Inhibition of Acyl-CoA Synthetase Long Chain Isozymes Decreases Multiple Myeloma Cell Proliferation and Causes Mitochondrial Dysfunction.

Multiple myeloma (MM) is an incurable cancer of plasma cells with a 5-year survival rate of 59%. Dysregulation of fatty acid (FA) metabolism is associated with MM development and progression; however, the underlying mechanisms remain unclear. Acyl-CoA synthetase long-chain family members (ACSLs) convert free long-chain fatty acids into fatty acyl-CoA esters and play key roles in catabolic and anabolic fatty acid metabolism. The Cancer Dependency Map data suggested that ACSL3 and ACSL4 were among the top 25% Hallmark Fatty Acid Metabolism genes that support MM fitness. Here, we show that inhibition of ACSLs in human myeloma cell lines using the pharmacological inhibitor Triascin C (TriC) causes apoptosis and decreases proliferation in a dose- and time-dependent manner. RNA-seq of MM.1S cells treated with TriC for 24 h showed a significant enrichment in apoptosis, ferroptosis, and ER stress. Proteomics of MM.1S cells treated with TriC for 48 h revealed that mitochondrial dysfunction and oxidative phosphorylation were significantly enriched pathways of interest, consistent with our observations of decreased mitochondrial membrane potential and increased mitochondrial superoxide levels. Interestingly, MM.1S cells treated with TriC for 24 h also showed decreased mitochondrial ATP production rates and overall lower cellular respiration.

Effects of a Global Rab27a Null Mutation on Murine PVAT and Cardiovascular Function.

BACKGROUND: RAB27A, a modulator of secretion, is expressed within vessels and perivascular adipose tissue. We hypothesized that loss of RAB27A would alter cardiovascular function. METHODS: Body weight of Rab27aash mice was measured from 2 to 18 months of age, along with glucose resorption at 6 and 12 months of age and glucose sensitivity at 18 months of age. Body weight and cellular and molecular features of perivascular adipose tissue and aortic tissue were examined in a novel C57BL/6J Rab27a null strain. Analyses included morphometric quantification and proteomic analyses. Wire myography measured vasoreactivity, and echocardiography measured cardiac function. Comparisons across ages and genotypes were evaluated via 2-way ANOVA with multiple comparison testing. Significance for myography was determined via 4-parameter nonlinear regression testing. RESULTS: Genome-wide association data linked rare human RAB27A variants with body mass index and glucose handling. Changes in glucose tolerance were observed in Rab27aash male mice at 18 months of age. In WT (wild-type) and Rab27a null male mice, body weight, adipocyte lipid area, and aortic area increased with age. In female mice, only body weight increased with age, independent of RAB27A presence. Protein signatures from male Rab27a null mice suggested greater associations with cardiovascular and metabolic phenotypes compared with female tissues. Wire myography results showed Rab27a null males exhibited increased vasoconstriction and reduced vasodilation at 8 weeks of age. Rab27a null females exhibited increased vasoconstriction and vasodilation at 20 weeks of age. Consistent with these vascular changes, male Rab27a null mice experienced age-related cardiomyopathy, with severe differences observed by 21 weeks of age. CONCLUSIONS: Global RAB27A loss impacted perivascular adipose tissue and thoracic aorta proteomic signatures, altered vasocontractile responses, and decreased left ventricular ejection fraction in mice.

Endothelial IL17RD promotes Western diet-induced aortic myeloid cell infiltration.

