Chloroquine Ingestion to Prevent SARS-CoV-2 Infection: A Report of Two Cases.

INTRODUCTION: Amid the global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), chloroquine and hydroxychloroquine were being studied as agents to prevent and treat coronavirus disease 2019. Information about these agents and their effects circulated throughout the general public media, raising the concern for self-directed consumption of both pharmaceutical and non-pharmaceutical products. CASE REPORT: We present two cases of chloroquine toxicity that occurred after ingestion of an aquarium disinfectant that contained chloroquine phosphate in a misguided attempt to prevent infection by SARS-CoV-2. One patient had repeated emesis and survived, while the other was unable to vomit, despite attempts, and suffered fatal cardiac dysrhythmias. CONCLUSION: These cases illustrate the spectrum of toxicity, varied presentations, and importance of early recognition and management of chloroquine poisoning. In addition, we can see the importance of sound medical guidance in an era of social confusion compounded by the extremes of public and social media.

miR-26 suppresses adipocyte progenitor differentiation and fat production by targeting Fbxl19.

Fat storage in adult mammals is a highly regulated process that involves the mobilization of adipocyte progenitor cells (APCs) that differentiate to produce new adipocytes. Here we report a role for the broadly conserved miR-26 family of microRNAs (miR-26a-1, miR-26a-2, and miR-26b) as major regulators of APC differentiation and adipose tissue mass. Deletion of all miR-26-encoding loci in mice resulted in a dramatic expansion of adipose tissue in adult animals fed normal chow. Conversely, transgenic overexpression of miR-26a protected mice from high-fat diet-induced obesity. These effects were attributable to a cell-autonomous function of miR-26 as a potent inhibitor of APC differentiation. miR-26 blocks adipogenesis, at least in part, by repressing expression of Fbxl19, a conserved miR-26 target without a previously known role in adipocyte biology that encodes a component of SCF-type E3 ubiquitin ligase complexes. These findings have therefore revealed a novel pathway that plays a critical role in regulating adipose tissue formation in vivo and suggest new potential therapeutic targets for obesity and related disorders.

Macrophage Smad3 Protects the Infarcted Heart, Stimulating Phagocytosis and Regulating Inflammation.

RATIONALE: TGF (transforming growth factor)-ß is critically involved in myocardial injury, repair, and fibrosis, activating both Smad (small mothers against decapentaplegic)-dependent and non-Smad pathways. The in vivo role of TGF-ß signaling in regulation of macrophage function is poorly understood. We hypothesized that in the infarcted myocardium, activation of TGF-ß/Smad signaling in macrophages may regulate repair and remodeling. OBJECTIVE: To investigate the role of macrophage-specific TGF-ß Smad3 signaling in a mouse model of myocardial infarction and to dissect the mechanisms mediating Smad-dependent modulation of macrophage function. METHODS AND RESULTS: TGF-ßs markedly activated Smad3 in macrophages, without affecting Smad-independent pathways. Phagocytosis rapidly and directly activated macrophage Smad3, in the absence of active TGF-ß release. MyS3KO (myeloid cell-specific Smad3 knockout) mice had no baseline defects but exhibited increased late mortality and accentuated dilative postmyocardial infarction remodeling. Adverse outcome in infarcted MyS3KO mice was associated with perturbations in phagocytic activity, defective transition of macrophages to an anti-inflammatory phenotype, scar expansion, and accentuated apoptosis of border zone cardiomyocytes. In vitro, Smad3 null macrophages exhibited reduced expression of genes associated with eat-me signals, such as Mfge8 (milk fat globule-epidermal growth factor factor 8), and reduced capacity to produce the anti-inflammatory mediators IL (interleukin)-10 and TGF-ß1, and the angiogenic growth factor VEGF (vascular endothelial growth factor). Mfge8 partly rescued the phagocytic defect of Smad3 null macrophages, without affecting inflammatory activity. Impaired anti-inflammatory actions of Smad3 null macrophages were associated with marked attenuation of phagocytosis-induced PPAR (peroxisome proliferator-activated receptor) expression. MyS3KO mice had no significant alterations in microvascular density and interstitial fibrosis in remodeling myocardial segments. CONCLUSIONS: We demonstrate that Smad3 critically regulates function of infarct macrophages, by mediating acquisition of a phagocytic phenotype and by contributing to anti-inflammatory transition. Smad3-dependent actions in macrophages protect the infarcted heart from adverse remodeling.

