Michael A. Horan

Topical Estrogen Accelerates Cutaneous Wound Healing in Aged Humans Associated with an Altered Inflammatory Response

The effects of intrinsic aging on the cutaneous wound healing process are profound, and the resulting acute and chronic wound morbidity imposes a substantial burden on health services. We have investigated the effects of topical estrogen on cutaneous wound healing in healthy elderly men and women, and related these effects to the inflammatory response and local elastase levels, an enzyme known to be up-regulated in impaired wound healing states. Eighteen health status-defined females (mean age, 74.4 years) and eighteen males (mean age, 70.7 years) were randomized in a double-blind study to either active estrogen patch or identical placebo patch attached for 24 hours to the upper inner arm, through which two 4-mm punch biopsies were made. The wounds were excised at either day 7 or day 80 post-wounding. Compared to placebo, estrogen treatment increased the extent of wound healing in both males and females with a decrease in wound size at day 7, increased collagen levels at both days 7 and 80, and increased day 7 fibronectin levels. In addition, estrogen enhanced the strength of day 80 wounds. Estrogen treatment was associated with a decrease in wound elastase levels secondary to reduced neutrophil numbers, and decreased fibronectin degradation. In vitro studies using isolated human neutrophils indicate that one mechanism underlying the altered inflammatory response involves both a direct inhibition of neutrophil chemotaxis by estrogen and an altered expression of neutrophil adhesion molecules. These data demonstrate that delays in wound healing in the elderly can be significantly diminished by topical estrogen in both male and female subjects.

Estrogen modulates cutaneous wound healing by downregulating macrophage migration inhibitory factor

Characteristic of both chronic wounds and acute wounds that fail to heal are excessive leukocytosis and reduced matrix deposition. Estrogen is a major regulator of wound repair that can reverse age-related impaired wound healing in human and animal models, characterized by a dampened inflammatory response and increased matrix deposited at the wound site. Macrophage migration inhibitory factor (MIF) is a candidate proinflammatory cytokine involved in the hormonal regulation of inflammation. We demonstrate that MIF is upregulated in a distinct spatial and temporal pattern during wound healing and its expression is markedly elevated in wounds of estrogen-deficient mice as compared with intact animals. Wound-healing studies in mice rendered null for the MIF gene have demonstrated that in the absence of MIF, the excessive inflammation and delayed-healing phenotype associated with reduced estrogen is reversed. Moreover, in vitro assays have shown a striking estrogen-mediated decrease in MIF production by activated murine macrophages, a process involving the estrogen receptor. We suggest that estrogen inhibits the local inflammatory response by downregulating MIF, suggesting a specific target for future therapeutic intervention in impaired wound-healing states.

Type 2 Diabetes Whole-Genome Association Study in Four Populations: The DiaGen Consortium

Type 2 diabetes (T2D) is a common, polygenic chronic disease with high heritability. The purpose of this whole-genome association study was to discover novel T2D-associated genes. We genotyped 500 familial cases and 497 controls with >300,000 HapMap-derived tagging single-nucleotide-polymorphism (SNP) markers. When a stringent statistical correction for multiple testing was used, the only significant SNP was at TCF7L2, which has already been discovered and confirmed as a T2D-susceptibility gene. For a replication study, we selected 10 SNPs in six chromosomal regions with the strongest association (singly or as part of a haplotype) for retesting in an independent case-control set including 2,573 T2D cases and 2,776 controls. The most significant replicated result was found at the AHI1-LOC441171 gene region.

Age-related differences in the temporal and spatial regulation of matrix metalloproteinases (MMPs) in normal skin and acute cutaneous wounds of healthy humans

Abstract.Despite the association of increasing age with chronic wound-healing disorders and an impaired rate of healing of acute cutaneous wounds, the role of matrix metalloproteinases (MMPs) is unknown. To determine the spatial and temporal patterns and activities of MMP-1, -2, -3 and -9, 132 healthy humans aged between 19 and 96 years underwent 4-mm punch biopsies followed by wound excision between day 1 and day 180 post-wounding. Wounds showed an age-related increase in MMP-2 and MMP-9 immunostaining from day 3; this was associated with degradation of gelatin as shown by zymograms and with increased proteinase activity as shown by azocoll assays. Distinct spatial localisations for each MMP were observed: MMP-2 was found in epidermal structures; MMP-9 was observed in inflammatory cells up to day 21; MMP-1 was localised to keratinocytes at the wound margin. Normal old skin showed pro-MMP-2 bands on zymography and increased MMP-2 immunostaining. These results indicate that: (1) intrinsic ageing is associated with the up-regulation of MMPs previously associated with chronic wound healing; (2) wound-tissue proteinases are essentially active up to day 21 postwounding; and (3) intrinsic ageing may predispose to tissue breakdown disorders because of MMP-2 up-regulation in normal skin.

