Skeletal muscle can regenerate from muscle stem cells and their myogenic precursor cell progeny, myoblasts. However, precise gene editing in human muscle stem cells for autologous cell replacement therapies of untreatable genetic muscle diseases has not yet been reported. Loss-of-function mutations in SGCA, encoding α-sarcoglycan, cause limb-girdle muscular dystrophy 2D/R3, an early onset, severe and rapidly progressive form of muscular dystrophy affecting equally girls and boys. Patients suffer from muscle degeneration and atrophy affecting the limbs, respiratory muscles, and the heart. We isolated human muscle stem cells from two donors with the common SGCA c.157G>A mutation affecting the last coding nucleotide of exon 2. We found that c.157G>A is an exonic splicing mutation that induces skipping of two co-regulated exons. Using adenine base editing, we corrected the mutation in the cells from both donors with >90% efficiency, thereby rescuing the splicing defect and α-sarcoglycan expression. Base edited patient cells regenerated muscle and contributed to the Pax7 positive satellite cell compartment in vivo in mouse xenografts. We hereby provide the first evidence that autologous gene repaired human muscle stem cells can be harnessed for cell replacement therapies of muscular dystrophies.
Helena Escobar, Anne Krause, Sandra Keiper, Janine Kieshauer, Stefanie Müthel, Manuel García de Paredes, Eric Metzler, Ralf Kühn, Florian Heyd, Simone Spuler
Bariatric surgery is the most effective method for weight loss in morbid obesity. There is significant individual variability in the weight loss outcomes, yet factors leading to postoperative weight loss or weight regain remain elusive. Alterations in the µ-opioid receptor (MOR) and dopamine D2 receptor (D2R) systems are associated with obesity and appetite control, and the magnitude of initial brain receptor system perturbation may predict long-term surgical weight loss outcomes. We tested this hypothesis by studying 19 morbidly obese women (mean BMI 40) scheduled to undergo bariatric surgery. We measured their preoperative MOR and D2R availabilities using positron emission tomography (PET) with [11C]carfentanil and [11C]raclopride, respectively, and then assessed their weight development association with regional MOR and D2R availabilities at 24-month follow-up. MOR availability in the amygdala consistently predicted weight development throughout the follow-up period, but no associations were found for D2R. This is the first study to demonstrate that neuroreceptor markers prior to bariatric surgery are associated with the postoperative weight loss. Postoperative weight regain may derive from dysfunction in the opioid system, and weight loss outcomes after bariatric surgery may be partially predicted based on preoperative brain receptor availability opening up new potential for treatment possibilities.
Henry K. Karlsson, Lauri Tuominen, Semi Helin, Paulina Salminen, Pirjo Nuutila, Lauri Nummenmaa
BACKGROUND. Idiopathic intracranial hypertension (IIH) is a condition predominantly affecting obese women of reproductive age. Recent evidence suggests that IIH is a disease of metabolic dysregulation, androgen excess and an increased risk of cardiovascular morbidity. Here we evaluate systemic and adipose specific metabolic determinants of the IIH phenotype. METHODS. In fasted, matched IIH (N=97) and control (N=43) patients, we assessed: glucose and insulin homeostasis and leptin levels. Body composition was assessed along with an interrogation of adipose tissue function via nuclear magnetic resonance metabolomics and RNA sequencing in paired omental and subcutaneous biopsies in a case control study. RESULTS. We demonstrate an insulin and leptin resistant phenotype in IIH in excess to that driven by obesity. Adiposity in IIH is preferentially centripetal and is associated with increased disease activity and insulin resistance. IIH adipocytes appear transcriptionally and metabolically primed towards depot-specific lipogenesis. CONCLUSIONS. These data show that IIH is a metabolic disorder in which adipose tissue dysfunction is a feature of the disease. Managing IIH as a metabolic disease could reduce disease morbidity and improving cardiovascular outcomes. FUNDING. This study was supported by the National Institute of Health Research UK (NIHR-CS-011-028), the Medical Research Council UK (MR/K015184/1) and the Midlands Neuroscience Teaching and Research Fund.
