Up‐regulation of fibroblast growth factor (FGF) 9 expression and FGF‐WNT/β‐catenin signaling in laser‐induced wound healing

Fibroblast growth factor (FGF) 9 is secreted by both mesothelial and epithelial cells, and plays important roles in organ development and wound healing via WNT/β‐catenin signaling. The aim of this study was to evaluate FGF9 expression and FGF‐WNT/β‐catenin signaling during wound healing of the skin. We investigated FGF9 expression and FGF‐WNT/β‐catenin signaling after laser ablation of mouse skin and adult human skin, as well as in cultured normal human epidermal keratinocytes (NHEKs) upon stimulation with recombinant human (rh) FGF9 and rh‐transforming growth factor (TGF)‐β1. Our results showed that laser ablation of both mouse skin and human skin leads to marked overexpression of FGF9 and FGF9 mRNA. Control NHEKs constitutively expressed FGF9, WNT7b, WNT2, and β‐catenin, but did not show Snail or FGF receptor (FGFR) 2 expression. We also found that FGFR2 was significantly induced in NHEKs by rhFGF9 stimulation, and observed that FGFR2 expression was slightly up‐regulated on particular days during the wound healing process after ablative laser therapy. Both WNT7b and WNT2 showed up‐regulated protein expression during the laser‐induced wound healing process in mouse skin; moreover, we discerned that the stimulatory effect of rhFGF9 and rhTGF‐β1 activates WNT/β‐catenin signaling via WNT7b in cultured NHEKs. Our data indicated that rhFGF9 and/or rhTGF‐β1 up‐regulate FGFR2, WNT7b, and β‐catenin, but not FGF9 and Snail; pretreatment with rh dickkopf‐1 significantly inhibited the up‐regulation of FGFR2, WNT7b, and β‐catenin. Our results suggested that FGF9 and FGF‐WNT/β‐catenin signaling may play important roles in ablative laser‐induced wound healing processes.     

071TGF‐ß‐Mediated Paracrine Regulation of Keratinocyte Wound Closure

The importance of stromal‐epithelial interactions in wound healing is well established. These interactions likely involve autocrine and paracrine action of multiple growth factors, including members of the TGF‐ß family. TGF‐ß1, ß2 and ß3 isoforms signal by sequentially binding to the TGF‐ß type II and type I receptors, respectively. We address the role of TGF‐ß signaling in dermal fibroblasts using a conditional fibroblastic TGF‐ß type II receptor knockout mouse model (termed FßKO). We found that the loss of TGF‐ß signaling in the dermal fibroblasts results in accelerated excision‐wound closure compared with similar wounds in wild type mice. The mechanism of the altered rate of re‐epitheliaization in the FßKO mice was examined with regard to keratiocyte motility and proliferation. The migration of keratinocytes through collagen I coated 8 μm pore filters in the presence or absence of fibroblast‐conditioned media was tested. These experiments showed increased keratinocyte migration when incubated with FßKO dermal fibroblast conditioned media compared to media conditioned in wild type fibroblasts. Immuno‐histochemical staining of paraffin embedded intact skin indicated both wild type and FßKO mice had similar low levels of keratinocyte proliferation, based on Ki67 staining. In healing wounds, only the distal wound edges of wild type mice were proliferative. In contrast, the FßKO mice exhibited elevated proliferation across the length of the wound, including the leading edge of epithelial closure. Together our results suggest TGF‐ß signaling by the dermal fibroblasts suppresses re‐epithelialization of excision wounds by regulating keratinocyte motility and proliferation through paracrine mechanisms.
Funding: DOD BC99184 and NIH CA85492.     

