Peroxisome proliferator-activated receptor gamma blunts endothelin-1-mediated contraction of the uterine artery in a murine model of high-altitude pregnancy.

The environmental hypoxia of high altitude (HA) increases the incidence of intrauterine growth restriction (IUGR) approximately threefold. The peroxisome proliferator-activated receptor γ (PPAR-γ), a ligand-activated nuclear receptor that promotes vasorelaxation by increasing nitric oxide and downregulating endothelin-1 (ET-1) production, has been implicated in IUGR. Based on our prior work indicating that pharmacologic activation of the PPARγ pathway protects against hypoxia-associated IUGR, we used an experimental murine model to determine whether such effects may be attributed to vasodilatory effects in the uteroplacental circulation. Using wire myography, ex vivo vasoreactivity studies were conducted in uterine arteries (UtA) isolated from pregnant mice exposed to hypoxia or normoxia from gestational day 14.5 to 18.5. Exposure to troglitazone, a high-affinity PPARγ agonist-induced vasorelaxation in UtA preconstricted with phenylephrine, with HA-UtA showing increased sensitivity. Troglitazone blunted ET-1-induced contraction of UtA in hypoxic and normoxic dams equivalently. Immunohistological analysis revealed enhanced staining for ET-1 receptors in the placental labyrinthine zone in hypoxic compared to normoxic dams. Our results suggest that pharmacologic PPAR-γ activation, via its vasoactive properties, may protect the fetal growth under hypoxic conditions by improving uteroplacental perfusion and thereby justify further investigation into PPARγ as a therapeutic target for IUGR in pregnancies complicated by hypoxia.

Using the novel pelvic organ prolapse histologic quantification system to identify phenotypes in uterosacral ligaments in women with pelvic organ prolapse.

BACKGROUND: Pelvic organ prolapse is common, but the underlying etiologies are poorly understood, which limits our current prevention and treatment options. OBJECTIVE: Our primary objective was to compare the uterosacral ligament histologic features in women with and without prolapse using the novel pelvic organ prolapse histologic quantification system. Our secondary aim was to determine whether composite histologic findings in uterosacral ligaments are associated with prolapse risk factors. STUDY DESIGN: This was a prospective cohort study in which paracervical uterosacral ligament biopsies were performed at the time of hysterectomy for primary prolapse or other benign gynecologic indications and processed for histologic evaluation. The pelvic organ prolapse quantification system was used to determine the prolapse stage. In this study, 9 prominent histologic features were semiquantitatively scored using the pelvic organ prolapse histologic quantification system in a blinded fashion and compared between prolapse and control groups. Unbiased principal component analysis of these scores was independently performed to identify potential relationships between histologic measures and prolapse risk factors. RESULTS: The histologic scores of 81 prolapse and 33 control ligaments were analyzed. Compared with the control group, women in the prolapse group were significantly older and more likely to be in the menopausal phase. There was no difference in the number of vaginal deliveries, body mass index, hormone use, or smoking status between the groups. To control for baseline differences, patients were also stratified by age over 40 years and menopausal status. Compared with the control group, the prolapse ligaments in the premenopausal group had significantly more loss of smooth muscle fibers within the fascicles (P<.001), increased inflammatory infiltrates of neutrophils within the tissue and perineural inflammatory cells (P<.01 and P=.04, respectively), and reduced neointimal hyperplasia (P=.02). Prolapse ligaments in the postmenopausal group exhibited elevated adipose content compared with that of the control group (P=.05). Amount of fibrillar collagen, total nonvascular smooth muscle, and muscle fiber vesicles of prolapse ligaments did not differ in either the premenopausal or postmenopausal group compared with that of the control group. Unbiased principal component analysis of the histologic scores separated the prolapse ligaments into 3 phenotypes: (1) increased adipose accumulation, (2) increased inflammation, and (3) abnormal vasculature, with variable overlap with controls. Posthoc analysis of these subgroups demonstrated a positive correlation between increasing number of vaginal deliveries and body mass index with increasing adipose content in the adipocyte accumulation and inflammatory phenotype and increasing neointimal hyperplasia in the vascular phenotype. However, only the relationship between vaginal delivery and adipocytes was significant in the adipose phenotype (R2=0.13; P=.04). CONCLUSION: Histologic phenotypes exist in pelvic support ligaments that can be distinguished using the pelvic organ prolapse histologic quantification system and principle component analysis. Vaginal delivery is associated with aberrant adipose accumulation in uterosacral ligaments. Our findings support a multifactorial etiology for pelvic organ prolapse contributing to altered smooth muscle, vasculature, and connective tissue content in crucial pelvic support structures. To confirm these associations and evaluate the biomechanical properties of histologic phenotypes of prolapse, larger studies are warranted. Closing this gap in knowledge will help optimize personalized medicine and help identify targets for prevention and treatment of this complex condition.

