Therapeutic Improvement of Scarring: Mechanisms of Scarless and Scar-Forming Healing and Approaches to the Discovery of New Treatments

Scarring in the skin after trauma, surgery, burn or sports injury is a major medical problem, often resulting in loss of function, restriction of tissue movement and adverse psychological effects. Whilst various studies have utilised a range of model systems that have increased our understanding of the pathways and processes underlying scar formation, they have typically not translated to the development of effective therapeutic approaches for scar management. Existing treatments are unreliable and unpredictable and there are no prescription drugs for the prevention or treatment of dermal scarring. As a consequence, scar improvement still remains an area of clear medical need. Here we describe the basic science of scar-free and scar-forming healing, the utility of pre-clinical model systems, their translation to humans, and our pioneering approach to the discovery and development of therapeutic approaches for the prophylactic improvement of scarring in ma

Human umbilical cord perivascular cells improve human pancreatic islet transplant function by increasing vascularization

Islet transplantation is an efficacious therapy for type 1 diabetes; however, islets from multiple donor pancreata are required, and a gradual attrition in transplant function is seen. Here, we manufactured human umbilical cord perivascular mesenchymal stromal cells (HUCPVCs) to Good Manufacturing Practice (GMP) standards. HUCPVCs showed a stable phenotype while undergoing rapid ex vivo expansion at passage 2 (p2) to passage 4 (p4) and produced proregenerative factors, strongly suppressing T cell responses in the resting state and in response to inflammation. Transplanting an islet equivalent (IEQ):HUCPVC ratio of 1:30 under the kidney capsule in diabetic NSG mice demonstrated the fastest return to normoglycemia by 3 days after transplant: Superior glycemic control was seen at both early (2.7 weeks) and later stages (7, 12, and 16 weeks) versus ratios of 1:0, 1:10, and 1:50, respectively. Syngeneic islet transplantation in immunocompetent mice using the clinically relevant hepatic portal route with a marginal islet mass showed that mice transplanted with an IEQ:HUCPVC ratio of 1:150 had superior glycemic control versus ratios of 1:0, 1:90, and 1:210 up to 6 weeks after transplant. Immunodeficient mice transplanted with human islets (IEQ:HUCPVC ratio of 1:150) exhibited better glycemic control for 7 weeks after transplant versus islet transplant alone, and islets transplanted via the hepatic portal vein in an allogeneic mouse model using a curative islet mass demonstrated delayed rejection of islets when cotransplanted with HUCPVCs (IEQ:HUCPVC ratio of 1:150). The immunosuppressive and proregenerative properties of HUCPVCs demonstrated long-term positive effects on graft function in vivo, indicating that they may improve long-term human islet allotransplantation outcomes

Long-lasting blood pressure lowering effects of nitrite are NO-independent and mediated by hydrogen peroxide, persulfides, and oxidation of protein kinase G1α redox signalling

Aims Under hypoxic conditions, nitrite (NO2-) can be reduced to nitric oxide (NO) eliciting vasorelaxation. However, nitrite also exerts vasorelaxant effects of potential therapeutic relevance under normal physiological conditions via undetermined mechanisms. We, therefore, sought to investigate the mechanism(s) by which nitrite regulates the vascular system in normoxia and, specifically, whether the biological effects are a result of NO generation (as in hypoxia) or mediated via alternative mechanisms involving classical downstream targets of NO [e.g. effects on protein kinase G1 alpha (PKG1 alpha)]. Methods and results Ex vivo myography revealed that, unlike in thoracic aorta (conduit vessels), the vasorelaxant effects of nitrite in mesenteric resistance vessels from wild-type (WT) mice were NO-independent. Oxidants such as H2O2 promote disulfide formation of PKG1 alpha, resulting in NO- cyclic guanosine monophosphate (cGMP) independent kinase activation. To explore whether the microvascular effects of nitrite were associated with PKG1 alpha oxidation, we used a Cys42Ser PKG1 alpha knock-in (C42S PKG1 alpha KI; 'redox-dead') mouse that cannot transduce oxidant signals. Resistance vessels from these C42S PKG1 alpha KI mice were markedly less responsive to nitrite-induced vasodilation. Intraperitoneal (i.p.) bolus application of nitrite in conscious WT mice induced a rapid yet transient increase in plasma nitrite and cGMP concentrations followed by prolonged hypotensive effects, as assessed using in vivo telemetry. In the C42S PKG1 alpha KI mice, the blood pressure lowering effects of nitrite were lower compared to WT. Increased H2O2 concentrations were detected in WT resistance vessel tissue challenged with nitrite. Consistent with this, increased cysteine and glutathione persulfide levels were detected in these vessels by mass spectrometry, matching the temporal profile of nitrite's effects on H2O2 and blood pressure. Conclusion Under physiological conditions, nitrite induces a delayed and long-lasting blood pressure lowering effect, which is NO-independent and occurs via a new redox mechanism involving H2O2, persulfides, and PKG1 alpha oxidation/activation. Targeting this novel pathway may provide new prospects for anti-hypertensive therapy