Impaired cutaneous wound healing in transforming growth factor‐β inducible early gene1 knockout mice

Transforming growth factor‐β inducible early gene (TIEG) is induced by transforming growth factor‐β (TGF‐β) and acts as the primary response gene in the TGF‐β/Smad pathway. TGF‐β is a multifunctional growth factor that affects dermal wound healing; however, the mechanism of how TGF‐β affects wound healing is still not well understood because of the complexity of its function and signaling pathways. We hypothesize that TIEG may play a role in dermal wound healing, with involvement in wound closure, contraction, and reepithelialization. In this study, we have shown that TIEG1 knockout (TIEG1^–/–) mice have a delay in wound closure related to an impairment in wound contraction, granulation tissue formation, collagen synthesis, and reepithelialization. We also found that Smad7 was increased in the wounds and appeared to play a role in this wound healing model in TIEG1^–/– mice.     

Activation of β‐Catenin Signaling in Articular Chondrocytes Leads to Osteoarthritis‐Like Phenotype in Adult β‐Catenin Conditional Activation Mice

Osteoarthritis (OA) is a degenerative joint disease, and the mechanism of its pathogenesis is poorly understood. Recent human genetic association studies showed that mutations in the Frzb gene predispose patients to OA, suggesting that the Wnt/β‐catenin signaling may be the key pathway to the development of OA. However, direct genetic evidence for β‐catenin in this disease has not been reported. Because tissue‐specific activation of the β‐catenin gene (targeted by Col2a1‐Cre) is embryonic lethal, we specifically activated the β‐catenin gene in articular chondrocytes in adult mice by generating β‐catenin conditional activation (cAct) mice through breeding of β‐catenin^{fx(Ex3)/fx(Ex3)} mice with Col2a1‐CreER^T2 transgenic mice. Deletion of exon 3 of the β‐catenin gene results in the production of a stabilized fusion β‐catenin protein that is resistant to phosphorylation by GSK‐3β. In this study, tamoxifen was administered to the 3‐ and 6‐mo‐old Col2a1‐CreER^T2;β‐catenin^fx(Ex3)/wt mice, and tissues were harvested for histologic analysis 2 mo after tamoxifen induction. Overexpression of β‐catenin protein was detected by immunostaining in articular cartilage tissues of β‐catenin cAct mice. In 5‐mo‐old β‐catenin cAct mice, reduction of Safranin O and Alcian blue staining in articular cartilage tissue and reduced articular cartilage area were observed. In 8‐mo‐old β‐catenin cAct mice, cell cloning, surface fibrillation, vertical clefting, and chondrophyte/osteophyte formation were observed. Complete loss of articular cartilage layers and the formation of new woven bone in the subchondral bone area were also found in β‐catenin cAct mice. Expression of chondrocyte marker genes, such as aggrecan, Mmp‐9, Mmp‐13, Alp, Oc, and colX, was significantly increased (3‐ to 6‐fold) in articular chondrocytes derived from β‐catenin cAct mice. Bmp2 but not Bmp4 expression was also significantly upregulated (6‐fold increase) in these cells. In addition, we also observed overexpression of β‐catenin protein in the knee joint samples from patients with OA. These findings indicate that activation of β‐catenin signaling in articular chondrocytes in adult mice leads to the premature chondrocyte differentiation and the development of an OA‐like phenotype. This study provides direct and definitive evidence about the role of β‐catenin in the development of OA.     

Transforming growth factor β1 regulates the expression of CCN2 in human keratinocytes via Smad‐ERK signalling

Connective tissue growth factor (CCN2/CTGF) and transforming growth factor β1 (TGF‐β1) are important regulators of skin wound healing, but controversy remains regarding their expression in epithelial cell lineages. Here, we investigate the expression of CCN2 in keratinocytes during reepithelialisation and its regulation by TGF‐β1. CCN2 was detected in the epidermis of healing full‐thickness porcine wounds. Human keratinocytes were incubated with or without 10 ng/ml TGF‐β1, and signalling pathways were blocked with 10‐μM SIS3 or 20‐μM PD98059. Semi‐quantitative real‐time PCR was used to study CCN2 mRNA expression, and western blot was used to measure CCN2, phosphorylated‐ERK1/2, ERK1/2, phosphorylated‐Smad3 and Smad2/3 proteins. CCN2 was transiently expressed in neoepidermis at the leading edge of the wound in vivo. In vitro, CCN2 expression was induced by TGF‐β1 at 2 hours (7·5 ± 1·9‐fold mRNA increase and 3·0 ± 0·6‐fold protein increase) and 12 hours (5·4 ± 1·9‐fold mRNA increase and 3·3 ± 0·6‐fold protein increase). Compared with inhibiting the SMAD pathway, inhibiting the mitogen‐activated protein kinase (MAPK) pathway was more effective in reducing TGF‐β1‐induced CCN2 mRNA and protein expression. Inhibition of the MAPK pathway had minimal impact on the activity of the SMAD pathway. CCN2 is expressed in keratinocytes in response to tissue injury or TGF‐β1. In addition, TGF‐β1 induces CCN2 expression in keratinocytes through the ras/MEK/ERK pathway. A complete understanding of CCN2 expression in keratinocytes is critical to developing novel therapies for wound healing and cutaneous malignancy.     

