Kimberly A. Mace

Sustained expression of Hif-1α in the diabetic environment promotes angiogenesis and cutaneous wound repair

Impaired wound healing in diabetic patients is associated with deficiencies in the production of factors involved in cell proliferation and migration, such as vascular endothelial growth factor. However, it remains unclear how the transcriptional regulation of the genes encoding these factors is affected by the diabetic environment. Hypoxia‐inducible factor‐1α (Hif‐1α), the regulatory subunit of the Hif‐1 transcription factor, plays an important role in activating many of these genes. Therefore, we tested whether Hif‐1α function is impaired in the diabetic wound environment and whether restoring Hif‐1 function improves wound healing. Here, we show that Hif‐1α protein levels are dramatically reduced in wounds of leptin receptor‐deficient diabetic mice compared with nondiabetic littermates. Reduction in Hif‐1α levels results in decreased DNA‐binding activity and in decreased expression of several Hif‐1 target genes, including vascular endothelial growth factor, heme oxygenase‐1, and inducible nitric oxide synthase. Furthermore, we demonstrate that sustained expression of Hif‐1α in leptin receptor‐deficient diabetic wounds restores expression of these factors, enhances angiogenesis, and significantly accelerates wound healing. Taken together, these results suggest that Hif‐1α function plays a significant role in wound healing and reduced levels of Hif‐1α may contribute to impaired healing.

HOXA3 induces cell migration in endothelial and epithelial cells promoting angiogenesis and wound repair

Wound repair requires both the recruitment and coordination of numerous cell types including inflammatory cells, fibroblasts, endothelial and epithelial cells. Each cell type has a distinct set of cell behavior such as formation of granulation tissue and basement membrane, migration, proliferation and redifferentiation. These processes are dependent on cell-cell and cell-ECM signaling, intracellular signal transduction cascades, and ultimately, changes in gene transcription. We have investigated the role of the transcription factor HOXA3 in wound repair and angiogenesis. Here we show that HOXA3 increases endothelial cell migration, induces angiogenesis in vivo, and leads to increased expression of the matrix metalloproteinase-14 (MMP-14) and urokinase-type plasminogen activator receptor (uPAR) genes in endothelial cells in culture and in vivo in response to injury. We find that HOXA3 gene expression is upregulated during wound healing in angiogenic endothelial cells and keratinocytes, and that HOXA3 is not induced in genetically diabetic mice that have impaired angiogenesis and wound repair. We demonstrate that gene transfer of HOXA3 into diabetic mouse wounds leads to dramatic improvements in both angiogenesis and wound closure. In addition, we show that HOXA3 promotes migration of endothelial cells and keratinocytes in a uPAR-dependent manner. Together these findings illustrate how the morphoregulatory protein, HOXA3 can facilitate tissue remodeling via coordinated changes in both epithelial and endothelial cell gene expression and behavior in adult tissues during wound repair.

Estrogen Receptor-Alpha Promotes Alternative Macrophage Activation during Cutaneous Repair

Efficient local monocyte/macrophage recruitment is critical for tissue repair. Recruited macrophages are polarized toward classical (proinflammatory) or alternative (prohealing) activation in response to cytokines, with tight temporal regulation crucial for efficient wound repair. Estrogen acts as a potent anti-inflammatory regulator of cutaneous healing. However, an understanding of estrogen/estrogen receptor (ER) contribution to macrophage polarization and subsequent local effects on wound healing is lacking. Here we identify, to our knowledge previously unreported, a role whereby estrogen receptor α (ERα) signaling preferentially polarizes macrophages from a range of sources to an alternative phenotype. Cell-specific ER ablation studies confirm an in vivo role for inflammatory cell ERα, but not ERβ, in poor healing associated with an altered cytokine profile and fewer alternatively activated macrophages. Furthermore, we reveal intrinsic changes in ERα-deficient macrophages, which are unable to respond to alternative activation signals in vitro. Collectively, our data reveal that inflammatory cell-expressed ERα promotes alternative macrophage polarization, which is beneficial for timely healing. Given the diverse physiological roles of ERs, these findings will likely be of relevance to many pathologies involving excessive inflammation.

