Sterile Injury Repair and Adhesion Formation at Serosal Surfaces.

Most multicellular organisms have a major body cavity containing vital organs. This cavity is lined by a mucosa-like serosal surface and filled with serous fluid which suspends many immune cells. Injuries affecting the major body cavity are potentially life-threatening. Here we summarize evidence that unique damage detection and repair mechanisms have evolved to ensure immediate and swift repair of injuries at serosal surfaces. Furthermore, thousands of patients undergo surgery within the abdominal and thoracic cavities each day. While these surgeries are potentially lifesaving, some patients will suffer complications due to inappropriate scar formation when wound healing at serosal surfaces defects. These scars called adhesions cause profound challenges for health care systems and patients. Therefore, reviewing the mechanisms of wound repair at serosal surfaces is of clinical importance. Serosal surfaces will be introduced with a short embryological and microanatomical perspective followed by a discussion of the mechanisms of damage recognition and initiation of sterile inflammation at serosal surfaces. Distinct immune cells populations are free floating within the coelomic (peritoneal) cavity and contribute towards damage recognition and initiation of wound repair. We will highlight the emerging role of resident cavity GATA6+ macrophages in repairing serosal injuries and compare serosal (mesothelial) injuries with injuries to the blood vessel walls. This allows to draw some parallels such as the critical role of the mesothelium in regulating fibrin deposition and how peritoneal macrophages can aggregate in a platelet-like fashion in response to sterile injury. Then, we discuss how serosal wound healing can go wrong, causing adhesions. The current pathogenetic understanding of and potential future therapeutic avenues against adhesions are discussed

The Placenta-A New Source of Bile Acids during Healthy Pregnancy? First Results of a Gene Expression Study in Humans and Mice.

Bile acids (BAs) are natural ligands for several receptors modulating cell activities. BAs are synthesized via the classic (neutral) and alternative (acidic) pathways. The classic pathway is initiated by CYP7A1/Cyp7a1, converting cholesterol to 7α-hydroxycholesterol, while the alternative pathway starts with hydroxylation of the cholesterol side chain, producing an oxysterol. In addition to originating from the liver, BAs are reported to be synthesized in the brain. We aimed at determining if the placenta potentially represents an extrahepatic source of BAs. Therefore, the mRNAs coding for selected enzymes involved in the hepatic BA synthesis machinery were screened in human term and CD1 mouse late gestation placentas from healthy pregnancies. Additionally, data from murine placenta and brain tissue were compared to determine whether the BA synthetic machinery is comparable in these organs. We found that CYP7A1, CYP46A1, and BAAT mRNAs are lacking in the human placenta, while corresponding homologs were detected in the murine placenta. Conversely, Cyp8b1 and Hsd17b1 mRNAs were undetected in the murine placenta, but these enzymes were found in the human placenta. CYP39A1/Cyp39a1 and cholesterol 25-hydroxylase (CH25H/Ch25h) mRNA expression were detected in the placentas of both species. When comparing murine placentas and brains, Cyp8b1 and Hsd17b1 mRNAs were only detected in the brain. We conclude that BA synthesis-related genes are placentally expressed in a species-specific manner. The potential placentally synthesized BAs could serve as endocrine and autocrine stimuli, which may play a role in fetoplacental growth and adaptation

Heat shock protein 90 (HSP90) inhibitors in gastrointestinal cancer: where do we currently stand?-A systematic review.