An extended set of PRDM1/BLIMP1 target genes links binding motif type to dynamic repression

The transcriptional repressor B lymphocyte-induced maturation protein-1 (BLIMP1) regulates gene expression and cell fate. The DNA motif bound by BLIMP1 in vitro overlaps with that of interferon regulatory factors (IRFs), which respond to inflammatory/immune signals. At such sites, BLIMP1 and IRFs can antagonistically regulate promoter activity. In vitro motif selection predicts that only a subset of BLIMP1 or IRF sites is subject to antagonistic regulation, but the extent to which antagonism occurs is unknown, since an unbiased assessment of BLIMP1 occupancy in vivo is lacking. To address this, we identified an extended set of promoters occupied by BLIMP1. Motif discovery and enrichment analysis demonstrate that multiple motif variants are required to capture BLIMP1 binding specificity. These are differentially associated with CpG content, leading to the observation that BLIMP1 DNA-binding is methylation sensitive. In occupied promoters, only a subset of BLIMP1 motifs overlap with IRF motifs. Conversely, a distinct subset of IRF motifs is not enriched amongst occupied promoters. Genes linked to occupied promoters containing overlapping BLIMP1/IRF motifs (e.g. AIM2, SP110, BTN3A3) are shown to constitute a dynamic target set which is preferentially activated by BLIMP1 knock-down. These data confirm and extend the competitive model of BLIMP1 and IRF interaction

VEGF-A isoforms differentially regulate ATF-2-dependent VCAM-1 gene expression and endothelial-leukocyte interactions

Vascular endothelial growth factor A (VEGF-A) regulates many aspects of vascular physiology. VEGF-A stimulates signal transduction pathways that modulate endothelial outputs such as cell migration, proliferation, tubulogenesis, and cell-cell interactions. Multiple VEGF-A isoforms exist, but the biological significance of this is unclear. Here we analyzed VEGF-A isoform-specific stimulation of VCAM-1 gene expression, which controls endothelial-leukocyte interactions, and show that this is dependent on both ERK1/2 and activating transcription factor-2 (ATF-2). VEGF-A isoforms showed differential ERK1/2 and p38 MAPK phosphorylation kinetics. A key feature of VEGF-A isoform-specific ERK1/2 activation and nuclear translocation was increased phosphorylation of ATF-2 on threonine residue 71 (T71). Using reverse genetics, we showed ATF-2 to be functionally required for VEGF-A-stimulated endothelial VCAM-1 gene expression. ATF-2 knockdown blocked VEGF-A-stimulated VCAM-1 expression and endothelial-leukocyte interactions. ATF-2 was also required for other endothelial cell outputs, such as cell migration and tubulogenesis. In contrast, VCAM-1 was essential only for promoting endothelial-leukocyte interactions. This work presents a new paradigm for understanding how soluble growth factor isoforms program complex cellular outputs and responses by modulating signal transduction pathways

Effects of fluoxetine on functional outcomes after acute stroke (FOCUS): a pragmatic, double-blind, randomised, controlled trial