Protective Effects of Activated Myofibroblasts in the Pressure-Overloaded Myocardium Are Mediated Through Smad-Dependent Activation of a Matrix-Preserving Program.

RATIONALE: The heart contains abundant interstitial and perivascular fibroblasts. Traditional views suggest that, under conditions of mechanical stress, cytokines, growth factors, and neurohumoral mediators stimulate fibroblast activation, inducing ECM (extracellular matrix) protein synthesis and promoting fibrosis and diastolic dysfunction. Members of the TGF (transforming growth factor)-ß family are upregulated and activated in the remodeling myocardium and modulate phenotype and function of all myocardial cell types through activation of intracellular effector molecules, the Smads (small mothers against decapentaplegic), and through Smad-independent pathways. OBJECTIVES: To examine the role of fibroblast-specific TGF-ß/Smad3 signaling in the remodeling pressure-overloaded myocardium. METHODS AND RESULTS: We examined the effects of cell-specific Smad3 loss in activated periostin-expressing myofibroblasts using a mouse model of cardiac pressure overload, induced through transverse aortic constriction. Surprisingly, FS3KO (myofibroblast-specific Smad3 knockout) mice exhibited accelerated systolic dysfunction after pressure overload, evidenced by an early 40% reduction in ejection fraction after 7 days of transverse aortic constriction. Accelerated systolic dysfunction in pressure-overloaded FS3KO mice was associated with accentuated matrix degradation and generation of collagen-derived matrikines, accompanied by cardiomyocyte myofibrillar loss and apoptosis, and by enhanced macrophage-driven inflammation. In vitro, TGF-ß1, TGF-ß2, and TGF-ß3 stimulated a Smad3-dependent matrix-preserving phenotype in cardiac fibroblasts, suppressing MMP (matrix metalloproteinase)-3 and MMP-8 synthesis and inducing TIMP (tissue inhibitor of metalloproteinases)-1. In vivo, administration of an MMP-8 inhibitor attenuated early systolic dysfunction in pressure-overloaded FS3KO mice, suggesting that the protective effects of activated cardiac myofibroblasts in the pressure-overloaded myocardium are, at least in part, because of suppression of MMPs and activation of a matrix-preserving program. MMP-8 stimulation induces a proinflammatory phenotype in isolated macrophages. CONCLUSIONS: In the pressure-overloaded myocardium, TGF-ß/Smad3-activated cardiac fibroblasts play an important protective role, preserving the ECM network, suppressing macrophage-driven inflammation, and attenuating cardiomyocyte injury. The protective actions of the myofibroblasts are mediated, at least in part, through Smad-dependent suppression of matrix-degrading proteases.

Circulating glucose levels inversely correlate with Drosophila larval feeding through insulin signaling and SLC5A11.

In mammals, blood glucose levels likely play a role in appetite regulation yet the mechanisms underlying this phenomenon remain opaque. Mechanisms can often be explored from Drosophila genetic approaches. To determine if circulating sugars might be involved in Drosophila feeding behaviors, we scored hemolymph glucose and trehalose, and food ingestion in larvae subjected to various diets, genetic mutations, or RNAi. We found that larvae with glucose elevations, hyperglycemia, have an aversion to feeding; however, trehalose levels do not track with feeding behavior. We further discovered that insulins and SLC5A11 may participate in glucose-regulated feeding. To see if food aversion might be an appropriate screening method for hyperglycemia candidates, we developed a food aversion screen to score larvae with abnormal feeding for glucose. We found that many feeding defective larvae have glucose elevations. These findings highlight intriguing roles for glucose in fly biology as a potential cue and regulator of appetite.