PURPOSE Dysregulated expression of heat shock proteins (HSP) plays a fundamental role in tumor development and progression. Consequently, HSP90 may be an effective tumor target in oncology, including the treatment of gastrointestinal cancers. METHODS We carried out a systematic review of data extracted from clinicaltrials.gov and pubmed.gov, which included all studies available until January 1st, 2022. The published data was evaluated using primary and secondary endpoints, particularly with focus on overall survival, progression-free survival, and rate of stable disease. RESULTS Twenty trials used HSP90 inhibitors in GI cancers, ranging from phase I to III clinical trials. Most studies assessed HSP90 inhibitors as a second line treatment. Seventeen of the 20 studies were performed prior to 2015 and only few studies have results pending. Several studies were terminated prematurely, due to insufficient efficacy or toxicity. Thus far, the data suggests that HSP90 inhibitor NVP-AUY922 might improve outcome for colorectal cancer and gastrointestinal stromal tumors. CONCLUSION It currently remains unclear which subgroup of patients might benefit from HSP90 inhibitors and at what time point these inhibitors may be beneficial. There are only few new or ongoing studies initiated during the last decade

LIM protein Ajuba promotes liver cell proliferation through its involvement in DNA replication and DNA damage control.

The LIM-domain protein Ajuba is associated with cell proliferation, a fundamental process of tissue regeneration and cancer. We report that in the liver, Ajuba expression is increased during regeneration and in tumor cells and tissues. Knockout of Ajuba using CRISPR/Cas9 is embryonic lethal in mice. shRNA targeting of Ajuba reduces cell proliferation, delays cell entry into S-phase, reduces cell survival and tumor growth in vivo, and increases expression of the DNA damage marker γH2AX. Ajuba binding partners include proteins involved in DNA replication and damage, such as SKP2, MCM2, MCM7 and RPA70. Taken together, our data support that Ajuba promotes liver cell proliferation associated with development, regeneration, and tumor growth and is involved in DNA replication and damage repair

Effective Treatment of Advanced Colorectal Cancer by Rapamycin and 5-FU/Oxaliplatin Monitored by TIMP-1

Aim: The mTOR-inhibitor rapamycin has shown antitumor activity in various tumors. Bedside observations have suggested that rapamycin may be effective as a treatment for colorectal carcinomatosis. Methods: We established an orthotopic syngenic model by transplanting CT26 peritoneal tumors in Balb/C mice and an orthotopic xenograft model by transplanting SW620 peritoneal tumors in nu/nu mice. Expression levels of tissue inhibitor of matrix-metalloproteinases 1 (TIMP-1) in the tumor and serum was determined by enzyme-linked immunosorbent assay. Results: Rapamycin significantly suppressed growth of syngenic and xenografted peritoneal tumors. The effect was similar with intraperitoneal or oral rapamycin administration. Tumor suppression was further enhanced when rapamycin was combined with 5-fluorouracil and/or oxaliplatin. The combination treatment showed no acute toxicity. TIMP-1 serum levels correlated well (CC = 0.75; P < 0.01) with rapamycin treatment. Conclusions: Rapamycin suppressed advanced stage colorectal cancer, even with oral administration. Combining rapamycin with current chemotherapy regimens significantly increased antitumor efficacy without apparent toxicity. The treatment efficacy correlated with serum TIMP-1 levels, suggesting its potential as a surrogate marker in future clinical trial

Primary Infection by E. multilocularis Induces Distinct Patterns of Cross Talk between Hepatic Natural Killer T Cells and Regulatory T Cells in Mice.

The larval stage of the helminthic cestode Echinococcus multilocularis can inflict tumor-like hepatic lesions that cause the parasitic disease alveolar echinococcosis in humans, with high mortality in untreated patients. Opportunistic properties of the disease have been established based on the increased incidence in immunocompromised patients and mouse models, indicating that an appropriate adaptive immune response is required for the control of the disease. However, cellular interactions and the kinetics of the local hepatic immune responses during the different stages of infection with E. multilocularis remain unknown. In a mouse model of oral infection that mimics the normal infection route in human patients, the networks of the hepatic immune response were assessed using single-cell RNA sequencing (scRNA-seq) of isolated hepatic CD3+ T cells at different infection stages. We observed an early and sustained significant increase in natural killer T (NKT) cells and regulatory T cells (Tregs). Early tumor necrosis factor (TNF)- and integrin-dependent interactions between these two cell types promote the formation of hepatic lesions. At late time points, downregulation of programmed cell death protein 1 (PD-1) and ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1)-dependent signaling suppress the resolution of parasite-induced pathology. The obtained data provide fresh insight into the adaptive immune responses and local regulatory pathways at different infection stages of E. multilocularis in mice

