Contributions of white adipose tissue to energy requirements for female reproduction.

Body composition impacts female fertility and there are established relationships between adipose tissue and the reproductive system. Maintaining functional adipose tissue is vital for meeting the energetic demands during the reproductive process, from ovulation to delivery and lactation. White adipose tissue (WAT) shows plastic responses to daily physiology and secretes diverse adipokines that affect the hypothalamic-pituitary-ovarian axis, but many other interorgan interactions remain to be determined. This review summarizes the current state of research on the dialogue between WAT and the female reproductive system, focusing on the impact of this crosstalk on ovarian and endometrial factors essential for fecundity.

N-acetylaspartate from fat cells regulates postprandial body temperature.

N-acetylaspartate (NAA), the brain's second most abundant metabolite, provides essential substrates for myelination through its hydrolysis. However, activities and physiological roles of NAA in other tissues remain unknown. Here, we show aspartoacylase (ASPA) expression in white adipose tissue (WAT) governs systemic NAA levels for postprandial body temperature regulation. Proteomics and mass spectrometry revealed NAA accumulation in WAT of Aspa knockout mice stimulated the pentose phosphate pathway and pyrimidine production. Stable isotope tracing confirmed higher incorporation of glucose-derived carbon into pyrimidine metabolites in Aspa knockout cells. Additionally, serum NAA positively correlates with the pyrimidine intermediate orotidine and this relationship predicted lower body mass index in humans. Using whole-body and tissue-specific knockout mouse models, we demonstrate that fat cells provided plasma NAA and suppressed postprandial body temperature elevation. Furthermore, exogenous NAA supplementation reduced body temperature. Our study unveils WAT-derived NAA as an endocrine regulator of postprandial body temperature and physiological homeostasis.

Loss of AMPKα2 promotes melanoma tumor growth and brain metastasis.

AMP-activated protein kinase (AMPK) is a critical cellular energy sensor at the interface of metabolism and cancer. However, the role of AMPK in carcinogenesis remains unclear. Here, through analysis of the TCGA melanoma dataset, we found that PRKAA2 gene that encodes the α2 subunit of AMPK is mutated in ∼9% of cutaneous melanomas, and these mutations tend to co-occur with NF1 mutations. Knockout of AMPKα2 promoted anchorage-independent growth of NF1-mutant melanoma cells, whereas ectopic expression of AMPKα2 inhibited their growth in soft agar assays. Moreover, loss of AMPKα2 accelerated tumor growth of NF1-mutant melanoma and enhanced their brain metastasis in immune-deficient mice. Our findings support that AMPKα2 serves as a tumor suppressor in NF1-mutant melanoma and suggest that AMPK could be a therapeutic target for treating melanoma brain metastasis.

Combined inhibition of HMGCoA reductase and mitochondrial complex I induces tumor regression of BRAF inhibitor-resistant melanomas.

BACKGROUND: Primary and posttreatment resistance to BRAFV600 mutation-targeting inhibitors leads to disease relapse in a majority of melanoma patients. In many instances, this resistance is promoted by upregulation of mitochondrial oxidative phosphorylation (OxPhos) in melanoma cells. We recently showed that a novel electron transport chain (ETC) complex I inhibitor, IACS-010759 (IACS), abolished OxPhos and significantly inhibited tumor growth of high-OxPhos, BRAF inhibitor (BRAFi)-resistant human melanomas. However, the inhibition was not uniform across different high OxPhos melanomas, and combination with BRAFi did not improve efficacy. METHODS: We performed a high-throughput unbiased combinatorial drug screen of clinically relevant small molecules to identify the most potent combination agent with IACS for inhibiting the growth of high-OxPhos, BRAFi-resistant melanomas. We performed bioenergetics and carbon-13 metabolite tracing to delineate the metabolic basis of sensitization of melanomas to the combination treatment. We performed xenograft tumor growth studies and Reverse-Phase Protein Array (RPPA)-based functional proteomics analysis of tumors from mice fed with regular or high-fat diet to evaluate in vivo molecular basis of sensitization to the combination treatment. RESULTS: A combinatorial drug screen and subsequent validation studies identified Atorvastatin (STN), a hydroxymethylglutaryl-coenzyme A reductase inhibitor (HMGCRi), as the most potent treatment combination with IACS to inhibit in vitro cell growth and induce tumor regression or stasis of some BRAFi-resistant melanomas. Bioenergetics analysis revealed a dependence on fatty acid metabolism in melanomas that responded to the combination treatment. RPPA analysis and carbon-13 tracing analysis in these melanoma cells showed that IACS treatment decreased metabolic fuel utilization for fatty acid metabolism, but increased substrate availability for activation of the mevalonate pathway by HMGCR, creating a dependence on this pathway. Functional proteomic analysis showed that IACS treatment inhibited MAPK but activated AKT pathway. Combination treatment with STN counteracted AKT activation. CONCLUSIONS: STN and other clinically approved HMGCRi could be promising combinatorial agents for improving the efficacy of ETC inhibitors like IACS in BRAFi-resistant melanomas.

