PTEN Regulates Myofibroblast Activation in Valvular Interstitials Cells based on Subcellular Localization.

Aortic valve stenosis (AVS) is characterized by altered mechanics of the valve leaflets, which disrupts blood flow through the aorta and can cause left ventricle hypotrophy. These changes in the valve tissue result in activation of resident valvular interstitial cells (VICs) into myofibroblasts, which have increased levels of αSMA in their stress fibers. The persistence of VIC myofibroblast activation is a hallmark of AVS. In recent years, the tumor suppressor gene phosphatase and tensin homolog (PTEN) has emerged as an important player in the regulation of fibrosis in various tissues (e.g., lung, skin), which motivated us to investigate PTEN as a potential protective factor against matrix-induced myofibroblast activation in VICs. In aortic valve samples from humans, we found high levels of PTEN in healthy tissue and low levels of PTEN in diseased tissue. Then, using pharmacological inducers to treat VIC cultures, we observed PTEN overexpression prevented stiffness-induced myofibroblast activation, whereas genetic and pharmacological inhibition of PTEN further activated myofibroblasts. We also observed increased nuclear PTEN localization in VICs cultured on stiff matrices, and nuclear PTEN also correlated with smaller nuclei, altered expression of histones and a quiescent fibroblast phenotype. Together, these results suggest that PTEN not only suppresses VIC activation, but functions to promote quiescence, and could serve as a potential pharmacological target for the treatment of AVS.

KLF4 in smooth muscle cell-derived progenitor cells is essential for angiotensin II-induced cardiac inflammation and fibrosis.

Cardiac fibrosis is defined by the excessive accumulation of extracellular matrix (ECM) material resulting in cardiac tissue scarring and dysfunction. While it is commonly accepted that myofibroblasts are the major contributors to ECM deposition in cardiac fibrosis, their origin remains debated. By combining lineage tracing and RNA sequencing, our group made the paradigm-shifting discovery that a subpopulation of resident vascular stem cells residing within the aortic, carotid artery, and femoral aartery adventitia (termed AdvSca1-SM cells) originate from mature vascular smooth muscle cells (SMCs) through an in situ reprogramming process. SMC-to-AdvSca1-SM reprogramming and AdvSca1-SM cell maintenance is dependent on induction and activity of the transcription factor, KLF4. However, the molecular mechanism whereby KLF4 regulates AdvSca1-SM phenotype remains unclear. In the current study, leveraging a highly specific AdvSca1-SM cell reporter system, single-cell RNA-sequencing (scRNA-seq), and spatial transcriptomic approaches, we demonstrate the profibrotic differentiation trajectory of coronary artery-associated AdvSca1-SM cells in the setting of Angiotensin II (AngII)-induced cardiac fibrosis. Differentiation was characterized by loss of stemness-related genes, including Klf4 , but gain of expression of a profibrotic phenotype. Importantly, these changes were recapitulated in human cardiac hypertrophic tissue, supporting the translational significance of profibrotic transition of AdvSca1-SM-like cells in human cardiomyopathy. Surprisingly and paradoxically, AdvSca1-SM-specific genetic knockout of Klf4 prior to AngII treatment protected against cardiac inflammation and fibrosis, indicating that Klf4 is essential for the profibrotic response of AdvSca1-SM cells. Overall, our data reveal the contribution of AdvSca1-SM cells to myofibroblasts in the setting of AngII-induced cardiac fibrosis. KLF4 not only maintains the stemness of AdvSca1-SM cells, but also orchestrates their response to profibrotic stimuli, and may serve as a therapeutic target in cardiac fibrosis.

Smooth muscle-derived adventitial progenitor cells direct atherosclerotic plaque composition complexity in a Klf4-dependent manner.

