OXYGENATION STATE AS A DRIVER OF MYOFIBROBLAST DIFFERENTIATION AND WOUND CONTRACTION: HYPOXIA IMPAIRS WOUND CLOSURE

Abstract

In the human body, myofibroblasts are ubiquitous and may be formed from the differentiation of fibroblasts and also from other cell types such as epithelial cells, endothelial cells, and mononuclear cells. The clinical significance of myofibroblast formation could be substantial depending on the biomedical context. Myofibroblasts help contraction of open skin wounds and is therefore beneficial. However, it could also be a key driver of fibrosis across a wide range of tissue systems in the human body as well as may support tumor invasiveness. Thus, understanding the molecular events underlying the formation of myofibroblasts is of extraordinary significance in the context of a wide range of human diseases. In this issue, Modarressi et al. address the significance of wound tissue hypoxia in impairing wound contraction by compromising the formation of myofibroblasts. The work represents an important contribution to the field of wound healing presenting compelling evidence to support that tissue hypoxia is in conflict with wound closure. We are again reminded that correction of wound tissue hypoxia is a critical factor that needs to be addressed while preparing the wound tissue to respond to other forms of therapeutic interventions.

In the human body, myofibroblasts are ubiquitous and may be formed from the differentiation of fibroblasts and also from other cell types such as epithelial cells, endothelial cells, and mononuclear cells. The clinical significance of myofibroblast formation could be substantial depending on the biomedical context. Myofibroblasts help contraction of open skin wounds and is therefore beneficial. However, it could also be a key driver of fibrosis across a wide range of tissue systems in the human body as well as may support tumor invasiveness. Thus, understanding the molecular events underlying the formation of myofibroblasts is of extraordinary significance in the context of a wide range of human diseases. In this issue, Modarressi et al. (Modarressi et al., 2010) address the significance of wound tissue hypoxia in impairing wound contraction by compromising the formation of myofibroblasts.

In 1977, Packer reported in Nature that human diploid fibroblasts grown at 10%O₂ have a longer life than cells grown at the routine 20%O₂ (Packer and Fuehr, 1977). Those were the days when the field of cellular senescence was in its infancy with the concept of “Hayflick limit” reported in 1961. In 2003, it was reported that growth arrest of fibroblasts caused by 20%O₂ was reversible (Roy et al., 2003a), consistent with the reports of the current study (Modarressi et al., 2010), and it was therefore concluded that exposure of cells to hyperoxic insult causes differentiation and not senescence. Although it is standard practice to culture cells at an ambient O₂ concentration of 20% (i.e. room air and balance of 5% CO₂) which corresponds to a pO₂ of approximately 140 mmHg at sea level, cells in the human body are exposed to much lower O₂ concentrations ranging from ~14% (100 mm Hg) in the pulmonary alveoli to 3–5% (35 mm Hg) in the heart and skin (Roy et al., 2003a; Sen et al., 2006). Thus, it is important to recognize that standard cell culture under conditions of 20%O₂ represents exposure of cells to hyperoxic insult, particularly for primary cells that have been freshly isolated from organs and were therefore adjusted to lower pO₂ as their physiological normoxic status (Roy et al., 2003a; Sen et al., 2006). For cell lines cultured at 20%O₂ over a long period of time, it is reasonable to assume that the overall cell population represents a hyperoxia-tolerant variety which has survived hyperoxic insult over time by accepting 20%O₂ as their normoxic state. While indeed 20%O₂ represents normoxia for these cells, it is important to appreciate that such state may have little to do with respect to resembling cells or cellular responses in vivo.

So, how do cells reset their normoxic set point? Cellular O₂ sensing enables physiological adjustments to variations in tissue pO₂. Under basal conditions, cells are adjusted to an O₂ environment biologically read as normoxia (Khanna et al., 2006). Any sharp departure from that state of normoxia triggers O₂-sensitive biological responses. The stabilization of hypoxia-inducible factor (HIF) signifies a robust biological read-out of hypoxia. In the presence of sufficient O₂, HIF is hydroxylated and degraded. HIF prolyl hydroxylation is catalyzed by prolyl hydroxylase (PHD) isoenzymes PHD1, 2 and 3. Using cells stably transfected with a HIF reporter construct, the hypothesis that biological cells are capable of resetting their normoxic set-point by O₂-sensitive changes in PHD expression has been tested (Khanna et al., 2006). Exposure of a cell line adjusted to growing in 20% O₂, to 5% O₂ resulted in HIF-driven transactivation. However, the same cells adjusted to growing in 5%O₂ did not report hypoxia as read by HIF transactivation. Of note, cells adjusted to growing in 30% O₂ reported hypoxia when acutely exposed to room air (20%O₂) culture conditions. When grown under elevated O₂ conditions, cells reset their normoxic set-point upwards by down-regulating the expression of PHDs. When grown under low O₂ conditions, cells reset their normoxic set-point downwards by inducing the expression of PHDs. Exposure of mice in vivo to a hypoxic 10% O₂ environment lowered blood as well as brain pO₂. Such hypoxic exposure induces PHDs. Exposure of mice to a hyperoxic 50% O₂ ambience repressed the expression of PHD1–3 indicating that O₂-sensitive regulation of PHD expression is effective in vivo as well. Studies employing knock-down of PHD expression reveal that O₂-sensitive regulation of PHD may contribute to tuning the normoxic set-point in biological cells (Khanna et al., 2006).