The Interleukin-17 (IL17) family is a group of cytokines implicated in the etiology of several inflammatory diseases. Interleukin-17 receptor D (IL17RD), also known as Sef (similar expression to fibroblast growth factor) belonging to the family of IL17 receptors, has been shown to modulate IL17A-associated inflammatory phenotypes. The objective of this study was to test the hypothesis that IL17RD promotes endothelial cell activation and consequent leukocyte adhesion. We utilized primary human aortic endothelial cells and demonstrated that RNAi targeting of IL17RD suppressed transcript levels by 83 % compared to non-targeted controls. Further, RNAi knockdown of IL17RD decreased the adhesion of THP-1 monocytic cells onto a monolayer of aortic endothelial cells in response to IL17A. Additionally, we determined that IL17A did not significantly enhance the activation of canonical MAPK and NFκB pathways in endothelial cells, and further did not significantly affect the expression of VCAM-1 and ICAM-1 in aortic endothelial cells, which is contrary to previous findings. We also determined the functional relevance of our findings in vivo by comparing the expression of endothelial VCAM-1 and ICAM-1 and leukocyte infiltration in the aorta in Western diet-fed Il17rd null versus wild-type mice. Our results showed that although Il17rd null mice do not have significant alteration in aortic expression of VCAM-1 and ICAM-1 in endothelial cells, they exhibit decreased accumulation of proinflammatory monocytes and neutrophils, suggesting that endothelial IL17RD induced in vivo myeloid cell accumulation is not dependent on upregulation of VCAM-1 and ICAM-1 expression. We further performed proteomics analysis to identify potential molecular mediators of the IL17A/IL17RD signaling axis. Collectively, our results underscore a critical role for Il17rd in the regulation of aortic myeloid cell infiltration in the context of Western diet feeding.

Purification of functional mouse skeletal muscle mitochondria using percoll density gradient centrifugation.

OBJECTIVE: Our goal was to isolate purified mitochondria from mouse skeletal muscle using a Percoll density gradient and to assess bioenergetic function and purity via Seahorse Extracellular Flux (XF) Analyses and mass spectrometry. RESULTS: Mitochondria isolated from murine quadriceps femoris skeletal muscle using a Percoll density gradient method allowed for minimally contaminated preparations with time from tissue harvest to mitochondrial isolation and quantification in about 3-4 h. Percoll purification from 100 to 200 mg fresh tissue yielded ~ 200-400 ug protein. Mitochondrial bioenergetics evaluated using the Seahorse XFe96 analyzer, a high-throughput respirometry platform, showed optimum mitochondrial input at 500 ng with respiratory control ratio ranging from 3.9 to 7.1 using various substrates demonstrating a high degree of functionality. Furthermore, proteomic analysis of Percoll-enriched mitochondria isolated from skeletal muscle using this method showed significant enrichment of mitochondrial proteins indicating high sample purity. This study established a methodology that ensures sufficient high quality mitochondria for downstream analyses such as mitochondrial bioenergetics and proteomics.

Exploratory mass spectrometry of cerebrospinal fluid from persons with autopsy-confirmed LATE-NC.

Background: Common neuropathologies associated with dementia include Alzheimer's disease neuropathologic change (ADNC) and limbic-predominant age-related TDP-43 encephalopathy neuropathologic change (LATE-NC). Biofluid proteomics provides a window into the pathobiology of dementia and the information from biofluid tests may help guide clinical management. Methods: Participants were recruited from a longitudinal cohort of older adults at the University of Kentucky AD Research Center. A convenience sample of clinically obtained lumbar puncture cerebrospinal fluid (CSF) samples was analyzed from 29 older adults that had autopsy confirmation of the presence or absence of LATE-NC. Nine of the participants had autopsy-confirmed LATE-NC. Antemortem CSF specimens were analyzed in two separate processes: From one group, aliquots were depleted of highly abundant proteins using affinity spin columns. Tryptic digests of sample proteins were subjected to liquid chromatographic separation and mass spectrometry using an Eksigent Ekspert nanoLC 400 system in line with a Sciex 6600+ mass spectrometer. Protein identification was performed using Protein Pilot (Sciex, ver. 5) software, and relative quantification was performed using the SWATH processing microApp in PeakView and MarkerView software (Sciex), respectively. Following data analyses, additional studies were performed using western blots. Results: A total of 830 proteins were identified in the samples depleted of abundant proteins, and 730 proteins were identified in the non-depleted samples. Whereas some dementia-related proteins were detected (Aß peptide and α-synuclein protein), others were not (TDP-43, TMEM106B, and tau proteins). When the Bonferroni correction was applied to correct for multiple comparisons, only 4 proteins showed differential expression (LATE-NC vs non-LATE-NC) in the nondepleted samples (RBP4, MIF, IGHG3 and ITM2B), whereas none showed statistically different changes in the depleted samples. Post-hoc western blots confirmed that RBP4 expression was higher in the LATE-NC cases at the group level, but there was overlap between the levels of RBP4 in LATE-NC and non-LATE-NC cases. Conclusions: An exploratory assessment of CSF proteomes of autopsy-confirmed LATE-NC and non-LATE-NC cases from a community-based cohort failed to demonstrate a clear-cut proteomic fingerprint that distinguished the two groups. There was intriguing increase in RBP4 protein levels in CSF from LATE-NC cases. This may provide clues about pathogenetic mechanisms in LATE-NC.