Human ageing impairs injury-induced in vivo expression of tissue inhibitor of matrix metalloproteinases (TIMP)-1 and -2 proteins and mRNA

Proteolysis is an essential component of wound healing but, if uncontrolled, it may lead to degradation of the neo‐matrix and a delay in wound repair. Despite numerous reports of impaired wound healing associated with increasing age, the control of proteolysis is completely unknown. Tissue inhibitor of matrix metalloproteinases (TIMP)‐1 and ‐2 inhibit the activity of matrix metalloproteinases and the pattern of regulation of these molecules determines in part the spatial and temporal regulation of proteolytic activity. This study reports on TIMP‐1 and ‐2 protein localization using immunocytochemistry in healing wounds of healthy subjects of different ages from day 1 to 6 months post‐wounding, and has quantified the mRNA levels for both inhibitors using reverse transcriptase‐polymerase chain reaction (RT‐PCR). TIMP‐1 and TIMP‐2 proteins are up‐regulated from 24 h post‐wounding, with a decrease in staining intensity by day 7 for TIMP‐2 and by day 14 for TIMP‐1. Steady‐state mRNA levels for both TIMPs were significantly greater in normal young skin than in aged skin. In the young, there was a significant increase in mRNA expression for TIMP‐1 and ‐2 by day 3 post‐wounding, which decreased by day 14 and had returned to basal levels at day 21. In the wounds of the aged subjects, basal levels were observed for TIMP‐1 and ‐2 at all time‐points. These results suggest that intrinsic cutaneous ageing is associated with reduced levels of TIMP mRNA both in normal skin and during acute wound repair. These levels may be instrumental in dermal tissue breakdown in normal skin, retarded wound healing, and the predisposition of the elderly to chronic wound healing states.

Predictors of outcome following hip fracture. Admission time predicts length of stay and in-hospital mortality

Many factors may contribute to the mortality and morbidity following hip fracture, including the provision of care. We wished to examine the contribution of potential factors to in-hospital mortality, length of hospital stay and 90-day mortality by statistical analyses of an audit database of all hip fractures admitted to a teaching hospital following the introduction of a fast track admission system. In-hospital mortality was predicted by ASA grade, the presence of any complications, cardiovascular complications, grade of surgeon, operation type and shorter admission time, a measure of time taken to admit a patient to a hospital bed (P<0.001). Length of hospital stay was predicted by increased age, presence of chronic cognitive impairment/dementia, presence of an implant complication, operation type, fracture type and longer admission time, r=0.455, P<0.001. Ninety-day mortality was predicted by the presence of chronic cognitive impairment/dementia, cardiovascular complications, pulmonary complications, ASA grade, grade of surgeon and admission day, P<0.001. Rapid admission following a hip fracture may not be the ideal management approach for all patient groups. Further study is required to identify factors in the process of care which are associated with better outcomes.

The effects of ageing on wound healing: immunolocalisation of growth factors and their receptors in a murine incisional model

A number of reports suggest that the process of ageing impairs wound repair and that strategies to manipulate the age‐related wound healing environment are necessary in order to stimulate repair. The process of cutaneous wound repair is controlled by growth factors in an autocrine and paracrine fashion: it is therefore surprising that the localisation of specific growth factors and their receptors has not been documented in wound healing with respect to chronological age. In this study the temporal profile of growth factor and receptor immunostaining was assessed within acute incisional wounds in an ageing mouse colony. A delay in appearance of platelet derived growth factor (PDGF) A and B isoforms, and PDGF‐α and ‐β receptors was evident with increasing animal age, paralleled by a similar finding for epidermal growth factor (EGF) and EGF receptor. Transforming growth factor (TGF)‐7beta;1 and 2 isoforms were increased at all time points in the wounds of younger animals, but the TGF‐β3 isoform increased in intensity from d 7 postwounding in the old mice wounds, and basic fibroblast growth factor (bFGF) from d 14. The quantity and distribution patterns of the various growth factors and their receptors may explain the age‐related differences in wound healing speed and quality, and possibly suggest new therapeutic targets for manipulating wound healing in the aged.

