The role of hair follicle nestin‐expressing stem cells during whisker sensory‐nerve growth in long‐term 3D culture

We have previously reported that nestin‐expressing hair follicle stem cells can differentiate into neurons, Schwann cells, and other cell types. In the present study, vibrissa hair follicles, including their sensory nerve stump, were excised from transgenic mice in which the nestin promoter drives green fluorescent protein (ND‐GFP mice), and were placed in 3D histoculture supported by Gelfoam®. β‐III tubulin‐positive fibers, consisting of ND‐GFP‐expressing cells, extended up to 500 µm from the whisker nerve stump in histoculture. The growing fibers had growth cones on their tips expressing F‐actin. These findings indicate that β‐III tubulin‐positive fibers elongating from the whisker follicle sensory nerve stump were growing axons. The growing whisker sensory nerve was highly enriched in ND‐GFP cells which appeared to play a major role in its elongation and interaction with other nerves in 3D culture, including the sciatic nerve, the trigeminal nerve, and the trigeminal nerve ganglion. The results of the present report suggest a major function of the nestin‐expressing stem cells in the hair follicle is for growth of the follicle sensory nerve. J. Cell. Biochem. 114: 1674–1684, 2013. © 2013 Wiley Periodicals, Inc.     

A transgenic red fluorescent protein‐expressing nude mouse for color‐coded imaging of the tumor microenvironment

The tumor microenvironment (TME) is critical for tumor growth and progression. We have previously developed color‐coded imaging of the TME using a green fluorescent protein (GFP) transgenic nude mouse as a host. However, most donor sources of cell types appropriate for study in the TME are from mice expressing GFP. Therefore, a nude mouse expressing red fluorescent protein (RFP) would be an appropriate host for transplantation of GFP‐expressing stromal cells as well as double‐labeled cancer cells expressing GFP in the nucleus and RFP in the cytoplasm, thereby creating a three‐color imaging model of the TME. The RFP nude mouse was obtained by crossing non‐transgenic nude mice with the transgenic C57/B6 mouse in which the β‐actin promoter drives RFP (DsRed2) expression in essentially all tissues. In crosses between nu/nu RFP male mice and nu/+ RFP female mice, the embryos fluoresced red. Approximately 50% of the offspring of these mice were RFP nude mice. In the RFP nude mouse, the organs all brightly expressed RFP, including the heart, lungs, spleen, pancreas, esophagus, stomach, duodenum, the male and female reproductive systems; brain and spinal cord; and the circulatory system, including the heart, and major arteries and veins. The skinned skeleton highly expressed RFP. The bone marrow and spleen cells were also RFP positive. GFP‐expressing human cancer cell lines, including HCT‐116‐GFP colon cancer and MDA‐MB‐435‐GFP breast cancer were orthotopically transplanted to the transgenic RFP nude mice. These human tumors grew extensively in the transgenic RFP nude mouse. Dual‐color fluorescence imaging enabled visualization of human tumor–host interaction. The RFP nude mouse model should greatly expand our knowledge of the TME. J. Cell. Biochem. 106: 279–284, 2009. © 2008 Wiley‐Liss, Inc.     

Multi-color palette of fluorescent proteins for imaging the tumor microenvironment of orthotopic tumorgraft mouse models of clinical pancreatic cancer specimens

Pancreatic-cancer-patient tumor specimens were initially established subcutaneously in NOD/SCID mice immediately after surgery. The patient tumors were then harvested from NOD/SCID mice and passaged orthotopically in transgenic nude mice ubiquitously expressing red fluorescent protein (RFP). The primary patient tumors acquired RFP-expressing stroma. The RFP-expressing stroma included cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs). Further passage to transgenic nude mice ubiquitously expressing green fluorescent protein (GFP) resulted in tumors that acquired GFP stroma in addition to their RFP stroma, including CAFs and TAMs as well as blood vessels. The RFP stroma persisted in the tumors growing in the GFP mice. Further passage to transgenic nude mice ubiquitously expressing cyan fluorescent protein (CFP) resulted in tumors acquiring CFP stroma in addition to persisting RFP and GFP stroma, including RFP- and GFP-expressing CAFs, TAMs and blood vessels. This model can be used to image progression of patient pancreatic tumors and to visually target stroma as well as cancer cells and to individualize patient therapy.     