AMP-activated protein kinase activator AICAR attenuates hypoxia-induced murine fetal growth restriction in part by improving uterine artery blood flow.

KEY POINTS: Pregnancy at high altitude is associated with a greater incidence of fetal growth restriction due, in part, to lesser uterine artery blood flow. AMP-activated protein kinase (AMPK) activation vasodilates arteries and may increase uterine artery blood flow. In this study, pharmacological activation of AMPK by the drug AICAR improved fetal growth and elevated uterine artery blood flow. These results suggest that AMPK activation is a potential strategy for improving fetal growth and raising uterine artery blood flow in pregnancy, which may be important in pregnancy disorders characterized by uteroplacental ischaemia and/or fetal hypoxia. ABSTRACT: Uteroplacental hypoxia is associated with pregnancy disorders such as intrauterine growth restriction and preeclampsia, which are characterized by uteroplacental ischaemia and/or fetal hypoxia. Activation of AMP-activated protein kinase (AMPK) results in vasodilatation and is therefore a potential therapeutic strategy for restoring uteroplacental perfusion in pregnancy disorders. In this study, C57Bl/6 mice were treated with subcutaneous pellets containing vehicle, the AMPK activator AICAR (200 mg kg-1 day-1 ), or the AMPK inhibitor Compound C (20 mg kg-1 day-1 ) beginning on gestational day 13.5, and were exposed to hypoxia starting on gestational day 14.5 that induced intrauterine growth restriction. Pharmacological AMPK activation by AICAR partially prevented hypoxia-induced fetal growth restriction (P < 0.01), due in part to increased uterine artery blood flow (P < 0.0001). The proportion of total cardiac output flowing through the uterine artery was increased with AICAR in hypoxic mice (P < 0.001), suggesting that the vasodilator effect of AICAR was selective for the uterine circulation. Further, pharmacological inhibition of AMPK with Compound C reduced uterine artery diameter and increased uterine artery contractility in normoxic mice, providing evidence that physiological levels of AMPK activation are necessary for vasodilatation in healthy pregnancy. Two-way ANOVA analyses indicated that hypoxia reduced AMPK activation in the uterine artery and placenta, and AICAR increased AMPK activation in these tissues compared to vehicle. These findings provide support for further investigation into the utility of pharmacological AMPK activation for treatment of fetal growth restriction.

AMPK activation in pregnant human myometrial arteries from high-altitude and intrauterine growth-restricted pregnancies.

High-altitude (>2,500 m) residence increases the incidence of intrauterine growth restriction (IUGR) due, in part, to reduced uterine artery blood flow and impaired myometrial artery (MA) vasodilator response. A role for the AMP-activated protein kinase (AMPK) pathway in protecting against hypoxia-associated IUGR is suggested by genomic and transcriptomic studies in humans and functional studies in mice. AMPK is a hypoxia-sensitive metabolic sensor with vasodilatory properties. Here we hypothesized that AMPK-dependent vasodilation was increased in MAs from high versus low-altitude (<1,700 m) Colorado women with appropriate for gestational age (AGA) pregnancies and reduced in IUGR pregnancies regardless of altitude. Vasoreactivity studies showed that, in AGA pregnancies, MAs from high-altitude women were more sensitive to vasodilation by activation of AMPK with A769662 due chiefly to increased endothelial nitric oxide production, whereas MA responses to AMPK activation in the low-altitude women were endothelium independent. MAs from IUGR compared with AGA pregnancies had blunted vasodilator responses to acetylcholine at high altitude. We concluded that 1) blunted vasodilator responses in IUGR pregnancies confirm the importance of MA vasodilation for normal fetal growth and 2) the increased sensitivity to AMPK activation in AGA pregnancies at high altitude suggests that AMPK activation helped maintain MA vasodilation and fetal growth. These results highlight a novel mechanism for vasodilation of MAs under conditions of chronic hypoxia and suggest that AMPK activation could provide a therapy for increasing uteroplacental blood flow and improving fetal growth in IUGR pregnancies.NEW & NOTEWORTHY Intrauterine growth restriction (IUGR) impairs infant well- being and increases susceptibility to later-in-life diseases for mother and child. Our study reveals a novel role for AMPK in vasodilating the myometrial artery (MA) from women residing at high altitude (>2,500 m) with appropriate for gestational age pregnancies but not in IUGR pregnancies at any altitude.