The state of tissue oxygenation serves as a major microenvironmental cue that is read by cells, integrated with other microenvironmental cues, as tissue decides to respond to any extracellular signal. While hypoxia has been extensively studied in the context of cell signaling, hyperoxia has been mostly studied in context of oxygen toxicity. Both in vitro as well as in vivo studies with fibroblasts have revealed that hyperoxia may serve as a potent inducer of differentiation to myofibroblast and it does so by turning on specific cell signaling events (Kuhn et al., 2007; Roy et al., 2010; Roy et al., 2003a; Roy et al., 2007; Roy et al., 2003b; Sen et al., 2006). In fibroblasts, hyperoxic insult causes growth arrest at G2/M phase. Such growth arrest is accompanied by induction of the expression of vimentin and α smooth muscle actin as well as increased contractility of cells in a collagen matrix (Roy et al., 2003a). Hyperoxia also enhances the stability of both Acta2 transcript as well as of α smooth muscle actin protein (Roy et al., 2007). The morophological/ cytoskeletal characteristics of fibroblasts observed in response to hyperoxic exposure match those of fibroblast cultured at normoxia but treated with TGFβ1, a classical inducer of fibroblast differentiation of fibroblast to myofibroblast. Interestingly, both hyperoxia as well as TGFβ signal through p38MAPK in causing the differentiation of fibroblasts (Roy et al., 2003a). Also, TGFβ activation may be caused by hyperoxia-induced oxidation of the latency associated peptide. Thus, hyperoxia may not only signal through the TGFβ pathway but may accentuate TGFβ signaling as well. Recent studies further support this contention by demonstrating that in fibroblasts all three isoforms of TGFβ are induced by hyperoxia (Roy et al., 2010). Deletion of any one or both of the activating protein-1 (AP-1) binding sites in the TGFβ reporter construct result in loss of O₂ sensitivity, demonstrating that AP-1 confers O₂ sensitivity to TGFβ transcription. Fos-related AP-1 transcription factor (Fra-2) and Ask-1 (apoptosis signal-regulating kinase-1) have been identified as key mediators of AP-1-dependent hyperoxia-sensitive TGFβ transcription. Knockdown of Fra-2 significantly blunted hyperoxia-induced expression of TGFβ1 as well as TGFβ3 in fibroblasts. Knockdown of Ask-1 blunted hyperoxia-induced Fra-2 gene expression and nuclear localization in fibroblasts. These observations point towards a central role of Ask-1 and Fra-2 in hyperoxia-inducible AP-1 activation and induction of TGFβ.

Transcriptome-wide profiling studies have identified hyperoxia-sensitive genes in fibroblasts and clustered them into functional groups (Roy et al., 2003b). The p21–p53 axis has emerged as a key hyperoxia-inducible pathway in fibroblast. Both p21 deficiency as well as knockdown blunts hyperoxia-induced Acta2 and smooth muscle actin expression. In vivo, reoxygenation-induced up-regulation of Acta2 is completely abrogated in p21-deficient mice. Strikingly, overexpression of p21 alone markedly induces differentiation of fibroblasts under normoxic basal conditions. Overexpression of p21 alone induced transcription of αsmooth muscle actin by down-regulating YB1 and independent of TGFβ1. Thus, studies aimed at understanding the significance of O₂ tension have discovered p21 as a key signaling mediator that regulates the differentiation of fibroblasts to myofibroblasts (Roy et al., 2010; Roy et al., 2003a).

The observations of Modarressi et al (Modarressi et al., 2010) establish key role of O₂-tension in driving wound contraction. A key contribution of this work is the demonstration that under hypoxic conditions compromised myofibroblast contraction is preceded by αsmooth muscle actin disassembly from stress fibers. Consistent with previous reports that the effects of changing O₂-tension on fibroblast differentiation are reversible (Roy et al., 2003a), Modarressi et al demonstrate that the negative effects of hypoxia on fibroblast differentiation may be corrected by restoration of normoxia. Interestingly, Modarressi et al (Modarressi et al., 2010) identify a facilitatory effect of mechanical stimulation in driving fibroblast differentiation. Such effect was most pronounced under conditions of high oxygenation. Such observation is relevant to negative pressure wound therapy which involves mechanical stimulation and during which improved tissue oxygenation is known to be followed by improved wound closure (Vikatmaa et al., 2008). The wound literature is often confused about the net impact of hypoxia on wound closure (Sen, 2009). Abundant literature, primarily tumor biology related, demonstrating that hypoxia is a cue for angiogenesis have misled many to conclude that hypoxia may be helpful for cutaneous wound healing. However, from a clinical standpoint we know that this is not true because ischemic wounds are clearly hypoxic yet refractory to closure. Acutely, hypoxia may help generate growth and repair factors necessary to lay the foundation for wound closure. However, unless there is enough supply to meet the oxygen cost of closure (Sen, 2009), a good foundation for healing would not deliver closure outcomes. HIF-dependent hypoxia-inducible microRNA miR210 impair wound epithelialization, a key aspect of overall wound closure (Biswas et al., 2010). Therefore, although stabilization of HIF may elicit angiogenic responses, it opposes wound closure by stalling wound re-epithelialization. The work by Modarressi et al. (Modarressi et al., 2010) represents an important contribution to the field of wound healing presenting compelling evidence to support that tissue hypoxia is in conflict with wound closure. We are again reminded that correction of wound tissue hypoxia is a critical factor that needs to be addressed while preparing the wound tissue to respond to other forms of therapeutic interventions.