Purification of functional mouse skeletal muscle mitochondria using Percoll density gradient centrifugation.

Objective: Our goal was to isolate purified mitochondria from mouse skeletal muscle using a Percoll density gradient and to assess bioenergetic function and purity via Seahorse Extracellular Flux (XF) Analyses and mass spectrometry. Results: Mitochondria isolated from murine quadriceps femoris skeletal muscle using a Percoll density gradient method allowed for minimally contaminated preparations with time from tissue harvest to mitochondrial isolation and quantification in about 3-4 hours. Percoll purification from 100-200 mg fresh tissue yielded ∼200-400 ug protein. Mitochondrial bioenergetics evaluated using the Seahorse XFe96 analyzer, a high-throughput respirometry platform, showed optimum mitochondrial input at 500 ng with respiratory control ratio ranging from 3.9-7.1 using various substrates demonstrating a high degree of functionality. Furthermore, proteomic analysis of Percoll-enriched mitochondria isolated from skeletal muscle using this method showed significant enrichment of mitochondrial proteins indicating high sample purity. This study established a methodology that ensures sufficient high quality mitochondria for downstream analyses such as mitochondrial bioenergetics and proteomics.

Targeting the fatty acid binding proteins disrupts multiple myeloma cell cycle progression and MYC signaling.

Multiple myeloma is an incurable plasma cell malignancy with only a 53% 5-year survival rate. There is a critical need to find new multiple myeloma vulnerabilities and therapeutic avenues. Herein, we identified and explored a novel multiple myeloma target: the fatty acid binding protein (FABP) family. In our work, myeloma cells were treated with FABP inhibitors (BMS3094013 and SBFI-26) and examined in vivo and in vitro for cell cycle state, proliferation, apoptosis, mitochondrial membrane potential, cellular metabolism (oxygen consumption rates and fatty acid oxidation), and DNA methylation properties. Myeloma cell responses to BMS309403, SBFI-26, or both, were also assessed with RNA sequencing (RNA-Seq) and proteomic analysis, and confirmed with western blotting and qRT-PCR. Myeloma cell dependency on FABPs was assessed using the Cancer Dependency Map (DepMap). Finally, MM patient datasets (CoMMpass and GEO) were mined for FABP expression correlations with clinical outcomes. We found that myeloma cells treated with FABPi or with FABP5 knockout (generated via CRISPR/Cas9 editing) exhibited diminished proliferation, increased apoptosis, and metabolic changes in vitro. FABPi had mixed results in vivo, in two pre-clinical MM mouse models, suggesting optimization of in vivo delivery, dosing, or type of FABP inhibitors will be needed before clinical applicability. FABPi negatively impacted mitochondrial respiration and reduced expression of MYC and other key signaling pathways in MM cells in vitro. Clinical data demonstrated worse overall and progression-free survival in patients with high FABP5 expression in tumor cells. Overall, this study establishes the FABP family as a potentially new target in multiple myeloma. In MM cells, FABPs have a multitude of actions and cellular roles that result in the support of myeloma progression. Further research into the FABP family in MM is warrented, especially into the effective translation of targeting these in vivo.

