In this study we have demonstrated that sustained expression of HOXA3 in diabetic wounds significantly alters BMDC recruitment in response to wounding. We show that local expression of HOXA3 in wound tissue not only results in increased mobilization and recruitment of EPCs, but also suppresses the excessive inflammatory response characteristic of diabetic wounds. Together, the enhanced angiogenic response and reduced inflammation lead to a significant acceleration of healing.
Previous attempts to improve healing in diabetic wounds have focused either on improving angiogenesis via direct activation and/or enhancing recruitment of EPCs, or reducing excessive inflammation, and have met with various degrees of success [24
]. Reduced angiogenesis in diabetic wounds has been attributed to reduced mobilization, function, and engraftment of EPCs into tissues [39
]. However, direct application of SDF-1/CXCL12 enhances mobilization of EPCs and improves healing, specifically enhancing recruitment of Cd34+
EPCs into the neovasculature of granulation tissue [39
]. In the current study we have shown that HOXA3 induces expression of MCP-1/Ccl2, a related member of the CxC/CC chemokine family that has previously been shown to directly recruit EPCs [43
] and stimulate angiogenesis [44
]. Although we did not detect any differences in VEGF expression itself, reduced VEGF signaling, as well as aberrant cell adhesion properties of EPCs to endothelial cells in diabetic wounds, also contributes to poor EPC recruitment and retention [45
]. VEGF itself has been shown sufficient for recruitment and retention of recruited bone marrow-derived circulating cells in wild type mice [5
]. VEGF can accelerate wound healing when topically applied to diabetic wounds by promoting angiogenesis and recruitment of Tie2+
]. However, VEGF and other growth factor therapy in patients has proven less successful than expected, most likely due to high levels of proteases and advanced glycation end products (AGEs), both of which are associated with excessive inflammation in the diabetic wound environment [16
]. High levels of glucose can react nonenzymatically with the amino groups of proteins (glycation). One consequence of this is reduced effectiveness of downstream components of the VEGF signaling pathway [49
]. Thus, treatment of damaged tissue, even with high levels of ligand, is ineffective. Furthermore, AGE-modified fibronectin significantly reduced EPC attachment [50
], whereas blockade of the receptor for AGE significantly improved vascularization in diabetic mice [16
]. Our findings that peripheral blood of HOXA3
-treated animals contained greater numbers of immature Cd34+
EPCs, whereas wounds contained predominantly more mature Cd34+
EPCs, suggest that HOXA3 not only increased mobilization of immature EPCs but also created an appropriate wound environment that was permissive to adhesion and subsequent maturation of the recruited EPCs. This environment may have also stimulated additional stem/progenitor cells not derived from the bone marrow, and thus not detectable by our GFP tracking methods, as these studies are based on cells derived from reconstituted bone marrow cells descended from hematopoietic stem cells.
Chronic wounds also exhibit high levels of proteases that can modify the provisional extracellular matrix necessary for proper EPC adhesion [51
]. The high levels of proteases present in diabetic and chronic wounds have been linked to excessive inflammation with recruited neutrophils and macrophages expressing high levels of a variety of matrix-degrading proteins [15
]. A role for limiting inflammation in improving tissue repair is also supported by studies in Pu.1
-null mice that lack neutrophils and macrophages. These mice healed faster and displayed no evidence of fibrosis compared with their wild type counterparts [14
]. Importantly, in diabetic wounds, neutralization of TNF-α activity resulted in improved repair and healing due to inactivation of macrophages [40
], and our present results show that in addition to improving EPC recruitment, HOXA3 also attenuates the TNF pathway, reducing inflammation. Specifically we show that HOXA3 inhibits hyperactivation of the NF-κB pathway, resulting in attenuation of Tnf-α
expression, and leading to an overall reduction in Cd45+
leukocytes within the diabetic wound tissue. Myd88
both significantly downregulated by HOXA3 treatment, encode central components of the NF-κB pathway, functioning as adaptor proteins required for most Toll-like receptor activation of NF-κB in both wound-resident cells and recruited BMDCs. Interestingly, complete loss of Myd88
function, however, results in impaired wound healing and poor neovascularization [53
]. Thus it seems that the correct balance of NF-κB activation must be achieved for efficient repair and regeneration. Concentration-dependent differential gene regulation by activated NF-κB is well documented (reviewed in Stathopoulos and Levine [54
] and Sur et al [55
]). We speculate that high levels of nuclear NF-κB result in transcription of genes promoting recruitment/retention of leukocytes, whereas moderate levels of nuclear NF-κB do not, thus facilitating healing.
Differential Recruitment of EPCs and Leukocytes?
The majority of EPCs as well as inflammatory cells recruited to the wound are derived form stem/progenitor populations within the bone marrow. However, it is not clear how different populations within the bone marrow are differentially mobilized. For example, MCP-1/Ccl2 can mobilize EPCs and monocyte populations. Although application of this factor has been linked to improved repair and increased EPC recruitment, Ccl2
-null mice also displayed reduced inflammation and skin fibrosis after wounding [56
]. Thus the precise mechanisms that allow for enhanced EPC recruitment but restrict leukocyte retention in HOXA3
-treated wounds are not clear. Whether enhanced EPC recruitment accelerates vascular maturation and stability, which in turn would reduce leukocyte extravasation, is not known. One possibility is that attenuated NF-κB activity results in a switch in phenotype of the recruited monocytes, resulting in their differentiation into endothelial cells or endothelial support cells instead of activated macrophages. This idea is supported by studies demonstrating that hematopoietic progenitor cell differentiation into an activated dendritic cell can be blocked by VEGF receptor activation on the progenitor cell and requires inhibition of NF-κB [57
]. Furthermore, environmental cues, such as continued proangiogenic stimulation by factors such as VEGF, promote the differentiation of monocytic progenitor cells into endothelial-like cells [58
]. Future studies will be focused on elucidating the precise gene regulatory networks downstream of HOXA3 function controlling the recruitment and behavior of EPCs to sites of injury.