Connar S.J. Westgate, Hannah F. Botfield, Zerin Alimajstorovic, Andreas Yiangou, Mark Walsh, Gabrielle Smith, Rishi Singhal, James L. Mitchell, Olivia Grech, Keira A. Markey, Daniel Hebenstreit, Daniel A. Tennant, Jeremy W. Tomlinson, Susan P. Mollan, Christian Ludwig, Ildem Akerman, Gareth G. Lavery, Alexandra J. Sinclair
Retinoic acid (RA) signaling is essential for enteric nervous system (ENS) development since vitamin A deficiency or mutations in RA signaling profoundly reduce bowel colonization by ENS precursors. These RA effects could occur because of RA activity within the ENS lineage or via RA activity in other cell types. To define cell-autonomous roles for retinoid signaling within the ENS lineage at distinct developmental time points, we activated a potent floxed dominant-negative RA receptor α (RarαDN) in the ENS using diverse CRE recombinase-expressing mouse lines. This strategy enabled us to block RA signaling at pre-migratory, migratory, and post-migratory stages for ENS precursors. We found that cell-autonomous loss of retinoic acid receptor (RAR) signaling dramatically affects ENS development. CRE activation of RarαDN expression at pre-migratory or migratory stages caused severe intestinal aganglionosis, but at later stages, RarαDN induced a broad range of phenotypes including hypoganglionosis, submucosal plexus loss, and abnormal neural differentiation. RNA-sequencing highlighted distinct RA-regulated gene sets at different developmental stages. These studies show complicated context-dependent RA-mediated regulation of ENS development.
Tao Gao, Elizabeth C. Wright-Jin, Rajarshi Sengupta, Jessica B. Anderson, Robert O. Heuckeroth
Transitions between cell fates commonly occur in development and disease. However, reversing an unwanted cell transition in order to treat disease remains an unexplored area. Here, we report a successful process of guiding ill-fated transitions toward normalization in vascular calcification. Vascular calcification is a severe complication that increases all-cause mortality of cardiovascular disease but lacks medical therapy. The vascular endothelium is a contributor of osteoprogenitor cells to vascular calcification through endothelial-mesenchymal transitions, in which endothelial cells (ECs) gain plasticity and ability to differentiate into osteoblast-like cells. We created a high throughput screening and identified SB216763, an inhibitor of glycogen synthase kinase 3 (GSK3), as an inducer of osteoblastic-endothelial transition. We demonstrated that SB216763 limits osteogenic differentiation in ECs at an early stage of vascular calcification. Lineage tracing showed that SB216763 redirected osteoblast-like cells to the endothelial lineage and reduced late-stage calcification. We also find that deletion of GSK3beta in osteoblasts recapitulated osteoblastic-endothelial transition and reduced vascular calcification. Overall, inhibition of GSK3beta promoted the transition of cells with osteoblastic characteristic to endothelial differentiation thereby ameliorating vascular calcification.
Jiayi Yao, Xiuju Wu, Xiaojing Qiao, Daoqin Zhang, Li Zhang, Jocelyn A. Ma, Xinjiang Cai, Kristina I. Boström, Yucheng Yao
Left ventricular hypertrophy (LVH) is a primary feature of cardiovascular complications in chronic kidney disease (CKD) patients. MiRNA-30 is an important posttranscriptional regulator of LVH, but it is unknown whether miRNA-30 participates in the process of CKD-induced LVH. In the present study, we found that CKD not only results in LVH but also suppresses miRNA-30 expression in the myocardium. Rescue of cardiomyocyte-specific miRNA-30 attenuates LVH in CKD rats without altering CKD progression. Importantly, in vivo and in vitro knockdown of miRNA-30 in cardiomyocytes leads to cardiomyocyte hypertrophy by upregulating the calcineurin signalling directly. Furthermore, CKD-related detrimental factors, such as fibroblast growth factor-23 (FGF-23), uraemic toxin, angiotensin-II (Ang-II) and transforming growth factor-β (TGF-β), suppress cardiac miRNA-30 expression, while miRNA-30 supplementation blunts cardiomyocyte hypertrophy induced by such factors. These results uncover a novel mechanism of CKD-induced LVH and provide a potential therapeutic target for CKD patients with LVH.