β‐catenin promotes bone formation and suppresses bone resorption in postnatal growing mice

Genetic studies in the mouse have demonstrated multiple roles for β‐catenin in the skeleton. In the embryo, β‐catenin is critical for the early stages of osteoblast differentiation. Postnatally, β‐catenin in mature osteoblasts and osteocytes indirectly suppresses osteoclast differentiation. However, a direct role for β‐catenin in regulating osteoblast number and/or function specifically in the postnatal life has not been demonstrated. Addressing this knowledge gap is important because low‐density lipoprotein receptor‐related protein 5 (LRP5), a coreceptor for WNT signaling proposed to function through β‐catenin, controls osteoblast number and function in postnatal mice or humans. To overcome the neonatal lethality caused by embryonic deletion of β‐catenin in early‐stage osteoblast‐lineage cells, we use the Osx‐CreER^T2 mouse strain to remove β‐catenin in Osterix (Osx)‐expressing cells by administering tamoxifen (TM) temporarily to postnatal mice. Lineage‐tracing experiments in the long bones demonstrate that Osx‐CreER^T2 targets predominantly osteoblast‐lineage cells on the bone surface, but also transient progenitors that contribute to bone marrow stromal cells and adipocytes. Deletion of β‐catenin by this strategy greatly reduces the bone formation activity of the targeted osteoblasts. However, the targeted osteoblasts rapidly turn over and are replaced by an excessive number of non‐targeted osteoblasts, causing an unexpected increase in bone formation, but an even greater increase in osteoclast number and activity produces a net effect of severe osteopenia. With time, the mutant mice also exhibit a marked increase in bone marrow adiposity. Thus, β‐catenin in postnatal Osx‐lineage cells critically regulates bone homeostasis by promoting osteoblast activity and suppressing osteoblast turnover, while restraining osteoclast and marrow fat formation. © 2013 American Society for Bone and Mineral Research.     

Leucine‐rich glioma inactivated 3 promotes HaCaT keratinocyte migration

Our finding that human skin expresses leucine‐rich glioma inactivated 3 (LGI3) raises the question of the function of this cytokine in keratinocytes. We have shown that LGI3 stimulates human HaCaT keratinocyte migration without affecting viability or proliferation. Western blot analysis showed that LGI3 induced focal adhesion kinase activation, Akt phosphorylation, and glycogen synthase kinase 3β (GSK3β) phosphorylation in these cells. Using the scratch wound assay and a modified Boyden chamber, we found that LY294002, a selective phosphatidylinositol 3‐kinase inhibitor, and LiCl, a selective GSK3β inhibitor, abolished LGI3‐induced cell migration. We tested β‐catenin levels after LGI3 treatment because the Akt‐GSK3β pathway regulates β‐catenin accumulation, and β‐catenin promotes cell migration. LGI3 treatment increased β‐catenin protein and nuclear localization, whereas LY294002 prevented LGI3‐induced focal adhesion kinase and Akt activation as well as β‐catenin accumulation. Overall, these data suggest that LGI3 stimulates HaCaT cell migration following β‐catenin accumulation through the Akt pathway.     

