Alteration Of Angiotensin Ⅱ Production And Its Receptor Expression During Mouse Cutaneous Wound Healing: Potential Function Of Angiotensin Ⅱ In Wound Repair

BackgroundSkin is the largest and outermost organ of human body. It functions in addition to as a biological barrier for pathogens and also plays a key role in maintaining homeostasis. Skin is not only an endocrine organ but also a target organ of hormone and neurotransmitter. The cutaneous neuroendocrine system plays a critical role in tissue repair and regeneration through their special receptors (neuro-endocrine mediators) in the paracrine or autocrine manner. Renin-angiotensin system (RAS) is one of several important hormonal systems which regulates blood pressure and maintains water-electrolyte homeostasis. During the last decade, completely new aspects in the field of RAS research had shown that, angiotensinⅡ(AngⅡ), a key peptide of RAS plays not only a role in regulation of blood pressure and body fluids but also generally in cell proliferation,apoptosis and differentiation. It had been demonstrated that skin is a target organ of renin-angiotensin system and this system participates in cutaneous wound repair and regeneration. This present study was undertaken to explore the roles of biologically active peptide of renin-angiotensin system-AngⅡand the expression of its corresponding receptors in cutaneous wound healing process and hope to shed lights on the improvements in treating obstinate wounds and scars. ObjectiveThis study was designed to explore the change in the local level of AngⅡand the expression of its corresponding receptors AT1 and AT2 during wound healing. We also observed the changes of proliferation and apoptosis of the repair cells in wounded sites after cutaneous wound, and tried to analyze the relationship between AngⅡas well as wound repair and regeneration. We hope that our study would provide new insight to the therapeutic strategy for obstinate wounds and scars.Methods1. To develop full-thickness cutaneous wound model:6-mm-diameter circular full-thickness wound was made at both sides of the lower dorsal trunk using scissor and scalpel, and left undressed. The wounds were separated by a minimum of 6 mm of uninjured skin. At the indicated time points after wounding, wounds were photographed using digital camera. Digital image software was used to measure the wound size. For histological examination and immunohistochemical staining, wounds with a 2-mm margin of normal skin surrounding them were excised and longitudinally cut in half through the least-healed portion. Both halves of the wound were fixed in 4% paraformaldehyde, embedded in paraffin, and serially sectioned (5-μm thickness) perpendicular to the wound surface. For PCR evaluation and enzyme-linked immunosorbent assay (ELISA), the wound area was removed at the indicated time points using surgical scissors. The skin was cut immediately (1-mm) adjacent to the wound edge including a thin layer of tissue below the wound bed. The recovered tissue was snap-frozen in liquid nitrogen then stored at-80℃.2. The change in the generation of AngⅡin wounded tissue during the healing process was detected with ELISA.3. The proliferation and the apoptosis of cells were detected by bromodeoxyuridine (Brdu) and terminal deoxyuncleotidyl transferase mediated deoxyuridine triphosphate nick end labeling (TUNEL) method in wounded skin during the healing process respectively.4. The cellular localization and the mRNA level change of AngⅡreceptors in wounded tissue during wound healing were detected with immunostaining and RT-PCR.ResultAnalysis of full-thickness cutaneous wound closure:The wounds were totally closed at about 9 days after injury. The average time of wound closure under macroscopic observation was 9.2±0.42 days.The change of AngⅡlevels in cutaneous wound during healing process:AngⅡproduced in wounded skin was gradually increased after wounding, reached to the peak at 7 days, and then gradually decreased.The localization of AT1 and AT2 receptors in cutaneous wound during healing process:In normal mice, AT1 and AT2 receptors were mainly expressed in the epidermal layer. While positive staining signals were also found in the endothelial cells of the capillary vessels within the dermal layer, and appendages of the skin, i.e. hair follicle, sweat gland and sebaceous gland respectively. Positive staining signal of both AT1 and AT2 receptors were increased after wounding, reach the peak at 7 days, and then gradually decreased. Expression of AT2R was increased again following the epithelization of wound.The change of mRNA of AT1 and AT2 receptors in cutaneous wound during healing process: The expression of both AT1 and AT2 receptors was detected by RT-PCR, and AT1 receptor was increased in the first 7 days to the peak, and then gradually decreased during wound healing, while the expression of AT2 receptor was reached to its peak value on day 7, then gradually decreased, and increased again following the epithelization of wound.The change of BrdU labeling index in cutaneous wound during healing process:BrdU labeling index was increased gradually in the first 7 days to reach the peak, and then gradually decreased. The change of TUNEL-positive cells in cutaneous wound during healing process:The number of TUNEL-positive cells was increased slowly in the first 7 days after wounding. The increase in the number of TUNEL-positive cells was more markedly after epithelization of the wound.ConclusionThese results indicate that AngⅡparticipate in wound repair and might be related to remolding at the late stage of wound healing through the change in the production of angiotensinⅡas well as the expression of AT1 and AT2 receptors. AT1 receptor might be closely associated with cell proliferation, while AT2 receptor might play a role in cell apoptosis and tissue remolding during wound healing.