Provisional matrix deposition in hemostasis and venous insufficiency: Tissue preconditioning for nonhealing venous ulcers

Significance: Chronic wounds represent a major burden on global healthcare systems and reduce the quality of life of those affected. Significant advances have been made in our understanding of the biochemistry of wound healing progression. However, knowledge regarding the specific molecular processes influencing chronic wound formation and persistence remains limited. Recent Advances: Generally, healing of acute wounds begins with hemostasis and the deposition of a plasma-derived provisional matrix into the wound. The deposition of plasma matrix proteins is known to occur around the microvasculature of the lower limb as a result of venous insufficiency. This appears to alter limb cutaneous tissue physiology and consequently drives the tissue into a ‘preconditioned’ state that negatively influences the response to wounding. Critical Issues: Processes, such as oxygen and nutrient suppression, edema, inflammatory cell trapping/extravasation, diffuse inflammation, and tissue necrosis are thought to contribute to the advent of a chronic wound. Healing of the wound then becomes difficult in the context of an internally injured limb. Thus, interventions and therapies for promoting healing of the limb is a growing area of interest. For venous ulcers, treatment using compression bandaging encourages venous return and improves healing processes within the limb, critically however, once treatment concludes ulcers often reoccur. Future Directions: Improved understanding of the composition and role of pericapillary matrix deposits in facilitating internal limb injury and subsequent development of chronic wounds will be critical for informing and enhancing current best practice therapies and preventative action in the wound care field.

Vascular remodeling in the internal mammary artery graft and association with in situ endothelin-1 and receptor expression

BACKGROUND: The vasoconstricting peptide endothelin-1 (ET-1) has been associated with atherosclerotic cardiovascular disease, vascular smooth muscle cell (VSMC) growth stimulation, and intimal thickening. ET-1 binds 2 receptor subtypes, endothelin A and B, and the ETA receptor mediates vasoconstriction and VSMC growth. This study aims to quantitatively assess arterial remodeling variables and compare them with changes in ET-1, ETA, and ETB expression in the internal mammary artery (IMA). METHODS AND RESULTS: Specimens from 55 coronary artery disease (CAD) patients (45 men, 10 women; mean age 65 years) and 14 control IMA specimens (from 7 men and 7 women; mean age 45 years) were collected. IMA cross sections were assessed by histochemical and immunohistochemical staining methods to quantify the levels of medionecrosis, fibrosis, VSMC growth, ET-1, ETA, ETB, and macrophage infiltration. The percentage area of medionecrosis in the patients was almost double that in the controls (31.85+/-14.52% versus 17.10+/-9.96%, P=0.0006). Total and type 1 collagen was significantly increased compared with controls (65.8+/-18.3% versus 33.7+/-13.7%, P=0.07, and 14.2+/-10.0% versus 4.8+/-2.8%, P=0.01, respectively). Despite ACE and/or statin therapy, ET-1 expression and cell cycling were significantly elevated in the patient IMAs relative to the controls (46.27+/-18.46 versus 8.56+/-8.42, P=0.0001, and 37.29+/-12.88 versus 11.06+/-8.18, P=0.0001, respectively). ETA and ETB staining was elevated in the patient vessels (46.88+/-11.52% versus 18.58+/-7.65%, P=0.0001, and 42.98+/-7.08% versus 34.73+/-5.20%, P=0.0067, respectively). A mild presence of macrophages was noted in all sections. CONCLUSIONS: Elevated distribution of collagen indicative of fibrosis coupled with increased cell cycling and high levels of ET-1 and ETA expression in the absence of chronic inflammation suggests altered IMA VSMC regulation is fundamental to the remodeling process.

Fetuin-A : a major fetal serum protein that promotes "wound closure" and scarless healing [Letter to the Editor]

Burn-wound healing is a dynamic, interactive process involving a number of cellular and molecular events and is characterized by inflammation, granulation tissue formation, re-epithelialization, and tissue remodeling (Greenhalgh, 2002; Linares, 2002). Unlike incisional-wound healing, it also requires extensive re-epithelialization due to a predominant horizontal loss of tissue and often heals with abnormal scarring when burns involve deep dermis. The early mammalian fetus has the remarkable ability to regenerate normal epidermis and dermis and to heal dermal incisional wounds with no signs of scarring. Extensive research has indicated that scarless healing appears to be intrinsic to fetal skin (McCallion and Ferguson, 1996; Ferguson and O’Kane, 2004). Previously, we reported a fetal burn model, in which 80-day-old ovine fetuses (gestation¼ 145–153 days) healed deep dermal partial thickness burns without scars, whereas postnatal lambs healed equal depth burns with significant scarring (Cuttle et al., 2005; Fraser et al., 2005). This burn model provided early evidence that fetal skin has the capacity to repair and restore dermal horizontal loss, not just vertical injuries.