Exogenous induction of unphosphorylated PTEN reduces TGFβ‐induced extracellular matrix expressions in lung fibroblasts

Transforming growth factor β (TGFβ) plays an important role in regulating aberrant extracellular matrix (ECM) production from alveolar/epithelial cells (AECs) and fibroblasts in pulmonary fibrosis. Although the tumor suppressor gene phosphatase and tensin homologue deleted from chromosome 10 (PTEN) can negatively control many TGFβ‐activated signaling pathways via the phosphatase activity, hyperactivation of the TGFβ‐related signaling pathways is often observed in fibrosis. Loss of PTEN expression might cause TGFβ‐induced ECM production. In addition, TGFβ was recently shown to induce loss of PTEN enzymatic activity by phosphorylating the PTEN C‐terminus. Therefore, we hypothesized that exogenous transfer of unphosphorylated PTEN (PTEN4A) might lead to reduce TGFβ‐induced ECM expression in not only epithelial cells but also fibroblasts. Adenovirus‐based exogenous PTEN4A induction successfully reduced TGFβ‐induced fibronectin expression and retained β‐catenin at the cell membrane in human epithelial cells. Exogenous unphosphorylated PTEN also attenuated TGFβ‐induced ECM production and inhibited TGFβ‐induced β‐catenin translocation in a human fibroblast cell line and in mouse primary isolated lung fibroblasts. Conversely, TGFβ‐induced α‐smooth muscle actin expression did not seem to be inhibited in these fibroblasts. Our data suggest that exogenous administration of unphosphorylated PTEN might be a promising strategy to restore TGFβ‐induced loss of PTEN activity and reduce aberrant TGFβ‐induced ECM production from epithelial cells and fibroblasts in lung fibrosis as compared with wild‐type PTEN induction.     

Loss of wnt/β‐catenin signaling causes cell fate shift of preosteoblasts from osteoblasts to adipocytes

Wnt signaling is essential for osteogenesis and also functions as an adipogenic switch, but it is not known if interrupting wnt signaling via knockout of β‐catenin from osteoblasts would cause bone marrow adiposity. Here, we determined whether postnatal deletion of β‐catenin in preosteoblasts, through conditional cre expression driven by the osterix promoter, causes bone marrow adiposity. Postnatal disruption of β‐catenin in the preosteoblasts led to extensive bone marrow adiposity and low bone mass in adult mice. In cultured bone marrow–derived cells isolated from the knockout mice, adipogenic differentiation was dramatically increased, whereas osteogenic differentiation was significantly decreased. As myoblasts, in the absence of wnt/β‐catenin signaling, can be reprogrammed into the adipocyte lineage, we sought to determine whether the increased adipogenesis we observed partly resulted from a cell‐fate shift of preosteoblasts that had to express osterix (lineage‐committed early osteoblasts), from the osteoblastic to the adipocyte lineage. Using lineage tracing both in vivo and in vitro we showed that the loss of β‐catenin from preosteoblasts caused a cell‐fate shift of these cells from osteoblasts to adipocytes, a shift that may at least partly contribute to the bone marrow adiposity and low bone mass in the knockout mice. These novel findings indicate that wnt/β‐catenin signaling exerts control over the fate of lineage‐committed early osteoblasts, with respect to their differentiation into osteoblastic versus adipocytic populations in bone, and thus offers potential insight into the origin of bone marrow adiposity. © 2012 American Society for Bone and Mineral Research.     

Matrix metalloproteinase inhibition delays wound healing and blocks the latent transforming growth factor‐β1‐promoted myofibroblast formation and function