We previously established that vascular smooth muscle-derived adventitial progenitor cells (AdvSca1-SM) preferentially differentiate into myofibroblasts and contribute to fibrosis in response to acute vascular injury. However, the role of these progenitor cells in chronic atherosclerosis has not been defined. Using an AdvSca1-SM cell lineage tracing model, scRNA-Seq, flow cytometry, and histological approaches, we confirmed that AdvSca1-SM-derived cells localized throughout the vessel wall and atherosclerotic plaques, where they primarily differentiated into fibroblasts, smooth muscle cells (SMC), or remained in a stem-like state. Krüppel-like factor 4 (Klf4) knockout specifically in AdvSca1-SM cells induced transition to a more collagen-enriched fibroblast phenotype compared with WT mice. Additionally, Klf4 deletion drastically modified the phenotypes of non-AdvSca1-SM-derived cells, resulting in more contractile SMC and atheroprotective macrophages. Functionally, overall plaque burden was not altered with Klf4 deletion, but multiple indices of plaque composition complexity, including necrotic core area, macrophage accumulation, and fibrous cap thickness, were reduced. Collectively, these data support that modulation of AdvSca1-SM cells through KLF4 depletion confers increased protection from the development of potentially unstable atherosclerotic plaques.

Confounding Effects of Tamoxifen: Cautionary and Practical Considerations for the Use of Tamoxifen-Inducible Mouse Models in Atherosclerosis Research-Brief Report.

BACKGROUND: In recent years, fate-mapping lineage studies in mouse models have led to major advances in vascular biology by allowing investigators to track specific cell populations in vivo. One of the most frequently used lineage tracing approaches involves tamoxifen-inducible CreERT-LoxP systems. However, tamoxifen treatment can also promote effects independent of Cre recombinase activation, many of which have not been fully explored. METHODS: To elucidate off-target effects of tamoxifen, male and female mice were either unmanipulated or injected with tamoxifen or corn oil. All mice received PCSK9 (proprotein convertase subtilisin/kexin type 9)-AAV (adeno-associated virus) injections and a modified Western diet to induce hypercholesterolemia. After 2 weeks, serum cholesterol and liver morphology were assessed. To determine the duration of any tamoxifen effects in long-term atherosclerosis experiments, mice received either 12 days of tamoxifen at baseline or 12 days plus 2 sets of 5-day tamoxifen boosters; all mice received PCSK9-AAV injections and a modified Western diet to induce hypercholesterolemia. After 24 weeks, serum cholesterol and aortic sinus plaque burden were measured. RESULTS: After 2 weeks of atherogenic treatment, mice injected with tamoxifen demonstrated significantly reduced serum cholesterol levels compared with uninjected- or corn oil-treated mice. However, there were no differences in PCSK9-mediated knockdown of LDL (low-density lipoprotein) receptors between the groups. Additionally, tamoxifen-treated mice exhibited significantly increased hepatic lipid accumulation compared with the other groups. Finally, the effects of tamoxifen remained for at least 8 weeks after completion of injections, with mice demonstrating persistent decreased serum cholesterol and impaired atherosclerotic plaque formation. CONCLUSIONS: In this study, we establish that tamoxifen administration results in decreased serum cholesterol, decreased plaque formation, and increased hepatic lipid accumulation. These alterations represent significant confounding variables in atherosclerosis research, and we urge future investigators to take these findings into consideration when planning and executing their own atherosclerosis experiments.

IL-6 mediates the hepatic acute phase response after prerenal azotemia in a clinically defined murine model.