Multiple myeloma is a type of blood cancer for which only a few treatments are available. Currently, only about half the patients with multiple myeloma survive for five years after diagnosis. Because obesity is a risk factor for multiple myeloma, researchers have been studying how fat cells or fatty acids affect multiple myeloma tumor cells to identify new treatment targets. Fatty acid binding proteins (FABPs) are one promising target. The FABPs shuttle fatty acids and help cells communicate. Previous studies linked FABPs to some types of cancer, including another blood cancer called leukemia, and cancers of the prostate and breast. A recent study showed that patients with multiple myeloma, who have high levels of FABP5 in their tumors, have worse outcomes than patients with lower levels. But, so far, no one has studied the effects of inhibiting FABPs in multiple myeloma tumor cells or animals with multiple myeloma. Farrell et al. show that blocking or eliminating FABPs kills myeloma tumor cells and slows their growth in a dish (in vitro) and in some laboratory mice. In the experiments, the researchers treated myeloma cells with drugs that inhibit FABPs or genetically engineered myeloma cells to lack FABPs. They also show that blocking FABPs reduces the activity of a protein called MYC, which promotes tumor cell survival in many types of cancer. It also changed the metabolism of the tumor cell. Finally, the team examined data collected from several sets of patients with multiple myeloma and found that patients with high FABP levels have more aggressive cancer. The experiments lay the groundwork for more studies to determine if drugs or other therapies targeting FABPs could treat multiple myeloma. More research is needed to determine why inhibiting FABPs worked in some mice with multiple myeloma but not others, and whether FABP inhibitors might work better if combined with other cancer therapies. There were no signs that the drugs were toxic in mice, but more studies must prove they are safe and effective before testing the drugs in humans with multiple myeloma. Designing better or more potent FABP-blocking drugs may also lead to better animal study results.

Development and characterization of three cell culture systems to investigate the relationship between primary bone marrow adipocytes and myeloma cells.

The unique properties of the bone marrow (BM) allow for migration and proliferation of multiple myeloma (MM) cells while also providing the perfect environment for development of quiescent, drug-resistant MM cell clones. BM adipocytes (BMAds) have recently been identified as important contributors to systemic adipokine levels, bone strength, hematopoiesis, and progression of metastatic and primary BM cancers, such as MM. Recent studies in myeloma suggest that BMAds can be reprogrammed by tumor cells to contribute to myeloma-induced bone disease, and, reciprocally, BMAds support MM cells in vitro. Importantly, most data investigating BMAds have been generated using adipocytes generated by differentiating BM-derived mesenchymal stromal cells (BMSCs) into adipocytes in vitro using adipogenic media, due to the extreme technical challenges associated with isolating and culturing primary adipocytes. However, if studies could be performed with primary adipocytes, then they likely will recapitulate in vivo biology better than BMSC-derived adipocytes, as the differentiation process is artificial and differs from in vivo differentiation, and progenitor cell(s) of the primary BMAd (pBMAds) may not be the same as the BMSCs precursors used for adipogenic differentiation in vitro. Therefore, we developed and refined three methods for culturing pBMAds: two-dimensional (2D) coverslips, 2D transwells, and three-dimensional (3D) silk scaffolds, all of which can be cultured alone or with MM cells to investigate bidirectional tumor-host signaling. To develop an in vitro model with a tissue-like structure to mimic the BM microenvironment, we developed the first 3D, tissue engineered model utilizing pBMAds derived from human BM. We found that pBMAds, which are extremely fragile, can be isolated and stably cultured in 2D for 10 days and in 3D for up to 4 week in vitro. To investigate the relationship between pBMAds and myeloma, MM cells can be added to investigate physical relationships through confocal imaging and soluble signaling molecules via mass spectrometry. In summary, we developed three in vitro cell culture systems to study pBMAds and myeloma cells, which could be adapted to investigate many diseases and biological processes involving the BM, including other bone-homing tumor types.

The Extracellular Matrix Environment of Clear Cell Renal Cell Carcinoma Determines Cancer Associated Fibroblast Growth.