The ability to regulate wound contraction is critical for wound healing as well as for pathological contractures. Matrix metalloproteinases (MMPs) have been demonstrated to be obligatory for normal wound healing. This study examined the effect that the broad‐spectrum MMP inhibitor BB‐94 has when applied topically to full‐thickness skin excisional wounds in rats and its ability to inhibit the promotion of myofibroblast formation and function by the latent transforming‐growth factor‐β1 (TGF‐β1). BB‐94 delayed wound contraction, as well as all other associated aspects of wound healing examined, including myofibroblast formation, stromal cell proliferation, blood vessel formation, and epithelial wound coverage. Interestingly, BB‐94 dramatically increased the level of latent and active MMP‐9. The increased levels of active MMP‐9 may eventually overcome the ability of BB‐94 to inhibit this MMP and may explain why wound contraction and other associated events of wound healing were only delayed and not completely inhibited. BB‐94 was also found to inhibit the ability of latent TGF‐β1 to promote the formation and function of myofibroblasts. These results suggest that BB‐94 could delay wound closure through a twofold mechanism; by blocking keratinocyte migration and thereby blocking the necessary keratinocyte–fibroblast interactions needed for myofibroblast formation and by inhibiting the activation of latent TGF‐β1.     

Different bone remodeling levels of trabecular and cortical bone in response to changes in Wnt/β‐catenin signaling in mice

Trabecular bone and cortical bone have different bone remodeling levels, and the underlying mechanisms are not fully understood. In the present study, the expression of Wnt/β‐catenin signaling and its downstream molecules along with bone mass in trabecular and cortical bone were compared in wild‐type mice, constitutive activation of β‐catenin (CA‐β‐catenin) mice and β‐catenin deletion mice. It was found that the expression level of most of the examined genes such as Wnt3a, β‐catenin, osteocalcin and RANKL/OPG ratio were significantly higher in trabecular bone than in cortical bone in wild‐type mice. CA‐β‐catenin resulted in up‐regulated expression of the above‐mentioned genes except for RANKL/OPG ratio, which were down‐regulated. Also, CA‐β‐catenin led to increased number of osteoblasts, decreased number of osteoclasts and increased bone mass in both the trabecular bone and cortical bone compared with wild‐type mice; however, the extent of changes was much greater in the trabecular bone than in the cortical bone. By contrast, null β‐catenin led to down‐regulated expression of the above‐mentioned genes except for RANKL/OPG ratio. Furthermore, β‐catenin deletion led to decreased number of osteoblasts, increased number of osteoclasts and decreased bone mass when compared with wild‐type mice. Again, the extent of these changes was more significant in trabecular bone than cortical bone. Taken together, we found that the expression level of Wnt/β‐catenin signaling and bone remodeling‐related molecules were different in cortical bone and trabecular bone, and the trabecular bone was more readily affected by changes in the Wnt/β‐catenin signaling pathway. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:812–819, 2017.

     

Indirubin‐3′‐Oxime Reverses Bone Loss in Ovariectomized and Hindlimb‐Unloaded Mice Via Activation of the Wnt/β‐Catenin Signaling

Osteoporosis is a major global health issue in elderly people. Because Wnt/β‐catenin signaling plays a key role in bone homeostasis, we screened activators of this pathway through cell‐based screening, and investigated indirubin‐3′‐oxime (I3O), one of the positive compounds known to inhibit GSK3β, as a potential anti‐osteoporotic agent. Here, we show that I3O activated Wnt/β‐catenin signaling via inhibition of the interaction of GSK3β with β‐catenin, and induced osteoblast differentiation in vitro and increased calvarial bone thickness ex vivo. Intraperitoneal injection of I3O increased bone mass and improved microarchitecture in normal mice and reversed bone loss in an ovariectomized mouse model of age‐related osteoporosis. I3O also increased thickness and area of cortical bone, indicating improved bone strength. Enhanced bone mass and strength correlated with activated Wnt/β‐catenin signaling, as shown by histological analyses of both trabecular and cortical bones. I3O also restored mass and density of bone in hindlimb‐unloaded mice compared with control, suspended mice, demonstrating bone‐restoration effects of I3O in non‐aged–related osteoporosis as well. Overall, I3O, a pharmacologically active small molecule, could be a potential therapeutic agent for the treatment and prevention of osteoporosis. © 2014 American Society for Bone and Mineral Research.     