Prerenal azotemia (PRA) is a major cause of acute kidney injury and uncommonly studied in preclinical models. We sought to develop and characterize a novel model of PRA that meets the clinical definition: acute loss of glomerular filtration rate (GFR) that returns to baseline with resuscitation. Adult male C57BL/6J wild-type (WT) and IL-6-/- mice were studied. Intraperitoneal furosemide (4 mg) or vehicle was administered at time = 0 and 3 h to induce PRA from volume loss. Resuscitation began at 6 h with 1 mL intraperitoneal saline for four times for 36 h. Six hours after furosemide administration, measured glomerular filtration rate was 25% of baseline and returned to baseline after saline resuscitation at 48 h. After 6 h of PRA, plasma interleukin (IL)-6 was significantly increased, kidney and liver histology were normal, kidney and liver lactate were normal, and kidney injury molecule-1 immunofluorescence was negative. There were 327 differentially regulated genes upregulated in the liver, and the acute phase response was the most significantly upregulated pathway; 84 of the upregulated genes (25%) were suppressed in IL-6-/- mice, and the acute phase response was the most significantly suppressed pathway. Significantly upregulated genes and their proteins were also investigated and included serum amyloid A2, serum amyloid A1, lipocalin 2, chemokine (C-X-C motif) ligand 1, and haptoglobin; hepatic gene expression and plasma protein levels were all increased in wild-type PRA and were all reduced in IL-6-/- PRA. This work demonstrates previously unknown systemic effects of PRA that includes IL-6-mediated upregulation of the hepatic acute phase response.NEW & NOTEWORTHY Prerenal azotemia (PRA) accounts for a third of acute kidney injury (AKI) cases yet is rarely studied in preclinical models. We developed a clinically defined murine model of prerenal azotemia characterized by a 75% decrease in measured glomerular filtration rate (GFR), return of measured glomerular filtration rate to baseline with resuscitation, and absent tubular injury. Numerous systemic effects were observed, such as increased plasma interleukin-6 (IL-6) and upregulation of the hepatic acute phase response.

Smooth muscle-derived adventitial progenitor cells promote key cell type transitions controlling plaque stability in atherosclerosis in a Klf4-dependent manner.

We previously established that vascular smooth muscle-derived adventitial progenitor cells (AdvSca1-SM) preferentially differentiate into myofibroblasts and contribute to fibrosis in response to acute vascular injury. However, the role of these progenitor cells in chronic atherosclerosis has not been defined. Using an AdvSca1-SM lineage tracing model, scRNA-Seq, flow cytometry, and histological approaches, we confirmed that AdvSca1-SM cells localize throughout the vessel wall and atherosclerotic plaques, where they primarily differentiate into fibroblasts, SMCs, or remain in a stem-like state. Klf4 knockout specifically in AdvSca1-SM cells induced transition to a more collagen-enriched myofibroblast phenotype compared to WT mice. Additionally, Klf4 depletion drastically modified the phenotypes of non-AdvSca1-SM-derived cells, resulting in more contractile SMCs and atheroprotective macrophages. Functionally, overall plaque burden was not altered with Klf4 depletion, but multiple indices of plaque vulnerability were reduced. Collectively, these data support that modulating the AdvSca1-SM population confers increased protection from the development of unstable atherosclerotic plaques.

Metformin protects against pulmonary hypertension-induced right ventricular dysfunction in an age- and sex-specific manner independent of cardiac AMPK.

Right ventricular (RV) function is the strongest predictor of survival in age-related heart failure as well as other clinical contexts in which aging populations suffer significant morbidity and mortality. However, despite the significance of maintaining RV function with age and disease, mechanisms of RV failure remain poorly understood and no RV-directed therapies exist. The antidiabetic drug and AMP-activated protein kinase (AMPK) activator metformin protects against left ventricular dysfunction, suggesting cardioprotective properties may translate to the RV. Here, we aimed to understand the impact of advanced age on pulmonary hypertension (PH)-induced right ventricular dysfunction. We further aimed to test whether metformin is cardioprotective in the RV and whether the protection afforded by metformin requires cardiac AMPK. We used a murine model of PH by exposing adult (4-6 mo) and aged (18 mo) male and female mice to hypobaric hypoxia (HH) for 4 wk. Cardiopulmonary remodeling was exacerbated in aged mice compared with adult mice as evidenced by elevated RV weight and impaired RV systolic function. Metformin attenuated HH-induced RV dysfunction but only in adult male mice. Metformin still protected the adult male RV even in the absence of cardiac AMPK. Together, we suggest that aging exacerbates PH-induced RV remodeling and that metformin may represent a therapeutic option for this disease in a sex- and age-dependent manner, but in an AMPK-independent manner. Ongoing efforts are aimed at elucidating the molecular basis for RV remodeling as well as delineating the mechanisms of cardioprotection provided by metformin in the absence of cardiac AMPK.NEW & NOTEWORTHY Right ventricular (RV) function predicts survival in age-related disease, yet mechanisms of RV failure are unclear. We show that aged mice undergo exacerbated RV remodeling compared with young. We tested the AMPK activator metformin to improve RV function and show that metformin attenuates RV remodeling only in adult male mice via a mechanism that does not require cardiac AMPK. Metformin is therapeutic for RV dysfunction in an age- and sex-specific manner independent of cardiac AMPK.