Increased uterine artery blood flow in hypoxic murine pregnancy is not sufficient to prevent fetal growth restriction†.

Incomplete maternal vascular responses to pregnancy contribute to pregnancy complications including intrauterine growth restriction (IUGR) and preeclampsia. We aimed to characterize maternal vascular dysfunction in a murine model of fetal growth restriction as an approach toward identifying targetable pathways for improving pregnancy outcomes. We utilized a murine model of late-gestation hypoxia-induced IUGR that reduced E18.5 fetal weight by 34%. Contrary to our hypothesis, uterine artery blood flow as measured in vivo by Doppler ultrasound was increased in mice housed under hypobaric hypoxia (385 mmHg; 5500 m) vs normoxia (760 mmHg; 0 m). Using wire myography, uterine arteries isolated from hypoxic mice had similar vasodilator responses to the two activators A769662 and acetylcholine as those from normoxic mice, although the contribution of an increase in nitric oxide production to uterine artery vasodilation was reduced in the hypoxic vs normoxic groups. Vasoconstrictor responses to phenylephrine and potassium chloride were unaltered by hypoxia. The levels of activated adenosine monophosphate-activated protein kinase (AMPK) were reduced with hypoxia in both the uterine artery and placenta as measured by western blot and immunohistochemistry. We concluded that the rise in uterine artery blood flow may be compensatory to hypoxia but was not sufficient to prevent fetal growth restriction. Although AMPK signaling was reduced by hypoxia, AMPK was still receptive to pharmacologic activation in the uterine arteries in which it was a potent vasodilator. Thus, AMPK activation may represent a new therapy for pregnancy complications involving reduced uteroplacental perfusion.

Perilipin-2 promotes obesity and progressive fatty liver disease in mice through mechanistically distinct hepatocyte and extra-hepatocyte actions.

KEY POINTS: Wild-type mice and mice with hepatocyte-specific or whole-body deletions of perilipin-2 (Plin2) were used to define hepatocyte and extra-hepatocyte effects of altered cellular lipid storage on obesity and non-alcoholic fatty liver disease (NAFLD) pathophysiology in a Western-diet (WD) model of these disorders. Extra-hepatocyte actions of Plin2 are responsible for obesity, adipose inflammation and glucose clearance abnormalities in WD-fed mice. Hepatocyte and extra-hepatic actions of Plin2 mediate fatty liver formation in WD-fed mice through distinct mechanisms. Hepatocyte-specific actions of Plin2 are primary mediators of immune cell infiltration and fibrotic injury in livers of obese mice. ABSTRACT: Non-alcoholic fatty liver disease (NAFLD) is an obesity- and insulin resistance-related metabolic disorder with progressive pathology. Perilipin-2 (Plin2), a ubiquitously expressed cytoplasmic lipid droplet scaffolding protein, is hypothesized to contribute to NAFLD in humans and rodent models through effects on cellular lipid metabolism. In this study, we delineate hepatocyte-specific and extra-hepatocyte Plin2 mechanisms regulating the effects of obesity and insulin resistance on NAFLD pathophysiology in mice fed an obesogenic Western-style diet (WD). Total Plin2 deletion (Plin2-Null) fully protected WD-fed mice from obesity, insulin resistance, adipose inflammation, steatohepatitis (NASH) and liver fibrosis found in WT animals. Hepatocyte-specific Plin2 deletion (Plin2-HepKO) largely protected against NASH and fibrosis and partially protected against steatosis in WD-fed animals, but it did not protect against obesity, insulin resistance, or adipose inflammation. Significantly, total or hepatocyte-specific Plin2 deletion impaired WD-induced monocyte recruitment and pro-inflammatory macrophage polarization found in livers of WT mice. Analyses of the molecular and cellular processes mediating steatosis, inflammation and fibrosis identified differences in total and hepatocyte-specific actions of Plin2 on the mechanisms promoting NAFLD pathophysiology. Our results demonstrate that hepatocyte-specific actions of Plin2 are central to the initiation and pathological progression of NAFLD in obese and insulin-resistant mice through effects on immune cell recruitment and fibrogenesis. Conversely, extra-hepatocyte Plin2 actions promote NAFLD pathophysiology through effects on obesity, inflammation and insulin resistance. Our findings provide new insight into hepatocyte and extra-hepatocyte mechanisms underlying NAFLD development and progression.

Obesity causes renal mitochondrial dysfunction and energy imbalance and accelerates chronic kidney disease in mice.

