A graded neonatal mouse model of necrotizing enterocolitis demonstrates that mild enterocolitis is sufficient to activate microglia and increase cerebral cytokine expression.

Background: Necrotizing enterocolitis (NEC) is an inflammatory gastrointestinal process that afflicts approximately 10% of preterm infants born in the United States each year, with a mortality rate of 30%. NEC severity is graded using Bell's classification system, from stage I mild NEC to stage III severe NEC. Over half of NEC survivors present with neurodevelopmental impairment during adolescence, a long-term complication that is poorly understood but can occur even after mild NEC. Although multiple animal models exist, none allow the experimenter to control nor represent the gradient of symptom severities seen in NEC patients. We bridge this knowledge gap by developing a graded murine model of NEC and studying its relationship with neuroinflammation across a range of NEC severities. Methods: Postnatal day 3 (P3) C57BL/6 mice were fed a formula containing different concentrations (0% control, 0.25%, 1%, 2%, and 3%) of dextran sodium sulfate (DSS). P3 mice were fed every 3 hours for 72-hours. We collected data on weight gain and behavior (activity, response, body color) during feeding. At the end of the experiment, we collected tissues (intestine, liver, plasma, brain) for immunohistochemistry, immunofluorescence, and cytokine and chemokine analysis. Results: Throughout NEC induction, mice fed higher concentrations of DSS died sooner, lost weight faster, and became sick or lethargic earlier. Intestinal characteristics (dilation, color, friability) were worse in mice fed with higher DSS concentrations. Histology revealed small intestinal disarray among mice fed all DSS concentrations, while higher DSS concentrations resulted in reduced small intestinal cellular proliferation and increased hepatic and systemic inflammation. In the brain, IL-2, G-CSF, and CXCL1 concentrations increased with higher DSS concentrations. Although the number of neurons and microglia in the CA1 hippocampal region did not differ, microglial branching was significantly reduced in DSS-fed mice. Conclusion: We characterize a novel graded model of NEC that recapitulates the full range of NEC severities. We show that mild NEC is sufficient to initiate neuroinflammation and microglia activation. This model will facilitate studies on the neurodevelopmental effects of NEC.

Activin A signaling stimulates neutrophil activation and macrophage migration in pancreatitis.

Acute Pancreatitis (AP) is associated with high mortality and current treatment options are limited to supportive care. We found that blockade of activin A (activin) in mice improves outcomes in two murine models of AP. To test the hypothesis that activin is produced early in response to pancreatitis and is maintained throughout disease progression to stimulate immune cells, we first performed digital spatial profiling (DSP) of human chronic pancreatitis (CP) patient tissue. Then, transwell migration assays using RAW264.7 mouse macrophages and qPCR analysis of "neutrophil-like" HL-60 cells were used for functional correlation. Immunofluorescence and western blots on cerulein-induced pancreatitis samples from pancreatic acinar cell-specific Kras knock-in (Ptf1aCreER™; LSL-KrasG12D) and functional WT Ptf1aCreER™ mouse lines mimicking AP and CP to allow for in vivo confirmation. Our data suggest activin promotes neutrophil and macrophage activation both in situ and in vitro, while pancreatic activin production is increased as early as 1 h in response to pancreatitis and is maintained throughout CP in vivo. Taken together, activin is produced early in response to pancreatitis and is maintained throughout disease progression to promote neutrophil and macrophage activation.

Acute Pancreatitis: Current Clinical Approaches, Molecular Pathophysiology, and Potential Therapeutics.

OBJECTIVE: Severe acute pancreatitis (SAP), pancreatic inflammation leading to multiorgan failure, is associated with high morbidity and mortality. There is a critical need to identify novel therapeutic strategies to improve clinical outcomes for SAP patients. MATERIALS AND METHODS: A comprehensive literature review was performed to identify current clinical strategies, known molecular pathophysiology, and potential therapeutic targets for SAP. RESULTS: Current clinical approaches focus on determining which patients will likely develop SAP. However, therapeutic options are limited to supportive care and fluid resuscitation. The application of a novel 5-cytokine panel accurately predicting disease outcomes in SAP suggests that molecular approaches will improve impact of future clinical trials in AP. CONCLUSIONS: Inflammatory outcomes in acute pancreatitis are driven by several unique molecular signals, which compound to promote both local and systemic inflammation. The identification of master cytokine regulators is critical to developing therapeutics, which reduce inflammation through several mechanisms.