Fibroblasts/myofibroblasts that participate in cutaneous wound healing are not derived from circulating progenitor cells

Dermal fibroblasts/myofibroblasts involved in the wound healing are thought to originate from the resident fibroblast progenitors. To test the hypothesis of an extra dermal origin of the dermal fibroblasts/myofibroblasts, bone marrow (BM) transplantation and parabiosis experiments were initiated utilizing a collagen promoter green fluorescent protein (GFP) reporter transgene as a visible marker for dermal fibroblasts/myofibroblasts. BM transplantation experiments using BM from Col3.6GFPsapph transgenic mice showed no evidence that BM derived progenitors differentiated into dermal fibroblasts/myofibroblasts at the wound site. Rather the GFP positive cells (GFP+) observed at the wound site were not dermal fibroblasts/myofibroblasts but immune cells. These GFP+ cells were also detected in the lung and spleen. Furthermore, GFP+ fibroblasts were not detected in primary dermal fibroblast cultures initiated from BM chimeras. Using the same transgenic mice, parabiotic pairs were generated. One partner in the parabiosis carried a GFP expressing transgene while the other partner was a non‐transgenic C57BL/6 mouse. Similar to the BM transplantation experiments, GFP+ immune cells were detected in the wound of the non‐transgenic parabiont, however, GFP expressing dermal fibroblasts/myofibroblasts were not observed. Collectively, these data suggest that dermal fibroblast/myofibroblast progenitors do not readily circulate. The expression of the Col3.6GFPsapph in the hematopoietic cells confirmed that our methods were sensitive enough to detect Col3.6GFP expressing dermal fibroblasts derived from the peripheral circulation if they had originated in the BM. J. Cell. Physiol. 222: 703–712, 2010. © 2009 Wiley‐Liss, Inc.     

Multipotent hair follicle stem cells promote repair of spinal cord injury and recovery of walking function

The mouse hair follicle is an easily accessible source of actively growing, pluripotent adult stem cells. C57BL transgenic mice, labeled with the fluorescent protein GFP, afforded follicle stem cells whose fate could be followed when transferred to recipient animals. These cells appear to be relatively undifferentiated since they are positive for the stem cell markers nestin and CD34 but negative for the keratinocyte marker keratin 15. These hair follicle stem cells can differentiate into neurons, glia, keratinocytes, smooth muscle cells, and melanocytes in vitro. Implanting hair follicle stem cells into the gap region of severed sciatic or tibial nerves greatly enhanced the rate of nerve regeneration and restoration of nerve function. The transplanted follicle cells transdifferentiated mostly into Schwann cells, which are known to support neuron regrowth. The treated mice regained the ability to walk essentially normally. In the present study, we severed the thoracic spinal chord of C57BL/6 immunocompetent mice and transplanted GFP-expressing hair follicle stem cells to the injury site. Most of the transplanted cells also differentiated into Schwann cells that apparently facilitated repair of the severed spinal cord. The rejoined spinal cord reestablished extensive hind-limb locomotor performance. These results suggest that hair follicle stem cells can promote the recovery of spinal cord injury. Thus, hair follicle stem cells provide an effective accessible, autologous source of stem cells for the promising treatment of peripheral nerve and spinal cord injury.     