Obesity and metabolic syndrome are well-known risk factors for chronic kidney disease (CKD); however, less is known about the mechanism(s) by which metabolic syndrome might accelerate kidney disease. We hypothesized that metabolic syndrome should accelerate the development of kidney disease and that it might be associated with alterations in energy metabolism. We studied the pound mouse (which develops early metabolic syndrome due to a leptin receptor deletion) and wild-type littermates and compared the level of renal injury and muscle wasting after equivalent injury with oral adenine. Renal function, histology, and biochemical analyses were performed. The presence of metabolic syndrome was associated with earlier development of renal disease (12 mo) and earlier mortality in pound mice compared with controls. After administration of adenine, kidney disease was worse in pound mice, and this was associated with greater tubular injury with a decrease in kidney mitochondria, lower tissue ATP levels, and worse oxidative stress. Pound mice with similar levels of renal function as adenine-treated wild-type mice also showed worse sarcopenia, with lower tissue ATP and intracellular phosphate levels. In summary, our data demonstrate that obesity and metabolic syndrome accelerate the progression of CKD and worsen CKD-dependent sarcopenia. Both conditions are associated with renal alterations in energy metabolism and lower tissue ATP levels secondary to mitochondrial dysfunction and reduced mitochondrial number.

Perilipin-2 deletion promotes carbohydrate-mediated browning of white adipose tissue at ambient temperature.

Mice lacking perilipin-2 (Plin2-null) are resistant to obesity, insulin resistance, and fatty liver induced by Western or high-fat diets. In the current study, we found that, compared with WT mice on Western diet, Plin2-null adipose tissue was more insulin sensitive and inguinal subcutaneous white adipose tissue (iWAT) exhibited profound browning and robust induction of thermogenic and carbohydrate-responsive genetic programs at room temperature. Surprisingly, these Plin2-null responses correlated with the content of simple carbohydrates, rather than fat, in the diet, and were independent of adipose Plin2 expression. To define Plin2 and sugar effects on adipose browning, WT and Plin2-null mice were placed on chow diets containing 20% sucrose in their drinking water for 6 weeks. Compared with WT mice, iWAT of Plin2-null mice exhibited pronounced browning and striking increases in the expression of thermogenic and insulin-responsive genes on this diet. Significantly, Plin2-null iWAT browning was associated with reduced sucrose intake and elevated serum fibroblast growth factor (FGF)21 levels, which correlated with greatly enhanced hepatic FGF21 production. These data identify Plin2 actions as novel mediators of sugar-induced adipose browning through indirect effects of hepatic FGF21 expression, and suggest that adipose browning mechanisms may contribute to Plin2-null resistance to obesity.

Perilipin-2 Deletion Impairs Hepatic Lipid Accumulation by Interfering with Sterol Regulatory Element-binding Protein (SREBP) Activation and Altering the Hepatic Lipidome.

Perilipin-2 (PLIN2) is a constitutively associated cytoplasmic lipid droplet coat protein that has been implicated in fatty liver formation in non-alcoholic fatty liver disease. Mice with or without whole-body deletion of perilipin-2 (Plin2-null) were fed either Western or control diets for 30 weeks. Perilipin-2 deletion prevents obesity and insulin resistance in Western diet-fed mice and dramatically reduces hepatic triglyceride and cholesterol levels in mice fed Western or control diets. Gene and protein expression studies reveal that PLIN2 deletion suppressed SREBP-1 and SREBP-2 target genes involved in de novo lipogenesis and cholesterol biosynthetic pathways in livers of mice on either diet. GC-MS lipidomics demonstrate that this reduction correlated with profound alterations in the hepatic lipidome with significant reductions in both desaturation and elongation of hepatic neutral lipid species. To examine the possibility that lipidomic actions of PLIN2 deletion contribute to suppression of SREBP activation, we isolated endoplasmic reticulum membrane fractions from long-term Western diet-fed wild type (WT) and Plin2-null mice. Lipidomic analyses reveal that endoplasmic reticulum membranes from Plin2-null mice are markedly enriched in ω-3 and ω-6 long-chain polyunsaturated fatty acids, which others have shown inhibit SREBP activation and de novo lipogenesis. Our results identify PLIN2 as a determinant of global changes in the hepatic lipidome and suggest the hypothesis that these actions contribute to SREBP-regulated de novo lipogenesis involved in non-alcoholic fatty liver disease.

Xanthine oxidoreductase mediates membrane docking of milk-fat droplets but is not essential for apocrine lipid secretion.