Opposing Actions of Fibroblast and Cardiomyocyte Smad3 Signaling in the Infarcted Myocardium.

BACKGROUND: Transforming growth factor-ßs regulate a wide range of cellular responses by activating Smad-dependent and Smad-independent cascades. In the infarcted heart, Smad3 signaling is activated in both cardiomyocytes and interstitial cells. We hypothesized that cell-specific actions of Smad3 regulate repair and remodeling in the infarcted myocardium. METHODS: To dissect cell-specific Smad3 actions in myocardial infarction, we generated mice with Smad3 loss in activated fibroblasts or cardiomyocytes. Cardiac function was assessed after reperfused or nonreperfused infarction using echocardiography. The effects of cell-specific Smad3 loss on the infarcted heart were studied using histological studies, assessment of protein, and gene expression levels. In vitro, we studied Smad-dependent and Smad-independent actions in isolated cardiac fibroblasts. RESULTS: Mice with fibroblast-specific Smad3 loss had accentuated adverse remodeling after reperfused infarction and exhibited an increased incidence of late rupture after nonreperfused infarction. The consequences of fibroblast-specific Smad3 loss were not a result of effects on acute infarct size but were associated with unrestrained fibroblast proliferation, impaired scar remodeling, reduced fibroblast-derived collagen synthesis, and perturbed alignment of myofibroblast arrays in the infarct. Polarized light microscopy in Sirius red-stained sections demonstrated that the changes in fibroblast morphology were associated with perturbed organization of the collagenous matrix in the infarcted area. In contrast, α-smooth muscle actin expression by infarct myofibroblasts was not affected by Smad3 loss. Smad3 critically regulated fibroblast function, activating integrin-mediated nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-2 (NOX-2) expression. Smad3 loss in cardiomyocytes attenuated remodeling and dysfunction after infarction. Cardiomyocyte-specific Smad3 loss did not affect acute infarct size but was associated with attenuated cardiomyocyte apoptosis in the remodeling myocardium, accompanied by decreased myocardial NOX-2 levels, reduced nitrosative stress, and lower matrix metalloproteinase-2 expression. CONCLUSIONS: In healing myocardial infarction, myofibroblast- and cardiomyocyte-specific activation of Smad3 has contrasting functional outcomes that may involve activation of an integrin/reactive oxygen axis.

Distinct cellular and molecular mechanisms for ß3 adrenergic receptor-induced beige adipocyte formation.

Beige/brite adipocytes are induced within white adipose tissues (WAT) and, when activated, consume glucose and fatty acids to produce heat. Classically, two stimuli have been used to trigger a beiging response: cold temperatures and ß3-adrenergic receptor (Adrb3) agonists. These two beiging triggers have been used interchangeably but whether these two stimuli may induce beiging differently at cellular and molecular levels remains unclear. Here, we found that cold-induced beige adipocyte formation requires Adrb1, not Adrb3, activation. Adrb1 activation stimulates WAT resident perivascular (Acta2+) cells to form cold-induced beige adipocytes. In contrast, Adrb3 activation stimulates mature white adipocytes to convert into beige adipocytes. Necessity tests, using mature adipocyte-specific Prdm16 deletion strategies, demonstrated that adipocytes are required and are predominant source to generate Adrb3-induced, but not cold-induced, beige adipocytes. Collectively, we identify that cold temperatures and Adrb3 agonists activate distinct cellular populations that express different ß-adrenergic receptors to induce beige adipogenesis.

A PPARγ transcriptional cascade directs adipose progenitor cell-niche interaction and niche expansion.

