A Dual-Acting Nitric Oxide Donor and Phosphodiesterase 5 Inhibitor Promotes Wound Healing in Normal Mice and Mice with Diabetes.

Chronic wounds affect a large percentage of the population worldwide and cause significant morbidity. Unfortunately, efficient compounds for the treatment of chronic wounds are yet not available. Endothelial dysfunction, which is at least in part a result of compromised nitric oxide production and concomitant reduction in cGMP levels, is a major pathologic feature of chronic wounds. Therefore, we designed and synthesized a compound with a unique dual-acting activity (TOP-N53), acting as a nitric oxide donor and phosphodiesterase 5 inhibitor, and applied it locally to full-thickness skin wounds in healthy and healing-impaired mice with diabetes. TOP-N53 promoted keratinocyte proliferation, angiogenesis, and collagen maturation in healthy mice without accelerating the wound inflammatory response or scar formation. Most importantly, it partially rescued the healing impairment of mice with genetically determined type II diabetes (db/db) by stimulating re-epithelialization and granulation tissue formation, including angiogenesis. In vitro studies with human and murine primary cells showed a positive effect of TOP-N53 on keratinocyte and fibroblast migration, keratinocyte proliferation, and endothelial cell migration and tube formation. These results demonstrate a remarkable healing-promoting activity of TOP-N53 by targeting the major resident cells in the wound tissue.

Comprehensive characterization of myeloid cells during wound healing in healthy and healing-impaired diabetic mice.

Wound healing involves the concerted action of various lymphoid and in particular myeloid cell populations. To characterize and quantitate different types of myeloid cells and to obtain information on their kinetics during wound healing, we performed multiparametric flow cytometry analysis. In healthy mice, neutrophil numbers increased early after injury and returned to near basal levels after completion of healing. Macrophages, monocyte-derived dendritic cells (DCs), and eosinophils were abundant throughout the healing phase, in particular in early wounds, and Langerhans cells increased after wounding and remained elevated after epithelial closure. Major differences in healing-impaired diabetic mice were a much higher percentage of immune cells in late wounds, mainly as a result of neutrophil, macrophage, and monocyte persistence; reduced numbers and percentages of macrophages and monocyte-derived DCs in early wounds; and of Langerhans cells, conventional DCs, and eosinophils throughout the healing process. Finally, unbiased cluster analysis (PhenoGraph) identified a large number of different clusters of myeloid cells in skin wounds. These results provide insight into myeloid cell diversity and dynamics during wound repair and highlight the abnormal inflammatory response associated with impaired healing.

Regulatory T cells are required for normal and activin-promoted wound repair in mice.

Healing of skin wounds is orchestrated by various types of immune cells, but little is known about the role of FoxP3+ regulatory T cells (Tregs) in this process. Here, we determined if Tregs are important for wound healing in normal mice and if they contribute to the accelerated healing of mice overexpressing the growth and differentiation factor activin. Diphtheria toxin induced Treg depletion prior to injury caused impaired healing characterized by delayed reepithelialization, reduced wound contraction, and impaired vessel maturation. The accelerated wound repair of activin-transgenic mice was also abrogated. Mechanistically, we found a strong increase in IL-4 levels combined with overrepresentation of T-bet+ and GATA-3+ αß T cells in Treg-depleted 7-day wounds. In addition, numbers of IFN-γ- or IL-17A-producing CD4+ and CD4- T cells were elevated. These results demonstrate that Treg depletion in wounds facilitates the expansion of an αß T-cell population with features of Th1 and Th2 cells, and suggest that concomitant changes in the cytokine milieu disturb the healing process.

The impact of enhanced recovery after surgery (ERAS) protocol compliance on morbidity from resection for primary lung cancer.

OBJECTIVE: The adoption of Enhanced Recovery After Surgery programs in thoracic surgery is relatively recent with limited outcome data. This study aimed to determine the impact of an Enhanced Recovery After Surgery pathway on morbidity and length of stay in patients undergoing lung resection for primary lung cancer. METHODS: This prospective cohort study collected data on consecutive patients undergoing lung resection for primary lung cancer between April 2012 and June 2014 at a regional referral center in the United Kingdom. All patients followed a standardized, 15-element Enhanced Recovery After Surgery protocol. Key data fields included protocol compliance with individual elements, pathophysiology, and operative factors. Thirty-day morbidity was taken as the primary outcome measure and classified a priori according to the Clavien-Dindo system. Logistic regression models were devised to identify independent risk factors for morbidity and length of stay. RESULTS: A total of 422 consecutive patients underwent lung resection over a 2-year period, of whom 302 (71.6%) underwent video-assisted thoracoscopic surgery. Lobectomy was performed in 297 patients (70.4%). Complications were experienced by 159 patients (37.6%). The median length of stay was 5 days (range, 1-67), and 6 patients (1.4%) died within 30 days of surgery. There was a significant inverse relationship between protocol compliance and morbidity after adjustment for confounding factors (odds ratio, 0.72; 95% confidence interval, 0.57-0.91; P < .01). Age, lobectomy or pneumonectomy, more than 1 resection, and delayed mobilization were independent predictors of morbidity. Age, lack of preoperative carbohydrate drinks, planned high dependency unit/intensive therapy unit admission, delayed mobilization, and open approach were independent predictors of delayed discharge (length of stay >5 days). CONCLUSIONS: Increased compliance with an Enhanced Recovery After Surgery pathway is associated with improved clinical outcomes after resection for primary lung cancer. Several elements, including early mobilization, appear to be more influential than others.