Background Results of small trials indicate that fluoxetine might improve functional outcomes after stroke. The FOCUS trial aimed to provide a precise estimate of these effects. Methods FOCUS was a pragmatic, multicentre, parallel group, double-blind, randomised, placebo-controlled trial done at 103 hospitals in the UK. Patients were eligible if they were aged 18 years or older, had a clinical stroke diagnosis, were enrolled and randomly assigned between 2 days and 15 days after onset, and had focal neurological deficits. Patients were randomly allocated fluoxetine 20 mg or matching placebo orally once daily for 6 months via a web-based system by use of a minimisation algorithm. The primary outcome was functional status, measured with the modified Rankin Scale (mRS), at 6 months. Patients, carers, health-care staff, and the trial team were masked to treatment allocation. Functional status was assessed at 6 months and 12 months after randomisation. Patients were analysed according to their treatment allocation. This trial is registered with the ISRCTN registry, number ISRCTN83290762. Findings Between Sept 10, 2012, and March 31, 2017, 3127 patients were recruited. 1564 patients were allocated fluoxetine and 1563 allocated placebo. mRS data at 6 months were available for 1553 (99·3%) patients in each treatment group. The distribution across mRS categories at 6 months was similar in the fluoxetine and placebo groups (common odds ratio adjusted for minimisation variables 0·951 [95% CI 0·839–1·079]; p=0·439). Patients allocated fluoxetine were less likely than those allocated placebo to develop new depression by 6 months (210 [13·43%] patients vs 269 [17·21%]; difference 3·78% [95% CI 1·26–6·30]; p=0·0033), but they had more bone fractures (45 [2·88%] vs 23 [1·47%]; difference 1·41% [95% CI 0·38–2·43]; p=0·0070). There were no significant differences in any other event at 6 or 12 months. Interpretation Fluoxetine 20 mg given daily for 6 months after acute stroke does not seem to improve functional outcomes. Although the treatment reduced the occurrence of depression, it increased the frequency of bone fractures. These results do not support the routine use of fluoxetine either for the prevention of post-stroke depression or to promote recovery of function. Funding UK Stroke Association and NIHR Health Technology Assessment Programme

Abstracts from the 8th International Conference on cGMP Generators, Effectors and Therapeutic Implications

This work was supported by a restricted research grant of Bayer AG

Duration of androgen deprivation therapy with postoperative radiotherapy for prostate cancer: a comparison of long-course versus short-course androgen deprivation therapy in the RADICALS-HD randomised trial

Background Previous evidence supports androgen deprivation therapy (ADT) with primary radiotherapy as initial treatment for intermediate-risk and high-risk localised prostate cancer. However, the use and optimal duration of ADT with postoperative radiotherapy after radical prostatectomy remains uncertain. Methods RADICALS-HD was a randomised controlled trial of ADT duration within the RADICALS protocol. Here, we report on the comparison of short-course versus long-course ADT. Key eligibility criteria were indication for radiotherapy after previous radical prostatectomy for prostate cancer, prostate-specific antigen less than 5 ng/mL, absence of metastatic disease, and written consent. Participants were randomly assigned (1:1) to add 6 months of ADT (short-course ADT) or 24 months of ADT (long-course ADT) to radiotherapy, using subcutaneous gonadotrophin-releasing hormone analogue (monthly in the short-course ADT group and 3-monthly in the long-course ADT group), daily oral bicalutamide monotherapy 150 mg, or monthly subcutaneous degarelix. Randomisation was done centrally through minimisation with a random element, stratified by Gleason score, positive margins, radiotherapy timing, planned radiotherapy schedule, and planned type of ADT, in a computerised system. The allocated treatment was not masked. The primary outcome measure was metastasis-free survival, defined as metastasis arising from prostate cancer or death from any cause. The comparison had more than 80% power with two-sided α of 5% to detect an absolute increase in 10-year metastasis-free survival from 75% to 81% (hazard ratio [HR] 0·72). Standard time-to-event analyses were used. Analyses followed intention-to-treat principle. The trial is registered with the ISRCTN registry, ISRCTN40814031, and ClinicalTrials.gov , NCT00541047 . Findings Between Jan 30, 2008, and July 7, 2015, 1523 patients (median age 65 years, IQR 60–69) were randomly assigned to receive short-course ADT (n=761) or long-course ADT (n=762) in addition to postoperative radiotherapy at 138 centres in Canada, Denmark, Ireland, and the UK. With a median follow-up of 8·9 years (7·0–10·0), 313 metastasis-free survival events were reported overall (174 in the short-course ADT group and 139 in the long-course ADT group; HR 0·773 [95% CI 0·612–0·975]; p=0·029). 10-year metastasis-free survival was 71·9% (95% CI 67·6–75·7) in the short-course ADT group and 78·1% (74·2–81·5) in the long-course ADT group. Toxicity of grade 3 or higher was reported for 105 (14%) of 753 participants in the short-course ADT group and 142 (19%) of 757 participants in the long-course ADT group (p=0·025), with no treatment-related deaths. Interpretation Compared with adding 6 months of ADT, adding 24 months of ADT improved metastasis-free survival in people receiving postoperative radiotherapy. For individuals who can accept the additional duration of adverse effects, long-course ADT should be offered with postoperative radiotherapy. Funding Cancer Research UK, UK Research and Innovation (formerly Medical Research Council), and Canadian Cancer Society

