Dynamic cell culture on porous biopolymer microcarriers in a spinner flask for bone tissue engineering: a feasibility study

Porous microspherical carriers have great promise for cell culture and tissue engineering. Dynamic cultures enable more uniform cell population and effective differentiation than static cultures. Here we applied dynamic spinner flask culture for the loading and multiplication of cells onto porous biopolymer microcarriers. The abilities of the microcarriers to populate cells and to induce osteogenic differentiation were examined and the feasibility of in vivo delivery of the constructs was addressed. Over time, the porous microcarriers enabled cell adhesion and expansion under proper dynamic culture conditions. Osteogenic markers were substantially expressed by the dynamic cell cultures. The cell-cultured microcarriers implanted in the mouse subcutaneous tissue for 4 weeks showed excellent tissue compatibility, with minimal inflammatory signs and significant induction of bone tissues. This first report on dynamic culture of porous biopolymer microcarriers providing an effective tool for bone tissue engineering.     

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.     

Effects of oxygen transport on 3-d human mesenchymal stem cell metabolic activity in perfusion and static cultures: experiments and mathematical model

Human mesenchymal stem cells (hMSCs) have unique potential to develop into functional tissue constructs to replace a wide range of tissues damaged by disease or injury. While recent studies have highlighted the necessity for 3-D culture systems to facilitate the proper biological, physiological, and developmental processes of the cells, the effects of the physiological environment on the intrinsic tissue development characteristics in the 3-D scaffolds have not been fully investigated. In this study, experimental results from a 3-D perfusion bioreactor system and the static culture are combined with a mathematical model to assess the effects of oxygen transport on hMSC metabolism and proliferation in 3-D constructs grown in static and perfusion conditions. Cells grown in the perfusion culture had order of magnitude higher metabolic rates, and the perfusion culture supports higher cell density at the end of cultivation. The specific oxygen consumption rate for the constructs in the perfusion bioreactor was found to decrease from 0.012 to 0.0017 micromol/10(6) cells/h as cell density increases, suggesting intrinsic physiological change at high cell density. BrdU staining revealed the noneven spatial distribution of the proliferating cells in the constructs grown under static culture conditions compared to the cells that were grown in the perfusion system. The hypothesis that the constructs in static culture grow under oxygen limitation is supported by higher Y(L/G) in static culture. Modeling results show that the oxygen tension in the static culture is lower than that of the perfusion unit, where the cell density was 4 times higher. The experimental and modeling results show the dependence of cell metabolism and spatial growth patterns on the culture environment and highlight the need to optimize the culture parameters in hMSC tissue engineering.     

Organizational behavior of human umbilical vein endothelial cells

Culture conditions that favor rapid multiplication of human umbilical vein endothelial cells (HUV-EC) also support long-term serial propagation of the cells. This is routinely achieved when HUV-EC are grown in Medium 199 (M-199) supplemented with fetal bovine serum (FBS) and endothelial cell growth factor (ECGF), on a human fibronectin (HFN) matrix. The HUV-EC can shift from a proliferative to an organized state when the in vitro conditions are changed from those favoring low density proliferation to those supporting high density survival. When ECGF and HFN are omitted, cultures fail to achieve confluence beyond the first or second passage: the preconfluent cultures organize into tubular structures after 4-6 wk. Some tubes become grossly visible and float in the culture medium, remaining tethered to the plastic dish at either end of the tube. On an ultrastructural level, the tubes consist of cells, held together by junctional complexes, arranged so as to form a lumen. The smallest lumens are formed by one cell folding over to form a junction with itself. The cells contain Weibel-Palade bodies and factor VIII-related antigen. The lumens contain granular, fibrillar and amorphous debris. Predigesting the HFN matrix with trypsin (10 min, 37 degrees C) or plasmin significantly accelerates tube formation. Thrombin and plasminogen activator had no apparent effect. Disruption of the largest tubes with trypsin/EDTA permits the cells to revert to a proliferative state if plated on HFN, in M-199, FBS, and ECGF. These observations indicate that culture conditions that do not favor proliferation permit attainment of a state of nonterminal differentiation (organization) by the endothelial cell. Furthermore, proteolytic modification of the HFN matrix may play an important role in endothelial organization.     