Mathematical investigation of the interactions between the inflammatory response and mechanical aspects of dermal wound repair

Dermal wound repair involves complex interactions between cells, cytokines and mechanics to close injuries to the skin. In particular, we investigate the contribution of fibroblasts, myofibroblasts, TGFβ, collagen and local tissue mechanics to wound repair in the human dermis. We develop a morphoelastic model where a realistic representation of tissue mechanics is key, and a fibrocontractive model that involves a reasonable approximation to the true kinetics of the important bioactive species. We use each of these descriptions to elucidate the mechanisms that generate pathologies such as hypertrophic scars, contractures and keloids. We find that for hypertrophic scar and contracture development, factors regulating the myofibroblast phenotype are critical, with heightened myofibroblast activation, reduced myofibroblast apoptosis or prolonged inflammation all predicted as mediators for scar hypertrophy and contractures. Prevention of these pathologies is predicted when myofibroblast apoptosis is induced, myofibroblast activation is blocked or TGFβ is neutralised. To investigate keloid invasion, we develop a caricature representation of the fibrocontractive model and find that TGFβ spread is the driving factor behind keloid growth. Blocking activation of TGFβ is found to cause keloid regression. Thus, we recommend myofibroblasts and TGFβ as targets for clinicians when developing intervention strategies for prevention and cure of fibrotic scars.

Effect of exercise training on skeletal muscle cytokine expression in the elderly

Aging is associated with increased circulating pro-inflammatory and lower anti-inflammatory cytokines. Exercise training, in addition to improving muscle function, reduces these circulating pro-inflammatory cytokines. Yet, few studies have evaluated changes in the expression of cytokines within skeletal muscle after exercise training. The aim of the current study was to examine the expression of cytokines both at rest and following a bout of isokinetic exercise performed before and after 12 weeks of resistance exercise training in young (n = 8, 20.3 ± 0.8 yr) and elderly men (n = 8, 66.9 ± 1.6 yr). Protein expression of various cytokines was determined in muscle homogenates. The expression of MCP-1, IL-8 and IL-6 (which are traditionally classified as ‘pro-inflammatory’) increased substantially after acute exercise. By contrast, the expression of the anti-inflammatory cytokines IL-4, IL-10 and IL-13 increased only slightly (or not at all) after acute exercise. These responses were not significantly different between young and elderly men, either before or after 12 weeks of exercise training. However, compared with the young men, the expression of pro-inflammatory cytokines 2 h post exercise tended to be greater in the elderly men prior to training. Training attenuated this difference. These data suggest that the inflammatory response to unaccustomed exercise increases with age. Furthermore, regular exercise training may help to normalize this inflammatory response, which could have important implications for muscle regeneration and adaptation in the elderly.

In vitro assessment of migratory behavior of two cell populations in a simple multichannel microdevice

Recent literature suggests that mesenchymal stem/stromal cells (MSC) could be used as Trojan Horses to deliver “death-signals” to cancer cells. Herein, we describe the development of a novel multichannel cell migration device, and use it to investigate the relative migration rates of bone marrow-derived MSC and breast cancer cells (MCF-7) towards each other. Confluent monolayers of MSC and MCF-7 were established in adjacent chambers separated by an array of 14 microchannels. Initially, culture chambers were isolated by air bubbles (air-valves) contained within each microchannel, and then bubbles were displaced to initiate the assay. The MCF-7 cells migrated preferentially towards MSC, whilst the MSC did not migrate preferentially towards the MCF-7 cells. Our results corroborate previous literature that suggests MSC migration towards cancer cells in vivo is in response to the associated inflammation rather than directly to signals secreted by the cancer cells themselves.