A Dermal Equivalent Engineered with TGF‐β3 Expressing Bone Marrow Stromal Cells and Amniotic Membrane: Cosmetic Healing of Full‐Thickness Skin Wounds in Rats

Transforming growth factor beta‐3 (TGF‐β3) has been shown to decrease scar formation after scheduled topical applications to the cutaneous wounds. This study aimed to continuously deliver TGF‐β3, during the early phase of wound healing, by engineering a dermal equivalent (DE) using TGF‐β3 expressing bone marrow stromal cells (BM‐SCs) and human dehydrated amniotic membrane (hDAM). To engineer a DE, rat BM‐SCs were seeded on the hDAM and TGF‐β3 was transiently transfected into the BM‐SCs using a plasmid vector. Pieces of the dermal equivalent were transplanted onto the full‐thickness excisional skin wounds in rats. The process of wound healing was assessed by image analysis, Manchester Scar Scale (MSS), and histopathological studies 7, 14, 21, and 85 days after the excision. The results confirmed accurate construction of recombinant pcDNA3.1‐TGF‐β3 expression system and showed that the transfected BM‐SCs seeded on hDAM expressed TGF‐β3 mRNA and protein from day 3 through day 7 after transfection. After implantation of the DE, contraction of the wounds was measured from day 7 through 21 and analyzed by linear regression, which revealed that the rate of wound contraction in all experimental groups was similar. Histologic evaluation demonstrated that transfected BM‐SCs decreased retention and recruitment of the cells during the early stage of wound healing, decreased the formation of vascular structures and led to formation of uniformly parallel collagen bundles. MSS scores showed that TGF‐β3 secreting cells significantly improved the cosmetic appearance of the healed skin and decreased the scar formation. From these results, it could be concluded that transient secretion of TGF‐β3, during the early phase of healing, by BM‐SCs seeded on hDAM can improve the cosmetic appearance of the scar in cutaneous wounds without negatively affecting the process of wound repair.     

Mechanical activation of β‐catenin regulates phenotype in adult murine marrow‐derived mesenchymal stem cells

Regulation of skeletal remodeling appears to influence the differentiation of multipotent mesenchymal stem cells (MSC) resident in the bone marrow. As murine marrow cultures are contaminated with hematopoietic cells, they are problematic for studying direct effects of mechanical input. Here we use a modified technique to isolate marrow‐derived MSC (mdMSC) from adult mice, yielding a population able to differentiate into adipogenic and osteogenic phenotypes that is devoid of hematopoietic cells. In pure mdMSC populations, a daily strain regimen inhibited adipogenic differentiation, suppressing expression of PPARγ and adiponectin. Strain increased β‐catenin and inhibition of adipogenesis required this effect. Under osteogenic conditions, strain activated β‐catenin signaling and increased expression of WISP1 and COX2. mdMSC were also generated from mice lacking caveolin‐1, a protein known to sequester β‐catenin: caveolin‐1^(?/?) mdMSC exhibited retarded differentiation along both adipogenic and osteogenic lineages but retained mechanical responses that involved β‐catenin activation. Interestingly, caveolin‐1^(?/?) mdMSC failed to express bone sialoprotein and did not form mineralized nodules. In summary, mdMSC from adult mice respond to both soluble factors and mechanical input, with mechanical activation of β‐catenin influencing phenotype. As such, these cells offer a useful model for studies of direct mechanical regulation of MSC differentiation and function. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:1531–1538, 2010     

Inhibition of beta‐catenin signaling by Pb leads to incomplete fracture healing

There is strong evidence in the clinical literature to suggest that elevated lead (Pb) exposure impairs fracture healing. Since Pb has been demonstrated to inhibit bone formation, and Wnt signaling is an important anabolic pathway in chondrocyte maturation and endochondral ossification, we investigated the impact of Wnt therapy on Pb‐exposed mice undergoing bone repair in a mouse tibial fracture model. We established that tibial fracture calluses from Pb‐treated mice were smaller and contained less mineralized tissue than vehicle controls. This resulted in the persistence of immature cartilage in the callus and decreased β‐catenin levels. Reduction of β‐catenin protein was concurrent with systemic elevation of LRP5/6 antagonists DKK1 and sclerostin in Pb‐exposed mice throughout fracture healing. β‐catenin stimulation by the GSK3 inhibitor BIO reversed these molecular changes and restored the amount of mineralized callus. Overall, Pb is identified as a potent inhibitor of endochondral ossification in vivo with correlated effects on bone healing with noted deficits in β‐catenin signaling, suggesting the Wnt/β‐catenin as a pivotal pathway in the influence of Pb on fracture repair. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:1397–1405, 2014.     