Comparison of a genetic algorithm neural network with logistic regression for predicting outcome after surgery for patients with nonsmall cell lung carcinoma

Neural networks have been used to predict outcome in cancer patients. Their accuracy compared with standard statistical methods has not been fully assessed.

Age-related changes in the temporal and spatial distributions of fibrillin and elastin mRNAs and proteins in acute cutaneous wounds of healthy humans

Elasticity and resilience of the skin are determined largely by the elastin framework, whose microfibrillar scaffold is composed of fibrillin. To date, the spatial and temporal patterns of expression of human elastin and fibrillin during wound healing have not been described. Ninety healthy human subjects underwent 4 mm cutaneous punch biopsy wounds from the upper inner arm, which were re‐excised from day 3 to 3 months post‐wounding. There were marked changes in the patterns of distribution and the amounts of elastin and fibrillin in sun‐protected skin with ageing. However, there were no major age‐related differences in the mRNA levels for elastin, fibrillin‐1 and fibrillin‐2 using in situ hybridization. Elastin and fibrillin appeared in greatest amounts in the wounds of the elderly, particularly in females. A regenerative pattern of elastin and fibrillin arcades at the dermo‐epidermal junction was observed in the wounds of aged subjects. mRNA expression of elastin was greatest in the wounds of the aged (from day 3 to day 14 post‐wounding) with a similar spatial and temporal pattern to fibrillin‐1 expression; this suggests that fibrillin‐1 is the major contributor to dermal elastic fibre construction during wound repair. Fibrillin‐2 was expressed only in the wounds of the aged and expression was confined to areas proximal to dermal blood vessels. The clear‐cut differences in the localization of the two members of the fibrillin family suggest that these have well‐defined roles in normal skin and wound tissue. In summary, these data indicate that ageing is associated with increased expression of fibrillin and elastin during acute wound healing and concomitant restoration of the papillary dermal architecture with an improved quality of scarring.

Genome-wide association study meta-analysis of chronic widespread pain: evidence for involvement of the 5p15.2 region

Background and objectives Chronic widespread pain (CWP) is a common disorder affecting ∼10% of the general population and has an estimated heritability of 48–52%. In the first large-scale genome-wide association study (GWAS) meta-analysis, we aimed to identify common genetic variants associated with CWP. Methods We conducted a GWAS meta-analysis in 1308 female CWP cases and 5791 controls of European descent, and replicated the effects of the genetic variants with suggestive evidence for association in 1480 CWP cases and 7989 controls. Subsequently, we studied gene expression levels of the nearest genes in two chronic inflammatory pain mouse models, and examined 92 genetic variants previously described associated with pain. Results The minor C-allele of rs13361160 on chromosome 5p15.2, located upstream of chaperonin-containing-TCP1-complex-5 gene (CCT5) and downstream of FAM173B, was found to be associated with a 30% higher risk of CWP (minor allele frequency=43%; OR=1.30, 95% CI 1.19 to 1.42, p=1.2×10−8). Combined with the replication, we observed a slightly attenuated OR of 1.17 (95% CI 1.10 to 1.24, p=4.7×10−7) with moderate heterogeneity (I2=28.4%). However, in a sensitivity analysis that only allowed studies with joint-specific pain, the combined association was genome-wide significant (OR=1.23, 95% CI 1.14 to 1.32, p=3.4×10−8, I2=0%). Expression levels of Cct5 and Fam173b in mice with inflammatory pain were higher in the lumbar spinal cord, not in the lumbar dorsal root ganglions, compared to mice without pain. None of the 92 genetic variants previously described were significantly associated with pain (p>7.7×10−4). Conclusions We identified a common genetic variant on chromosome 5p15.2 associated with joint-specific CWP in humans. This work suggests that CCT5 and FAM173B are promising targets in the regulation of pain.