Toll-like receptor and pro-inflammatory cytokine expression during prolonged hyperinsulinaemia in horses : implications for laminitis

Equine laminitis, a disease of the lamellar structure of the horse’s hoof, can be incited by numerous factors that include inflammatory and metabolic aetiologies. However, the role of inflammation in hyperinsulinaemic laminitis has not been adequately defined. Tolllike receptor (TLR) activation results in up-regulation of inflammatory pathways and the release of pro-inflammatory cytokines, including interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-�), and may be a pathogenic factor in laminitis. The aim of this study was to determine whether TLR4 expression and subsequent pro-inflammatory cytokine production is increased in lamellae and skeletal muscle during equine hyperinsulinaemia. Standardbred horses were treated with either a prolonged, euglycaemic hyperinsulinaemic clamp (p-EHC) or a prolonged, glucose infusion (p-GI), which induced marked and moderate hyperinsulinaemia, respectively. Age-matched control horses were treated simultaneously with a balanced electrolyte solution. Treated horses developed clinical (p-EHC) or subclinical (p-GI) laminitis, whereas controls did not. Skeletal muscle and lamellar protein extracts were analysed by Western blotting for TLR4, IL-6, TNF-� and suppressor of cytokine signalling 3 (SOCS3) expression. Lamellar protein expression of TLR4 and TNF-�, but not IL-6, was increased by the p-EHC, compared to control horses. A significant positive correlation was found between lamellar TLR4 and SOCS3. Skeletal muscle protein expression of TLR4 signalling parameters did not differ between control and p-EHC-treated horses. Similarly, the p-GI did not result in up-regulation of lamellar protein expression of any parameter. The results suggest that insulin-sensitive tissues may not accurately reflect lamellar pathology during hyperinsulinaemia. While TLR4 is present in the lamellae, its activation appears unlikely to contribute significantly to the developmental pathogenesis of hyperinsulinaemic laminitis. However, inflammation may have a role to play in the later stages (e.g., repair or remodelling) of the disease.

Mechanical tension as a driver of connective tissue growth in vitro

We propose the progressive mechanical expansion of cell-derived tissue analogues as a novel, growth-based approach to in vitro tissue engineering. The prevailing approach to producing tissue in vitro is to culture cells in an exogenous “scaffold” that provides a basic structure and mechanical support. This necessarily pre-defines the final size of the implantable material, and specific signals must be provided to stimulate appropriate cell growth, differentiation and matrix formation. In contrast, surgical skin expansion, driven by increments of stretch, produces increasing quantities of tissue without trauma or inflammation. This suggests that connective tissue cells have the innate ability to produce growth in response to elevated tension. We posit that this capacity is maintained in vitro, and that order-of-magnitude growth may be similarly attained in self-assembling cultures of cells and their own extracellular matrix. The hypothesis that growth of connective tissue analogues can be induced by mechanical expansion in vitro may be divided into three components: (1) tension stimulates cell proliferation and extracellular matrix synthesis; (2) the corresponding volume increase will relax the tension imparted by a fixed displacement; (3) the repeated application of static stretch will produce sustained growth and a tissue structure adapted to the tensile loading. Connective tissues exist in a state of residual tension, which is actively maintained by resident cells such as fibroblasts. Studies in vitro and in vivo have demonstrated that cellular survival, reproduction, and matrix synthesis and degradation are regulated by the mechanical environment. Order-of-magnitude increases in both bone and skin volume have been achieved clinically through staged expansion protocols, demonstrating that tension-driven growth can be sustained over prolonged periods. Furthermore, cell-derived tissue analogues have demonstrated mechanically advantageous structural adaptation in response to applied loading. Together, these data suggest that a program of incremental stretch constitutes an appealing way to replicate tissue growth in cell culture, by harnessing the constituent cells’ innate mechanical responsiveness. In addition to offering a platform to study the growth and structural adaptation of connective tissues, tension-driven growth presents a novel approach to in vitro tissue engineering. Because the supporting structure is secreted and organised by the cells themselves, growth is not restricted by a “scaffold” of fixed size. This also minimises potential adverse reactions to exogenous materials upon implantation. Most importantly, we posit that the growth induced by progressive stretch will allow substantial volumes of connective tissue to be produced from relatively small initial cell numbers.