Prevalence, burden and treatment effects of vaginal bleeding in women with (suspected) congenital platelet disorders throughout life: a cross-sectional study.

Congenital platelet disorders (CPDs) are rare bleeding disorders that are associated with mucocutaneous bleeds. However, data on vaginal bleeding in women with CPDs are scarce. A set of generic and bleeding-specific questionnaires were used to evaluate the prevalence of vaginal bleeding, its impact on quality of life (QoL) and sexual functioning and the consequences for pregnancy, miscarriage and delivery in a cohort of women who were referred for diagnostic evaluation for CPDs. A total of 78 women included in the study were either diagnosed with a CPD (n = 35) or were clinically suspected of a CPD (n = 43). Heavy menstrual bleeding (HMB) was reported by a large proportion of women, which mainly started at menarche. In all, 76% of women received any kind of HMB treatment, often leading to surgical prodecures. HMB was shown to have a high impact on QoL, which improved upon treatment. Even though women reported that vaginal bleeding affects sexuality, this topic is not frequently discussed with physicians. Heavy blood loss frequently occurred after miscarriage/delivery, often requiring treatment. Women with (suspected) CPDs frequently encounter HMB, negatively impacting daily life and sexual functioning. Together with peripartum bleeding, these data highlight the burden of vaginal bleeding in CPDs and importance of adequate treatment.

Regulation of von Willebrand factor-platelet interactions.

The formation of thrombi is a multistep process involving several components, including von Willebrand factor (VWF). VWF is an adhesive multimeric protein, which acts as a molecular bridge between the subendothelial matrix and the glycoprotein Ib/IX/V receptor complex. Furthermore, VWF promotes the expansion of the platelet plug by cross-linking platelets via binding to integrin alphaIIbbeta3. In terms of thrombus formation, it is essential that VWF-platelet interactions occur timely, that is: it should happen not too early or too late. Given the co-existence of VWF and platelets in the circulation, this implies that there must be regulatory mechanisms that prevent premature formation of VWF-rich platelet aggregates that could occlude the vasculature. Indeed, several mechanisms have been identified at the level of VWF, which are dedicated to the prevention of excessive VWF-platelet interactions following endothelial release of VWF (which may include limited exposure to shear stress, the presence of Mg2+ ions, inhibition of VWF-platelet interactions by endothelial proteins, ADAMTS13-mediated proteolysis) and of circulating VWF-platelet aggregates during normal circulation (shielding of the platelet-binding A1 domain by other regions of the VWF molecule, inhibition of VWF-platelet interactions by beta2-glycoprotein I). In the present review an overview of these mechanisms will be discussed.

A novel binding site for ADAMTS13 constitutively exposed on the surface of globular VWF.

ADAMTS13 metalloprotease regulates the multimeric size of von Willebrand factor (VWF) by cleaving the Tyr1605-Met1606 bond in the VWF A2 domain. The mechanisms of VWF recognition by ADAMTS13 have yet to be fully resolved. Most studies have focused on the role of exosites within the VWF A2 domain, involved in interaction with the ADAMTS13 spacer domain. In the present study, we expressed different C-terminal domain VWF fragments and evaluated their binding to ADAMTS13 and its truncated mutants, MDTCS and del(TSP5-CUB). Using plate binding assay and surface plasmon resonance, we identified a novel ADAMTS13 binding site (K(D) approximately 86 nM) in the region of VWF spanning residues 1874 to 2813, which includes the VWF D4 domain and that interacts with the C-terminal domains of ADAMTS13. We show that the interaction occurs even when VWF is in static conditions, assumed to be globular and where the VWF A2 domain is hidden. We demonstrate that C-terminal VWF fragments, as well as an antibody specifically directed toward the VWF D4 domain, inhibit VWF proteolysis by ADAMTS13 under shear conditions. We propose that this novel VWF C-terminal binding site may participate as the initial step of a multistep interaction ultimately leading to proteolysis of VWF by ADAMTS13.