Adipose progenitor cells (APCs) reside in a vascular niche, located within the perivascular compartment of adipose tissue blood vessels. Yet, the signals and mechanisms that govern adipose vascular niche formation and APC niche interaction are unknown. Here we show that the assembly and maintenance of the adipose vascular niche is controlled by PPARγ acting within APCs. PPARγ triggers a molecular hierarchy that induces vascular sprouting, APC vessel niche affinity and APC vessel occupancy. Mechanistically, PPARγ transcriptionally activates PDGFRß and VEGF. APC expression and activation of PDGFRß promotes the recruitment and retention of APCs to the niche. Pharmacologically, targeting PDGFRß disrupts APC niche contact thus blocking adipose tissue expansion. Moreover, enhanced APC expression of VEGF stimulates endothelial cell proliferation and expands the adipose niche. Consequently, APC niche communication and retention are boosted by VEGF thereby impairing adipogenesis. Our data indicate that APCs direct adipose tissue niche expansion via a PPARγ-initiated PDGFRß and VEGF transcriptional axis.

Form(ul)ation of adipocytes by lipids.

Lipids have the potential to serve as bio-markers, which allow us to analyze and to identify cells under various experimental settings, and to serve as a clinical diagnostic tool. For example, diagnosis according to specific lipids that are associated with diabetes and obesity. The rapid development of mass-spectrometry techniques enables identification and profiling of multiple types of lipid species. Together, lipid profiling and data interpretation forge the new field of lipidomics. Lipidomics can be used to characterize physiologic and pathophysiological processes in adipocytes, since lipid metabolism is at the core of adipocyte physiology and energy homeostasis. A significant bulk of lipids are stored in adipocytes, which can be released and used to produce energy, used to build membranes, or used as signaling molecules that regulate metabolism. In this review, we discuss how exhaust of lipidomes can be used to study adipocyte differentiation, physiology and pathophysiology.

SMAD3 Regulates Follicle-stimulating Hormone Synthesis by Pituitary Gonadotrope Cells in Vivo.

Pituitary follicle-stimulating hormone (FSH) is an essential regulator of fertility in females and of quantitatively normal spermatogenesis in males. Pituitary-derived activins are thought to act as major stimulators of FSH synthesis by gonadotrope cells. In vitro, activins signal via SMAD3, SMAD4, and forkhead box L2 (FOXL2) to regulate transcription of the FSHß subunit gene (Fshb). Consistent with this model, gonadotrope-specific Smad4 or Foxl2 knock-out mice have greatly reduced FSH and are subfertile. The role of SMAD3 in vivo is unresolved; however, residual FSH production in Smad4 conditional knock-out mice may derive from partial compensation by SMAD3 and its ability to bind DNA in the absence of SMAD4. To test this hypothesis and determine the role of SMAD3 in FSH biosynthesis, we generated mice lacking both the SMAD3 DNA binding domain and SMAD4 specifically in gonadotropes. Conditional knock-out females were hypogonadal, acyclic, and sterile and had thread-like uteri; their ovaries lacked antral follicles and corpora lutea. Knock-out males were fertile but had reduced testis weights and epididymal sperm counts. These phenotypes were consistent with those of Fshb knock-out mice. Indeed, pituitary Fshb mRNA levels were nearly undetectable in both male and female knock-outs. In contrast, gonadotropin-releasing hormone receptor mRNA levels were significantly elevated in knock-outs in both sexes. Interestingly, luteinizing hormone production was altered in a sex-specific fashion. Overall, our analyses demonstrate that SMAD3 is required for FSH synthesis in vivo.

Cellular Aging Contributes to Failure of Cold-Induced Beige Adipocyte Formation in Old Mice and Humans.

Cold temperatures induce progenitor cells within white adipose tissue to form beige adipocytes that burn energy and generate heat; this is a potential anti-diabesity therapy. However, the potential to form cold-induced beige adipocytes declines with age. This creates a clinical roadblock to potential therapeutic use in older individuals, who constitute a large percentage of the obesity epidemic. Here we show that aging murine and human beige progenitor cells display a cellular aging, senescence-like phenotype that accounts for their age-dependent failure. Activating the senescence pathway, either genetically or pharmacologically, in young beige progenitors induces premature cellular senescence and blocks their potential to form cold-induced beige adipocytes. Conversely, genetically or pharmacologically reversing cellular aging by targeting the p38/MAPK-p16Ink4a pathway in aged mouse or human beige progenitor cells rejuvenates cold-induced beiging. This in turn increases glucose sensitivity. Collectively, these data indicate that anti-aging or senescence modalities could be a strategy to induce beiging, thereby improving metabolic health in aging humans.