Diabetes induces stable intrinsic changes to myeloid cells that contribute to chronic inflammation during wound healing in mice

SUMMARY Acute inflammation in response to injury is a tightly regulated process by which subsets of leukocytes are recruited to the injured tissue and undergo behavioural changes that are essential for effective tissue repair and regeneration. The diabetic wound environment is characterised by excessive and prolonged inflammation that is linked to poor progression of healing and, in humans, the development of diabetic foot ulcers. However, the underlying mechanisms contributing to excessive inflammation remain poorly understood. Here we show in a murine model that the diabetic environment induces stable intrinsic changes in haematopoietic cells. These changes lead to a hyper-responsive phenotype to both pro-inflammatory and anti-inflammatory stimuli, producing extreme M1 and M2 polarised cells. During early wound healing, myeloid cells in diabetic mice show hyperpolarisation towards both M1 and M2 phenotypes, whereas, at late stages of healing, when non-diabetic macrophages have transitioned to an M2 phenotype, diabetic wound macrophages continue to display an M1 phenotype. Intriguingly, we show that this population predominantly consists of Gr-1+ CD11b+ CD14+ cells that have been previously reported as ‘inflammatory macrophages’ recruited to injured tissue in the early stages of wound healing. Finally, we show that this phenomenon is directly relevant to human diabetic ulcers, for which M2 polarisation predicts healing outcome. Thus, treatments focused at targeting this inflammatory cell subset could prove beneficial for pathological tissue repair.

The dual roles of homeobox genes in vascularization and wound healing.

Homeobox genes represent a family of highly conserved transcription factors originally discovered to regulate organ patterning during development. More recently, several homeobox genes were shown to affect processes in adult tissue, including angiogenesis and wound healing. Whereas a subset of members of the Hox-family of homeobox genes activate growth and migration to promote angiogenesis or wound healing, other Hox genes function to restore or maintain quiescent, differentiated tissue function. Pathological tissue remodeling is linked to differential expression of activating or stabilizing Hox genes and dysregulation of Hox expression can contribute to disease progression. Studies aimed at understanding the role and regulation of Hox genes have provided insight into how these potent morphoregulatory genes can be applied to enhance tissue engineering or limit cancer progression.

HOXA3 Modulates Injury‐Induced Mobilization and Recruitment of Bone Marrow‐Derived Cells

The regulated recruitment and differentiation of multipotent bone marrow‐derived cells (BMDCs) to sites of injury are critical for efficient wound healing. Previously we demonstrated that sustained expression of HOXA3 both accelerated wound healing and promoted angiogenesis in diabetic mice. In this study, we have used green fluorescent protein‐positive bone marrow chimeras to investigate the effect of HOXA3 expression on recruitment of BMDCs to wounds. We hypothesized that the enhanced neovascularization induced by HOXA3 is due to enhanced mobilization, recruitment, and/or differentiation of BMDCs. Here we show that diabetic mice treated with HOXA3 displayed a significant increase in both mobilization and recruitment of endothelial progenitor cells compared with control mice. Importantly, we also found that HOXA3‐treated mice had significantly fewer inflammatory cells recruited to the wound compared with control mice. Microarray analyses of HOXA3‐treated wounds revealed that indeed HOXA3 locally increased expression of genes that selectively promote stem/progenitor cell mobilization and recruitment while also suppressing expression of numerous members of the proinflammatory nuclear factor κB pathway, including myeloid differentiation primary response gene 88 and toll‐interacting protein. Thus HOXA3 accelerates wound repair by mobilizing endothelial progenitor cells and attenuating the excessive inflammatory response of chronic wounds. STEM CELLS 2009;27:1654–1665

Hoxa3 promotes the differentiation of hematopoietic progenitor cells into proangiogenic Gr-1+CD11b+ myeloid cells.