ADAMTS13 deficiency with elevated levels of ultra-large and active von Willebrand factor in P. falciparum and P. vivax malaria.

A deficiency in ADAMTS13 (a von Willebrand factor [VWF] cleaving protease) is associated with accumulation of prothrombogenic unusually large VWF multimers (UL-VWF) in plasma. We studied VWF release and proteolysis in patients with symptomatic Plasmodium falciparum or P. vivax malaria on the Indonesian island Sumba. Malaria patients had significantly lower platelet counts and higher VWF concentrations and VWF activation factors than healthy hospital staff controls. The latter indicates that a higher amount of circulating VWF was in a conformation enabling spontaneous platelet binding. In addition, ADAMTS13 activity and antigen levels were reduced in both malaria groups, and this was associated with the appearance of UL-VWF. The mechanism behind this reduction and the role in malaria pathogenesis needs to be further elucidated. In malaria, endothelial cell activation with increased circulating amounts of active and ultra-large VWF, together with reduced VWF inactivation by ADAMTS13, may result in intravascular platelet aggregation, thrombocytopenia, and microvascular disease.

Macrophages contribute to the cellular uptake of von Willebrand factor and factor VIII in vivo.

Von Willebrand factor (VWF) and factor VIII (FVIII) circulate in a tight noncovalent complex. At present, the cells that contribute to the removal of FVIII and VWF are of unknown identity. Here, we analyzed spleen and liver tissue sections of VWF-deficient mice infused with recombinant VWF or recombinant FVIII. This analysis revealed that both proteins were targeted to cells of macrophage origin. When applied as a complex, both proteins were codirected to the same macrophages. Chemical inactivation of macrophages using gadolinium chloride resulted in doubling of endogenous FVIII levels in VWF-null mice, and of VWF levels in wild-type mice. Moreover, the survival of infused VWF was prolonged almost 2-fold in VWF-deficient mice after gadolinium chloride treatment. VWF and FVIII also bound to primary human macrophages in in vitro tests. In addition, radiolabeled VWF bound to human THP1 macrophages in a dose-dependent, specific, and saturable manner (half-maximal binding at 0.014 mg/mL). Binding to macrophages was followed by a rapid uptake and subsequent degradation of the internalized protein. This process was also visualized using a VWF-green fluorescent protein fusion protein. In conclusion, our data strongly indicate that macrophages play a prominent role in the clearance of the VWF/FVIII complex.

Association between thrombotic microangiopathy and reduced ADAMTS13 activity in malignant hypertension.

The thrombotic microangiopathy observed in malignant hypertension is similar to that of thrombotic thrombocytopenic purpura, which is associated with a deficiency of ADAMTS13, a von Willebrand factor (VWF)-cleaving protease that cleaves large prothrombogenic multimers. We hypothesized that ADAMTS13 is deficient in malignant hypertension and that the severity of thrombotic microangiopathy is associated with decreased ADAMTS13 activity. We included 20 patients with malignant and 20 patients with severe hypertension, and 20 matched normotensive individuals served as control subjects. VWF, active VWF, and free hemoglobin were assessed to explore predictors of ADAMTS13 activity. Patients with malignant hypertension had lower ADAMTS13 activity (80%; interquartile range: 53% to 130%) compared with control subjects (99% interquartile range: 82% to 129%; P<0.01) but not compared with patients with severe hypertension (P=0.14). ADAMTS13 activity negatively correlated with lactic dehydrogenase levels after logarithmic transformation (r=-0.65; P<0.001) and was associated with platelet count (r=0.34; P=0.04) and the presence of schistocytes (r=-0.37; P=0.02). Apart from the association with thrombotic microangiopathy, ADAMTS13 was inversely associated with creatinine (r=-0.42; P=0.008). Increasing levels of VWF were associated with a decrease in ADAMTS13 activity (r=-0.34; P=0.03). There was no significant association between ADAMTS13 activity and other parameters, including blood pressure. In conclusion, ADAMTS13 is decreased in malignant hypertension and associated with the severity of thrombotic microangiopathy, likely because of the release of VWF after endothelium stimulation. A severe deficiency could not be demonstrated. More studies are needed to identify the role of ADAMTS13 in the thrombotic microangiopathy and ischemic complications of malignant hypertension.