Jingfu Bao, Yinghui Lu, Qin-ying She, Weijuan Dou, Rong Tang, Xiaodong Xu, Mingchao Zhang, Ling Zhu, Qing Zhou, Hui Li, Guohua Zhou, Zhongzhou Yang, Shaolin Shi, Zhihong Liu, Chunxia Zheng
Endothelial cells are important in the maintenance of healthy blood vessels and in the development of vascular diseases. However, the origin and dynamics of endothelial precursors and remodeling at the single-cell level have been difficult to study in vivo due to technical limitations. We aimed to develop a direct visual approach to track the fate and function of single endothelial cells over several days-weeks in the same vascular bed in vivo using multiphoton microscopy (MPM) of transgenic Cdh5-Confetti mice and the kidney glomerulus as a model. Individual cells of the vascular endothelial lineage were identified and tracked due to their unique color combination, based on the random expression of cyan/green/yellow/red fluorescent proteins. Experimental hypertension, hyperglycemia, and laser-induced endothelial cell ablation rapidly increased the number of new glomerular endothelial cells that appeared in clusters of the same color, suggesting clonal cell remodeling by local precursors at the vascular pole. Furthermore, intravital MPM allowed the detection of distinct structural and functional alterations of proliferating endothelial cells. No circulating Cdh5-Confetti+ cells were found in the renal cortex. The heart, lung, and kidneys showed more significant clonal endothelial cell expansion compared to the brain, pancreas, liver and spleen. Serial MPM of Cdh5-Confetti mice in vivo is a powerful new technical advance to study endothelial remodeling and repair in the kidney and other organs under physiological and disease conditions.
Dorinne Desposito, Ina Maria Schiessl, Georgina Gyarmati, Anne Riquier-Brison, Audrey Izuhara, Hiroyuki Kadoya, Balint Der, Urvi Nikhil Shroff, Young-Kwon Hong, Janos Peti-Peterdi
Extensive activation of glial cells during a latent period has been well documented in various animal models of epilepsy. However, it remains unclear whether activated glial cells contribute to epileptogenesis; i.e., the chronically persistent process leading to epilepsy. Particularly, it is not clear whether inter-glial communication between different types of glial cells contributes to epileptogenesis, as past literature mainly focused on one type of glial cell. Here, we show that temporally distinct activation profiles of microglia and astrocytes collaboratively contribute to epileptogenesis in a drug-induced status epilepticus model. We found that reactive microglia appeared first, followed by reactive astrocytes and increased susceptibility to seizures. Reactive astrocytes exhibited larger Ca2+ signals mediated by IP3R2, whereas deletion of this type of Ca2+ signaling reduced seizure susceptibility after status epilepticus. Immediate, but not late, pharmacological inhibition of microglial activation prevented subsequent reactive astrocytes, aberrant astrocyte Ca2+ signaling, and the enhanced seizure susceptibility. These findings indicate that the sequential activation of glial cells constitutes a cause of epileptogenesis after status epilepticus. Thus, our findings suggest that the therapeutic target to prevent epilepsy after status epilepticus should be shifted from microglia (early phase) to astrocytes (late phase).