AUM302, a novel triple kinase PIM/PI3K/mTOR inhibitor, is a potent in vitro pancreatic cancer growth inhibitor.

Pancreatic cancer is one of the leading causes of cancer deaths, with pancreatic ductal adenocarcinoma (PDAC) being the most common subtype. Advanced stage diagnosis of PDAC is common, causing limited treatment opportunities. Gemcitabine is a frequently used chemotherapeutic agent which can be used as a monotherapy or in combination. However, tumors often develop resistance to gemcitabine. Previous studies show that the proto-oncogene PIM kinases (PIM1 and PIM3) are upregulated in PDAC compared to matched normal tissue and are related to chemoresistance and PDAC cell growth. The PIM kinases are also involved in the PI3K/AKT/mTOR pathway to promote cell survival. In this study, we evaluate the effect of the novel multikinase PIM/PI3K/mTOR inhibitor, AUM302, and commercially available PIM inhibitor, TP-3654. Using five human PDAC cell lines, we found AUM302 to be a potent inhibitor of cell proliferation, cell viability, cell cycle progression, and phosphoprotein expression, while TP-3654 was less effective. Significantly, AUM302 had a strong impact on the viability of gemcitabine-resistant PDAC cells. Taken together, these results demonstrate that AUM302 exhibits antitumor activity in human PDAC cells and thus has the potential to be an effective drug for PDAC therapy.

Krüppel-like Factor 5 Plays an Important Role in the Pathogenesis of Chronic Pancreatitis.

Chronic pancreatitis results in the formation of pancreatic intraepithelial neoplasia (PanIN) and poses a risk of developing pancreatic cancer. Our previous study demonstrated that Krüppel-like factor 5 (KLF5) is necessary for forming acinar-to-ductal metaplasia (ADM) in acute pancreatitis. Here, we investigated the role of KLF5 in response to chronic injury in the pancreas. Human tissues originating from chronic pancreatitis patients showed increased levels of epithelial KLF5. An inducible genetic model combining the deletion of Klf5 and the activation of KrasG12D mutant expression in pancreatic acinar cells together with chemically induced chronic pancreatitis was used. The chronic injury resulted in increased levels of KLF5 in both control and KrasG12D mutant mice. Furthermore, it led to numerous ADM and PanIN lesions and extensive fibrosis in the KRAS mutant mice. In contrast, pancreata with Klf5 loss (with or without KrasG12D) failed to develop ADM, PanIN, or significant fibrosis. Furthermore, the deletion of Klf5 reduced the expression level of cytokines and fibrotic components such as Il1b, Il6, Tnf, Tgfb1, Timp1, and Mmp9. Notably, using ChIP-PCR, we showed that KLF5 binds directly to the promoters of Il1b, Il6, and Tgfb1 genes. In summary, the inactivation of Klf5 inhibits ADM and PanIN formation and the development of pancreatic fibrosis.

Computational immunohistochemical mapping adds immune context to histological phenotypes in mouse models of colitis.

Inflammatory bowel disease (IBD) is characterized by chronic, dysregulated inflammation in the gastrointestinal tract. The heterogeneity of IBD is reflected through two major subtypes, Crohn's Disease (CD) and Ulcerative Colitis (UC). CD and UC differ across symptomatic presentation, histology, immune responses, and treatment. While colitis mouse models have been influential in deciphering IBD pathogenesis, no single model captures the full heterogeneity of clinical disease. The translational capacity of mouse models may be augmented by shifting to multi-mouse model studies that aggregate analysis across various well-controlled phenotypes. Here, we evaluate the value of histology in multi-mouse model characterizations by building upon a previous pipeline that detects histological disease classes in hematoxylin and eosin (H&E)-stained murine colons. Specifically, we map immune marker positivity across serially-sectioned slides to H&E histological classes across the dextran sodium sulfate (DSS) chemical induction model and the intestinal epithelium-specific, inducible Villin-CreERT2;Klf5fl/fl (Klf5ΔIND) genetic model. In this study, we construct the beginning frameworks to define H&E-patch-based immunophenotypes based on IHC-H&E mappings.