Clear cell renal cell carcinoma (ccRCC) is the most common kidney cancer and is often caused by mutations in the oxygen-sensing machinery of kidney epithelial cells. Due to its pseudo-hypoxic state, ccRCC recruits extensive vasculature and other stromal components. Conventional cell culture methods provide poor representation of stromal cell types in primary cultures of ccRCC, and we hypothesized that mimicking the extracellular environment of the tumor would promote growth of both tumor and stromal cells. We employed proteomics to identify the components of ccRCC extracellular matrix (ECM) and found that in contrast to healthy kidney cortex, laminin, collagen IV, and entactin/nidogen are minor contributors. Instead, the ccRCC ECM is composed largely of collagen VI, fibronectin, and tenascin C. Analysis of single cell expression data indicates that cancer-associated fibroblasts are a major source of tumor ECM production. Tumor cells as well as stromal cells bind efficiently to a nine-component ECM blend characteristic of ccRCC. Primary patient-derived tumor cells bind the nine-component blend efficiently, allowing to us to establish mixed primary cultures of tumor cells and stromal cells. These miniature patient-specific replicas are conducive to microscopy and can be used to analyze interactions between cells in a model tumor microenvironment.

Sclerostin antibody increases trabecular bone and bone mechanical properties by increasing osteoblast activity damaged by whole-body irradiation in mice.

Irradiation therapy causes bone deterioration and increased risk for skeletal-related events. Irradiation interferes with trabecular architecture through increased osteoclastic activity, decreased osteoblastic activity, and increased adipocyte expansion in the bone marrow (BM), which further compounds bone-related disease. Neutralizing antibodies to sclerostin (Scl-Ab) increase bone mass and strength by increasing bone formation and reducing bone resorption. We hypothesized that treatment with Scl-Ab would attenuate the adverse effects of irradiation by increasing bone volume and decreasing BM adipose tissue (BMAT), resulting in better quality bone. In this study, 12-week-old female C57BL/6J mice were exposed to 6 Gy whole-body irradiation or were non-irradiated, then administered Scl-Ab (25 mg/kg) or vehicle weekly for 5 weeks. Femoral µCT analysis confirmed that the overall effect of IR significantly decreased trabecular bone volume/total volume (Tb.BV/TV) (2-way ANOVA, p < 0.0001) with a -43.8% loss in Tb.BV/TV in the IR control group. Scl-Ab independently increased Tb.BV/TV by 3.07-fold in non-irradiated and 3.6-fold in irradiated mice (2-way ANOVA, p < 0.0001). Irradiation did not affect cortical parameters, although Scl-Ab increased cortical thickness and area significantly in both irradiated and non-irradiated mice (2-way ANOVA, p < 0.0001). Femoral mechanical testing confirmed Scl-Ab significantly increased bending rigidity and ultimate moment independently of irradiation (2-way ANOVA, p < 0.0001). Static and dynamic histomorphometry of the femoral metaphysis revealed osteoblast vigor, not number, was significantly increased in the irradiated mice treated with Scl-Ab. Systemic alterations were assessed through serum lipidomic analysis, which showed that Scl-Ab normalized lipid profiles in the irradiated group. This data supports the theory of sclerostin as a novel contributor to the regulation of osteoblast activity after irradiation. Overall, our data support the hypothesis that Scl-Ab ameliorates the deleterious effects of whole-body irradiation on bone and adipose tissue in a mouse model. Our findings suggest that future research into localized and systemic therapies after irradiation exposure is warranted.

An RGDKGE-Containing Cryptic Collagen Fragment Regulates Phosphorylation of Large Tumor Suppressor Kinase-1 and Controls Ovarian Tumor Growth by a Yes-Associated Protein-Dependent Mechanism.