Fumikazu Sano, Eiji Shigetomi, Youichi Shinozaki, Haruka Tsuzukiyama, Kozo Saito, Katsuhiko Mikoshiba, Hiroshi Horiuchi, Dennis Lawrence Cheung, Junichi Nabekura, Kanji Sugita, Masao Aihara, Schuichi Koizumi
Idiopathic pulmonary fibrosis is a progressive fibrotic lung disease. We previously identified fibrogenic mesenchymal progenitor cells (MPCs) in the lungs of IPF patients that serve as an obligatory driver of progressive fibrosis. Recent single cell RNA sequencing work revealed that IPF MPCs with the highest transcriptomic network entropy differ the most from control MPCs; and that CD44 was a marker of these IPF MPCs. We hypothesize that IPF MPCs with high CD44 (CD44hi) expression will display enhanced fibrogenicity. We demonstrate that CD44 expressing MPCs are present at the periphery of the IPF fibroblastic focus, placing them in regions of active fibrogenesis. In a humanized mouse xenograft model, CD44hi IPF MPCs are more fibrogenic than CD44lo IPF MPCs and knock-down of CD44 diminishes their fibrogenicity. CD44hi IPF MPCs display increased expression of pluripotency markers and self-renewal compared to CD44lo IPF MPCs; properties potentiated by IL-8. The mechanism involves the accumulation of CD44 within the nucleus where it associates with the chromatin modulator protein BRG1 and the Zeb1 transcription factor. This CD44/BRG1/Zeb1 nuclear protein complex targets the Sox2 gene promoting its upregulation and self-renewal. Our data implicates CD44 interaction with the epigenetic modulator protein Brg1 in conveying IPF MPCs with cell-autonomous fibrogenicity.
Libang Yang, Hong Xia, Karen A. Smith, Adam Gilbertsen, Daniel Beisang, Jonathan Kuo, Peter B. Bitterman, Craig A. Henke
T cell receptor (TCR) triggering by antigen results in metabolic reprogramming that, in turn, facilitates T cells’ exit from quiescence. The increased nutrient requirements of activated lymphocytes are met in part by upregulation of cell surface transporters and enhanced uptake of amino acids, fatty acids and glucose from the environment. However, the role of intracellular pathways of amino acid biosynthesis in T cell activation is relatively unexplored. Asparagine (Asn) is a non-essential amino acid that can be synthesized intracellularly through the glutamine-hydrolyzing enzyme asparagine synthetase (ASNS). We set out to define the requirements for uptake of extracellular Asn and ASNS activity in CD8+ T cell activation. At early timepoints of activation in vitro, CD8+ T cells expressed little or no ASNS and, as a consequence, viability and TCR-stimulated growth, activation and metabolic reprogramming were substantially impaired under conditions of Asn deprivation. At later timepoints (>24h of activation), TCR-induced mTOR-dependent signals resulted in upregulation of ASNS, that endowed CD8+ T cells with the capacity to function independently of extracellular Asn. Thus, our data suggest that the coordinated upregulation of ASNS expression and uptake of extracellular Asn is involved in optimal T cell effector responses.
Helen Carrasco Hope, Rebecca J. Brownlie, Christopher M. Fife, Lynette Steele, Mihaela Lorger, Robert J. Salmond
Telomerase catalyzes chromosome end replication in stem cells and other long-lived cells. Mutations in telomerase or telomere-related genes result in diseases known as telomeropathies. Telomerase is recruited to chromosome ends by the ACD/TPP1 protein (TPP1 hereafter), a component of the shelterin complex that protects chromosome ends from unwanted end-joining. TPP1 facilitates end-protection by binding shelterin proteins POT1 and TIN2. TPP1 variants have been associated with telomeropathies, but remain poorly characterized in vivo. Disease variants and mutagenesis scans provide efficient avenues to interrogate the distinct physiological roles of TPP1. Here, we conduct mutagenesis in the TIN2- and POT1-binding domains of TPP1 to discover mutations that dissect TPP1’s functions. Our results extend upon current structural data to reveal that the TPP1-TIN2 interface is more extensive than previously thought, and highlight the robustness of the POT1-TPP1 interface. Introduction of separation-of-function mutants alongside known TPP1 telomeropathy mutations in mouse hematopoietic stem cells (mHSCs) lacking endogenous TPP1 demonstrated a clear phenotypic demarcation. TIN2- and POT1-binding mutants were unable to rescue mHSC failure resulting from end-deprotection. In contrast, TPP1 telomeropathy mutations sustained mHSC viability, consistent with them selectively impacting end-replication. These results highlight the power of scanning mutagenesis in revealing structural interfaces and dissecting multifunctional genes.