Sonic Hedgehog and WNT Signaling Regulate a Positive Feedback Loop Between Intestinal Epithelial and Stromal Cells to Promote Epithelial Regeneration.

BACKGROUND AND AIMS: Active intestinal stem cells are prone to injury by ionizing radiation. We previously showed that upon radiation-induced injury, normally quiescent reserve intestinal stem cells (rISCs) (marked by BMI1) are activated by Musashi-1 (MSI1) and exit from the quiescent state to regenerate the intestinal epithelium. This study aims to further establish the mechanism that regulates activation of Bmi1-CreER;Rosa26eYFP (Bmi1-CreER) rISCs following γ radiation-induced injury. METHODS: Bmi1-CreER mice were treated with tamoxifen to initiate lineage tracing of BMI1 (eYFP+) cells and exposed to 12 Gy of total body γ irradiation or sham. Intestinal tissues were collected and analyzed by immunofluorescence, Western blot, reverse-transcription quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, and chromatin immunoprecipitation real-time polymerase chain reaction. RESULTS: After irradiation, increased expression of Msi1 in eYFP+ cells was accompanied by increased expression of Axin2, a WNT marker. Promoter studies of the Msi1 gene indicated that Msi1 is a WNT target gene. Coculture of stromal cells isolated from irradiated mice stimulated Bmi1-CreER-derived organoid regeneration more effectively than those from sham mice. Expression of WNT ligands, including Wnt2b, Wnt4, Wnt5a, and Rspo3, was increased in irradiated stromal cells compared with sham-treated stromal cells. Moreover, expression of the Sonic hedgehog (SHH) effector Gli1 was increased in stromal cells from irradiated mice. This was correlated with an increased expression of SHH in epithelial cells postirradiation, indicating epithelial-stromal interaction. Finally, preinjury treatment with SHH inhibitor cyclopamine significantly reduced intestinal epithelial regeneration and Msi1 expression postirradiation. CONCLUSIONS: Upon ionizing radiation-induced injury, intestinal epithelial cells increase SHH secretion, stimulating stromal cells to secrete WNT ligands. WNT activators induce Msi1 expression in the Bmi1-CreER cells. This stromal-epithelial interaction leads to Bmi1-CreER rISCs induction and epithelial regeneration.

Krüppel-like Factors 4 and 5 in Colorectal Tumorigenesis.

Krüppel-like factors (KLFs) are transcription factors regulating various biological processes such as proliferation, differentiation, migration, invasion, and homeostasis. Importantly, they participate in disease development and progression. KLFs are expressed in multiple tissues, and their role is tissue- and context-dependent. KLF4 and KLF5 are two fascinating members of this family that regulate crucial stages of cellular identity from embryogenesis through differentiation and, finally, during tumorigenesis. They maintain homeostasis of various tissues and regulate inflammation, response to injury, regeneration, and development and progression of multiple cancers such as colorectal, breast, ovarian, pancreatic, lung, and prostate, to name a few. Recent studies broaden our understanding of their function and demonstrate their opposing roles in regulating gene expression, cellular function, and tumorigenesis. This review will focus on the roles KLF4 and KLF5 play in colorectal cancer. Understanding the context-dependent functions of KLF4 and KLF5 and the mechanisms through which they exert their effects will be extremely helpful in developing targeted cancer therapy.

The Role of Krüppel-like Factors in Pancreatic Physiology and Pathophysiology.