The growth and spread of malignant tumors, such as ovarian carcinomas, are governed in part by complex interconnected signaling cascades occurring between stromal and tumor cells. These reciprocal cross-talk signaling networks operating within the local tissue microenvironment may enhance malignant tumor progression. Understanding how novel bioactive molecules generated within the tumor microenvironment regulate signaling pathways in distinct cellular compartments is critical for the development of more effective treatment paradigms. Herein, we provide evidence that blocking cellular interactions with an RGDKGE-containing collagen peptide that selectively binds integrin ß3 on ovarian tumor cells enhances the phosphorylation of the hippo effector kinase large tumor suppressor kinase-1 and reduces nuclear accumulation of yes-associated protein and its target gene c-Myc. Selectively targeting this RGDKGE-containing collagen fragment inhibited ovarian tumor growth and the development of ascites fluid in vivo. These findings suggest that this bioactive collagen fragment may represent a previously unknown regulator of the hippo effector kinase large tumor suppressor kinase-1 and regulate ovarian tumor growth by a yes-associated protein-dependent mechanism. Taken together, these data not only provide new mechanistic insight into how a unique collagen fragment may regulate ovarian cancer, but in addition may help provide a useful new alternative strategy to control ovarian tumor progression based on selectively disrupting a previously unappreciated signaling cascade.

Understanding Mechanisms Underlying Non-Alcoholic Fatty Liver Disease (NAFLD) in Mental Illness: Risperidone and Olanzapine Alter the Hepatic Proteomic Signature in Mice.

Patients with severe mental illness have increased mortality, often linked to cardio-metabolic disease. Non-alcoholic fatty liver disease (NAFLD) incidence is higher in patients with schizophrenia and is exacerbated with antipsychotic treatment. NAFLD is associated with obesity and insulin resistance, both of which are induced by several antipsychotic medications. NAFLD is considered an independent risk factor for cardiovascular disease, the leading cause of death for patients with severe mental illness. Although the clinical literature clearly defines increased risk of NAFLD with antipsychotic therapy, the underlying mechanisms are not understood. Given the complexity of the disorder as well as the complex pharmacology associated with atypical antipsychotic (AA) medications, we chose to use a proteomic approach in healthy mice treated with a low dose of risperidone (RIS) or olanzapine (OLAN) for 28 days to determine effects on development of NAFLD and to identify pathways impacted by AA medications, while removing confounding intrinsic effects of mental illness. Both AA drugs caused development of steatosis in comparison with vehicle controls (p < 0.01) and affected multiple pathways relating to energy metabolism, NAFLD, and immune function. AA-associated alteration in autonomic function appears to be a unifying theme in the regulation of hepatic pathology.

Targeting Bone Cells During Sexual Maturation Reveals Sexually Dimorphic Regulation of Endochondral Ossification.

In endochondral ossification, chondroblasts become embedded in their matrix and become chondrocytes, which are mature cells that continue to proliferate, eventually becoming hypertrophic. Hypertrophic chondrocytes produce cartilage that is then resorbed by osteoclasts prior to bone matrix replacement via osteoblasts. Although sexually dimorphic bone phenotypes have long been characterized, specific modulation of the growth plate during a critical window in sexual maturation has not been evaluated. Here we report that specific depletion of osteocalcin- (OCN-) expressing cells in vivo during sexual maturation leads to dimorphic bone phenotypes in males and females. At 6 to 8 weeks of age, OCN-Cre;iDTR (inducible diphtheria toxin receptor-expressing) mice were treated with diphtheria toxin (DT) for 2 weeks to deplete OCN+ cells. At the end of the study, long bones were collected for µCT and histomorphometry, and serum was collected for proteomic and lipidomic analyses. Ablation of OCN+ cells in mice leads to consistent trends for weight loss after 2 weeks of treatment. Females exhibited decreased skeletal parameters in response to OCN+ cell ablation treatment, as expected. However, OCN+ cell ablation in males uniquely displayed an expansion of hypertrophic chondrocytes, a widening of the growth plate, and an abnormal "clubbing" anatomy of the distal femur. Following DT treatment, mice from both sexes also underwent metabolic cage analysis, in which both sexes exhibited decreased energy expenditure. We conclude that skewing endochondral bone formation during longitudinal growth has a profound effect on body weight and energy expenditure with sex-specific effects on developing bone. © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

PHOSPHO1 is a skeletal regulator of insulin resistance and obesity.