Inhibition of SIRT1 Impairs the Accumulation and Transcriptional Activity of HIF-1α Protein under Hypoxic Conditions

Sirtuins and hypoxia-inducible transcription factors (HIF) have well-established roles in regulating cellular responses to metabolic and oxidative stress. Recent reports have linked these two protein families by demonstrating that sirtuins can regulate the activity of HIF-1 and HIF-2. Here we investigated the role of SIRT1, a NAD+-dependent deacetylase, in the regulation of HIF-1 activity in hypoxic conditions. Our results show that in hepatocellular carcinoma (HCC) cell lines, hypoxia did not alter SIRT1 mRNA or protein expression, whereas it predictably led to the accumulation of HIF-1α and the up-regulation of its target genes. In hypoxic models in vitro and in in vivo models of systemic hypoxia and xenograft tumor growth, knockdown of SIRT1 protein with shRNA or inhibition of its activity with small molecule inhibitors impaired the accumulation of HIF-1α protein and the transcriptional increase of its target genes. In addition, endogenous SIRT1 and HIF-1α proteins co-immunoprecipitated and loss of SIRT1 activity led to a hyperacetylation of HIF-1α. Taken together, our data suggest that HIF-1α and SIRT1 proteins interact in HCC cells and that HIF-1α is a target of SIRT1 deacetylase activity. Moreover, SIRT1 is necessary for HIF-1α protein accumulation and activation of HIF-1 target genes under hypoxic conditions

Autologous transplantation of adipose-derived stem cells improves functional recovery of skeletal muscle without direct participation in new myofiber formation.

BACKGROUND Skeletal muscle has a remarkable regenerative capacity. However, extensive damage that exceeds the self-regenerative ability of the muscle can lead to irreversible fibrosis, scarring, and significant loss of function. Adipose-derived stem cells (ADSC) are a highly abundant source of progenitor cells that have been previously reported to support the regeneration of various muscle tissues, including striated muscles. The aim of this study was to evaluate the effect of ADSC transplantation on functional skeletal muscle regeneration in an acute injury model. METHODS Mouse ADSC were isolated from subcutaneous fat tissue and transplanted with a collagen hydrogel into the crushed tibialis anterior muscle of mice. Recovering muscles were analyzed for gene and protein expression by real-time quantitative polymerase chain reaction and immunohistochemistry. The muscle contractility was assessed by myography in an organ bath system. RESULTS Intramuscular transplantation of ADSC into crushed tibialis anterior muscle leads to an improved muscle regeneration with ADSC residing in the damaged area. We did not observe ADSC differentiation into new muscle fibers or endothelial cells. However, the ADSC-injected muscles had improved contractility in comparison with the collagen-injected controls 28 days post-transplantation. Additionally, an increase in fiber cross-sectional size and in the number of mature fibers with centralized nuclei was observed. CONCLUSIONS ADSC transplantation into acute damaged skeletal muscle significantly improves functional muscle tissue regeneration without direct participation in muscle fiber formation. Cellular therapy with ADSC represents a novel approach to promote skeletal muscle regeneration

A new mouse model of radiation-induced liver disease reveals mitochondrial dysfunction as an underlying fibrotic stimulus.