The TNFΔARE Mouse as a Model of Intestinal Fibrosis.

Crohn disease (CD) is a highly morbid chronic inflammatory disease. Although many patients with CD also develop fibrostenosing complications, there are no medical therapies for intestinal fibrosis. This is due, in part, to a lack of high-fidelity biomimetic models to enhance understanding and drug development, which highlights the need for developing in vivo models of inflammatory bowel disease-related intestinal fibrosis. This study investigates whether the TNFΔARE mouse, a model of ileal inflammation, also develops intestinal fibrosis. Several clinically relevant outcomes were studied, including features of structural fibrosis, histologic fibrosis, and gene expression. These include the use of a new luminal casting technique, traditional histologic outcomes, use of second harmonic imaging, and quantitative PCR. These features were studied in aged TNFΔARE mice as well as in cohorts of numerous ages. At >24 weeks of age, TNFΔARE mice developed structural, histologic, and transcriptional changes of ileal fibrosis. Protein and RNA expression profiles showed changes as early as 6 weeks, coinciding with histologic changes as early as 14 to 15 weeks. Overt structural fibrosis was delayed until at least 16 weeks and was most developed after 24 weeks. This study found that the TNFΔARE mouse is a viable and highly tractable model of ileal fibrosis. This model and the techniques used herein can be leveraged for both mechanistic studies and therapeutic development for the treatment of intestinal fibrosis.

Redistribution of the chromatin remodeler Brg1 directs smooth muscle-derived adventitial progenitor-to-myofibroblast differentiation and vascular fibrosis.

Vascular smooth muscle-derived Sca1+ adventitial progenitor (AdvSca1-SM) cells are tissue-resident, multipotent stem cells that contribute to progression of vascular remodeling and fibrosis. Upon acute vascular injury, AdvSca1-SM cells differentiate into myofibroblasts and are embedded in perivascular collagen and the extracellular matrix. While the phenotypic properties of AdvSca1-SM-derived myofibroblasts have been defined, the underlying epigenetic regulators driving the AdvSca1-SM-to-myofibroblast transition are unclear. We show that the chromatin remodeler Smarca4/Brg1 facilitates AdvSca1-SM myofibroblast differentiation. Brg1 mRNA and protein were upregulated in AdvSca1-SM cells after acute vascular injury, and pharmacological inhibition of Brg1 by the small molecule PFI-3 attenuated perivascular fibrosis and adventitial expansion. TGF-ß1 stimulation of AdvSca1-SM cells in vitro reduced expression of stemness genes while inducing expression of myofibroblast genes that was associated with enhanced contractility; PFI blocked TGF-ß1-induced phenotypic transition. Similarly, genetic knockdown of Brg1 in vivo reduced adventitial remodeling and fibrosis and reversed AdvSca1-SM-to-myofibroblast transition in vitro. Mechanistically, TGF-ß1 promoted redistribution of Brg1 from distal intergenic sites of stemness genes and recruitment to promoter regions of myofibroblast-related genes, which was blocked by PFI-3. These data provide insight into epigenetic regulation of resident vascular progenitor cell differentiation and support that manipulating the AdvSca1-SM phenotype will provide antifibrotic clinical benefits.

Durable responses to alectinib in murine models of EML4-ALK lung cancer requires adaptive immunity.