Generation of Two-color Transgenic Zebrafish Using the Green and Red Fluorescent Protein Reporter Genes gfp and rfp

Two tissue-specific promoters were used to express both green fluorescent protein (GFP) and red fluorescent protein (RFP) in transgenic zebrafish embryos. One promoter (CK), derived from a cytokeratin gene, is active specifically in skin epithelia in embryos, and the other promoter (MLC) from a muscle-specific gene encodes a myosin light chain 2 polypeptide. When the 2 promoters drove the 2 reporter genes to express in the same embryos, both genes were faithfully expressed in the respective tissues, skin or muscle. When the 2 fluorescent proteins were expressed in the same skin or muscle cells under the same promoter, GFP fluorescence appeared earlier than RFP fluorescence in both skin and muscle tissues, probably owing to a higher detection sensitivity of GFP. However, RFP appeared to be more stable as its fluorescence steadily increased during development. Finally, F₁ transgenic offspring were obtained expressing GFP in skin cells under the CK promoter and RFP in muscle cells under the MLC promoter. Our study demonstrates the feasibility of monitoring expression of multiple genes in different tissues in the same transgenic organism.     

Dynamic multiphoton imaging of acellular dermal matrix scaffolds seeded with mesenchymal stem cells in diabetic wound healing

Significantly effective therapies need to be developed for chronic nonhealing diabetic wounds. In this work, the topical transplantation of mesenchymal stem cell (MSC) seeded on an acellular dermal matrix (ADM) scaffold is proposed as a novel therapeutic strategy for diabetic cutaneous wound healing. GFP‐labeled MSCs were cocultured with an ADM scaffold that was decellularized from normal mouse skin. These cultures were subsequently transplanted as a whole into the full‐thickness cutaneous wound site in streptozotocin‐induced diabetic mice. Wounds treated with MSC‐ADM demonstrated an increased percentage of wound closure. The treatment of MSC‐ADM also greatly increased angiogenesis and rapidly completed the reepithelialization of newly formed skin on diabetic mice. More importantly, multiphoton microscopy was used for the intravital and dynamic monitoring of collagen type I (Col‐I) fibers synthesis via second harmonic generation imaging. The synthesis of Col‐I fibers during diabetic wound healing is of great significance for revealing wound repair mechanisms. In addition, the activity of GFP‐labeled MSCs during wound healing was simultaneously traced via two‐photon excitation fluorescence imaging. Our research offers a novel advanced nonlinear optical imaging method for monitoring the diabetic wound healing process while the ADM and MSCs interact in situ. Schematic of dynamic imaging of ADM scaffolds seeded with mesenchymal stem cells in diabetic wound healing using multiphoton microscopy. PMT, photo‐multiplier tube.      

Vessel destruction by tumor-targeting Salmonella typhimurium A1-R is enhanced by high tumor vascularity

Our laboratory has previously developed a tumor-targeting double-auxotrophic mutant of Salmonella typhimurium termed A1-R. The present report demonstrates that S. typhimurium A1-R destroys tumor blood vessels and this is enhanced in tumors with high vascularity. Red fluorescent protein (RFP)-expressing Lewis lung cancer cells (LLC-RFP) were transplanted subcutaneously in the ear, back skin and footpad of nestin-driven green fluorescent protein (ND-GFP) transgenic nude mice, which selectively express GFP in nascent blood vessels. Color-coded in vivo imaging demonstrated that the LLC-RFP ear tumor had the highest cell density and the footpad tumor had the least. The ear tumor had more abundant blood vessels than that on the back or footpad. The tumor-bearing mice were treated with A1-R bacteria via tail-vein injection. Tumors in the ear were the earliest responders to bacterial therapy and hemorrhaged severely the day after A1-R administration. Tumors growing in the back were the second fastest responders to bacterial treatment and appeared necrotic 3 days after A1-R administration. Tumors growing in the footpad had the least vascularity and were the last responders to A1-R. Therefore, tumor vascularity correlated positively with tumor efficacy of A1-R. The present study suggests that bacteria efficacy on tumors involves vessel destruction which depends on the extent of vascularity of the tumor.Key words: tumor targeting bacteria, Salmonella typhimurium A1-R, Lewis lung carcinoma, RFP, GFP, nestin, nude mice     