KEY POINTS: Xanthine oxidoreductase (XOR) modulates milk lipid secretion and lactation initiation. XOR is required for butyrophilin1a1 clustering in the membrane during milk lipid secretion. XOR mediates apical membrane reorganization during milk lipid secretion. Loss of XOR delays milk fat globule secretion. XOR loss alters the proteome of milk fat globules. ABSTRACT: Apocrine secretion is utilized by epithelial cells of exocrine glands. These cells bud off membrane-bound particles into the lumen of the gland, losing a portion of the cytoplasm in the secretion product. The lactating mammary gland secretes milk lipid by this mechanism, and xanthine oxidoreductase (XOR) has long been thought to be functionally important. We generated mammary-specific XOR knockout (MGKO) mice, expecting lactation to fail. Histology of the knockout glands showed very large lipid droplets enclosed in the mammary alveolar cells, but milk analysis showed that these large globules were secreted. Butyrophilin, a membrane protein known to bind to XOR, was clustered at the point of contact of the cytoplasmic lipid droplet with the apical plasma membrane, in the wild-type gland but not in the knockout, suggesting that XOR mediates 'docking' to this membrane. Secreted milk fat globules were isolated from mouse milk of wild-type and XOR MGKO dams, and subjected to LC-MS/MS for analysis of protein component. Proteomic results showed that loss of XOR leads to an increase in cytoplasmic, cytoskeletal, Golgi apparatus and lipid metabolism proteins associated with the secreted milk fat globule. Association of XOR with the lipid droplet results in membrane docking and more efficient retention of cytoplasmic components by the secretory cell. Loss of XOR then results in a reversion to a more rudimentary, less efficient, apocrine secretion mechanism, but does not prevent milk fat globule secretion.

Perilipin-2-null mice are protected against diet-induced obesity, adipose inflammation, and fatty liver disease.

The cytoplasmic lipid droplet (CLD) protein perilipin-2 (Plin2) is expressed in multiple nonadipose tissues, where it is thought to play a role in regulating their lipid storage properties. However, the extent to which Plin2 functions in nutrient utilization and metabolism, or how it influences the consequences of over-feeding, remains unclear. In this study, we demonstrate that the absence of Plin2 prevents high-fat diet(HFD)-induced obesity in male and female mice. This response is associated with increased formation of subcutaneous beige adipocyte cells with uncoupling protein 1 expression, and amelioration of inflammatory foci formation in white adipose tissue and steatosis in the liver. Experiments demonstrate that Plin2 loss results in reduced energy intake and increased physical activity in response to HFD feeding. Our study provides the first evidence that Plin2 contributes to HFD-induced obesity by modulating food intake, and that its absence prevents obesity-associated adipose tissue inflammatory foci and liver steatosis.

Fibronectin Contributes to a BRAF Inhibitor-driven Invasive Phenotype in Thyroid Cancer through EGR1, Which Can Be Blocked by Inhibition of ERK1/2.

Mutations in BRAF are common in advanced papillary and anaplastic thyroid cancer (PTC and ATC). However, patients with BRAF-mutant PTC currently lack therapies targeting this pathway. Despite the approved combination of BRAF and MEK1/2 inhibition for patients with BRAF-mutant ATC, these patients often progress. Thus, we screened a panel of BRAF-mutant thyroid cancer cell lines to identify new therapeutic strategies. We showed that thyroid cancer cells resistant to BRAF inhibition (BRAFi) exhibit an increase in invasion and a proinvasive secretome in response to BRAFi. Using reverse-phase protein array (RPPA), we identified a nearly 2-fold increase in expression of the extracellular matrix protein, fibronectin, in response to BRAFi treatment, and a corresponding 1.8- to 3.0-fold increase in fibronectin secretion. Accordingly, the addition of exogenous fibronectin phenocopied the BRAFi-induced increase in invasion while depletion of fibronectin in resistant cells resulted in loss of increased invasion. We further showed that BRAFi-induced invasion can be blocked by inhibition of ERK1/2. In a BRAFi-resistant patient-derived xenograft model, we found that dual inhibition of BRAF and ERK1/2 slowed tumor growth and decreased circulating fibronectin. Using RNA sequencing, we identified EGR1 as a top downregulated gene in response to combined BRAF/ERK1/2 inhibition, and we further showed that EGR1 is necessary for a BRAFi-induced increase in invasion and for induction of fibronectin in response to BRAFi. IMPLICATIONS: Together, these data show that increased invasion represents a new mechanism of resistance to BRAF inhibition in thyroid cancer that can be targeted with an ERK1/2 inhibitor.

Expression and T cell regulatory action of the PD-1 immune checkpoint in the ovary and fallopian tube.