Dynamic culture of droplet‐confined cell arrays

Responding to the need of creating an accurate and controlled microenvironment surrounding the cell while meeting the requirements for biological processes or pharmacological screening tests, we aimed at designing and developing a microscaled culture system suitable for analyzing the synergic effects of extracellular matrix proteins and soluble environments on cell phenotype in a high‐throughput fashion. We produced cell arrays deposing micrometer‐scale protein islands on hydrogels using a robotic DNA microarrayer, constrained the culture media in a droplet‐like volume and developed a suitable perfusion system. The droplet‐confined cell arrays were used either with conventional culture methods (batch operating system) or with automated stable and constant perfusion (steady‐state operating system). Mathematical modeling assisted the experimental design and assessed efficient mass transport and proper fluidodynamic regimes. Cells cultured on arrayed islands (500 μm diameter) maintained the correct phenotype both after static and perfused conditions, confirmed by immunostaining and gene expression analyses through total RNA extraction. The mathematical model, validated using a particle tracking experiment, predicted the constant value of velocities over the cell arrays (less than 10% variation) ensuring the same mass transport regime. BrdU analysis on an average of 96 cell spots for each experimental condition showed uniform expression inside each cell island and low variability in the data (average of 13%). Perfused arrays showed longer doubling times when compared with static cultures. In addition, perfused cultures showed a reduced variability in the collected data, allowing to detect statistically significant differences in cell behavior depending on the spotted ECM protein. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Vascular smooth muscle cell growth-promoting factor/F-spondin inhibits angiogenesis via the blockade of integrin alphavbeta3 on vascular endothelial cells

Vascular smooth muscle cell growth-promoting factor (VSGP) was originally isolated from bovine ovarian follicular fluid as a stimulator of vascular smooth muscle cell proliferation. Homology searches indicate that bovine and human VSGPs are orthologs of rat F-spondin. Here, we examined whether recombinant human VSGP/F-spondin affected the biological activities of endothelial cells. VSGP/F-spondin did not affect the proliferation of human umbilical vein endothelial cells (HUVECs); however, it did inhibit VEGF- or bFGF-stimulated HUVEC migration. To clarify the mechanism of this inhibitory effect, we examined the adhesion of HUVECs to extracellular matrix proteins. VSGP/F-spondin specifically inhibited the spreading of HUVECs on vitronectin via the functional blockade of integrin alphavbeta3. As a result, VSGP/F-spondin inhibited the tyrosine phosphorylation of focal adhesion kinase (FAK) when HUVECs were plated on vitronectin. Moreover, VSGP/F-spondin inhibited the activation of Akt when HUVECs on vitronectin were stimulated with VEGF. VSGP/F-spondin inhibited tube formation by HUVECs in vitro and neovascularization in the rat cornea in vivo. These results indicate that VSGP/F-spondin inhibits angiogenesis at least in part by the blockade of endothelial integrin alphavbeta3.     

Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases

Advances in microfabrication techniques have enabled the production of inexpensive and reproducible microfluidic systems for conducting biological and biochemical experiments at the micro- and nanoscales ^1,2. In addition, microfluidics have also been specifically used to quantitatively analyze hematologic and microvascular processes, because of their ability to easily control the dynamic fluidic environment and biological conditions^3-6. As such, researchers have more recently used microfluidic systems to study blood cell deformability, blood cell aggregation, microvascular blood flow, and blood cell-endothelial cell interactions^6-13.However, these microfluidic systems either did not include cultured endothelial cells or were larger than the sizescale relevant to microvascular pathologic processes. A microfluidic platform with cultured endothelial cells that accurately recapitulates the cellular, physical, and hemodynamic environment of the microcirculation is needed to further our understanding of the underlying biophysical pathophysiology of hematologic diseases that involve the microvasculature.Here, we report a method to create an "endothelialized" in vitro model of the microvasculature, using a simple, single mask microfabrication process in conjunction with standard endothelial cell culture techniques, to study pathologic biophysical microvascular interactions that occur in hematologic disease. This "microvasculature-on-a-chip" provides the researcher with a robust assay that tightly controls biological as well as biophysical conditions and is operated using a standard syringe pump and brightfield/fluorescence microscopy. Parameters such as microcirculatory hemodynamic conditions, endothelial cell type, blood cell type(s) and concentration(s), drug/inhibitory concentration etc., can all be easily controlled. As such, our microsystem provides a method to quantitatively investigate disease processes in which microvascular flow is impaired due to alterations in cell adhesion, aggregation, and deformability, a capability unavailable with existing assays.     