Numerical estimation of 3D mechanical forces exerted by cells on non-linear materials

The exchange of physical forces in both cell-cell and cell-matrix interactions play a significant role in a variety of physiological and pathological processes, such as cell migration, cancer metastasis, inflammation and wound healing. Therefore, great interest exists in accurately quantifying the forces that cells exert on their substrate during migration. Traction Force Microscopy (TFM) is the most widely used method for measuring cell traction forces. Several mathematical techniques have been developed to estimate forces from TFM experiments. However, certain simplifications are commonly assumed, such as linear elasticity of the materials and/or free geometries, which in some cases may lead to inaccurate results. Here, cellular forces are numerically estimated by solving a minimization problem that combines multiple non-linear FEM solutions. Our simulations, free from constraints on the geometrical and the mechanical conditions, show that forces are predicted with higher accuracy than when using the standard approaches.

Proteases : common culprits in human skin disorders

Recent findings from the clinic and the laboratory have transformed the way proteases and their inhibitors are perceived in the outermost layer of the skin, the epidermis. It now appears that an integrated proteolytic network operates within the epidermis, comprising more than 30 enzymes that carry out a growing list of essential functions. Equally, defective regulation or execution of protease-mediated processes is emerging as a key contributor to diverse human skin pathologies, and in recent years the number of diseases attributable to aberrant proteolytic activity has more than doubled. Here, we survey the different roles of proteases in epidermal homeostasis (from processing enzymes to signalling molecules) and explore the spectrum of rare and common human skin disorders where proteolytic pathways are dysregulated.

Proteases and proteomics : cutting to the core of human skin pathologies

Preserving the integrity of the skin's outermost layer (the epidermis) is vital for humans to thrive in hostile surroundings. Covering the entire body, the epidermis forms a thin but impenetrable cellular cordon that repels external assaults and blocks escape of water and electrolytes from within. This structure exists in a perpetual state of regeneration where the production of new cellular subunits at the base of the epidermis is offset by the release of terminally differentiated corneocytes from the surface. It is becoming increasingly clear that proteases hold vital roles in assembling and maintaining the epidermal barrier. More than 30 proteases are expressed by keratinocytes or infiltrating immune cells and the activity of each must be maintained within narrow limits and confined to the correct time and place. Accordingly, over- or under-exertion of proteolytic activity is a common factor in a multitude of skin disorders that range in severity from relatively mild to life-threatening. This review explores the current state of knowledge on the involvement of proteases in skin diseases and the latest findings from proteomic and transcriptomic studies focused on uncovering novel (patho)physiological roles for these enzymes.

Antibodies mediate formation of neutrophil extracellular traps in the middle ear and facilitate secondary pneumococcal otitis media

Otitis media (OM) (a middle ear infection) is a common childhood illness that can leave some children with permanent hearing loss. OM can arise following infection with a variety of different pathogens, including a coinfection with influenza A virus (IAV) and Streptococcus pneumoniae (the pneumococcus). We and others have demonstrated that coinfection with IAV facilitates the replication of pneumococci in the middle ear. Specifically, we used a mouse model of OM to show that IAV facilitates the outgrowth of S. pneumoniae in the middle ear by inducing middle ear inflammation. Here, we seek to understand how the host inflammatory response facilitates bacterial outgrowth in the middle ear. Using B cell-deficient infant mice, we show that antibodies play a crucial role in facilitating pneumococcal replication. We subsequently show that this is due to antibody-dependent neutrophil extracellular trap (NET) formation in the middle ear, which, instead of clearing the infection, allows the bacteria to replicate. We further demonstrate the importance of these NETs as a potential therapeutic target through the transtympanic administration of a DNase, which effectively reduces the bacterial load in the middle ear. Taken together, these data provide novel insight into how pneumococci are able to replicate in the middle ear cavity and induce disease.

Tailored interventions based on sputum eosinophils versus clinical symptoms for asthma in children and adults