Sherilyn Grill, Shilpa Padmanaban, Ann Friedman, Eric Perkey, Frederick Allen, Valerie M. Tesmer, Jennifer Chase, Rami Khoriaty, Catherine E. Keegan, Ivan Maillard, Jayakrishnan Nandakumar
BACKGROUND. Methodology for estimation of cerebrospinal fluid (CSF) tracer clearance could have wide clinical application in predicting excretion of intrathecal drugs and metabolic solutes from brain metabolism, and for diagnostic work-up of cerebrospinal fluid disturbances. METHODS. The magnetic resonance imaging (MRI) contrast agent gadobutrol (Gadovist) was utilized as CSF tracer and injected into the lumbar CSF. Gadobutrol is contained outside blood vessels of the central nervous system (CNS) and is thus eliminated along extra-vascular pathways, analogous to many CNS metabolites and intrathecal drugs. Tracer enrichment was verified and assessed in CSF by MRI at level of the cisterna magna in parallel with obtaining blood samples through 48 hours. RESULTS. In a reference patient cohort (REF; n=29), both enrichment within CSF and blood coincided in time. Blood concentration profiles of gadobutrol through 48 hours varied between patients diagnosed with CSF leakage (n=4), idiopathic normal pressure hydrocephalus dementia (iNPH; n=7), pineal cysts (n=8), and idiopathic intracranial hypertension (IIH; n=4). CONCLUSION. Assessment of CSF tracer clearance is clinically feasible and may provide a way to predict extra-vascular clearance of intrathecal drugs and endogenous metabolites from the CNS. The peak concentration in blood (at about 10 hrs) preceded by far peak tracer enhancement at MRI in extra-cranial lymphatic structures (at about 24 hrs) as shown in previous studies, indicating a major role of the spinal canal in CSF clearance capacity. FUNDING. The work was supported by Department of Neurosurgery, Oslo university hospital, and Norwegian Institute for Air Research, Kjeller, Norway, and University of Oslo.
Per K. Eide, Espen Mariussen, Hilde Uggerud, Are H. Pripp, Aslan Lashkarivand, Bjørnar Hassel, Hege Christensen, Markus Herberg Hovd, Geir Ringstad
Roughly one year after the first case of COVID-19 was identified and less than one year after the sequencing of SARS-CoV-2, multiple SARS-CoV-2 vaccines with demonstrated safety and efficacy in phase III clinical trials are available. The most promising vaccines have targeted the surface glycoprotein (S-protein) of SARS-CoV-2 and achieved an approximate 85-95% reduction in the risk of symptomatic COVID-19, while retaining excellent safety profiles and modest side effects in the phase III clinical trials. The mRNA, replication-incompetent viral vector, and protein subunit vaccine technologies have all been successfully employed. Some novel SARS-CoV-2 variants evade but do not appear to fully overcome the potent immunity induced by these vaccines. Emerging real-world effectiveness data add evidence for protection from severe COVID-19. This is an impressive first demonstration of the effectiveness of the mRNA vaccine and vector vaccine platforms. The success of SARS-CoV-2 vaccine development should be credited to open science, industry partnerships, harmonization of clinical trials, and the altruism of study participants. The manufacturing and distribution of the emergency use-authorized SARS-CoV-2 vaccines are ongoing challenges. What remains now is to ensure broad and equitable global vaccination against COVID-19.