Nanoliter scale microbioreactor array for quantitative cell biology

A nanoliter scale microbioreactor array was designed for multiplexed quantitative cell biology. An addressable 8 x 8 array of three nanoliter chambers was demonstrated for observing the serum response of HeLa human cancer cells in 64 parallel cultures. The individual culture unit was designed with a "C" shaped ring that effectively decoupled the central cell growth regions from the outer fluid transport channels. The chamber layout mimics physiological tissue conditions by implementing an outer channel for convective "blood" flow that feeds cells through diffusion into the low shear "interstitial" space. The 2 microm opening at the base of the "C" ring established a differential fluidic resistance up to 3 orders of magnitude greater than the fluid transport channel within a single mold microfluidic device. Three-dimensional (3D) finite element simulation were used to predict fluid transport properties based on chamber dimensions and verified experimentally. The microbioreactor array provided a continuous flow culture environment with a Peclet number (0.02) and shear stress (0.01 Pa) that approximated in vivo tissue conditions without limiting mass transport (10 s nutrient turnover). This microfluidic design overcomes the major problems encountered in multiplexing nanoliter culture environments by enabling uniform cell loading, eliminating shear, and pressure stresses on cultured cells, providing stable control of fluidic addressing, and permitting continuous on-chip optical monitoring.     

预血管化细胞膜片的构建

为探索新的体外获得毛细血管样网络结构来解决工程化组织预血管化的问题,该研究将人骨髓间充质干细胞（humanmesenchymalstemcells,hMSCs）以9×10^4／cm。细胞密度体外连续培养形成细胞膜片,将培养的脐静脉血内皮细胞（humanumbilicalveinendothelialcells,HUVECs）v25x104／cm2细胞密度接种到上述间充质干细胞膜片上,并培养在内皮细胞培养介质中。在设计的时间点用倒置相差显微镜观察,发现内皮细胞在膜片上迁移,细胞重排,膜片上的基质蛋白也发生重排．导致微槽和空泡出现。CD31免疫荧光染色观察到进行性管腔形成的过程;CD90免疫荧光染色显示膜片上的hMSCs围绕着HUVECs周边排列,说明hMSCs作为周细胞支持了HUVECs的生长;在培养第10d可见少量的α-SMA的表达,暗示着在此种培养模式下,hMSCs具有较低的向肌细胞分化的潜能。这些结果表明,将内皮细胞接种在未分化干细胞膜片上,可以在体外形成具有血管网络结构的预血管化膜片,为构建血管化工程化组织提供了新的思路。     

A multichamber fluidic device for 3D cultures under interstitial flow with live imaging: Development,characterization, and applications