Krüppel-like factors (KLFs) belong to the family of transcription factors with three highly conserved zinc finger domains in the C-terminus. They regulate homeostasis, development, and disease progression in many tissues. It has been shown that KLFs play an essential role in the endocrine and exocrine compartments of the pancreas. They are necessary to maintain glucose homeostasis and have been implicated in the development of diabetes. Furthermore, they can be a vital tool in enabling pancreas regeneration and disease modeling. Finally, the KLF family contains proteins that act as tumor suppressors and oncogenes. A subset of members has a biphasic function, being upregulated in the early stages of oncogenesis and stimulating its progression and downregulated in the late stages to allow for tumor dissemination. Here, we describe KLFs' function in pancreatic physiology and pathophysiology.

5-FU-miR-15a Inhibits Activation of Pancreatic Stellate Cells by Reducing YAP1 and BCL-2 Levels In Vitro.

Chronic pancreatitis is characterized by chronic inflammation and fibrosis, processes heightened by activated pancreatic stellate cells (PSCs). Recent publications have demonstrated that miR-15a, which targets YAP1 and BCL-2, is significantly downregulated in patients with chronic pancreatitis compared to healthy controls. We have utilized a miRNA modification strategy to enhance the therapeutic efficacy of miR-15a by replacing uracil with 5-fluorouracil (5-FU). We demonstrated increased levels of YAP1 and BCL-2 (both targets of miR-15a) in pancreatic tissues obtained from Ptf1aCreERTM and Ptf1aCreERTM;LSL-KrasG12D mice after chronic pancreatitis induction as compared to controls. In vitro studies showed that delivery of 5-FU-miR-15a significantly decreased viability, proliferation, and migration of PSCs over six days compared to 5-FU, TGFß1, control miR, and miR-15a. In addition, treatment of PSCs with 5-FU-miR-15a in the context of TGFß1 treatment exerted a more substantial effect than TGFß1 alone or when combined with other miRs. Conditioned medium obtained from PSC cells treated with 5-FU-miR-15a significantly inhibits the invasion of pancreatic cancer cells compared to controls. Importantly, we demonstrated that treatment with 5-FU-miR-15a reduced the levels of YAP1 and BCL-2 observed in PSCs. Our results strongly suggest that ectopic delivery of miR mimetics is a promising therapeutic approach for pancreatic fibrosis and that 5-FU-miR-15a shows specific promise.

The Role of MicroRNAs in Pancreatitis Development and Progression.

Pancreatitis (acute and chronic) is an inflammatory disease associated with significant morbidity, including a high rate of hospitalization and mortality. MicroRNAs (miRs) are essential post-transcriptional modulators of gene expression. They are crucial in many diseases' development and progression. Recent studies have demonstrated aberrant miRs expression patterns in pancreatic tissues obtained from patients experiencing acute and chronic pancreatitis compared to tissues from unaffected individuals. Increasing evidence showed that miRs regulate multiple aspects of pancreatic acinar biology, such as autophagy, mitophagy, and migration, impact local and systemic inflammation and, thus, are involved in the disease development and progression. Notably, multiple miRs act on pancreatic acinar cells and regulate the transduction of signals between pancreatic acinar cells, pancreatic stellate cells, and immune cells, and provide a complex interaction network between these cells. Importantly, recent studies from various animal models and patients' data combined with advanced detection techniques support their importance in diagnosing and treating pancreatitis. In this review, we plan to provide an up-to-date summary of the role of miRs in the development and progression of pancreatitis.

FABP5 Inhibition against PTEN-Mutant Therapy Resistant Prostate Cancer.