Genetic analysis of the two zebrafish patched homologues identifies novel roles for the hedgehog signaling pathway.

BACKGROUND: Aberrant activation of the Hedgehog (Hh) signaling pathway in different organisms has shown the importance of this family of morphogens during development. Genetic screens in zebrafish have assigned specific roles for Hh in proliferation, differentiation and patterning, but mainly as a result of a loss of its activity. We attempted to fully activate the Hh pathway by removing both receptors for the Hh proteins, called Patched1 and 2, which are functioning as negative regulators in this pathway. RESULTS: Here we describe a splice-donor mutation in Ptc1, called ptc1hu1602, which in a homozygous state results in a subtle eye and somite phenotype. Since we recently positionally cloned a ptc2 mutant, a ptc1;ptc2 double mutant was generated, showing severely increased levels of ptc1, gli1 and nkx2.2a, confirming an aberrant activation of Hh signaling. As a consequence, a number of phenotypes were observed that have not been reported previously using Shh mRNA overexpression. Somites of ptc1;ptc2 double mutants do not express anteroposterior polarity markers, however initial segmentation of the somites itself is not affected. This is the first evidence that segmentation and anterior/posterior (A/P) patterning of the somites are genetically uncoupled processes. Furthermore, a novel negative function of Hh signaling is observed in the induction of the fin field, acting well before any of the previously reported function of Shh in fin formation and in a way that is different from the proposed early role of Gli3 in limb/fin bud patterning. CONCLUSION: The generation and characterization of the ptc1;ptc2 double mutant assigned novel and unexpected functions to the Hh signaling pathway. Additionally, these mutants will provide a useful system to further investigate the consequences of constitutively activated Hh signaling during vertebrate development.

Thrombocytopenia and release of activated von Willebrand Factor during early Plasmodium falciparum malaria.

BACKGROUND: Thrombocytopenia occurs early during malarial infection, but its underlying mechanism is unclear. Secretion of von Willebrand factor (vWF) occurs on endothelial cell activation, and it plays an important role in platelet agglutination. METHODS: In 14 healthy human volunteers who were experimentally infected with Plasmodium falciparum, we studied vWF secretion and proteolysis as well as the relationship between changes in circulating platelet numbers and plasma levels of vWF and activated vWF. RESULTS: Platelet numbers started to decrease between days 7 and 9 after infection, which corresponded to the earliest phase of blood-stage infection. With the decrease in platelet numbers, levels of vWF, vWF propeptide (markers of chronic and acute endothelial cell activation, respectively), and activated vWF (exposing the glycoprotein Ib alpha platelet-binding domain) increased proportionally. A strong, reciprocal relationship was observed between platelet numbers and levels of both vWF and activated vWF. Activity of the vWF-cleaving protease ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) -- a regulator of vWF activity -- remained unchanged. CONCLUSIONS: P. falciparum induces systemic acute endothelial cell activation and release of activated vWF immediately after the onset of blood-stage infection. The resulting platelet agglutination may result in early thrombocytopenia and may play a role in the pathogenesis of malaria.

The presence of active von Willebrand factor under various pathological conditions.

PURPOSE OF REVIEW: To highlight mechanisms that regulate the balance between latent and active von Willebrand factor (VWF), and describe pathological conditions leading to increased levels of active VWF. RECENT FINDINGS: Levels of circulating active VWF are increased in von Willebrand disease type 2B, HELLP syndrome, malaria and antiphospholipid syndrome. SUMMARY: Freshly secreted VWF consists of ultra-large multimers that interact spontaneously with platelets at the endothelial cell surface. Proteolysis of ultra-large VWF by a member of the disintegrin and metalloprotease with thrombospondin motif family (ADAMTS13) reduces both multimeric size and accessibility of platelet-adhesion sites. The resulting VWF molecules circulate as inactive multimers, which regain their platelet-adhesion capacity upon binding to the subendothelial matrix, in particular under conditions of high shear. Unfortunately, mechanisms responsible for suppression of circulating plasma levels of active VWF are hampered in a number of pathological conditions, leading to VWF-platelet aggregates associated with thrombotic complications or thrombocytopenia. A recently developed assay allowed us to monitor the presence of circulating active VWF and we found that several diseases are characterized by increased levels. Further analysis provided insight into mechanisms contributing to the presence of active VWF, which revealed that beta2-glycoprotein I may act as a natural regulator of VWF-platelet interactions.