Interstitial flow is an important biophysical cue that can affect capillary morphogenesis, tumor cell migration, and fibroblast remodeling of the extracellular matrix, among others. Current models that incorporate interstitial flow and that are suitable for live imaging lack the ability to perform multiple simultaneous experiments, for example, to compare effects of growth factors, extracellular matrix composition, etc. We present a nine‐chamber radial flow device that allows simultaneous 3D fluidic experiments for relatively long‐term culture with live imaging capabilities. Flow velocity profiles were characterized by fluorescence recovery after photobleaching (FRAP) for flow uniformity and estimating the hydraulic conductivity. We demonstrate lymphatic and blood capillary morphogenesis in fibrin gels over 10 days, comparing flow with static conditions as well as the effects of an engineered variant of VEGF that binds fibrin via Factor XIII. We also demonstrate the culture of contractile fibroblasts and co‐cultures with tumor cells for modeling the tumor microenvironment. Therefore, this device is useful for studies of capillary morphogenesis, cell migration, contractile cells like fibroblasts, and multicellular cultures, all under interstitial flow. Biotechnol. Bioeng. 2010;105: 982–991. © 2009 Wiley Periodicals, Inc.     

uPA and MMP‐2 were involved in self‐assembled network formation in a two dimensional co‐culture model of bone marrow stromal cells and endothelial cells

Two dimensional (2D) co‐cultures of human bone marrow stromal cells (HBMSCs) and human umbilical vein endothelial cells (HUVECs) stimulate osteoblastic differentiation of HBMSCs, induce the formation of self‐assembled network and cell interactions between the two cell types involving many vascular molecules. Because of their strong activities on angiogenesis and tissue remodeling, urokinase plasminogen activator (uPA), plasminogen activator inhibitor‐1 (PAI‐1), matrix metalloproteinase‐2 (MMP‐2) as well tissue inhibitors of matrix metalloproteinase‐2 (TIMP‐2) were investigated in this 2D co‐culture model. We found that the expression of uPA, MMP‐2 in the co‐cultured cells was significantly higher than those in mono‐cultured cells. In opposite, PAI‐1, expressed only by HUVECs is not regulated in the co‐culture. Inhibition assays confirm that uPA played a critical role in the formation of self‐assembled network as neutralization of uPA disturbed this network. In the same context, inhibition of MMP‐2 prevented the formation of self‐assembled network, while the inhibition of uPA abolished the over expression and the activity of MMP‐2. This upregulation could initiate the uPA expression and proteolysis processes through the MMP‐2 activity, and may contribute to endothelial cell migration and the formation of this self‐assembled network observed in these 2D co‐cultured cells. J. Cell. Biochem. 114: 650–657, 2013. © 2012 Wiley Periodicals, Inc.     

A bioreactor test system to mimic the biological and mechanical environment of oral soft tissues and to evaluate substitutes for connective tissue grafts

Gingival cells of the oral connective tissue are exposed to complex mechanical forces during mastication, speech, tooth movement and orthodontic treatments. Especially during wound healing following surgical procedures, internal and external forces may occur, creating pressure upon the newly formed tissue. This clinical situation has to be considered when developing biomaterials to augment soft tissue in the oral cavity. In order to pre‐evaluate a collagen sponge intended to serve as a substitute for autogenous connective tissue grafts (CTGs), a dynamic bioreactor system was developed. Pressure and shear forces can be applied in this bioreactor in addition to a constant medium perfusion to cell‐material constructs. Three‐dimensional volume changes and stiffness of the matrices were analyzed. In addition, cell responses such as cell vitality and extracellular matrix (ECM) production were investigated. The number of metabolic active cells constantly increased under fully dynamic culture conditions. The sponges remained elastic even after mechanical forces were applied for 14 days. Analysis of collagen type I and fibronectin revealed a statistically significant accumulation of these ECM molecules (P < 0.05–0.001) when compared to static cultures. An increased expression of tenascin‐c, indicating tissue remodeling processes, was observed under dynamic conditions only. The results indicate that the tested in vitro cell culture system was able to mimic both the biological and mechanical environments of the clinical situation in a healing wound. Biotechnol. Bioeng. 2010;107: 1029–1039. © 2010 Wiley Periodicals, Inc.     