Roxadustat promotes angiogenesis through HIF‐1α/VEGF/VEGFR2 signaling and accelerates cutaneous wound healing in diabetic rats

Diabetic foot ulcers are a major health‐care burden worldwide. One primary cause of the delayed wound healing in diabetic patients is impaired function of the hypoxia‐inducible factor‐1α/vascular endothelial growth factor (HIF‐1α/VEGF) axis, which results in compromised neovascularization in response to hypoxia. In the present study, we aimed to investigate the effect of roxadustat, a novel HIF prolyl‐4‐hydroxylase inhibitor, on angiogenesis and its therapeutic effect on cutaneous wound healing in diabetic rats. In vitro, we found that roxadustat could promote the angiogenic activity of human umbilical vein endothelial cells, accompanied by up‐regulation of HIF‐1α/VEGF/VEGFR2 signaling. Next, we demonstrated that Ki8751, a VEGFR2‐specific inhibitor, could inhibit the increased angiogenic activity of human umbilical vein endothelial cells induced by roxadustat. In vivo, we performed a Matrigel plug assay and demonstrated that roxadustat induced vascularization of the Matrigel plugs, and this effect could be partially inhibited by Ki8751. Finally, we utilized a streptozotocin‐induced diabetic rat model and found that roxadustat could accelerate cutaneous wound healing and promote angiogenesis in the wound sites. In conclusion, roxadustat promotes angiogenesis via activation of the HIF‐1α/VEGF/VEGFR2 pathway and exhibits therapeutic effects on diabetic wound healing by increasing angiogenesis. Our findings suggest that roxadustat can be a promising strategy to promote diabetic cutaneous wound healing.     

Deletion of a Single β‐Catenin Allele in Osteocytes Abolishes the Bone Anabolic Response to Loading

The Wnt/β‐catenin signaling pathway is essential for bone cell viability and function and for skeletal integrity. To determine if β‐catenin in osteocytes plays a role in the bone anabolic response to mechanical loading, 18‐ to 24‐week‐old osteocyte β‐catenin haploinsufficient mice (Dmp1‐Cre × β‐catenin fl/ + ; HET cKO) were compared with their β‐catenin fl/fl (control) littermates. Trabecular bone volume (BV/TV) was significantly less (58.3%) in HET cKO females versus controls, whereas male HET cKO and control mice were not significantly different. Trabecular number was significantly less in HET cKO mice compared with controls for both genders, and trabecular separation was greater in female HET cKO mice. Osteoclast surface was significantly greater in female HET cKO mice. Cortical bone parameters in males and females showed subtle or no differences between HET cKO and controls. The right ulnas were loaded in vivo at 100 cycles, 2 Hz, 2500 µ?, 3 days per week for 3 weeks, and the left ulnas served as nonloaded controls. Calcein and alizarin complexone dihydrate were injected 10 days and 3 days before euthanization, respectively. Micro‐computed tomography (µCT) analysis detected an 8.7% and 7.1% increase in cortical thickness in the loaded right ulnas of male and female control mice, respectively, compared with their nonloaded left ulnas. No significant increase in new cortical bone formation was observed in the HET cKO mice. Histomorphometric analysis of control mice showed a significant increase in endocortical and periosteal mineral apposition rate (MAR), bone‐formation rate/bone surface (BFR/BS), BFR/BV, and BFR/TV in response to loading, but no significant increases were detected in the loaded HET cKO mice. These data show that deleting a single copy of β‐catenin in osteocytes abolishes the anabolic response to loading, that trabecular bone in females is more severely affected and suggest that a critical threshold of β‐catenin is required for bone formation in response to mechanical loading. © 2014 American Society for Bone and Mineral Research