Emerging Roles of Adipose Progenitor Cells in Tissue Development, Homeostasis, Expansion and Thermogenesis.

Stem or progenitor cells are an essential component for the development, homeostasis, expansion, and regeneration of many tissues. Within white adipose tissue (WAT) reside vascular-resident adipose progenitor cells (APCs) that can proliferate and differentiate into either white or beige/brite adipocytes, which may control adiposity. Recent studies have begun to show that APCs can be manipulated to control adiposity and counteract 'diabesity'. However, much remains unknown about the identity of APCs and how they may control adiposity in response to homeostatic and external cues. Here, we discuss recent advances in our understanding of adipose progenitors and cover a range of topics, including the stem cell/progenitor lineage, their niche, their developmental and adult roles, and their role in cold-induced beige/brite adipocyte formation.

FGFR2IIIb-MAPK Activity Is Required for Epithelial Cell Fate Decision in the Lower Müllerian Duct.

Cell fate of lower Müllerian duct epithelium (MDE), to become uterine or vaginal epithelium, is determined by the absence or presence of ΔNp63 expression, respectively. Previously, we showed that SMAD4 and runt-related transcription factor 1 (RUNX1) were independently required for MDE to express ΔNp63. Here, we report that vaginal mesenchyme directs vaginal epithelial cell fate in MDE through paracrine activation of fibroblast growth factor (FGF) receptor-MAPK pathway. In the developing reproductive tract, FGF7 and FGF10 were enriched in vaginal mesenchyme, whereas FGF receptor 2IIIb was expressed in epithelia of both the uterus and vagina. When Fgfr2 was inactivated, vaginal MDE underwent uterine cell fate, and this differentiation defect was corrected by activation of MEK-ERK pathway. In vitro, FGF10 in combination with bone morphogenetic protein 4 and activin A (ActA) was sufficient to induce ΔNp63 in MDE, and ActA was essential for induction of RUNX1 through SMAD-independent pathways. Accordingly, inhibition of type 1 receptors for activin in neonatal mice induced uterine differentiation in vaginal epithelium by down-regulating RUNX1, whereas conditional deletion of Smad2 and Smad3 had no effect on vaginal epithelial differentiation. In conclusion, vaginal epithelial cell fate in MDE is induced by FGF7/10-MAPK, bone morphogenetic protein 4-SMAD, and ActA-RUNX1 pathway activities, and the disruption in any one of these pathways results in conversion from vaginal to uterine epithelial cell fate.

Exercise-Induced Skeletal Muscle Adaptations Alter the Activity of Adipose Progenitor Cells.

Exercise decreases adiposity and improves metabolic health; however, the physiological and molecular underpinnings of these phenomena remain unknown. Here, we investigate the effect of endurance training on adipose progenitor lineage commitment. Using mice with genetically labeled adipose progenitors, we show that these cells react to exercise by decreasing their proliferation and differentiation potential. Analyses of mouse models that mimic the skeletal muscle adaptation to exercise indicate that muscle, in a non-autonomous manner, regulates adipose progenitor homeostasis, highlighting a role for muscle-derived secreted factors. These findings support a humoral link between skeletal muscle and adipose progenitors and indicate that manipulation of adipose stem cell function may help address obesity and diabetes.

Mouse strains to study cold-inducible beige progenitors and beige adipocyte formation and function.

Cold temperatures induce formation of beige adipocytes, which convert glucose and fatty acids to heat, and may increase energy expenditure, reduce adiposity and lower blood glucose. This therapeutic potential is unrealized, hindered by a dearth of genetic tools to fate map, track and manipulate beige progenitors and 'beiging'. Here we examined 12 Cre/inducible Cre mouse strains that mark adipocyte, muscle and mural lineages, three proposed beige origins. Among these mouse strains, only those that marked perivascular mural cells tracked the cold-induced beige lineage. Two SMA-based strains, SMA-Cre(ERT2) and SMA-rtTA, fate mapped into the majority of cold-induced beige adipocytes and SMA-marked progenitors appeared essential for beiging. Disruption of the potential of the SMA-tracked progenitors to form beige adipocytes was accompanied by an inability to maintain body temperature and by hyperglycaemia. Thus, SMA-engineered mice may be useful to track and manipulate beige progenitors, beige adipocyte formation and function.