The bulge area is the origin of nestin-expressing pluripotent stem cells of the hair follicle

Nestin-expressing pluripotent stem cells have been found both in the bulge area (BA) as well as the dermal papilla (DP). Nestin-expressing stem cells of both the BA and DP have been previously shown to be able to form neurons and other non-follicle cell types. The nestin-expressing stem cells from the DP have been termed skin precursor or SKP cells. Both nestin-expressing DP and BA cells have been previously shown to effect repair of the injured spinal cord and peripheral nerve, with the BA being the greater and more constant source of the stem cells. The BA contains nestin-expressing stem cells throughout the hair cycle, whereas nestin-expressing dermal papillae stem cells were found in early and mid-anagen only. Our previous studies have shown that the nestin-expressing stem cells in the BA and DP have similar morphological features. The cells from both regions have a small body diameter of approximately 7 μm with long extrusions, as shown by 2-photon imaging. In the present study, using 2-photon imaging of whisker follicles from transgenic mice expressing nestin-driven green fluorescent protein (ND-GFP), we demonstrate that the BA is the source of the nestin-expressing stem cells of the hair follicle. The nestin-expressing stem cells migrate from the BA to the DP as well as into the surrounding skin tissues including the epidermis, and during wound healing, suggesting that the BA may be the source of the stem cells of the skin itself.     

Long-Term Extensive Ectopic Hair Growth on the Spinal Cord of Mice from Transplanted Whisker Follicles

We have previously demonstrated that hair follicles contain nestin-expressing pluripotent stem cells that can effect nerve and spinal cord repair upon transplantation. In the present study, isolated whisker follicles from nestin-driven green fluorescent protein (ND-GFP) mice were histocultured on Gelfoam for 3 weeks for the purpose of transplantation to the spinal cord to heal an induced injury. The hair shaft was cut off from Gelfoam-histocultured whisker follicles, and the remaining part of the whisker follicles containing GFP-nestin expressing pluripotent stem cells were transplanted into the injured spinal cord of nude mice, along with the Gelfoam. After 90 days, the mice were sacrificed and the spinal cord lesion was observed to have healed. ND-GFP expression was intense at the healed area of the spinal cord, as observed by fluorescence microscopy, demonstrating that the hair follicle stem cells were involved in healing the spinal cord. Unexpectedly, the transplanted whisker follicles sprouted out remarkably long hair shafts in the spinal cord during the 90 days after transplantation of Gelfoam whisker histocultures to the injured spine. The pigmented hair fibers, grown from the transplanted whisker histocultures, curved and enclosed the spinal cord. The unanticipated results demonstrate the great potential of hair growth after transplantation of Gelfoam hair follicle histocultures, even at an ectopic site.     

The role of activins and follistatins in skin and hair follicle development and function

Investigations of the signalling between epithelial and mesenchymal compartments of skin during hair follicle initiation in utero and hair cycling have revealed the importance of the TGFβ superfamily in ectodermal organogenesis and morphogenesis. In particular the activins, their receptors and binding proteins such as follistatin, have been shown to be important regulators of cell proliferation, differentiation and apoptosis in hair follicle initiation, hair cycling, normal skin homeostasis and wound healing. Transgenic mice lacking various components of the activin signalling pathways display varying ectodermal pathologies including altered pelage hair follicle initiation. This review summarises the activin signal transduction pathways and the interactions between activins and other TGFβ signalling systems during hair follicle formation, hair growth cycling, skin function and wound healing.     

