. Self-association of collagen triple helix peptides into higher order structures. Journal of Biological Chemistry. 281:33283-33290. 116 73 Tschank, G., Raghunath, M., Gunzler, V., Hanauske-Abel, H.M. 1987. Pyridinedicarboxylates, the first mechanism-derived inhibitors for prolyl 4-hydroxylase, selectively suppress cellular hydroxyprolyl biosynthesis. Biochem J. 248:625-633. 74 Majamaa, J., Hanauske-Abel, H.M., Gunzler, V., Kivirikko, K.I. 1984. The 2oxoglutarate binding site of prolyl 4-hydroxylase. Eur J Biochem. 138:239-245. 75 Ju, H., Hao, J., Zhao, S, Dixon, I.M.C. 1998. Antiproliferative and antifibrotic effects of mimosine on adult cardiac fibroblasts. Biochimica et Biophysica Acta. 1448:51-60. 76 Blumenkrantz, J., Chistiansen, A.H., Asboe-Hansen, G. 1979. Effect of hydralazine and dihydralazine on connective tissue and binding to serum protein. Scan J Rheumatology. 8:177-183. 77 Murad, S., Tajim, S., Pinnell, S.R. 1985. A paradoxical effect of hydralazine on prolyl and lysyl hydroxylase activities in cultured human skin fibroblasts. Archives of Biochemistry and Biophysics. 241:356-363. 78 Yeowell, H.N., Murad, S., Pinnell, S.R. 1991. Hydralazine differentially increases mRNAs for the α and β subunits of prolyl 4-hydroxylase whereas it decreases proα1(I) collagen mRNAs in human skin fibroblasts. Archives of Biochemistry and Biophysics. 289:399-404. 79 Franklin, T.J., Morris, W.P., Edwards, P.N., Large, M.S., Stephenson, R. 2001. Inhibition of prolyl 4-hydroxylase in vitro and in vivo by members of a novel series of phenanthrolinones. Biochem J. 353:333-338. 80 Hanauske-Abel, H.M. 1991. Prolyl 4-hydroxylase, a target enzyme for drug development. J Hepatology. 13:S8-S16. 81 Kagan, H.M. 2000. Intra and extracellular enzymes of collagen biosynthesis as biological and chemical targets in the control of fibrosis. Acta Tropica. 77:147-152. 82 Kim, I., Mogford, J.E., Aitschi, C., Nafissi, M., Mustoe, T.A. 2003. Inhibition of prolyl-4-hydroxylase reduces scar hypertrophy in a rabbit model of cutaneous scarring. Wound Rep Reg. 11:368-372. 83 Heazell, A.E.P., Mahomoud, S., Pirie, A.M. 2004. The treatment of severe hypertension in pregnancy: a review of current practice and knowledge in West-Midlands maternity units. Journal of Obstetrics and Gynaecology. 24:897-898. 84 Jue, S.G., Dawson, G.W., Brogden, R.N. 1985. Ciclopirox olamine 1% cream. A preliminary review of its antimicrobial activity and therapeutic use. Drugs. 29:330-41. 85 Melnikoca, I. Anaemia therapies. Nature Reviews Drug Discovery. 2006;5:627-628. 117 86 Hsieh, M.M., Linde, N.S., Wynter, A., Metzger, M., Wong, C., Langsetmo, I., Lin, A., Smith, R., Rodgers, G.P., Donahue, R.E., Klaus, S.J., Tisdale, J.F. HIF-prolyl hydroxylase inhibition results in endogenous erythropoietin induction, erythrocytosis, and fetal hemoglobin expression in rhesus macaques. Blood. 2007; Jun (Epub ahead of print). 87 Lareu, R.R., Arsianti, I., Subramhanya, K.H., Peng, Y., Raghunath, M. 2007. In vitro enhancement of collagen matrix formation and crosslinking for applications in tissue engineering. Tissue Engineering 13, 385-391. 88 Westerfield, M. 1993. The Zebrafish Book: A guide for the laboratory use of zebrafish. Eugene, Oregon: The University of Oregon Press. 89 Takano, S., Gately, S., Neville, M.E., Herblin, W., Gross, J.L., Engelhard, H., Perricone, M., Edisvoog, K., Brem, S. 1994. Anticancer and angiosuppressive agent, inhibits endothelial cell binding of basic fibroblast growth factor, migration, proliferation and induction of urokinase-type plasminogen activator. Cancer Research. 54:2654-2660. Kimura, S. 1992. Wide distribution of the skin type I collagen α3 chain in bony fish. Comp Biochem Physiol B. 102:255-260. 90 91 Rosso, F., Giordano, A., Barbarisi, M., Barbarisi, A. 2004. From Cell-ECM Interactions to Tissue Engineering. Journal of Cellular Physiology. 199:174-180. 92 Berthod, F., Germain, L., Tremblay, N., Auger, F.A. 2006. Extracellular matrix deposition by fibroblasts is necessary to promote capillary-like tube formation in vitro. Journal of Cellular Physiology. 207:491-498. 93 Ashino, H., Shimamura, M., Nakajima, H., Dombou, M., Kawanaka, S., Oikawa, T., Iwagushi, T., Kawashima, S. 2003. Novel function of ascorbic acid as an angiostatic factor. Angiogenesis. 6:259-269. 94 Epstein, S.E., Kornowski, R., Fuchs, S., Dvorak, H.F. 2002. Angiogenesis Therapy: Amidst the hype, the neglected potential for serious side effects. Circulation. 104:115119. 95 Stetler-Stevenson, W.G. 1999. Matrix metalloproteinases in angiogenesis: A moving target for therapeutic intervention. The Journal of Clinical Investigation. 103:1237-1241. 96 Barleon, B., Sozzani, S., Zhou, D., Weich, H.A., Mantovani, A., Marme, D. 1996. Migration of human monocytes in response to vascular endothelial growth factor (VEGF) is mediated via theVEGF receptor flt-1. Blood. 87:3336-3343. 118 97 Tremblay, B.L., Hudon, V., Berthod, F., Germain, L., Auger, F.A. 2005. Inosculation of tissue-engineered capillaries with the host’s vasculature in a reconstructed skin transplanted on mice. Am J Transplantation. 