Martyn Lewis

Relaxin induces vascular endothelial growth factor expression and angiogenesis selectively at wound sites.

Relaxin is a reproductive hormone that has historically been characterized as being responsible for pubic ligament loosening and cervical ripening. Recently, relaxin has been associated with neovascularization of the endometrial lining of the uterus, potentially via specific induction of vascular endothelial growth factor. Previously conducted clinical studies using partially purified porcine relaxin have described relaxins ability to stimulate the healing of ischemic wounds, suggesting that relaxin may also have angiogenic effects at sites of ischemic wound healing. In the present study, relaxins angiogenic effects in the context of wound repair were tested in rodent models of angiogenesis and wound healing. Relaxin showed an ability to stimulate new blood vessel formation, particularly at ischemic wound sites, and to induce both vascular endothelial growth factor and basic fibroblast growth factor specifically in cells, presumably including macrophages, collected from wound sites. Resident macrophages collected from nonwound sites, such as the lung, did not show altered expression of these cytokines following relaxin administration. Because angiogenic wound cells are frequently macrophages, THP‐1 cells, a cell line of monocyte lineage that binds relaxin specifically, were tested for and shown to induce vascular endothelial growth factor and basic fibroblast growth factor in response to relaxin. In conclusion, relaxin may be useful in the treatment of ischemic wounds by stimulating angiogenesis via the induction of vascular endothelial growth factor and basic fibroblast growth factor in wound macrophages.

Relaxin activates the MAP kinase pathway in human endometrial stromal cells

The reproductive hormone, relaxin, is structurally similar to insulin and insulin‐like growth factor (IGF). Although a number of cellular responses to relaxin have been described, intracellular signaling mechanisms that link relaxin receptor engagement to alterations in gene expression remain uncharacterized. In the present study, relaxin treatment of a well‐characterized target, human endometrial stromal cells, resulted in rapid activation of p42/44 mitogen‐activated protein (MAP) kinase, as well as of MAPK (or ERK) kinase (MEK). Using a selective chemical inhibitor of MEK, it was further demonstrated that MEK phosphorylation is critical for relaxin‐induced MAP kinase activation. Relaxin treatment also induced MAP kinase activation in THP‐1 monocytic cells and in human smooth muscle cells, indicating that it may be a major signaling transducer utilized by the relaxin receptor. In contrast to insulin or IGF‐1, relaxin did not trigger the PI 3‐kinase/Akt pathway, perhaps accounting in part for relaxins unique biological profile. Relaxin was also found to cause activation of the transcription factor CREB, a substrate of the MAP kinase pathway. Finally, activation of the MAP kinase pathway was shown to be essential for optimal stimulation of expression of the gene for vascular endothelial growth factor. J. Cell. Biochem. 85: 536–544, 2002.

Transcriptional inhibition of stromelysin by interferon-gamma in normal human fibroblasts is mediated by the AP-1 domain.

The expression of the major matrix‐degrading metalloproteinase, stromelysin (SL), is modulated by a variety of cytokines and growth factors. Interferon‐γ (IFN‐γ) is a potent modulator of SL expression, either inhibiting or activating expression in a cell‐specific manner. We have investigated the mechanisms involved in the regulation of SL gene expression in cultured human fibroblasts by IFN‐γ. Reverse transcription‐polymerase chain reaction (RT‐PCR) assays confirmed the previously reported profound inhibitory response of SL mRNA expression to IFN‐γ [Amaldi et al., 1989]. For evaluation in transient gene expression assays, 1.2‐kilobase (kb) pairs (−1214 to +14 relative to the transcription start site), and shorter, deletion mutant fragments of the SL promoter were cloned into appropriate chloramphenicol acetyltransferase transferase (CAT) expression vectors. The SL promoter along this region contains an active polyomavirus enhancer A‐binding protein‐3 (PEA‐3) site at −216 and an activator protein‐1 (AP‐1) site at −70. Treatment of transfected neonatal foreskin fibroblasts with 300–500 U/ml IFN‐γ resulted in down‐regulation of both basal and IL‐1β‐induced CAT gene expression. IFN‐γ also decreased CAT expression when placed under the control of a synthetic multimeric AP‐1 site construct. Gel‐shift assay data indicate a decrease in specific binding to AP‐1 oligonucleotide of nuclear extract from IFN‐γ and PMA/IFN‐γ‐treated cells. The suppression of SL expression by IFN‐γ, in human fibroblasts therefore is mediated through the AP‐1 element. J. Cell. Biochem. 72:373–386, 1999.

Systemic relaxin administration stimulates angiogenic cytokine expression and vessel formation in a rat myocardial infarct model

The myocardium consists of three integrated components: myocytes, extracellular matrix, and a capillary microcirculation. Myocardial injury following vessel occlusion results in the formation of an ischemic zone whose size and position in the myocardium is determined by the anatomical region over which the capillary bed, supplied by the occluded vessel, lies [1]. Ischemia results in the onset of myocyte necrosis and generates an inflammatory response driven by infiltrating neutrophils and macrophages [2,3]. Neutrophils initiate local degradation of matrix at infarct sites by secretion of metalloproteases (MMPs) [4,5]. In an attempt to initiate repair, macrophages and dying myocytes then release TGFβ1 and vascular endothelial growth factor (VEGF) [6]. An early increase in levels of TGFβ1 in the infarct zone results in macrophage and fibroblast chemotaxis and fibroblast proliferation [2,7–9]. These ischemic-induced changes in turn increase VEGF and basic fibroblast growth factor (bFGF) levels [10]. Concomitantly, stress-induced changes to endothelium results in the expression of monocyte chemoattractant protein-1 (MCP-1) and endothelial integrins, which facilitate increased macrophage infiltration [2,11]. The early increase in MMPs, and subsequent increases in VEGF and bFGF levels, are essential requirements for the onset of neoangiogenesis of microvascular endothelial cells in the myocardium.

Effect of relaxin on normal and impaired wound healing in rodents

Wound healing is a well orchestrated complex series of events and can be divided into three phases: inflammation, granulation tissue formation and re-epithelialization, and final tissue remodeling [1]. The common element that governs all three phases is local vascular supply [2]. One of the few axioms of surgery is that damaged tissue will heal and resist infection in direct proportion to its local perfusion. Feet heal more slowly and become infected more commonly than chests, necks, and heads in that order. Impaired local or regional perfusion seems to be the major factor in the failure to heal [2]. Common vascular conditions that lead to chronic wounds are diabetes, venous stasis, arterio-sclerosis, pressure sores, steroid hormone use, radionecrosis and ischemic ulcers.

Relaxin induces specific alterations in gene expression in the human endometrium

Tight, coordinate regulation of numerous genes is necessary for the endometrium’s sole function, to provide a bed compatible with embryonic implantation and the maintenance of a successful pregnancy. In vitro, relaxin has been shown to regulate the expression of a number of proteins believed to be critical for, or contribute to, endometrial receptivity. The list of genes upregulated by relaxin in the endometrial stroma include insulin-like growth factor binding protein-1 [1], prolactin [2], and HOXA-10 [3]. Because relaxin is present in the circulation and in the endometrium itself during the peri-implantation period, it may play a major role in determining endometrial receptivity.