PROBLEM: Immune cell trafficking and surveillance within the ovary and fallopian tube are thought to impact fertility and also tumorigenesis in those organs. However, little is known of how native cells of the ovary and fallopian tube interact with resident immune cells. Interaction of the Programmed Cell Death Protein-1 (PD-1/PDCD-1/CD279) checkpoint with PD-L1 is associated with downregulated immune response. We have begun to address the question of whether PD-1 ligand or its receptors (PD-L1/-L2) can regulate immune cell function in these tissues of the female reproductive tract. METHOD OF STUDY: PD-1 and ligand protein expression was evaluated in human ovary and fallopian tube specimens, the latter of which included stages of tubal cell transformation and early tumorigenesis. Ovarian expression analysis included the determination of the proteins in human follicular fluid (HFF) specimens collected during in vitro fertilization procedures. Finally, checkpoint bioactivity of HFF was determined by treatment of separately-isolated human T cells and the measurement of interferon gamma (IFNγ). RESULTS: We show that membrane bound and soluble variants of PD-1 and ligands are expressed by permanent constituent cell types of the human ovary and fallopian tube, including granulosa cells and oocytes. PD-1 and soluble ligands were present in HFF at bioactive levels that control T cell PD-1 activation and IFNγ production; full-length checkpoint proteins were found to be highly enriched in HFF exosome fractions. CONCLUSION: The detection of PD-1 checkpoint proteins in the human ovary and fallopian tube suggests that the pathway is involved in immunomodulation during folliculogenesis, the window of ovulation, and subsequent egg and embryo immune-privilege. Immunomodulatory action of receptor and ligands in HFF exosomes is suggestive of an acute checkpoint role during ovulation. This is the first study in the role of PD-1 checkpoint proteins in human tubo-ovarian specimens and the first examination of its potential regulatory action in the contexts of normal and assisted reproduction.

Lenvatinib Plus Anti-PD-1 Combination Therapy for Advanced Cancers: Defining Mechanisms of Resistance in an Inducible Transgenic Model of Thyroid Cancer.

Background: Combination therapy with lenvatinib plus programmed death-1 (PD-1) immune checkpoint blockades (ICBs) is under investigation in many solid tumors, including thyroid cancer. Lenvatinib is known to reduce angiogenesis and may overturn the immunosuppressive effects of vascular endothelial growth factor in the tumor microenvironment. Previous studies investigating the effects of VEGF receptor inhibition on the immune response were performed in rapidly growing tumor models where immune equilibrium is not established before treatment. We hypothesize that physiologically relevant preclinical models are necessary to define mechanisms of resistance to immune-targeted combination therapies. Methods: We utilized the TPO-CreER/BrafV600E/wt/Trp53Δex2-10/Δex2-10 inducible transgenic model of advanced thyroid cancer to investigate lenvatinib treatment in the context of an anti-PD-1 ICB. Following tumor establishment, 3.5 months postinduction, mice were treated with high- (10 mg/kg) or low-dose (2 mg/kg) lenvatinib, anti-PD-1, or combination of lenvatinib with anti-PD-1. Tumor volume and lung metastases were assessed in each group. Immune infiltrate was characterized by flow cytometry and immunohistochemistry, and TCRß sequencing was performed to further investigate the T cell response. Results: Both low- and high-dose lenvatinib reduced tumor volume, while anti-PD-1 had no effect, alone or in combination. Although both low- and high-dose lenvatinib reduced vascular density, low-dose lenvatinib was superior in controlling tumor size. Lung metastases and survival were not improved with therapy despite the effects of lenvatinib on primary tumor size. Low-dose lenvatinib treatment led to a subtle reduction in the dominant Ly6G+CD11b+ myeloid cell population and was associated with increased CD4+ T cell infiltrate and enrichment in 4-1BB+ and granzyme B+ CD4+ T cells and FoxP3+ regulatory T cells. Polyclonal T cell expansion was evident in the majority of mice, suggesting that a tumor-specific T cell response was generated. Conclusions: The effects of lenvatinib on the immune response were most pronounced in mice treated with low-dose lenvatinib, suggesting that dose should be considered in clinical application. While the immune-modulating potential of lenvatinib is encouraging, alterations in the immune milieu and T cell activation status were insufficient to sustain durable tumor regression, even with added anti-PD-1. Additional studies are necessary to develop more effective combination approaches in low-mutation burden tumors, such as thyroid cancer.

Chronic ethanol consumption in mice alters hepatocyte lipid droplet properties.