Lung cancers bearing oncogenic EML4-ALK fusions respond to targeted tyrosine kinase inhibitors (TKIs; e.g., alectinib), with variation in the degree of shrinkage and duration of treatment (DOT). However, factors that control this response are not well understood. While the contribution of the immune system in mediating the response to immunotherapy has been extensively investigated, less is known regarding the contribution of immunity to TKI therapeutic responses. We previously demonstrated a positive association of a TKI-induced interferon gamma (IFNγ) transcriptional response with DOT in EGFR-mutant lung cancers. Herein, we used three murine models of EML4-ALK lung cancer to test the role for host immunity in the alectinib therapeutic response. The cell lines (EA1, EA2, EA3) were propagated orthotopically in the lungs of immunocompetent and immunodeficient mice and treated with alectinib. Tumor volumes were serially measured by µCT and immune cell content was measured by flow cytometry and multispectral immunofluorescence. Transcriptional responses to alectinib were assessed by RNAseq and secreted chemokines were measured by ELISA. All cell lines were similarly sensitive to alectinib in vitro and as orthotopic tumors in immunocompetent mice, exhibited durable shrinkage. However, in immunodeficient mice, all tumor models rapidly progressed on TKI therapy. In immunocompetent mice, EA2 tumors exhibited a complete response, whereas EA1 and EA3 tumors retained residual disease that rapidly progressed upon termination of TKI treatment. Prior to treatment, EA2 tumors had greater numbers of CD8+ T cells and fewer neutrophils compared to EA1 tumors. Also, RNAseq of cancer cells recovered from untreated tumors revealed elevated levels of CXCL9 and 10 in EA2 tumors, and higher levels of CXCL1 and 2 in EA1 tumors. Analysis of pre-treatment patient biopsies from ALK+ tumors revealed an association of neutrophil content with shorter time to progression. Combined, these data support a role for adaptive immunity in durability of TKI responses and demonstrate that the immune cell composition of the tumor microenvironment is predictive of response to alectinib therapy.

The TNF ΔARE mouse as a model of intestinal fibrosis.

Background & Aims: Crohn's disease (CD) is a highly morbid chronic inflammatory disease. The majority of CD patients also develop fibrostenosing complications. Despite this, there are no medical therapies for intestinal fibrosis. This is in part due to lack of high-fidelity biomimetic models to enhance understanding and drug development. There is a need to develop in vivo models of inflammatory bowel disease-related intestinal fibrosis. We sought to determine if the TNF ΔARE mouse, a model of ileal inflammation, may also develop intestinal fibrosis. Methods: Several clinically relevant outcomes were studied including features of structural fibrosis, histological fibrosis, and gene expression. These include the use of a luminal casting technique we developed, traditional histological outcomes, use of second harmonic imaging, and quantitative PCR. These features were studied in aged TNF ΔARE mice as well as in cohorts of numerous ages. Results: At ages of 24+ weeks, TNF ΔARE mice develop structural, histological, and genetic changes of ileal fibrosis. Genetic expression profiles have changes as early as six weeks, followed by histological changes occurring as early as 14-15 weeks, and overt structural fibrosis delayed until after 24 weeks. Discussion: The TNF ΔARE mouse is a viable and highly tractable model of intestinal fibrosis. This model and the techniques employed can be leveraged for both mechanistic studies and therapeutic development for the treatment of intestinal fibrosis.

Novel EGFR-mutant mouse models of lung adenocarcinoma reveal adaptive immunity requirement for durable osimertinib response.