Pathological lymph node involvement is not a predictor of adverse outcomes in patients undergoing thoracoscopic lobectomy for lung cancer†.

OBJECTIVES: As the practice of video-assisted thoracoscopic surgery (VATS) lobectomy gains widespread acceptance, the complexity of procedures attempted increases and the stage of tumour that may be safely approached remains controversial. We examined the impact of nodal involvement with respect to perioperative outcomes after VATS lobectomy. METHODS: All patients listed for VATS lobectomy for non-small-cell lung cancer at our institution from 2012 to 2016 were analysed. Bronchoplastic or chest wall resections and tumours over 7 cm were considered a contraindication to a thoracoscopic approach. RESULTS: Of the 489 patients identified, 97 (19.8%) patients had pathological nodal involvement. The overall conversion rate was 6.1%, reoperation rate was 5.3% and readmission rate was 5.9%. Median hospital stay was 5 days, 30-day mortality was 0.6% and 90-day mortality was 1.6%. No significant difference was identified between the nodal-negative or -positive groups in terms of preoperative demographics, hospital stay, postoperative complications, conversion rate, reoperation rate or readmission rate. Univariate logistic regression identified gender, Thoracoscore, dyspnoea score, performance status, chronic obstructive pulmonary disease, previous stroke, preoperative lung function and non-adenocarcinoma as predictors of postoperative complications. A multivariate model including nodal status identified Thoracoscore (odds ratio 1.57, 95% confidence interval 1.16-2.18; P < 0.001) and preoperative transfer factor (odds ratio 0.97, 95% confidence interval 0.96-0.98; P < 0.001) as the only predictors of complications. CONCLUSIONS: In non-small-cell lung cancer patients with pathological hilar or mediastinal lymph node involvement, VATS lobectomy can be safely performed, as there does not appear to be an adverse effect on the incidence of perioperative complications, length of stay or readmissions.

Nrf2 is highly expressed in neutrophils, but myeloid cell-derived Nrf2 is dispensable for wound healing in mice.

Immune cells of the myeloid lineage are key players in skin wound healing, since they secrete various cytokines and growth factors that orchestrate the repair process. In addition, they are crucial for the defense against invading pathogens through their capacity to produce high levels of reactive oxygen species (ROS). To limit the toxicity of ROS, cells have developed antioxidant defense strategies, including expression of the cytoprotective NRF2 transcription factor. Here we show that murine neutrophils and to a lesser extent macrophages strongly express Nrf2 already when present in the circulation and in particular at the wound site. To determine the role of Nrf2 in neutrophils and macrophages for wound repair, we generated mice with a gain- or loss-of-function of this transcription factor in the myeloid cell lineage. Expression of a constitutively active Nrf2 mutant in myeloid cells did not further enhance the overall Nrf2 activity in these cells due to the already high steady-state activity of endogenous Nrf2. Surprisingly, deletion of Nrf2 in myeloid cells only mildly affected the levels of ROS and the expression of pro-inflammatory cytokines by these cells. In particular, various parameters of wound healing, including wound closure, reepithelialization, wound contraction and the presence of myeloid cells at the wound site were not affected. These results reveal that Nrf2 in myeloid cells is dispensable for wound healing and suggest the presence of additional antioxidant defense strategies of these cells that compensate for the loss of Nrf2, even in the harsh environment of skin wounds.

Lung inflammation promotes metastasis through neutrophil protease-mediated degradation of Tsp-1.

Inflammation is inextricably associated with primary tumor progression. However, the contribution of inflammation to tumor outgrowth in metastatic organs has remained underexplored. Here, we show that extrinsic inflammation in the lungs leads to the recruitment of bone marrow-derived neutrophils, which degranulate azurophilic granules to release the Ser proteases, elastase and cathepsin G, resulting in the proteolytic destruction of the antitumorigenic factor thrombospondin-1 (Tsp-1). Genetic ablation of these neutrophil proteases protected Tsp-1 from degradation and suppressed lung metastasis. These results provide mechanistic insights into the contribution of inflammatory neutrophils to metastasis and highlight the unique neutrophil protease-Tsp-1 axis as a potential antimetastatic therapeutic target.

Bone marrow-derived Gr1+ cells can generate a metastasis-resistant microenvironment via induced secretion of thrombospondin-1.