BACKGROUND: Hepatosteatosis is a common pathological feature of impaired hepatic metabolism following chronic alcohol consumption. Although often benign and reversible, it is widely believed that steatosis is a risk factor for development of advanced liver pathologies, including steatohepatitis and fibrosis. The hepatocyte alterations accompanying the initiation of steatosis are not yet clearly defined. METHODS: Induction of hepatosteatosis by chronic ethanol consumption was investigated using the Lieber-DeCarli (LD) high fat diet model. Effects were assessed by immunohistochemistry and blood and tissue enzymatic assays. Cell culture models were employed for mechanistic studies. RESULTS: Pair feeding mice ethanol (LD-Et) or isocaloric control (LD-Co) diets for 6 weeks progressively increased hepatocyte triglyceride accumulation in morphological, biochemical, and zonally distinct cytoplasmic lipid droplets (CLD). The LD-Et diet induced zone 2-specific triglyceride accumulation in large CLD coated with perilipin, adipophilin (ADPH), and TIP47. In LD-Co-fed mice, CLD were significantly smaller than those in LD-Et-fed mice and lacked perilipin. A direct role of perilipin in formation of large CLD was further suggested by cell culture studies showing that perilipin-coated CLD were significantly larger than those coated with ADPH or TIP47. LD-Co- and LD-Et-fed animals also differed in hepatic metabolic stress responses. In LD-Et but not LD-Co-fed mice, inductions were observed in the following: microsomal ethanol-oxidizing system [cytochrome P-4502E1 (CYP2E1)], hypoxia response pathway (hypoxia-inducible factor 1 alpha, HIF1α), endoplasmic reticulum stress pathway (calreticulin), and synthesis of lipid peroxidation products [4-hydroxynonenal (4-HNE)]. CYP2E1 and HIF1 α immunostaining localized to zone 3 and did not correlate with accumulation of large CLD. In contrast, calreticulin and 4-HNE immunostaining closely correlated with large CLD accumulation. Importantly, 4-HNE staining significantly colocalized with ADPH and perilipin on the CLD surface. CONCLUSIONS: These data suggest that ethanol contributes to macrosteatosis by both altering CLD protein composition and inducing lipid peroxide adduction of CLD-associated proteins.

High altitude regulates the expression of AMPK pathways in human placenta.

INTRODUCTION: High-altitude (>2500 m) residence augments the risk of intrauterine growth restriction (IUGR) and preeclampsia likely due, in part, to uteroplacental hypoperfusion. Previous genomic and transcriptomic studies in humans and functional studies in mice and humans suggest a role for AMP-activated protein kinase (AMPK) pathway in protecting against hypoxia-associated IUGR. AMPK is a metabolic sensor activated by hypoxia that is ubiquitously expressed in vascular beds and placenta. METHODS: We measured gene expression and protein levels of AMPK and its upstream regulators and downstream targets in human placentas from high (>2500 m) vs. moderate (~1700 m) and low (~100 m) altitude. RESULTS: We found that phosphorylated AMPK protein levels and its downstream target TSC2 were increased in placentas from high and moderate vs. low altitude, whereas the phosphorylated form of the downstream target translation repressor protein 4E-BP1 was increased in high compared to moderate as well as low altitude placentas. Mean birth weights progressively fell with increasing altitude but no infants, by study design, were clinically growth-restricted. Gene expression analysis showed moderate increases in PRKAG2, encoding the AMPK γ2 subunit, and mechanistic target of rapamycin, MTOR, expression. DISCUSSION: These results highlight a differential regulation of placental AMPK pathway activation in women residing at low, moderate or high altitude during pregnancy, suggesting AMPK may be serving as a metabolic regulator for integrating hypoxic stimuli with placental function.

Control of Murine Primordial Follicle Growth Activation by IκB/NFκB Signaling.

The transcription factor NFκB has been associated with the timing of menopause in a large human genome-wide association study. Furthermore, preclinical studies demonstrate that loss of Tumor necrosis factor alpha (Tnfα) or its receptor Tnfr2 slows primordial follicle growth activation (PFGA). Although Tnfα:receptor signaling stimulates NFκB and may mechanistically link these findings, very little is known about NFκB signaling in PFGA. Because signaling downstream of Tnfα/Tnfr2 ligand/receptor interaction has not been interrogated as relates to PFGA, we evaluated the expression of key NFκB signaling proteins in primordial and growing follicles, as well as during ovarian aging. We show that key members of the NFκB pathway, including subunits, activating kinases, and inhibitory proteins, are expressed in the murine ovary. Furthermore, the subunits p65 and p50, and the cytosolic inhibitory proteins IκBα and IκBß, are present in ovarian follicles, including at the primordial stage. Finally, we assessed PFGA in genetically modified mice (AKBI) previously demonstrated to be resistant to inflammatory stress-induced NFκB activation due to overexpression of the NFκB inhibitory protein IκBß. Consistent with the hypothesis that NFκB plays a key role in PFGA, AKBI mice exhibit slower PGFA than wild-type (WT) controls, and their ovaries contain nearly twice the number of primordial follicles as WT both at early and late reproductive ages. These data provide mechanistic insight on the control of PFGA and suggest that targeting NFκB at the level of IκB proteins may be a tractable route to slowing the rate of PFGA in women faced with early ovarian demise.