Embryonic dermal condensation and adult dermal papilla induce hair follicles in adult glabrous epidermis through different mechanisms

Hair induction in the adult glabrous epidermis by the embryonic dermis was compared with that by the adult dermis. Recombinant skin, composed of the adult sole epidermis and the embryonic dermis containing dermal condensations (DC), was transplanted onto the back of nude mice. The epidermis of transplants formed hairs. Histology on the induction process demonstrated the formation of placode-like tissues, indicating that the transplant produces hair follicles through a mechanism similar to that underlying hair follicle development in the embryonic skin. An isolated adult rat sole skin piece, inserted with either an aggregate of cultured dermal papilla (DP) cells or an intact DP between its epidermis and dermis, was similarly transplanted. The transplant produced hair follicles. Histology showed that the epidermis in both cases surrounded the aggregates of DP cells. The epidermis never formed placode-like tissues. Thus, it was concluded that the adult epidermal cells recapitulate the embryonic process of hair follicle development when exposed to DC, whereas they get directly into the anagen of the hair cycle when exposed to DP. The expression pattern of Edar and Shh genes, and P-cadherin protein during the hair follicle development in the two types of transplants supported the above conclusion.     

Nestin-Expressing Stem Cells Promote Nerve Growth in Long-Term 3-Dimensional Gelfoam?-Supported Histoculture

We have previously reported that hair follicles contain multipotent stem cells which express nestin. The nestin-expressing cells form the hair follicle sensory nerve. In vitro, the nestin-expressing hair follicle cells can differentiate into neurons, Schwann cells, and other cell types. In the present study, the sciatic nerve was excised from transgenic mice in which the nestin promoter drives green fluorescent protein (ND-GFP mice). The ND-GFP cells of the sciatic nerve were also found to be multipotent as the ND-GFP cells in the hair follicle. When the ND-GFP cells in the mouse sciatic nerve cultured on Gelfoam® and were imaged by confocal microscopy, they were observed forming fibers extending the nerve. The fibers consisted of ND-GFP-expressing spindle cells, which co-expressed the neuron marker β-III tubulin, the immature Schwann-cell marker p75^NTR and TrkB which is associated with neurons. The fibers also contain nestin-negative spherical cells expressing GFAP, a Schwann-cell marker. The β-III tubulin-positive fibers had growth cones on their tips expressing F-actin, indicating they are growing axons. When the sciatic nerve from mice ubiquitously expressing red fluorescent protein (RFP) was co-cultured on Gelfoam® with the sciatic nerve from ND-GFP transgenic mice, the interaction of nerves was observed. Proliferating nestin-expressing cells in the injured sciatic nerve were also observed in vivo. Nestin-expressing cells were also observed in posterior nerves but not in the spinal cord itself, when placed in 3-D Gelfoam® culture. The results of the present report suggest a critical function of nestin-expressing cells in peripheral nerve growth and regeneration.     

Vessel destruction by tumor-targeting Salmonella typhimurium A1-R is enhanced by high tumor vascularity

Our laboratory has previously developed a tumor-targeting double-auxotrophic mutant of Salmonella typhimurium termed A1-R. The present report demonstrates that S. typhimurium A1-R destroys tumor blood vessels and this is enhanced in tumors with high vascularity. Red fluorescent protein (RFP)-expressing Lewis lung cancer cells (LLC-RFP) were transplanted subcutaneously in the ear, back skin, and footpad of nestin-driven green fluorescent protein (ND-GFP) transgenic nude mice, which selectively express GFP in nascent blood vessels. Color-coded in vivo imaging demonstrated that the LLC-RFP ear tumor had the highest cell density and the footpad tumor had the least with the ear tumor having more abundant blood vessels than that on the back or footpad. The tumor-bearing mice were treated with A1-R bacteria via tail-vein injection. Tumors in the ear were the earliest responders to bacterial therapy and hemorrhaged severely the day after A1-R administration. Tumors growing in the back were the second fastest responders to bacterial treatment and appeared necrotic 3 days after A1-R administration. Tumors growing in the footpad had the least vascularity and were the last responders to A1-R. Therefore, tumor vascularity correlated positively with tumor efficacy of A1-R. The present study suggests that bacteria efficacy on tumors involved vessel destruction which depends on the extent of vascularity of the tumor.     