Adding 6 months of androgen deprivation therapy to postoperative radiotherapy for prostate cancer: a comparison of short-course versus no androgen deprivation therapy in the RADICALS-HD randomised controlled trial

Background Previous evidence indicates that adjuvant, short-course androgen deprivation therapy (ADT) improves metastasis-free survival when given with primary radiotherapy for intermediate-risk and high-risk localised prostate cancer. However, the value of ADT with postoperative radiotherapy after radical prostatectomy is unclear. Methods RADICALS-HD was an international randomised controlled trial to test the efficacy of ADT used in combination with postoperative radiotherapy for prostate cancer. Key eligibility criteria were indication for radiotherapy after radical prostatectomy for prostate cancer, prostate-specific antigen less than 5 ng/mL, absence of metastatic disease, and written consent. Participants were randomly assigned (1:1) to radiotherapy alone (no ADT) or radiotherapy with 6 months of ADT (short-course ADT), using monthly subcutaneous gonadotropin-releasing hormone analogue injections, daily oral bicalutamide monotherapy 150 mg, or monthly subcutaneous degarelix. Randomisation was done centrally through minimisation with a random element, stratified by Gleason score, positive margins, radiotherapy timing, planned radiotherapy schedule, and planned type of ADT, in a computerised system. The allocated treatment was not masked. The primary outcome measure was metastasis-free survival, defined as distant metastasis arising from prostate cancer or death from any cause. Standard survival analysis methods were used, accounting for randomisation stratification factors. The trial had 80% power with two-sided α of 5% to detect an absolute increase in 10-year metastasis-free survival from 80% to 86% (hazard ratio [HR] 0·67). Analyses followed the intention-to-treat principle. The trial is registered with the ISRCTN registry, ISRCTN40814031, and ClinicalTrials.gov, NCT00541047. Findings Between Nov 22, 2007, and June 29, 2015, 1480 patients (median age 66 years [IQR 61–69]) were randomly assigned to receive no ADT (n=737) or short-course ADT (n=743) in addition to postoperative radiotherapy at 121 centres in Canada, Denmark, Ireland, and the UK. With a median follow-up of 9·0 years (IQR 7·1–10·1), metastasis-free survival events were reported for 268 participants (142 in the no ADT group and 126 in the short-course ADT group; HR 0·886 [95% CI 0·688–1·140], p=0·35). 10-year metastasis-free survival was 79·2% (95% CI 75·4–82·5) in the no ADT group and 80·4% (76·6–83·6) in the short-course ADT group. Toxicity of grade 3 or higher was reported for 121 (17%) of 737 participants in the no ADT group and 100 (14%) of 743 in the short-course ADT group (p=0·15), with no treatment-related deaths. Interpretation Metastatic disease is uncommon following postoperative bed radiotherapy after radical prostatectomy. Adding 6 months of ADT to this radiotherapy did not improve metastasis-free survival compared with no ADT. These findings do not support the use of short-course ADT with postoperative radiotherapy in this patient population

Predicting protein interaction sites: binding hot-spots in protein-protein and protein-ligand interfaces

MOTIVATION: Protein assemblies are currently poorly represented in structural databases and their structural elucidation is a key goal in biology. Here we analyse clefts in protein surfaces, likely to correspond to binding “hot-spots”, and rank them according to sequence conservation and simple measures of physical properties including hydrophobicity, desolvation, electrostatics and van der Waals energies, to predict which are involved in binding in the native complex. RESULTS: The resulting differences between predicting binding-sites at protein-protein and protein-ligand interfaces are striking. There is a high level of prediction accuracy (C93%) for protein-ligand interactions, based on the following attributes: van der Waals potential, electrostatic potential, desolvation and surface conservation. Generally, the prediction accuracy for protein-protein interactions is lower, with the exception of enzymes. Our results show that the ease of cleft desolvation or “de-wetting ” is strongly predictive of interfaces and strongly maintained across all classes of protein binding interface