UNLABELLED: Metastatic tumors have been shown to establish permissive microenvironments for metastases via recruitment of bone marrow-derived cells. Here, we show that metastasis-incompetent tumors are also capable of generating such microenvironments. However, in these situations, the otherwise prometastatic Gr1(+) myeloid cells create a metastasis-refractory microenvironment via the induction of thrombospondin-1 (Tsp-1) by tumor-secreted prosaposin. Bone marrow-specific genetic deletion of Tsp-1 abolished the inhibition of metastasis, which was restored by bone marrow transplant from Tsp-1(+) donors. We also developed a 5-amino acid peptide from prosaposin as a pharmacologic inducer of Tsp-1 in Gr1(+) bone marrow cells, which dramatically suppressed metastasis. These results provide mechanistic insights into why certain tumors are deficient in metastatic potential and implicate recruited Gr1(+) myeloid cells as the main source of Tsp-1. The results underscore the plasticity of Gr1(+) cells, which, depending on the context, promote or inhibit metastasis, and suggest that the peptide could be a potential therapeutic agent against metastatic cancer. SIGNIFICANCE: The mechanisms of metastasis suppression are poorly understood. Here, we have identified a novel mechanism whereby metastasis-incompetent tumors generate metastasis-suppressive microenvironments in distant organs by inducing Tsp-1 expression in the bone marrow­derived Gr1+myeloid cells. A 5-amino acid peptide with Tsp-1­inducing activity was identified as a therapeutic agent against metastatic cancer.

Suppression of miRNA-708 by polycomb group promotes metastases by calcium-induced cell migration.

The progression of cancer to metastatic disease is a major cause of death. We identified miR-708 being transcriptionally repressed by polycomb repressor complex 2-induced H3K27 trimethylation in metastatic breast cancer. miR-708 targets the endoplasmic reticulum protein neuronatin to decrease intracellular calcium level, resulting in reduction of activation of ERK and FAK, decreased cell migration, and impaired metastases. Ectopic expression of neuronatin refractory to suppression by miR-708 rescued cell migration and metastasis defects. In patients with breast cancer, miR-708 expression was decreased in lymph node and distal metastases, suggesting a metastasis-suppressive role. Our findings uncover a mechanistic role for miR-708 in metastasis and provide a rationale for developing miR-708 as a therapeutic agent against metastatic breast cancer.

Myeloid progenitor cells in the premetastatic lung promote metastases by inducing mesenchymal to epithelial transition.

Tumors systemically initiate metastatic niches in distant target metastatic organs. These niches, composed of bone marrow-derived hematopoietic cells, provide permissive conditions for future metastases. However, the mechanisms by which these cells mediate outgrowth of metastatic tumor cells are not completely known. Using mouse models of spontaneous breast cancer, we show enhanced recruitment of bone marrow-derived CD11b(+)Gr1(+) myeloid progenitor cells in the premetastatic lungs. Gene expression profiling revealed that the myeloid cells from metastatic lungs express versican, an extracellular matrix proteoglycan. Notably, versican in metastatic lungs was mainly contributed by the CD11b(+)Ly6C(high) monocytic fraction of the myeloid cells and not the tumor cells or other stromal cells. Versican knockdown in the bone marrow significantly impaired lung metastases in vivo, without impacting their recruitment to the lungs or altering the immune microenvironment. Versican stimulated mesenchymal to epithelial transition of metastatic tumor cells by attenuating phospho-Smad2 levels, which resulted in elevated cell proliferation and accelerated metastases. Analysis of clinical specimens showed elevated versican expression within the metastatic lung of patients with breast cancer. Together, our findings suggest that selectively targeting tumor-elicited myeloid cells or versican represents a potential therapeutic strategy for combating metastatic disease.

Discovery of novel human breast cancer microRNAs from deep sequencing data by analysis of pri-microRNA secondary structures.

MicroRNAs (miRNAs) are key regulators of gene expression and contribute to a variety of biological processes. Abnormal miRNA expression has been reported in various diseases including pathophysiology of breast cancer, where they regulate protumorigenic processes including vascular invasiveness, estrogen receptor status, chemotherapy resistance, invasion and metastasis. The miRBase sequence database, a public repository for newly discovered miRNAs, has grown rapidly with approximately >10,000 entries to date. Despite this rapid growth, many miRNAs have not yet been validated, and several others are yet to be identified. A lack of a full complement of miRNAs has imposed limitations on recognizing their important roles in cancer, including breast cancer. Using deep sequencing technology, we have identified 189 candidate novel microRNAs in human breast cancer cell lines with diverse tumorigenic potential. We further show that analysis of 500-nucleotide pri-microRNA secondary structure constitutes a reliable method to predict bona fide miRNAs as judged by experimental validation. Candidate novel breast cancer miRNAs with stem lengths of greater than 30 bp resulted in the generation of precursor and mature sequences in vivo. On the other hand, candidates with stem length less than 30 bp were less efficient in producing mature miRNA. This approach may be used to predict which candidate novel miRNA would qualify as bona fide miRNAs from deep sequencing data with approximately 90% accuracy.