Background & Aims High-dose irradiation is an essential tool to help control the growth of hepatic tumors, but it can cause radiation-induced liver disease (RILD). This life-threatening complication manifests itself months following radiation therapy and is characterized by fibrosis of the pericentral sinusoids. In this study, we aimed to establish a mouse model of RILD to investigate the underlying mechanism of radiation-induced liver fibrosis. Methods Using a small animal image-guided radiation therapy platform, an irradiation scheme delivering 50 Gy as a single dose to a focal point in mouse livers was designed. Tissues were analyzed 1 and 6 days, and 6 and 20 weeks post-irradiation. Irradiated livers were assessed by histology, immunohistochemistry, imaging mass cytometry and RNA sequencing. Mitochondrial function was assessed using high-resolution respirometry. Results At 6 and 20 weeks post-irradiation, pericentral fibrosis was visible in highly irradiated areas together with immune cell infiltration and extravasation of red blood cells. RNA sequencing analysis showed gene signatures associated with acute DNA damage, p53 activation, senescence and its associated secretory phenotype and fibrosis. Moreover, gene profiles of mitochondrial damage and an increase in mitochondrial DNA heteroplasmy were detected. Respirometry measurements of hepatocytes in vitro confirmed irradiation-induced mitochondrial dysfunction. Finally, the highly irradiated fibrotic areas showed markers of reactive oxygen species such as decreased glutathione and increased lipid peroxides and a senescence-like phenotype. Conclusions Based on our mouse model of RILD, we propose that irradiation-induced mitochondrial DNA instability contributes to the development of fibrosis via the generation of excessive reactive oxygen species, p53 pathway activation and a senescence-like phenotype. Lay summary Irradiation is an efficient cancer therapy, however, its applicability to the liver is limited by life-threatening radiation-induced hepatic fibrosis. We have developed a new mouse model of radiation-induced liver fibrosis, that recapitulates the human disease. Our model highlights the role of mitochondrial DNA instability in the development of irradiation-induced liver fibrosis. This new model and subsequent findings will help increase our understanding of the hepatic reaction to irradiation and to find strategies that protect the liver, enabling the expanded use of radiotherapy to treat hepatic tumors

An optimized protocol for the generation and monitoring of conditional orthotopic lung cancer in the KP mouse model using an adeno-associated virus vector compatible with biosafety level 1.

BACKGROUND The inducible Kras/p53 lung adenocarcinoma mouse model, which faithfully recapitulates human disease, is routinely initiated by the intratracheal instillation of a virus-based Cre recombinase delivery system. Handling virus-based delivery systems requires elevated biosafety levels, e.g., biosafety level 2 (BSL-2). However, in experimental animal research facilities, following exposure to viral vectors in a BSL-2 environment, rodents may not be reclassified to BSL-1 according to standard practice, preventing access to small animal micro-computed tomography (micro-CT) scanners that are typically housed in general access areas such as BSL-1 rooms. Therefore, our goal was to adapt the protocol so that the Cre-induced KP mouse model could be handled under BSL-1 conditions during the entire procedure. RESULTS The Kras-Lox-STOP-Lox-G12D/p53 flox/flox (KP)-based lung adenocarcinoma mouse model was activated by intratracheal instillation of either an adenoviral-based or a gutless, adeno-associated viral-based Cre delivery system. Tumor growth was monitored over time by micro-CT. We have successfully substituted the virus-based Cre delivery system with a commercially available, gutless, adeno-associated, Cre-expressing vector that allows the KP mouse model to be handled and imaged in a BSL-1 facility. By optimizing the anesthesia protocol and switching to a microscope-guided vector instillation procedure, productivity was increased and procedure-related complications were significantly reduced. In addition, repeated micro-CT analysis of individual animals allowed us to monitor tumor growth longitudinally, dramatically reducing the number of animals required per experiment. Finally, we documented the evolution of tumor volume for different doses, which revealed that individual tumor nodules induced by low-titer AAV-Cre transductions can be monitored over time by micro-CT. CONCLUSION Modifications to the anesthesia and instillation protocols increased the productivity of the original KP protocol. In addition, the switch to a gutless, adeno-associated, Cre-expressing vector allowed longitudinal monitoring of tumor growth under BSL-1 conditions, significantly reducing the number of animals required for an experiment, in line with the 3R principles