Drosophila glucome screening identifies Ck1alpha as a regulator of mammalian glucose metabolism.

Circulating carbohydrates are an essential energy source, perturbations in which are pathognomonic of various diseases, diabetes being the most prevalent. Yet many of the genes underlying diabetes and its characteristic hyperglycaemia remain elusive. Here we use physiological and genetic interrogations in D. melanogaster to uncover the 'glucome', the complete set of genes involved in glucose regulation in flies. Partial genomic screens of ∼1,000 genes yield ∼160 hyperglycaemia 'flyabetes' candidates that we classify using fat body- and muscle-specific knockdown and biochemical assays. The results highlight the minor glucose fraction as a physiological indicator of metabolism in Drosophila. The hits uncovered in our screen may have conserved functions in mammalian glucose homeostasis, as heterozygous and homozygous mutants of Ck1alpha in the murine adipose lineage, develop diabetes. Our findings demonstrate that glucose has a role in fly biology and that genetic screenings carried out in flies may increase our understanding of mammalian pathophysiology.

SMAD2 and p38 signaling pathways act in concert to determine XY primordial germ cell fate in mice.

The sex of primordial germ cells (PGCs) is determined in developing gonads on the basis of cues from somatic cells. In XY gonads, sex-determining region Y (SRY) triggers fibroblast growth factor 9 (FGF9) expression in somatic cells. FGF signaling, together with downstream nodal/activin signaling, promotes male differentiation in XY germ cells by suppressing retinoic acid (RA)-dependent meiotic entry and inducing male-specific genes. However, the mechanism by which nodal/activin signaling regulates XY PGC fate is unknown. We uncovered the roles of SMAD2/3 and p38 MAPK, the putative downstream factors of nodal/activin signaling, in PGC sexual fate decision. We found that conditional deletion of Smad2, but not Smad3, from XY PGCs led to a loss of male-specific gene expression. Moreover, suppression of RA signaling did not rescue male-specific gene expression in Smad2-mutant testes, indicating that SMAD2 signaling promotes male differentiation in a RA-independent manner. By contrast, we found that p38 signaling has an important role in the suppression of RA signaling. The Smad2 deletion did not disrupt the p38 signaling pathway even though Nodal expression was significantly reduced, suggesting that p38 was not regulated by nodal signaling in XY PGCs. Additionally, the inhibition of p38 signaling in the Smad2-mutant testes severely impeded XY PGC differentiation and induced meiosis. In conclusion, we propose a model in which p38 and SMAD2 signaling coordinate to determine the sexual fate of XY PGCs.

Independent stem cell lineages regulate adipose organogenesis and adipose homeostasis.

Adipose tissues have striking plasticity, highlighted by childhood and adult obesity. Using adipose lineage analyses, smooth muscle actin (SMA)-mural cell-fate mapping, and conditional PPARγ deletion to block adipocyte differentiation, we find two phases of adipocyte generation that emanate from two independent adipose progenitor compartments: developmental and adult. These two compartments are sequentially required for organ formation and maintenance. Although both developmental and adult progenitors are specified during the developmental period and express PPARγ, they have distinct microanatomical, functional, morphogenetic, and molecular profiles. Furthermore, the two compartments derive from different lineages; whereas adult adipose progenitors fate-map from an SMA+ mural lineage, developmental progenitors do not. Remarkably, the adult progenitor compartment appears to be specified earlier than the developmental cells and then enters the already developmentally formed adipose depots. Thus, two distinct cell compartments control adipose organ development and organ homeostasis, which may provide a discrete therapeutic target for childhood and adult obesity.