High Altitude Reduces NO-Dependent Myometrial Artery Vasodilator Response During Pregnancy.

The chronic hypoxia of high-altitude (HA) residence reduces uterine artery blood flow during pregnancy, likely contributing to an increased frequency of preeclampsia and intrauterine growth restriction. We hypothesized that this lesser pregnancy blood flow rise was due, in part, to reduced vasodilation of myometrial arteries (MAs). Here, we assessed MA vasoreactivity in healthy residents of high (2902±39 m) or low altitude (LA; 1669±10 m). MA contractile responses to potassium chloride, phenylephrine, or the thromboxane A2 agonist U46619 did not differ between LA and HA women. Acetylcholine vasodilated phenylephrine or U466119 preconstricted MAs at LA, yet had no effect on HA MAs. In contrast, another vasodilator, bradykinin, relaxed MAs from both altitudes similarly. At LA, the NO synthase inhibitor L-NG-nitroarginine methyl ester decreased both acetylcholine and bradykinin vasodilation by 56% and 33%, respectively. L-NG-nitroarginine methyl ester plus the COX (cyclooxygenase) inhibitor indomethacin had similar effects on acetylcholine and bradykinin vasodilation (68% and 42% reduction, respectively) as did removing the endothelium (78% and 50% decrease, respectively), suggesting a predominantly NO-dependent vasodilation at LA. However, at HA, L-NG-nitroarginine methyl ester did not change bradykinin vasodilation, whereas indomethacin or endothelium removal decreased it by 28% and 72%, respectively, indicating impaired NO signaling at HA. Suggesting that the impairment was downstream of eNOS (endothelial NO synthase), HA attenuated the vasodilation elicited by the NO donor sodium nitroprusside. We concluded that reduced NO-dependent MA vasodilation likely contributes to diminished uteroplacental perfusion in HA pregnancies.

Maternal obesity during lactation may protect offspring from high fat diet-induced metabolic dysfunction.

BACKGROUND/OBJECTIVES: The current obesity epidemic has spurred exploration of the developmental origin of adult heath and disease. A mother's dietary choices and health can affect both the early wellbeing and lifelong disease-risk of the offspring. SUBJECTS/METHODS: To determine if changes in the mother's diet and adiposity have long-term effects on the baby's metabolism, independently from a prenatal insult, we utilized a mouse model of diet-induced-obesity and cross-fostering. All pups were born to lean dams fed a low fat diet but were fostered onto lean or obese dams fed a high fat diet. This study design allowed us to discern the effects of a poor diet from those of mother's adiposity and metabolism. The weaned offspring were placed on a high fat diet to test their metabolic function. RESULTS: In this feeding challenge, all male (but not female) offspring developed metabolic dysfunction. We saw increased weight gain in the pups nursed on an obesity-resistant dam fed a high fat diet, and increased pathogenesis including liver steatosis and adipose tissue inflammation, when compared to pups nursed on either obesity-prone dams on a high fat diet or lean dams on a low fat diet. CONCLUSION: Exposure to maternal over-nutrition, through the milk, is sufficient to shape offspring health outcomes in a sex- and organ-specific manner, and milk from a mother who is obesity-prone may partially protect the offspring from the insult of a poor diet.

The low molecular weight cyclin E isoforms augment angiogenesis and metastasis of human melanoma cells in vivo.

Immunohistochemical analysis has consistently shown that cyclin E is up-regulated in human malignant melanomas in vivo. Here we analyzed such expression in more detail and show that cyclin E is overexpressed and present in low molecular weight (LMW) isoforms in metastatic melanoma and in a subset of primary invasive melanoma tumor tissues, but not in benign nevi. Human metastatic melanoma cell lines, but not normal melanocytes, also expressed the LMW cyclin E forms. The biological significance of these findings was established by showing that overexpression of two LMW cyclin E forms named cyclin E truncated 1 [cyclinE(T1)] and cyclin E truncated 2 [cyclinE(T2)] in a low tumorigenic and non-metastatic primary cutaneous melanoma cell line generated angiogenic tumors with prominent perineural invasion compared with full-length cyclin E. In addition, cyclin E(T1)- and cyclin E(T2)-expressing melanoma cells displayed a dramatic increase in the incidence and number of metastases in an experimental lung metastasis assay. Together, these results indicate that the LMW cyclin E forms are functional and likely act as regulators of human melanoma tumor progression and invasion.