Jonathan L. Golob, Njira Lugogo, Adam S. Lauring, Anna S. Lok
Background: COVID-19 is more benign in children compared to adults for unknown reasons. This contrasts with other respiratory viruses where disease manifestations are often more severe in children. We hypothesize that a more robust early innate immune response to SARS-CoV-2 protects against severe disease. Methods: Clinical outcomes, SARS-CoV-2 viral copies and cellular gene expression were compared in nasopharyngeal swabs obtained at the time of presentation to the Emergency Department from 12 children and 27 adults using bulk RNA sequencing and quantitative reverse transcription PCR. Total protein, cytokines and anti-SARS-CoV-2 IgG and IgA were quantified in nasal fluid. Results: SARS-CoV-2 copies, ACE2 and TMPRSS2 gene expression were similar in children and adults, but children displayed higher expression of genes associated with interferon signaling, NLRP3 inflammasome, and other innate pathways. Higher levels of IFN-α2, IFN-γ, IP-10, IL-8, and IL-1β protein were detected in nasal fluid in children versus adults. Children also expressed higher levels of genes associated with immune cells whereas expression of those associated with epithelial cells did not differ in children versus adults. Anti-SARS-CoV-2 IgA and IgG were detected at similar levels in nasal fluid from both groups. None of the children required supplemental oxygen whereas 7 adults did (p=0.03); four adults died. Conclusions: These findings provide direct evidence of a more vigorous early mucosal immune response in children compared to adults and suggest that this contributes to favorable clinical outcomes.
Carl A. Pierce, Sharlene Sy, Benjamin Galen, Doctor Y. Goldstein, Erika P. Orner, Marla J. Keller, Kevan C. Herold, Betsy C. Herold
Age-related macular degeneration (AMD) damages the retinal pigment epithelium (RPE), the tissue that safeguards photoreceptor health, leading to irreversible vision loss. Polymorphisms in cholesterol and complement genes are implicated in AMD, yet mechanisms linking risk variants to RPE injury remain unclear. We sought to determine how allelic variants in the apolipoprotein E cholesterol transporter modulate RPE homeostasis and function. Using live-cell imaging, we show that inefficient cholesterol transport by the AMD risk-associated ApoE2 increases RPE ceramide, leading to autophagic defects and complement-mediated mitochondrial damage. Mitochondrial injury drives redox state-sensitive cysteine-mediated phase separation of ApoE2, forming biomolecular condensates that could nucleate drusen. The protective ApoE4 isoform lacks these cysteines and is resistant to phase separation and condensate formation. In Abca4-/- Stargardt macular degeneration mice, mitochondrial dysfunction induces liquid-liquid phase separation of p62/SQSTM1, a multifunctional protein that regulates autophagy. Drugs that decrease RPE cholesterol or ceramide prevent mitochondrial injury and phase separation in vitro and in vivo. In AMD donor RPE, mitochondrial fragmentation correlates with ApoE and p62 condensates. Our studies demonstrate that major AMD genetic and biological risk pathways converge upon RPE mitochondria, and identify mitochondrial stress-mediated protein phase separation as an important pathogenic mechanism and promising therapeutic target in AMD.