Inhibition of Endothelial Cell Differentiation on a Glycosylated Reconstituted Basement Membrane Complex

The vascular basement membrane is involved in the regulation of endothelial cell differentiation. The accumulation of advanced glycosylation endproducts (AGEs) has been demonstrated on these basement membranes in patients with diabetes. We examined the effect of AGEs on endothelial cell behavior on reconstituted basement membrane, Matrigel. Human umbilical vein-derived endothelial cells (HUVECs) stopped proliferating and differentiated into capillary-like tube-shaped structures on Matrigel. Laminin antibody partially blocked this process. HUVECs cultured on glycosylated Matrigel, however, proliferated and formed a monolayer without tube formation. The inclusion of aminoguanidine, an inhibitor of AGE formation, during the glycosylation of Matrigel restored HUVEC differentiation. Although the laminin adsorbed onto the plastic culture wells promoted HUVEC attachment and spreading, glycosylated laminin reduced HUVEC attachment by 50% and abolished cellular spreading. These effects were restored by aminoguanidine. HUVEC attachment to glycosylated laminin was further reduced by AGE-modified albumin, poly I, acetylated low-density lipoprotein, or maleylated albumin, ligands for a scavenger receptor. Coating the culture dishes with the laminin peptides RGD, YIGSR, and SIKVAV supported the attachment of HUVECs that was unaffected by glycosylation. Results suggest that AGE accumulation on the basement membranes inhibits endothelial cell differentiation by impairing the normal interactions of endothelial cell receptors with their specific matrix ligands. This process may be involved in diabetic angiopathy.     

Flow-regulated glucose and lipid metabolism in adipose tissue,endothelial cell and hepatocyte cultures in a modular bioreactor

Static cell culture has serious limitations in its ability to represent cellular behaviour within a live organism. In vivo, cells are constantly exposed to the flow of bodily fluids and contact with other cell types. Bioreactors provide the opportunity to study cells in an environment that more closely resembles the in vivo setting because cell cultures can be exposed to dynamic flow in contact with or in proximity to other cell types. In this study we compared the metabolic profile of a dynamic cell culture system to that of a static cell culture in three different cellular phenotypes: adipocytes, endothelial cells and hepatocytes. Albumin, glucose, free fatty acids, glycerol, and lactate were measured over 48 h. We show that all three cell types have increased glucose uptake in the presence of flow; lactate release was also significantly affected. We provide robust evidence that the presence of flow significantly modifies cellular metabolism. While flow provides a more uniform nutrient distribution and increases metabolite turnover, our results indicate that different cell types have specific metabolic responses to flow, suggesting cell-specific flow-regulated activation of metabolite signalling pathways.     

Immunoelectron microscopic localization of type 1 plasminogen activator inhibitor on the surface of activated endothelial cells

Immunogold EM was employed to compare the distribution of type 1 plasminogen activator inhibitor (PAI-1) on the surface of agonist-activated human umbilical vein endothelial cells (HUVECs) with that of control, unactivated cells. As previously observed, (Schleef, R.R., T.J. Podor, E. Dunne, J. Mimuro, and D.J. Loskutoff. J. Cell Biol. 110:155-163), analysis of cross-sections of nonpermeabilized control HUVEC monolayers stained first with affinity-purified rabbit antibodies to PAI-1 and then with gold-conjugated goat anti-rabbit IgG, revealed the presence of relatively few gold particles (less than 1-2% of the total) on the apical cell surface. The majority of gold particles were detected primarily in the extracellular matrix between the culture substratum and the cell membrane. In contrast, treatment of HUVECs with tumor necrosis factor alpha (TNF alpha; 200 U/ml, 24 h) or with lipopolysaccharide (LPS; 10 micrograms/ml, 24 h) resulted in an increased staining of PAI-1 not only in the extracellular matrix, but also on the apical cell surface (10-fold increase). Immunoabsorption of the rabbit anti-PAI-1 with purified PAI-1, or treatment of HUVECs with tissue-type plasminogen activator (2.5 micrograms/ml, 2 h, 4 degrees C) reduced the amount of staining both on the apical surface and in the extracellular matrix of agonist-activated HUVECs by 80-95%. The topographical location of PAI-1 on the cell surface was examined further by coupling immunogold staining with high resolution surface replication. Transmission EM of surface replicas from TNF alpha- or LPS-activated HUVECs revealed a general increase in PAI-1 staining both on planar regions and within indentations of the apical cell surface. Nonactivated HUVECs revealed PAI-1-specific immunogold particles only in areas of exposed extracellular matrix between the cells and occasionally at regions of cell-cell contacts. Analysis of activated bovine aortic endothelial cells by immuno-electron microscopy, immunologic assays, and flow cytometry revealed similar increases in surface PAI-1. These increases in surface PAI-1 could be detected by 3 h and continued over a 24-h period. The expression of PAI-1 on the luminal surface of endothelial cells during immune or inflammatory reactions could reduce endothelial fibrinolytic activity, thus, promoting the localized, pathologic formation of intravascular thrombi.     