Nerve-derived sonic hedgehog defines a niche for hair follicle stem cells capable of becoming epidermal stem cells

In adult skin, stem cells in the hair follicle bulge cyclically regenerate the follicle, whereas a distinct stem cell population maintains the epidermis. The degree to which all bulge cells have equal regenerative potential is not known. We found that Sonic hedgehog (Shh) from neurons signals to a population of cells in the telogen bulge marked by the Hedgehog response gene Gli1. Gli1-expressing bulge cells function as multipotent stem cells in their native environment and repeatedly regenerate the anagen follicle. Shh-responding perineural bulge cells incorporate into healing skin wounds where, notably, they can change their lineage into epidermal stem cells. The perineural niche (including Shh) is dispensable for follicle contributions to acute wound healing and skin homeostasis, but is necessary to maintain bulge cells capable of becoming epidermal stem cells. Thus, nerves cultivate a microenvironment where Shh creates a molecularly and phenotypically distinct population of hair follicle stem cells.     

Direct transplantation of uncultured hair‐follicle pluripotent stem (hfPS) cells promotes the recovery of peripheral nerve injury

We previously showed that the stem cell marker nestin is expressed in hair follicle stem cells which suggested their pluripotency. We subsequently showed that the nestin‐expressing hair‐follicle pluripotent stem (hfPS) cells can differentiate in culture to neurons, glial cells, keratinocytes, and other cell types and can promote regeneration of peripheral nerve and spinal cord injuries upon injection to the injured nerve or spinal cord. The location of the hfPS cells has been termed the hfPS cell area (hfPSCA). Previously, hfPS cells were cultured for 1–2 months before transplantation to the injured nerve or spinal cord which would not be optimal for clinical application of these cells for nerve or spinal cord repair, since the patient should be treated soon after injury. In the present study, we addressed this issue by directly using the upper part of the hair follicle containing the hfPSCA, without culture, for injection into the severed sciatic nerve in mice. After injection of hfPSCA, the implanted hfPS cells grew and promoted joining of the severed nerve. The transplanted hfPS cells differentiated mostly to glial cells forming myelin sheaths, which promoted axonal growth and functional recovery of the severed nerve. These results suggest that the direct transplantation of the uncultured upper part of the hair follicle containing the hfPSA is an important method to promote the recovery of peripheral nerve injuries and has significant clinical potential. J. Cell. Biochem. 110: 272–277, 2010. © 2010 Wiley‐Liss, Inc.     

Development of new inbred transgenic strains of rats with LacZ or GFP

The ideal goal of regeneration medicine is to restore form and function to damaged tissues. While stem cell transplantation is considered a promising therapeutic approach, knowing the fate of transplanted cells using appropriate markers is essential. We developed new inbred transgenic rat strains with lacZ and GFP based on the transgenic (Tg) animal technique in rats. These Tg animals expressed most of their marker genes ubiquitously, compared to previous Tg rats. Immunological antigenicity against marker proteins was evaluated using conventional skin grafting, and results suggested lacZ-Tg-derived skin was much less immunogenic than that of GFP-Tg. However, GFP-positive cells from parental transgenic rats were still potential candidates for the study of cellular fate in immune privilege sites, such as the brain. Taking advantage of less immunogenic lacZ, we also examined the role of bone marrow-derived cells (BMDCs) in skin wound healing using an in vivo biological imaging system. Although transplantation of BMDCs enhanced wound healing at the injection site, BMDCs were detected only for a short time, suggesting a transient contribution of autologous BMDC-transplantation in wound healing. Our Tg-rat system may provide great benefits for the elucidation of the cellular process of regenerative medicine, including cell and tissue transplantation.     