Nilsa La Cunza, Li Xuan Tan, Thushara Thamban, Colin J Germer, Gurugirijha Rathnasamy, Kimberly Toops, Aparna Lakkaraju
Archana S. Nagaraja, Robert L. Dood, Guillermo Armaiz-Pena, Yu Kang, Sherry Y. Wu, Julie K. Allen, Nicholas B. Jennings, Lingegowda S. Mangala, Sunila Pradeep, Yasmin Lyons, Monika Haemmerle, Kshipra M. Gharpure, Nouara C. Sadaoui, Cristian Rodriguez-Aguayo, Cristina Ivan, Ying Wang, Keith Baggerly, Prahlad Ram, Gabriel Lopez-Berestein, Jinsong Liu, Samuel C. Mok, Lorenzo Cohen, Susan K. Lutgendorf, Steve W. Cole, and Anil K. Sood Original citation: JCI Insight. 2017;2(16):e93076. https://doi.org/10.1172/jci.insight.93076. Citation for this corrigendum: JCI Insight. 2021;6(7):e149895. https://doi.org/10.1172/jci.insight.149895. The Editors previously posted an Expression of Concern for this article regarding images in Figure 5E that appeared to be the same (siControl Stress and siNHBA Control). An institutional review committee concluded that the error in Figure 5E was inadvertent and recommended correction. The authors provided the correct image for the siControl Stress sample from the original study. The authors regret the error.
JCI Insight Staff
Lingegowda S. Mangala, Hongyu Wang, Dahai Jiang, Sherry Y. Wu, Anoma Somasunderam, David E. Volk, Ganesh L. R. Lokesh, Xin Li, Sunila Pradeep, Xianbin Yang, Monika Haemmerle, Cristian Rodriguez-Aguayo, Archana S Nagaraja, Rajesha Rupaimoole, Emine Bayraktar, Recep Bayraktar, Li Li, Takemi Tanaka, Wei Hu, Cristina Ivan, Kshipra M Gharpure, Michael H. McGuire, Varatharasa Thiviyanathan, Xinna Zhang, Sourindra N. Maiti, Nataliya Bulayeva, Hyun-Jin Choi, Piotr L. Dorniak, Laurence J.N. Cooper, Kevin P. Rosenblatt, Gabriel Lopez-Berestein, David G. Gorenstein, and Anil K. Sood Original citation: JCI Insight. 2016;1(17):e87754. https://doi.org/10.1172/jci.insight.87754. Citation for this corrigendum: JCI Insight. 2021;6(7):e149896. https://doi.org/10.1172/jci.insight.149896. The Editors previously posted an Expression of Concern for this article regarding images in Supple-mental 4B that appeared similar (Endo40 and Endo42 samples) and images in Supplemental Figure 9 that appeared to be the same (CH and CH/Endo28-NPs for heart tissue). No changes were necessary for Supplemental Figure 4B, as the Endo40 and Endo42 samples were determined to be derived from adjacent tumor sections. An institutional review committee concluded that the error in Supplemental Figure 9 was inadvertent and recommended correction. The authors provided the correct image for the CH/Endo28-NPs sample from the original study. An updated supplemental file that includes the correct version of Supplemental Figure 9 is now posted. The authors regret the error.
JCI Insight Staff
Anastomotic leakage (AL) accounts for a major part of in-house mortality in patients undergoing colorectal surgery. Local ischemia and abdominal sepsis are common risk factors contributing to AL and are characterized by upregulation of the hypoxia-inducible factor (HIF) pathway. The HIF pathway is critically regulated by HIF-prolyl hydroxylases (PHDs). Here, we investigated the significance of PHDs and the effects of pharmacologic PHD inhibition (PHI) during anastomotic healing. Ischemic or septic colonic anastomoses were created in mice by ligation of mesenteric vessels or lipopolysaccharide-induced abdominal sepsis, respectively. Genetic PHD-deficiency (Phd1-/-, Phd2+/-, and Phd3-/-) or PHI were applied to manipulate PHD activity. Pharmacologic PHI and genetic PHD2-haplodeficiency (Phd2+/-) significantly improved healing of ischemic or septic colonic anastomoses, as indicated by increased bursting pressure and reduced AL rates. Only Phd2+/- (but not PHI or Phd1-/-) protected from sepsis-related mortality. Mechanistically, PHI and Phd2+/- induced immuno-modulatory (M2) polarization of macrophages, resulting in increased collagen content and attenuated inflammation-driven immune cell recruitment. We conclude that PHI improves healing of colonic anastomoses in ischemic or septic conditions by Phd2+/--mediated M2 polarization of macrophages, conferring a favourable microenvironment for anastomotic healing. Patients with critically perfused colorectal anastomosis or abdominal sepsis could benefit from pharmacologic PHI.