BACKGROUND Asthma severity and control can be measured both subjectively and objectively. Sputum analysis for evaluation of percentage of sputum eosinophilia directly measures airway inflammation, and is one method of objectively monitoring asthma. Interventions for asthma therapies have been traditionally based on symptoms and spirometry. OBJECTIVES To evaluate the efficacy of tailoring asthma interventions based on sputum analysis in comparison to clinical symptoms (with or without spirometry/peak flow) for asthma related outcomes in children and adults. SEARCH STRATEGY We searched the Cochrane Airways Group Specialised Register of Trials, the Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE, EMBASE and reference lists of articles. The last search was on 31 October 2006. SELECTION CRITERIA All randomised controlled comparisons of adjustment of asthma therapy based on sputum eosinophils compared to traditional methods (primarily clinical symptoms and spirometry/peak flow). DATA COLLECTION AND ANALYSIS Results of searches were reviewed against pre-determined criteria for inclusion. Three sets of reviewers selected relevant studies.Two review authors independently assessed trial quality extracted data. Authors were contacted for further information but none were received. Data was analysed as "treatment received" and sensitivity analyses performed. MAIN RESULTS Three adult studies were included; these studies were clinically and methodologically heterogenous (use of medications, cut off for percentage of sputum eosinophils and definition of asthma exacerbation). There were no eligible paediatric studies. Of 246 participants randomised, 221 completed the trials. In the meta-analysis, a significant reduction in number of participants who had one or more asthma exacerbations occurred when treatment was based on sputum eosinophils in comparison to clinical symptoms; pooled odds ratio (OR) was 0.49 (95% CI 0.28 to 0.87); number needed to treat to benefit (NNTB) was 6 (95% CI 4 to 32).There were also differences between groups in the rate of exacerbation (any exacerbation per year) and severity of exacerbations defined by requirement for use of oral corticosteroids but the reduction in hospitalisations was not statistically significant. Data for clinical symptoms, quality of life and spirometry were not significantly different between groups. The mean dose of inhaled corticosteroids per day was similar in both groups and no adverse events were reported. However sputum induction was not always possible. AUTHORS' CONCLUSIONS Tailored asthma interventions based on sputum eosinophils is beneficial in reducing the frequency of asthma exacerbations in adults with asthma. This review supports the use of sputum eosinophils to tailor asthma therapy for adults with frequent exacerbations and severe asthma. Further studies need to be undertaken to strengthen these results and no conclusion can be drawn for children with asthma.

Tailored interventions based on exhaled nitric oxide versus clinical symptoms for asthma in children and adults

Background The measurement of severity and control of asthma in both children and adults can be based on subjective or objective measures. It has been advocated that fractional exhaled nitric oxide (FeNO) can be used to monitor airway inflammation as it correlates with some markers of asthma. Interventions for asthma therapies have been traditionally based on symptoms and/or spirometry. Objectives To evaluate the efficacy of tailoring asthma interventions based on exhaled nitric oxide in comparison to clinical symptoms (with or without spirometry/peak flow) for asthma related outcomes in children and adults. Search methods We searched the Cochrane Airways Group Specialised Register of Trials, the Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE, EMBASE and reference lists of articles. The last search was completed in February 2009. Selection criteria All randomised controlled comparisons of adjustment of asthma therapy based on exhaled nitric oxide compared to traditional methods (primarily clinical symptoms and spirometry/peak flow). Data collection and analysis Results of searches were reviewed against pre-determined criteria for inclusion. Relevant studies were independently selected in duplicate. Two authors independently assessed trial quality and extracted data. Authors were contacted for further information with response from one. Main results Two studies have been added for this update, which now includes six (2 adults and 4 children/adolescent) studies; these studies differed in a variety of ways including definition of asthma exacerbations, FeNO cut off levels, the way in which FeNO was used to adjust therapy and duration of study. Of 1053 participants randomised, 1010 completed the trials. In the meta-analysis, there was no significant difference between groups for the primary outcome of asthma exacerbations or for other outcomes (clinical symptoms, FeNO level and spirometry). In post-hoc analysis, a significant reduction in mean final daily dose inhaled corticosteroid per adult was found in the group where treatment was based on FeNO in comparison to clinical symptoms, (mean difference -450 mcg; 95% CI -677 to -223 mcg budesonide equivalent/day). However, the total amount of inhaled corticosteroid used in one of the adult studies was 11% greater in the FeNO arm. In contrast, in the paediatric studies, there was a significant increase in inhaled corticosteroid dose in the FeNO strategy arm (mean difference of 140 mcg; 95% CI 29 to 251, mcg budesonide equivalent/day). Authors' conclusions Tailoring the dose of inhaled corticosteroids based on exhaled nitric oxide in comparison to clinical symptoms was carried out in different ways in the six studies and found only modest benefit at best and potentially higher doses of inhaled corticosteroids in children. The role of utilising exhaled nitric oxide to tailor the dose of inhaled corticosteroids cannot be routinely recommended for clinical practice at this stage and remains uncertain.