Resistance to standard of care taxane and androgen deprivation therapy (ADT) causes the vast majority of prostate cancer (PC) deaths worldwide. We have developed RapidCaP, an autochthonous genetically engineered mouse model of PC. It is driven by the loss of PTEN and p53, the most common driver events in PC patients with life-threatening diseases. As in human ADT, surgical castration of RapidCaP animals invariably results in disease relapse and death from the metastatic disease burden. Fatty Acid Binding Proteins (FABPs) are a large family of signaling lipid carriers. They have been suggested as drivers of multiple cancer types. Here we combine analysis of primary cancer cells from RapidCaP (RCaP cells) with large-scale patient datasets to show that among the 10 FABP paralogs, FABP5 is the PC-relevant target. Next, we show that RCaP cells are uniquely insensitive to both ADT and taxane treatment compared to a panel of human PC cell lines. Yet, they share an exquisite sensitivity to the small-molecule FABP5 inhibitor SBFI-103. We show that SBFI-103 is well tolerated and can strongly eliminate RCaP tumor cells in vivo. This provides a pre-clinical platform to fight incurable PC and suggests an important role for FABP5 in PTEN-deficient PC.

Acute postoperative pain and dorsal root ganglia transcriptomic signatures following total knee arthroplasty (TKA) in rats: An experimental study.

Total knee arthroplasty (TKA) is the final treatment option for patients with advanced knee osteoarthritis (OA). Unfortunately, TKA surgery is accompanied by acute postoperative pain that is more severe than arthroplasty performed in other joints. Elucidating the molecular mechanisms specific to post-TKA pain necessitates an animal model that replicates clinical TKA procedures, induces acute postoperative pain, and leads to complete functional recovery. Here, we present a new preclinical TKA model in rats and report on functional and behavioral outcomes indicative of pain, analgesic efficacy, serum cytokine levels, and dorsal root ganglia (DRG) transcriptomes during the acute postoperative period. Following TKA, rats exhibited marked deficits in weight bearing that persisted for 28 days. Home cage locomotion, rearing, and gait were similarly impacted and recovered by day 14. Cytokine levels were elevated on postoperative days one and/or two. Treatment with morphine, ketorolac, or their combination improved weight bearing while gabapentin lacked efficacy. When TKA was performed in rats with OA, similar functional deficits and comparable recovery time courses were observed. Analysis of DRG transcriptomes revealed upregulation of transcripts linked to multiple molecular pathways including inflammation, MAPK signaling, and cytokine signaling and production. In summary, we developed a clinically relevant rat TKA model characterized by resolution of pain and functional recovery within five weeks and with pain-associated behavioral deficits that are partially alleviated by clinically administered analgesics, mirroring the postoperative experience of TKA patients.

SP and KLF Transcription Factors in Cancer Metabolism.

Tumor development and progression depend on reprogramming of signaling pathways that regulate cell metabolism. Alterations to various metabolic pathways such as glycolysis, oxidative phosphorylation, lipid metabolism, and hexosamine biosynthesis pathway are crucial to sustain increased redox, bioenergetic, and biosynthesis demands of a tumor cell. Transcription factors (oncogenes and tumor suppressors) play crucial roles in modulating these alterations, and their functions are tethered to major metabolic pathways under homeostatic conditions and disease initiation and advancement. Specificity proteins (SPs) and Krüppel-like factors (KLFs) are closely related transcription factors characterized by three highly conserved zinc fingers domains that interact with DNA. Studies have demonstrated that SP and KLF transcription factors are expressed in various tissues and regulate diverse processes such as proliferation, differentiation, apoptosis, inflammation, and tumorigenesis. This review highlights the role of SP and KLF transcription factors in the metabolism of various cancers and their impact on tumorigenesis. A better understanding of the role and underlying mechanisms governing the metabolic changes during tumorigenesis could provide new therapeutic opportunities for cancer treatment.

Intestinal Epithelial Regeneration in Response to Ionizing Irradiation.

The intestinal epithelium consists of a single layer of cells yet contains multiple types of terminally differentiated cells, which are generated by the active proliferation of intestinal stem cells located at the bottom of intestinal crypts. However, during events of acute intestinal injury, these active intestinal stem cells undergo cell death. Gamma irradiation is a widely used colorectal cancer treatment, which, while therapeutically efficacious, has the side effect of depleting the active stem cell pool. Indeed, patients frequently experience gastrointestinal radiation syndrome while undergoing radiotherapy, in part due to active stem cell depletion. The loss of active intestinal stem cells in intestinal crypts activates a pool of typically quiescent reserve intestinal stem cells and induces dedifferentiation of secretory and enterocyte precursor cells. If not for these cells, the intestinal epithelium would lack the ability to recover from radiotherapy and other such major tissue insults. New advances in lineage-tracing technologies allow tracking of the activation, differentiation, and migration of cells during regeneration and have been successfully employed for studying this in the gut. This study aims to depict a method for the analysis of cells within the mouse intestinal epithelium following radiation injury.