Moritz J. Strowitzki, Gwendolyn Kimmer, Julian S. Wehrmann, Alina S. Ritter, Praveen Radhakrishnan, Vanessa M. Opitz, Christopher Tuffs, Marvin Biller, Julia Kugler, Ulrich Keppler, Jonathan M. Harnoss, Johannes Klose, Thomas Schmidt, Alfonso Blanco, Cormac T. Taylor, Martin Schneider
Despite the recent launch of Tolvaptan, the search for safer polycystic kidney disease (PKD) drugs continues. Ciclopirox (CPX) or its olamine salt (CPX-O) are contained in number of commercially available antifungal agents. CPX is also reported to possess anticancer activity. Several mechanisms of action have been proposed including chelation of iron and inhibition of iron dependent enzymes. Here, we show that CPX-O inhibited in vitro cystogenesis of primary human PKD cyst-lining epithelial cells cultured in a 3D collagen matrix. To assess in vivo role of CPX-O, we treated PKD mice with CPX-O. CPX-O reduced the kidney- to-body weight ratios of PKD mice. This was also associated with decreased cell proliferation, decreased cystic area and improved renal function. Ferritin levels were significantly elevated in cystic kidneys of PKD mice, and CPX-O treatment reduced renal ferritin levels. The reduction in ferritin was associated with increased ferritinophagy marker, NCOA4 which reversed upon CPX-O treatment in PKD mice. Interestingly, these effects on ferritin appeared independent of iron. These data suggest that CPX-O can induce ferritin degradation via ferritinophagy which is associated with decreased cyst growth progression in PKD mice. Most importantly these data indicate that CPX-O has the potential to treat autosomal dominant PKD.
Priyanka S. Radadiya, Mackenzie M. Thornton, Rajni V. Puri, Sireeesha Yerrathota, Johnny Dinh-Phan, Brenda Magenheimer, Dharmalingam Subramaniam, Pamela V. Tran, Hao Zhu, Subhashini Bolisetty, James P. Calvet, Darren P. Wallace, Madhulika Sharma
The retinal pigment epithelium (RPE) provides vital metabolic support for retinal photoreceptor cells and also is an important player in numerous retinal diseases. Gene manipulation in mice using the Cre-LoxP system is an invaluable tool for studying the genetic basis of these retinal diseases. However, existing RPE-targeted Cre mouse lines have critical limitations that restrict their reliability for studies of disease pathogenesis and treatment, including mosaic Cre expression, inducer-independent activity, off-target Cre expression, and intrinsic toxicity. Here, we report the generation and characterization of a knock-in mouse line in which a P2A-CreERT2 coding sequence is fused with the native RPE-specific 65 kDa protein (Rpe65) gene for co-translational expression of CreERT2. Cre+/- mice were able to recombine a stringent Cre reporter allele with >99% efficiency and absolute RPE specificity upon tamoxifen induction at both post-natal days (PD) 21 and 50. Tamoxifen-independent Cre activity was negligible at PD64. Moreover, tamoxifen-treated Cre+/- mice displayed no signs of structural or functional retinal pathology up to 4 months of age. Despite weak RPE65 expression from the knock-in allele, visual cycle function was normal in Cre+/- mice. These data indicate that Rpe65CreERT2 mice are well-suited for studies of gene function and pathophysiology in the RPE.
Elliot H. Choi, Susie Suh, David E. Einstein, Henri Leinonen, Zhiqian Dong, Sriganesh Ramachandra Rao, Steven J. Fliesler, Seth Blackshaw, Minzhong Yu, Neal S. Peachey, Krzysztof Palczewski, Philip D. Kiser