Real-time confocal imaging of trafficking of nestin-expressing multipotent stem cells in mouse whiskers in long-term 3-D histoculture

We have previously demonstrated that nestin-expressing multipotent hair follicle stem cells are located above the hair follicle bulge and can differentiate into neurons and other cell types in vitro. The nestin-expressing hair follicle stem cells promoted the recovery of pre-existing axons when they were transplanted to the severed sciatic nerve or injured spinal cord. We have also previously demonstrated that the whisker hair follicle contains nestin-expressing stem cells in the dermal papilla (DP) as well as in the bulge area (BA), but that their origin is in the BA. In the present study, we established the technique of long-term Gelfoam? histoculture of whiskers isolated from transgenic mice in which nestin drives green fluorescent protein (ND-GFP). Confocal imaging was used to monitor ND-GFP-expressing stem cells trafficking in real time between the BA and DP to determine the fate of the stem cells. It was observed over a 2-week period that the stem cells trafficked from the BA toward the DP area and extensively grew out onto Gelfoam? forming nerve-like structures. This new method of long-term histoculture of whiskers from ND-GFP mice will enable the extensive study of the behavior of nestin-expressing multipotent stem cells of the hair follicle.     

Transplantation of bone-marrow-derived mesenchymal and epidermal stem cells contribute to wound healing with different regenerative features

A new concept for wound therapy is the initiation of the regeneration of epidermal and dermal layers with appendages for skin function recovery. Bone-marrow-derived mesenchymal and epidermal stem cells (BMSCs and SSCs) are hypothesized to be able to home toward or to be transplanted to wound sites for skin repair and regeneration, but this awaits confirmation by further experimental and clinical evidence. In this study, the influence of the transplantation of BMSCs and SSCs with porous gelatin-β-tricalcium phosphate sponge as scaffolds on wound re-epithelization, collagen synthesis, skin tensile strength recovery, and skin appendage regeneration has been investigated. The transplantation of BMSCs or SSCs significantly accelerates wound re-epithelization, stimulates dermal collagen synthesis, and exhibits the trend to enhance the tensile strength recovery of skin. Furthermore, regenerative features of BMSCs and SSCs have been identified in activating blood vessel and hair follicle formation, respectively. These results not only provide experimental evidence for the application of BMSCs and SSCs as promising therapeutics for clinical wound treatment, but also display their characteristics in activating distinct skin appendage regeneration, which might have novel applications in skin tissue engineering.     

Delayed cutaneous wound closure in HO‐2 deficient mice despite normal HO‐1 expression

Impaired wound healing can lead to scarring, and aesthetical and functional problems. The cytoprotective haem oxygenase (HO) enzymes degrade haem into iron, biliverdin and carbon monoxide. HO‐1 deficient mice suffer from chronic inflammatory stress and delayed cutaneous wound healing, while corneal wound healing in HO‐2 deficient mice is impaired with exorbitant inflammation and absence of HO‐1 expression. This study addresses the role of HO‐2 in cutaneous excisional wound healing using HO‐2 knockout (KO) mice. Here, we show that HO‐2 deficiency also delays cutaneous wound closure compared to WT controls. In addition, we detected reduced collagen deposition and vessel density in the wounds of HO‐2 KO mice compared to WT controls. Surprisingly, wound closure in HO‐2 KO mice was accompanied by an inflammatory response comparable to WT mice. HO‐1 induction in HO‐2 deficient skin was also similar to WT controls and may explain this protection against exaggerated cutaneous inflammation but not the delayed wound closure. Proliferation and myofibroblast differentiation were similar in both two genotypes. Next, we screened for candidate genes to explain the observed delayed wound closure, and detected delayed gene and protein expression profiles of the chemokine (C‐X‐C) ligand‐11 (CXCL‐11) in wounds of HO‐2 KO mice. Abnormal regulation of CXCL‐11 has been linked to delayed wound healing and disturbed angiogenesis. However, whether aberrant CXCL‐11 expression in HO‐2 KO mice is caused by or is causing delayed wound healing needs to be further investigated.