Deep learning-based approach to the characterization and quantification of histopathology in mouse models of colitis.

Inflammatory bowel disease (IBD) is a chronic immune-mediated disease of the gastrointestinal tract. While therapies exist, response can be limited within the patient population. Researchers have thus studied mouse models of colitis to further understand pathogenesis and identify new treatment targets. Flow cytometry and RNA-sequencing can phenotype immune populations with single-cell resolution but provide no spatial context. Spatial context may be particularly important in colitis mouse models, due to the simultaneous presence of colonic regions that are involved or uninvolved with disease. These regions can be identified on hematoxylin and eosin (H&E)-stained colonic tissue slides based on the presence of abnormal or normal histology. However, detection of such regions requires expert interpretation by pathologists. This can be a tedious process that may be difficult to perform consistently across experiments. To this end, we trained a deep learning model to detect 'Involved' and 'Uninvolved' regions from H&E-stained colonic tissue slides. Our model was trained on specimens from controls and three mouse models of colitis-the dextran sodium sulfate (DSS) chemical induction model, the recently established intestinal epithelium-specific, inducible Klf5ΔIND (Villin-CreERT2;Klf5fl/fl) genetic model, and one that combines both induction methods. Image patches predicted to be 'Involved' and 'Uninvolved' were extracted across mice to cluster and identify histological classes. We quantified the proportion of 'Uninvolved' patches and 'Involved' patch classes in murine swiss-rolled colons. Furthermore, we trained linear determinant analysis classifiers on these patch proportions to predict mouse model and clinical score bins in a prospectively treated cohort of mice. Such a pipeline has the potential to reveal histological links and improve synergy between various colitis mouse model studies to identify new therapeutic targets and pathophysiological mechanisms.

KLF5 protects the intestinal epithelium against Th17 immune response in a murine colitis model.

Inflammatory bowel disease (IBD) is a chronic illness characterized by dysregulated immune cascades in the intestines, in which the Th17 immune response plays an important role. We demonstrated that mice with intestinal epithelium-specific deletion of Krüppel-like factor 5 (Klf5) developed Th17-dependent colonic inflammation. In the absence of KLF5, there was aberrant cellular localization of phosphorylated STAT3, an essential mediator of the Th17-associated cytokine, IL-22, which is required for epithelial tissue regeneration. In contrast, mitigation of IL-17A with anti-IL-17A neutralizing antibody attenuated colitis in Klf5-deficient mice. There was also a considerable shift in the colonic microbiota of Klf5-deficient mice that phenocopied human IBD. Notably, the inflammatory response due to Klf5 deletion was alleviated by antibiotic treatment, implicating the role of microbiota in pathogenesis. Finally, human colitic tissues had reduced KLF5 levels when compared with healthy tissues. Together, these findings demonstrated the importance of KLF5 in protecting the intestinal epithelium against Th17-mediated immune and inflammatory responses. The mice described herein may serve as a potential model for human IBD.

IL-17RA-signaling in Lgr5+ intestinal stem cells induces expression of transcription factor ATOH1 to promote secretory cell lineage commitment.

