All PhD Theses

N.A.J. Cremers

Cytoprotective mechanisms, palatogenesis, and wound repair


A scientific essay in Medical Sciences

DOCTORAL THESIS defended in public on 16th of March 2019


Chapter 1 introduces the topic of cleft lip and/ or palate (CL/P), its incidence and related problems, the process of palatogenesis, and the etiology and treatment strategies of CL/P. The close similarity between biological processes involved in palatogenesis and wound repair was discussed. We postulated that the balance between injurious and protective signaling determines wound repair, the level of scar formation, and the risk of congenital abnormalities, such as CL/P. Therefore, we split this thesis into three parts:

I). Decreased protective mechanisms hamper tissue repair and craniofacial development,

II). Activation of protective mechanisms to enhance tissue regeneration, and

III). General discussion and Summary

Part I consists of three studies investigating the effects of decreased protective mechanisms on wound repair and palatogenesis.

Chapter 2 evaluated the contribution of the cytoprotective enzyme heme oxygenase (HO) during excisional wound repair using HO-2 knockout (KO) and wild-type (WT) mice. HO-1 has previously shown to be very important during wound repair and embryonic development, and lack of HO-1 results in severe problems. The mild phenotype in HO-2 KO animals results in attenuated corneal wound repair, but more data investigating the role of HO-2 in other morbidities is scarce. Here, we showed that lack of HO-2 expression resulted in hampered wound repair, and reduced collagen deposition and vessel density when compared to WT mice. Although there were no differences between the two genotypes in inflammation, HO-1 expression, and proliferation and differentiation of myofibroblasts, CXCL11 expression was delayed in HO-2 KO mice. Abnormal CXCL11regulation has been linked to hampered wound repair and disturbed angiogenesis. This may help explain the observed hampered wound repair following HO-2 depletion.

In chapter 3, the disappearance of the midline epithelial seam (MES), a crucial process during palatal fusion and CL/P formation, was investigated. During palatogenesis several processes are similar to wound repair processes, including mesenchymal-epithelial cross-talk, chemokine signaling, and cell proliferation, differentiation and apoptosis. Therefore, we postulated that chemokine CXCL11, its receptor CXCR3, and HO, which all are important factors during wound repair, also play a decisive role during palatogenesis. The contribution of HO-2 in this process was studied using HO-2 KO and WT mice. HO-2 KO embryos at day 15-16 of intrauterine gestation suffered from fetal growth restriction and craniofacial abnormalities but had no problems with palatal fusion. In both WT and HO-2 KO mice, CXCR3-positive macrophages were recruited towards the CXCL11 expressing MES, and contained multiple apoptotic DNA fragments, supporting the hypothesis that the MES was disintegrated by epithelial apoptosis. Since macrophages located near the MES were HO-1 positive, and more HO-1 positive cells were present in HO-2 KO mice, HO-1 induction forms likely a compensatory mechanism to handle oxidative stressors. HO and CXCL11/CXCR3 signaling play thus also an important role in embryogenesis and palatal fusion. 

In chapter 4, since CL/P patients after cleft surgery experience mechanical stress during wound repair from myofibroblasts and the growing head, static mechanical stress was induced in a mouse model, to simulate the increased injurious environment and the effects of decreased protective pathways. Mechanical stress during wound repair was applied using a splinted excisional wound model. HO-1 is thought to orchestrate the defense against inflammatory and oxidative insults that drive fibrosis, and therefore we investigated the activation of the HO-system in splinted and non-splinted excisional wound models using HO-1 luc transgenic mice. These mice co-express luciferase when HO-1 gets induced, which can be measured in vivo by administration of the substrate luciferin which results in the release of photons. After seven days, splinting had delayed cutaneous wound closure and HO-1 protein expression, whereas the number of F4/80-positive macrophages, αSMApositive myofibroblasts, and pro-inflammatory signals IL-1β, TNF-α, and COX-2 were increased after application of mechanical stress. The pro-inflammatory environment following splinting may explain the higher myofibroblast numbers and increased risk of fibrosis and scar formation when compared to non-splinted wounds. 

Part II consists of three studies investigating the effects of activation of protective pathways using preconditioning strategies on mesenchymal stem cell (MSC) apoptosis, wound repair, and organs. In the latter study, we designed a novel “tissue survival theory” that could help explain how tissue gets first warned by tissue survival factors (TSFs) following injurious insults.