5:1002-1010. 98 Supp, D.M., Wilson-Landy, K., Boyce, S.T. 2002. Human dermal microvascular endothelial cells form vascular analogs in cultured skin substitues after grafting to athymic mice. FASEB J. 16:797-804. 119 [...]... growth factor-depleted formulations of Matrigel are available to allow some room for stimulation of angiogenesis, angiogenesis still proceeds rapidly Extensive and time-consuming analysis is required to measure the length of tubule formation and the number of nodes to quantify angiogeneis, although some software algorithms have been developed for this purpose Furthermore, there is the issue of cost as... survival and function of implanted tissue constructs, controlled drug delivery devices and sensors To tackle this problem, some researchers have explored the use of scaffold free tissue constructs, while many more are involved with surface modifications of the biomaterials to limit foreign body encapsulation The use of anti- fibrotic substances to minimize and reduce the formation of a fibrotic sheath around... growth factors such as basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), insulin-like growth factor-1 (IGF-1) and transforming growth factor-β (TGF-β) are present in various quantities and contribute to spontaneous induction of angiogenesis This is useful for testing anti- angiogenic drug effects, but presents difficulties in showing augmentation of angiogenic effects Although... proliferation, stimulation of angiogenesis and synthesis of collagen, proteoglycans and other extracellular matrix proteins to form granulation tissue 67,68,69 19 In addition to the wound healing response, implantation of biomaterials invokes a host immune response, also known as the foreign body reaction Macrophages adhere to the surface of the biomaterial and spread as they try to phagocytose the foreign body... limited to endothelial cell proliferation alone, it was important to further investigate and compare the potential angiogenic effects of some of these PHi in vitro 1.8 In vitro Angiogenesis Assays Existing in vitro angiogenic assays have been mainly developed and used for testing effect of anti- angiogenic compounds Although some of these assays are able to show pro-angiogenesis induced by growth factors... – A Result of Foreign Body Reaction and Wound Healing Regenerative medicine and tissue engineering often inevitably require surgery for implantation of tissue engineered constructs Surgery creates wounds, therefore provoking a wound healing response at the surgical site Wound healing involves phases of inflammation, proliferation and remodeling In the inflammatory phase, clotting leads to hemostasis,... analogue of mimosine, is used topically for treatment of fungal and yeast infection of the skin or mucosa51,53,84 PDCA is a well established and effective inhibitor of collagen prolyl 4-hydroxylase Several analogues of PDCA were developed and patented by the former Hoechst AG These patents were subsequently purchased by Fibrogen, Inc (South San Francisco) and some of these compounds have been found to prevent... FG-4592 and FG-2216, for example, are oral compounds currently tested in phase I and phase II clinical trials for treatment of anaemia and are thought to stimulate erythropoiesis by stabilizing HIF85,86 As the use of PHi to improve local vascularization for tissue engineering applications has not been reported, the results of this research study will be regarded as a novel approach towards this problem... soluble in water A 100 mM stock solution was prepared by dissolving 20mg per ml of ultrapure water A primary stock solution of 0.5 M CPX (Molecular Weight of 268.35 g) was prepared by dissolving 67 mg of CPX in 500 µl methanol and stored at -20°C in aliquots A secondary stock solution of 1 mM CPX was prepared prior to use, by (1:500) dilution of 0.5 M CPX with * k denotes 1,000 For example, 150k is 150,000... derivatives79 have been shown to inhibit collagen deposition by disrupting this hydroxylation step As a result, the procollagen molecules do not assemble into triple helical chains and are thus retained intracellularly instead of being secreted into the extracellular matrix73 This function of prolyl hydroxylase inhibitors has been exploited towards the design of antifibrotic drugs80,81, most of which are currently . use of anti- fibrotic substances to minimize and reduce the formation of a fibrotic sheath around an implanted tissue engineered construct would therefore be of great benefit in tissue engineering. . science. Most of these efforts converge into tissue engineering, an exciting field which offers a means of developing biological substitutes for maintaining, restoring and improving tissue function 1 useful for testing anti- angiogenic drug effects, but presents difficulties in showing augmentation of angiogenic effects. Although growth factor-depleted formulations of Matrigel are available to