Injury induces the recruitment of bone marrow-derived cells (BMDCs) that contribute to the repair and regeneration process. The behavior of BMDCs in injured tissue has a profound effect on repair, but the regulation of BMDC behavior is poorly understood. Aberrant recruitment/retention of these cells in wounds of diabetic patients and animal models is associated with chronic inflammation and impaired healing. BMD Gr-1(+)CD11b(+) cells function as immune suppressor cells and contribute significantly to tumor-induced neovascularization. Here we report that Gr-1(+)CD11b(+) cells also contribute to injury-induced neovascularization, but show altered recruitment/retention kinetics in the diabetic environment. Moreover, diabetic-derived Gr-1(+)CD11b(+) cells fail to stimulate neovascularization in vivo and have aberrant proliferative, chemotaxis, adhesion, and differentiation potential. Previously we demonstrated that gene transfer of HOXA3 to wounds of diabetic mice is taken up by and expressed by recruited BMDCs. This is associated with a suppressed inflammatory response, enhanced neovascularization, and accelerated wound healing. Here we show that sustained expression of Hoxa3 in diabetic-derived BMD Gr-1(+)CD11b(+) cells reverses their diabetic phenotype. These findings demonstrate that manipulation of adult stem/progenitor cells ex vivo could be used as a potential therapy in patients with impaired wound healing.

Secretion of SDF-1α by bone marrow-derived stromal cells enhances skin wound healing of C57BL/6 mice exposed to ionizing radiation

Patients treated for cancer therapy using ionizing radiation (IR) have delayed tissue repair and regeneration. The mechanisms mediating these defects remain largely unknown at present, thus limiting the development of therapeutic approaches. Using a wound healing model, we here investigate the mechanisms by which IR exposure limits skin regeneration. Our data show that induction of the stromal cell‐derived growth factor 1α (SDF‐1α) is severely impaired in the wounded skin of irradiated, compared to non‐irradiated, mice. Hence, we evaluated the potential of bone marrow‐derived multipotent stromal cells (MSCs), which secrete high levels of SDF‐1α, to improve skin regeneration in irradiated mice. Injection of MSCs into the wound margin led to remarkable enhancement of skin healing in mice exposed to IR. Injection of irradiated MSCs into the wound periphery of non‐irradiated mice delayed wound closure, also suggesting an important role for the stromal microenvironment in skin repair. The beneficial actions of MSCs were mainly paracrine, as the cells did not differentiate into keratinocytes. Specific knockdown of SDF‐1α expression led to drastically reduced efficiency of MSCs in improving wound closure, indicating that SDF‐1α secretion by MSCs is largely responsible for their beneficial action. We also found that one mechanism by which SDF‐1α enhances wound closure likely involves increased skin vascularization. Our findings collectively indicate that SDF‐1α is an important deregulated cytokine in irradiated wounded skin, and that the decline in tissue regeneration potential following IR can be reversed, given adequate microenvironmental support

Effects of decreased insulin-like growth factor-1 stimulation on hypoxia inducible factor 1-α protein synthesis and function during cutaneous repair in diabetic mice

Insulin‐like growth factor‐1 (Igf‐1), a critical mediator of tissue repair, is significantly decreased in diabetic wounds. Furthermore, decreased levels of hypoxia‐inducible factor 1‐α (Hif‐1α) and its target genes are also associated with impaired wound healing in diabetic mice. The aim of our study was to examine whether the reduced levels of Igf‐1 are responsible for the reduction in Hif‐1α protein synthesis and activity in diabetic wounds. We provide evidence that Igf‐1 regulates Hif‐1α protein synthesis and activity during wound repair. In addition, Igf‐1 stimulated phosphytidylinositol 3‐kinase activity in diabetic fibroblasts, which, in turn, increased activation of the translational regulatory protein, p70 S6 kinase. Moreover, improved healing of diabetic wounds by addition of recombinant IGF‐1 protein was associated with an increase in Hif‐1α protein synthesis and function in vivo.

Myeloid cell dysfunction and the pathogenesis of the diabetic chronic wound

Diabetes can promote a state of chronic inflammation associated with serious complications that are difficult to treat, including ulceration of the lower extremities and chronic wounds. Chronic wounds are often incurable and contribute to both a reduced quality of life for patients and an enormous burden for healthcare services. In diabetes, the inflammatory response early in wound healing is inappropriately amplified and prolonged, leading to the persistent presence in the wound of vastly elevated numbers of dysfunctional, hyperpolarised macrophages that fail to transition to a pro-healing phenotype. Recent evidence suggests that systemic chronic inflammation induces intrinsic defects in monocytes via chromatin modifications that may pre-programme monocytes to a pro-inflammatory phenotype, while the local wound environment inhibits differentiation to a pro-healing phenotype. Current understanding remains incomplete, and careful dissection of how local and systemic inflammation combine to negatively influence myeloid cell development will be key to developing effective therapies aimed at healing the diabetic wound.