Chapter 5 investigated the effects of the cytoprotective HO-system on MSC survival following pharmacological preconditioning. MSC therapy is considered a promising strategy for a wide diversity of injuries and diseases. However, the survival of MSCs after administration is limited due to the hostile wound microenvironment displaying an accumulation of oxidative and inflammatory stressors. A prolonged MSC survival by preconditioning strategies could improve their therapeutic efficacy. In this study, the antioxidative and anti-apoptotic effects of HO-1, HO-2 and its effector molecules were investigated on adipose-derived MSC (ASC) survival. HO-1 was induced following curcumin treatment, and this provided potent protection against oxidative stress, which was mediated by hydrogen peroxide (H2O2). This protection was abrogated by simultaneous treatment with the selective HO-1 activity inhibitor QC15, supporting an HO-1-dependent protective mechanism. Also, exposure to anti-oxidant N-acetylcysteine protected against H2O2-induced ASC apoptosis, while the HO-effector molecules and anti-oxidants bilirubin and biliverdin did not have an effect. Interestingly, HO-effector molecule CO did also rescue H2O2-induced ASC death, suggesting that HO-1 provided protection against apoptosis via CO signaling. No differences were found between WT, and HO-2 KO derived ASCs in sensitivity towards H2O2, or curcumin and HO-effector molecules-mediated protection. Thus, induction of cytoprotective molecules by pharmacological preconditioning led to better protection against oxidative stress in an in vitro model of stem cell survival.

Next, in chapter 6, we analyzed the effects of remote ischemic preconditioning (RIPC) on cutaneous tissue injury. RIPC has shown that transient occlusion of the blood flow to a limb will induce a stress response that protects the body against a secondary more harmful stress, e.g., transplantation and organ injuries. We aimed to improve excisional wound repair using early and late RIPC (respectively 5 min and 24h before wounding) and investigated the role of HO-1 using HO-1 luc transgenic mice. HO-1 promotor activity was induced dorsally, and locally in the kidneys, following RIPC treatment. On mRNA level, HO-1 was increased in the ligated muscle, heart, and kidneys, but surprisingly not in the skin. Early and late RIPC did not change HO-1 mRNA and protein levels in the wounds seven days after cutaneous injury, nor did it accelerate cutaneous wound closure or did it affect collagen deposition. Thus, the used RIPC protocol induces HO-1 expression in several organs, but not the skin, and did not improve excisional wound repair, suggesting that the skin is insensitive to RIPC-mediated protection. However, other studies showed that RIPC improved transplanted skin flap survival and improved healing of diabetic ulcers. Possibly other signals play a decisive role.

Lastly, chapter 7 proposes a novel molecular mechanism that explains how the tissue is protected from injurious agents or collateral damage following the attack by an activated immune system. TSFs mediate tissue protection while danger signals alert the immune system. Hemopexin (hpx) can scavenge heme, and form the heme-hpx complex that can bind to LRP-1 having a protective effect. When hpx levels are exhausted or depleted, the excess of free heme can bind to TLR-4 and alarm the immune system resulting in a pro-inflammatory effect. We proposed that the presence of hpx is the discriminating factor between tissue repair and tissue damage, and provided a new concept of how RIPC can induce protective HO-1 signaling. RIPC led to the release of low levels of heme, and the induction of HO-1, which was co-expressed with LRP-1. We propose that systemic protection mediated by RIPC is orchestrated via binding of the heme-hpx complex, a tissue survival factor (TSF), to LRP-1, subsequently activating protective responses, including HO-1. Therefore, the binding of heme by hpx may regulate tissue protection or immune activation.

In Part III, the most important results of this thesis and their clinical relevance were summarized and discussed, and future perspectives for therapeutical approaches were proposed. The induction of cytoprotective pathways, such as HO-1, by pharmacological therapy or by application of RIPC or administration of hpx, may harness inflammatory and oxidative insults. Therefore, these strategies may prevent CL/P formation and improve the outcome of tissue repair in patients with CL/P or burn wounds. However, more mechanistic and translational research is necessary before it can be used in the clinic.

In conclusion, decreased activity of protective pathways can hamper wound repair and craniofacial development, whereas induction of protective signaling cascades, with a focus on pathways that mediate the resolution of oxidative and inflammatory stress, may provide a regenerative microenvironment that improves wound repair and regeneration.