Gp38k, a protein synthesized by vascular smooth muscle cells, stimulates directional migration of human umbilical vein endothelial cells.

Gp38k is a 383-amino-acid secreted glycoprotein expressed by cultured vascular smooth muscle cells during the time of transition from a proliferating monolayer culture to a nonproliferating multilayered (differentiated) culture. Expression continues as the cell culture forms multicellular nodules. Because this transition period involves active cell migration, we evaluated the effects of exogenously added gp38k on vascular endothelial cell (HUVEC) migration and chemotaxis. Here we demonstrate that gp38k acts as a chemoattractant for HUVECs and stimulates cell migration in Boyden chambers at a level comparable to that achieved with the known endothelial cell chemoattractant bFGF. The migration effect is neutralized by the presence of a polyclonal anti-gp38k antibody. Because gp38k expression is also correlated with changes in culture morphology, we also assessed its ability to act as an agonist of HUVEC morphology using cultures growing on Matrigel. We report that gp38k stimulates endothelial cell tubulogenesis in this assay system. These results provide the first evidence that gp38k may function in angiogenesis by stimulating the migration and reorganization of vascular endothelial cells.     

Mathematical modeling of cell growth in a 3D scaffold and validation of static and dynamic cultures

Tissue engineering, an immensely important field in contemporary clinical practices, aims at the repair or replacement of damaged tissues. The mathematical model proposed herein shows the distribution and growth of cells in their characteristic time in a 3D scaffold model. This study contributes to the progress of simulation techniques in static and dynamic cultures of bone tissue. Brinkman, nutrient transport, and cell growth equations are brought together to quantify the growth behavior of cells. However, when a static culture is being studied, the Brinkman equation is eliminated. The model was validated by experimental cell culture using 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay and scanning electron microscopy. Then, static and dynamic cultures were compared to assess the cell density and cell distribution in the scaffold. Cell counting after 21 days of cell culture showed that the number of cells increased 42‐fold in static and 53.5‐fold in dynamic cultures, which was in good agreement with our model estimations (37‐fold increase in the number of cells in static and 49‐fold increase in dynamic cultures). In conclusion, our mathematical model could predict cell distribution and growth in the scaffold.     

Repression of heme oxygenase-1 by hypoxia in vascular endothelial cells

Heme oxygenase 1 (HO-1), a rate-limiting enzyme in heme catabolism, has been reported to be induced by hypoxia. Unexpectedly, here we show that expression of HO-1 mRNA is repressed by hypoxia in primary cultures of human umbilical vein endothelial cells (HUVECs), but is increased by cobalt chloride (CoCl(2)) that is known to mimic hypoxia. Under the culture conditions used, the DNA-binding and transactivation activities of hypoxia-inducible factor 1 were increased in HUVECs by hypoxia or CoCl(2). Therefore, hypoxia and cobalt showed opposing effects on HO-1 mRNA expression, despite activation of hypoxia-inducible factor 1. The half-life of HO-1 mRNA was not changed by hypoxia, but was significantly prolonged by CoCl(2). Hypoxia also represses HO-1 mRNA expression in human coronary artery endothelial cells and astrocytes. The repression of HO-1 expression may represent the adaptation to hypoxia in certain cell types.