Objective: To ascertain the spatial and temporal relation of wound hypoxia to the cell types involved, expression of selected angiogenic cytokines, the proliferative status of cells in the wound site, and angiogenesis.
Summary Background Data: Hypoxia is considered to drive the angiogenic response by upregulating angiogenic cytokines observed during wound healing. But this correlation has not been shown on a cell-to-cell basis in vivo because of limitations in measuring tissue PO2 at the cellular level.
Methods: Using punch biopsy wounds in rats as a wound healing model, the distributions of vascular endothelial growth factor, transforming growth factor-beta, tumor necrosis factor-alpha, and pimonidazole adducts (as a hypoxia marker) were followed immunohistochemically during the healing process.
Results: Hypoxia was absent on day 1 after wounding, even though angiogenesis and maximal expression of cytokines were observed in the wounds. Hypoxia peaked in the granulation tissue stage at day 4 and correlated with increased cellularity and cellular proliferation. Hypoxia started to decrease after day 4 and was limited to the remnant blood vessels and epithelial layer in the scar tissue.
Conclusions: Induction of angiogenic cytokines early during wound healing may be due to triggering mechanisms other than hypoxia. Alternatively, the unique pattern of development and decline of cellular hypoxia as wound cellularity and proliferation regress suggest its involvement in initiating vascular regression during the later stages of healing.
Tissue hypoxia is considered a major signal that initiates and regulates angiogenic processes such as wound healing and tumor growth. 1-3 Hypoxia has been shown (in vitro) to induce several major cytokines such as vascular endothelial growth factor (VEGF), 4 transforming growth factor-beta (TGFβ), 5 tumor necrosis factor-alpha (TNFα), 6 and interleukin-8 7 from a wide variety of cells involved in tissue repair, including fibroblasts, endothelial cells, and macrophages. During wound healing, tissue PO2 is considered to be low at the center of the wound, but it increases as the wound heals. 8-10 Wound-induced hypoxia, as suggested by the in vitro data, is thought to be a major determinant of all phases of wound healing by regulating cellular proliferation, cell migration, and extracellular matrix protein synthesis through the induction of cytokines and diverse intracellular signaling pathways. In vivo studies have demonstrated decreased PO2 in wounds, 9-11 suggesting that conditions favorable to hypoxic stimulation of cytokine production exist. 12,13 These studies of wound tissue PO2 have provided useful information on the kinetics of change in oxygenation after wounding. However, the methods have measured overall average tissue PO2, whereas it is known that the diffusion distance of oxygen is on the order of a few tens of microns. 14,15 To elucidate the relation between wound hypoxia, cytokine expression, and cellular responses to wounding, we used immunohistochemical methods that allowed us to evaluate these responses on a cell-to-cell basis. The object of our study was to ascertain the spatial and temporal relation of wound hypoxia to the cell types involved, expression of selected angiogenic cytokines, the proliferative status of cells in the wound site, and angiogenesis.
Pimonidazole hydrochloride belongs to a group of compounds known as 2-nitroimidazole hypoxia markers that form protein adducts under conditions of low oxygen tension (i.e., ≤10 mmHg) by the action of cellular nitroreductases. 16,17 The introduction of immunochemical reagents that recognize marker adducts 18 allowed the nonradioactive detection of tissue hypoxia. 19-21 Although originally developed for use in animal tumors 22,23 and human tumors, 24-26 pimonidazole hydrochloride has also been applied to the study of hypoxia and hypoxia-associated pathophysiologic changes in normal rat liver and kidney. 27-30 Of particular interest to the present study is the binding of pimonidazole to suprabasal cells in epithelial structures, including skin. 31 This binding is consistent with radiobiologic data that indicate that the skin is hypoxic. 32 The binding of pimonidazole to skin epithelium serves as a useful positive control in the present study of wound healing. Another advantage of the immunohistochemical hypoxia marker approach is that it measures cellular events with spatial resolution at the cell level without physically disturbing the tissue during the accumulation of the hypoxia signal. This is essential to the present study, and no other assay can do this in vivo. Validation of the immunohistochemical technique has been carried out in rodent tumors and human tumor xenografts tumors where correlations between pimonidazole adduct formation, oxygen electrode measurements, and radiation response have been demonstrated. 23,33 In a discussion of the scope and limitation of pimonidazole as a hypoxia marker, 23 it was noted that the bioreductive activation of pimonidazole was not dependent on specialized enzymes, nor does the concentration of P450 cytochrome enzymes in the perivascular region of rat livers, for example, override the oxygen dependence of pimonidazole activation. A cell type of specific interest to the present study is the macrophage and its bioreductive properties in terms of the oxygen-dependent activation of 2-nitroimidazole binding. Although relatively little appears to be known about this topic, Olive 34 has shown that host macrophages in rodent tumors behave as tumor cells with respect to binding hypoxia markers in the sense that binding occurred only in macrophages that were in hypoxic regions of the tumors.
We report the absence of pimonidazole adduct formation in the wound site and surrounding normal skin at day 1 after wounding, indicating the absence of tissue regions with PO2 ≤ 10 mmHg. Hypoxia marker intensity peaked at day 4 after wounding, which coincided with the greatest cellularity and proliferation. Hypoxia diminished as the cellularity of the wounds regressed and mature scar tissue was generated by day 8. In contrast, hypoxia-inducible cytokines such as VEGF, TGFβ, and TNFα exhibited maximal immunoreactivity at day 1, a time point where we saw no evidence of hypoxia. These results suggest there are likely to be signals present other than hypoxia that initiate tissue repair and angiogenesis during the early time points after wounding. Hypoxia reached its maximal intensity in granulation tissue at a critical juncture in the healing process when apoptosis and remodeling were being initiated. This pattern of development of hypoxia suggests that it may play a role in triggering the apoptosis and remodeling of the granulation tissue, as opposed to providing the initial stimulus for proangiogenic cytokine production in the early phases of wound healing.