The Th17 cell-lineage-defining cytokine IL-17A contributes to host defense and inflammatory disease by coordinating multicellular immune responses. The IL-17 receptor (IL-17RA) is expressed by diverse intestinal cell types, and therapies targeting IL-17A induce adverse intestinal events, suggesting additional tissue-specific functions. Here, we used multiple conditional deletion models to identify a role for IL-17A in secretory epithelial cell differentiation in the gut. Paneth, tuft, goblet, and enteroendocrine cell numbers were dependent on IL-17A-mediated induction of the transcription factor ATOH1 in Lgr5+ intestinal epithelial stem cells. Although dispensable at steady state, IL-17RA signaling in ATOH1+ cells was required to regenerate secretory cells following injury. Finally, IL-17A stimulation of human-derived intestinal organoids that were locked into a cystic immature state induced ATOH1 expression and rescued secretory cell differentiation. Our data suggest that the cross talk between immune cells and stem cells regulates secretory cell lineage commitment and the integrity of the mucosa.

Podocyte-specific KLF4 is required to maintain parietal epithelial cell quiescence in the kidney.

Podocyte loss triggering aberrant activation and proliferation of parietal epithelial cells (PECs) is a central pathogenic event in proliferative glomerulopathies. Podocyte-specific Krüppel-like factor 4 (KLF4), a zinc-finger transcription factor, is essential for maintaining podocyte homeostasis and PEC quiescence. Using mice with podocyte-specific knockdown of Klf4, we conducted glomerular RNA-sequencing, tandem mass spectrometry, and single-nucleus RNA-sequencing to identify cell-specific transcriptional changes that trigger PEC activation due to podocyte loss. Integration with in silico chromatin immunoprecipitation identified key ligand-receptor interactions, such as fibronectin 1 (FN1)­αVß6, between podocytes and PECs dependent on KLF4 and downstream signal transducer and activator of transcription 3 (STAT3) signaling. Knockdown of Itgb6 in PECs attenuated PEC activation. Additionally, podocyte-specific induction of human KLF4 or pharmacological inhibition of downstream STAT3 activation reduced FN1 and integrin ß 6 (ITGB6) expression and mitigated podocyte loss and PEC activation in mice. Targeting podocyte-PEC crosstalk might be a critical therapeutic strategy in proliferative glomerulopathies.

KLF5 Is Induced by FOXO1 and Causes Oxidative Stress and Diabetic Cardiomyopathy.

RATIONALE: Diabetic cardiomyopathy (DbCM) is a major complication in type-1 diabetes, accompanied by altered cardiac energetics, impaired mitochondrial function, and oxidative stress. Previous studies indicate that type-1 diabetes is associated with increased cardiac expression of KLF5 (Krüppel-like factor-5) and PPARα (peroxisome proliferator-activated receptor) that regulate cardiac lipid metabolism. OBJECTIVE: In this study, we investigated the involvement of KLF5 in DbCM and its transcriptional regulation. METHODS AND RESULTS: KLF5 mRNA levels were assessed in isolated cardiomyocytes from cardiovascular patients with diabetes and were higher compared with nondiabetic individuals. Analyses in human cells and diabetic mice with cardiomyocyte-specific FOXO1 (Forkhead box protein O1) deletion showed that FOXO1 bound directly on the KLF5 promoter and increased KLF5 expression. Diabetic mice with cardiomyocyte-specific FOXO1 deletion had lower cardiac KLF5 expression and were protected from DbCM. Genetic, pharmacological gain and loss of KLF5 function approaches and AAV (adeno-associated virus)-mediated Klf5 delivery in mice showed that KLF5 induces DbCM. Accordingly, the protective effect of cardiomyocyte FOXO1 ablation in DbCM was abolished when KLF5 expression was rescued. Similarly, constitutive cardiomyocyte-specific KLF5 overexpression caused cardiac dysfunction. KLF5 caused oxidative stress via direct binding on NADPH oxidase (NOX)4 promoter and induction of NOX4 (NADPH oxidase 4) expression. This was accompanied by accumulation of cardiac ceramides. Pharmacological or genetic KLF5 inhibition alleviated superoxide formation, prevented ceramide accumulation, and improved cardiac function in diabetic mice. CONCLUSIONS: Diabetes-mediated activation of cardiomyocyte FOXO1 increases KLF5 expression, which stimulates NOX4 expression, ceramide accumulation, and causes DbCM.