Lung cancers bearing oncogenically-mutated EGFR represent a significant fraction of lung adenocarcinomas (LUADs) for which EGFR-targeting tyrosine kinase inhibitors (TKIs) provide a highly effective therapeutic approach. However, these lung cancers eventually acquire resistance and undergo progression within a characteristically broad treatment duration range. Our previous study of EGFR mutant lung cancer patient biopsies highlighted the positive association of a TKI-induced interferon γ transcriptional response with increased time to treatment progression. To test the hypothesis that host immunity contributes to the TKI response, we developed novel genetically-engineered mouse models of EGFR mutant lung cancer bearing exon 19 deletions (del19) or the L860R missense mutation. Both oncogenic EGFR mouse models developed multifocal LUADs from which transplantable cancer cell lines sensitive to the EGFR-specific TKIs, gefitinib and osimertinib, were derived. When propagated orthotopically in the left lungs of syngeneic C57BL/6 mice, deep and durable shrinkage of the cell line-derived tumors was observed in response to daily treatment with osimertinib. By contrast, orthotopic tumors propagated in immune deficient nu/nu or Rag1-/- mice exhibited modest tumor shrinkage followed by rapid progression on continuous osimertinib treatment. Importantly, osimertinib treatment significantly increased intratumoral T cell content and decreased neutrophil content relative to diluent treatment. The findings provide strong evidence supporting the requirement for adaptive immunity in the durable therapeutic control of EGFR mutant lung cancer.

Mammalian organ regeneration in spiny mice.

Fibrosis-driven solid organ failure is a major world-wide health burden with few therapeutic options. Spiny mice (genus: Acomys) are terrestrial mammals that regenerate severe skin wounds without fibrotic scars to evade predators. Recent studies have shown that spiny mice also regenerate acute ischemic and traumatic injuries to kidney, heart, spinal cord, and skeletal muscle. A common feature of this evolved wound healing response is a lack of formation of fibrotic scar tissue that degrades organ function, inhibits regeneration, and leads to organ failure. Complex tissue regeneration is an extremely rare property among mammalian species. In this article, we discuss the evidence that Acomys represents an emerging model organism that offers a unique opportunity for the biomedical community to investigate and clinically translate molecular mechanisms of scarless wound healing and regeneration of organ function in a mammalian species.

The adventitia in arterial development, remodeling, and hypertension.

The adventitia receives input signals from the vessel wall, the immune system, perivascular nerves and from surrounding tissues to generate effector responses that regulate structural and mechanical properties of blood vessels. It is a complex and dynamic tissue that orchestrates multiple functions for vascular development, homeostasis, repair, and disease. The purpose of this review is to highlight recent advances in our understanding of the origins, phenotypes, and functions of adventitial and perivascular cells with particular emphasis on hypertensive vascular remodeling.

Heterogeneous subpopulations of adventitial progenitor cells regulate vascular homeostasis and pathological vascular remodelling.

Cardiovascular diseases are characterized by chronic vascular dysfunction and provoke pathological remodelling events, such as neointima formation, atherosclerotic lesion development, and adventitial fibrosis. While lineage-tracing studies have shown that phenotypically modulated smooth muscle cells (SMCs) are the major cellular component of neointimal lesions, the cellular origins and microenvironmental signalling mechanisms that underlie remodelling along the adventitial vascular layer are not fully understood. However, a growing body of evidence supports a unique population of adventitial lineage-restricted progenitor cells expressing the stem cell marker, stem cell antigen-1 (Sca1; AdvSca1 cells) as important effectors of adventitial remodelling and suggests that they are at least partially responsible for subsequent pathological changes that occur in the media and intima. AdvSca1 cells are being studied in murine models of atherosclerosis, perivascular fibrosis, and neointima formation in response to acute vascular injury. Depending on the experimental conditions, AdvSca1 cells exhibit the capacity to differentiate into SMCs, endothelial cells, chondrocytes, adipocytes, and pro-remodelling cells, such as myofibroblasts and macrophages. These data indicate that AdvSca1 cells may be a targetable cell population to influence the outcomes of pathologic vascular remodelling. Important questions remain regarding the origins of AdvSca1 cells and the essential signalling mechanisms and microenvironmental factors that regulate both maintenance of their stem-like, progenitor phenotype and their differentiation into lineage-specified cell types. Adding complexity to the story, recent data indicate that the collective population of adventitial progenitor cells is likely composed of several smaller, lineage-restricted subpopulations, which are not fully defined by their transcriptomic profile and differentiation capabilities. The aim of this review is to outline the heterogeneity of Sca1+ adventitial progenitor cells, summarize their role in vascular homeostasis and remodelling, and comment on their translational relevance in humans.

15-Lipoxygenase worsens renal fibrosis, inflammation, and metabolism in a murine model of ureteral obstruction.

15-Lipoxygenase (15-LO) is a nonheme iron-containing dioxygenase that has both pro- and anti-inflammatory roles in many tissues and disease states. 15-LO is thought to influence macrophage phenotype, and silencing 15-LO reduces fibrosis after acute inflammatory triggers. The goal of the present study was to determine whether altering 15-LO expression influences inflammation and fibrogenesis in a murine model of unilateral ureteral obstruction (UUO). C57BL/6J mice, 15-LO knockout (Alox15-/-) mice, and 15-LO transgenic overexpressing (15LOTG) mice were subjected UUO, and kidneys were analyzed at 3, 10, and 14 days postinjury. Histology for fibrosis, inflammation, cytokine quantification, flow cytometry, and metabolomics were performed on injured tissues and controls. PD146176, a specific 15-LO inhibitor, was used to complement experiments involving knockout animals. Compared with wild-type animals undergoing UUO, Alox15-/- mouse kidneys had less proinflammatory, profibrotic message along with less fibrosis and macrophage infiltration. PD146176 inhibited 15-LO and resulted in reduced fibrosis and macrophage infiltration similar to Alox15-/- mice. Flow cytometry revealed that Alox15-/- UUO-injured kidneys had a dynamic change in macrophage phenotype, with an early blunting of CD11bHiLy6CHi "M1" macrophages and an increase in anti-inflammatory CD11bHiLy6CInt "M2c" macrophages and reduced expression of the fractalkine receptor chemokine (C-X3-C motif) receptor 1. Many of these findings were reversed when UUO was performed on 15LOTG mice. Metabolomics analysis revealed that wild-type kidneys developed a glycolytic shift postinjury, while Alox15-/- kidneys exhibited increased oxidative phosphorylation. In conclusion, 15-LO manipulation by genetic or pharmacological means induces dynamic changes in the inflammatory microenvironment in the UUO model and appears to be critical in the progression of UUO-induced fibrosis.NEW & NOTEWORTHY 15-Lipoxygenase (15-LO) has both pro- and anti-inflammatory functions in leukocytes, and its role in kidney injury and repair is unexplored. Our study showed that 15-LO worsens inflammation and fibrosis in a rodent model of chronic kidney disease using genetic and pharmacological manipulation. Silencing 15-LO promotes an increase in M2c-like wound-healing macrophages in the kidney and alters kidney metabolism globally, protecting against anaerobic glycolysis after injury.

Upregulation of complement proteins in lung cancer cells mediates tumor progression.

Introduction: In vivo, cancer cells respond to signals from the tumor microenvironment resulting in changes in expression of proteins that promote tumor progression and suppress anti-tumor immunity. This study employed an orthotopic immunocompetent model of lung cancer to define pathways that are altered in cancer cells recovered from tumors compared to cells grown in culture. Methods: Studies used four murine cell lines implanted into the lungs of syngeneic mice. Cancer cells were recovered using FACS, and transcriptional changes compared to cells grown in culture were determined by RNA-seq. Results: Changes in interferon response, antigen presentation and cytokine signaling were observed in all tumors. In addition, we observed induction of the complement pathway. We previously demonstrated that activation of complement is critical for tumor progression in this model. Complement can play both a pro-tumorigenic role through production of anaphylatoxins, and an anti-tumorigenic role by promoting complement-mediated cell killing of cancer cells. While complement proteins are produced by the liver, expression of complement proteins by cancer cells has been described. Silencing cancer cell-specific C3 inhibited tumor growth In vivo. We hypothesized that induction of complement regulatory proteins was critical for blocking the anti-tumor effects of complement activation. Silencing complement regulatory proteins also inhibited tumor growth, with different regulatory proteins acting in a cell-specific manner. Discussion: Based on these data we propose that localized induction of complement in cancer cells is a common feature of lung tumors that promotes tumor progression, with induction of complement regulatory proteins protecting cells from complement mediated-cell killing.