BACKGROUND: The classical functions of the skeleton encompass locomotion, protection and mineral homeostasis. However, cell-specific gene deletions in the mouse and human genetic studies have identified the skeleton as a key endocrine regulator of metabolism. The bone-specific phosphatase, Phosphatase, Orphan 1 (PHOSPHO1), which is indispensable for bone mineralisation, has been recently implicated in the regulation of energy metabolism in humans, but its role in systemic metabolism remains unclear. Here, we probe the mechanism underlying metabolic regulation by analysing Phospho1 mutant mice. RESULTS: Phospho1-/- mice exhibited improved basal glucose homeostasis and resisted high-fat-diet-induced weight gain and diabetes. The metabolic protection in Phospho1-/- mice was manifested in the absence of altered levels of osteocalcin. Osteoblasts isolated from Phospho1-/- mice were enriched for genes associated with energy metabolism and diabetes; Phospho1 both directly and indirectly interacted with genes associated with glucose transport and insulin receptor signalling. Canonical thermogenesis via brown adipose tissue did not underlie the metabolic protection observed in adult Phospho1-/- mice. However, the decreased serum choline levels in Phospho1-/- mice were normalised by feeding a 2% choline rich diet resulting in a normalisation in insulin sensitivity and fat mass. CONCLUSION: We show that mice lacking the bone mineralisation enzyme PHOSPHO1 exhibit improved basal glucose homeostasis and resist high-fat-diet-induced weight gain and diabetes. This study identifies PHOSPHO1 as a potential bone-derived therapeutic target for the treatment of obesity and diabetes.

Overexpression of Activin Receptor-Like Kinase 1 in Endothelial Cells Suppresses Development of Arteriovenous Malformations in Mouse Models of Hereditary Hemorrhagic Telangiectasia.

RATIONALE: Hereditary hemorrhagic telangiectasia (HHT) is a genetic disease caused by mutations in ENG, ALK1, or SMAD4. Since proteins from all 3 HHT genes are components of signal transduction of TGF-ß (transforming growth factor ß) family members, it has been hypothesized that HHT is a disease caused by defects in the ENG-ALK1-SMAD4 linear signaling. However, in vivo evidence supporting this hypothesis is scarce. OBJECTIVE: We tested this hypothesis and investigated the therapeutic effects and potential risks of induced-ALK1 or -ENG overexpression (OE) for HHT. METHODS AND RESULTS: We generated a novel mouse allele (ROSA26Alk1) in which HA (human influenza hemagglutinin)-tagged ALK1 and bicistronic eGFP expression are induced by Cre activity. We examined whether ALK1-OE using the ROSA26Alk1 allele could suppress the development of arteriovenous malformations (AVMs) in wounded adult skin and developing retinas of Alk1- and Eng-inducible knockout (iKO) mice. We also used a similar approach to investigate whether ENG-OE could rescue AVMs. Biochemical and immunofluorescence analyses confirmed the Cre-dependent OE of the ALK1-HA transgene. We could not detect any pathological signs in ALK1-OE mice up to 3 months after induction. ALK1-OE prevented the development of retinal AVMs and wound-induced skin AVMs in Eng-iKO as well as Alk1-iKO mice. ALK1-OE normalized expression of SMAD and NOTCH target genes in ENG-deficient endothelial cells (ECs) and restored the effect of BMP9 (bone morphogenetic protein 9) on suppression of phosphor-AKT levels in these endothelial cells. On the other hand, ENG-OE could not inhibit the AVM development in Alk1-iKO models. CONCLUSIONS: These data support the notion that ENG and ALK1 form a linear signaling pathway for the formation of a proper arteriovenous network during angiogenesis. We suggest that ALK1 OE or activation can be an effective therapeutic strategy for HHT. Further research is required to study whether this therapy could be translated into treatment for humans.

Pathological Conversion of Mouse Perivascular Adipose Tissue by Notch Activation.

OBJECTIVE: Perivascular adipose tissue (PVAT) surrounding arteries supports healthy vascular function. During obesity, PVAT loses its vasoprotective effect. We study pathological conversion of PVAT, which involves molecular changes in protein profiles and functional changes in adipocytes. Approach and Results: C57BL6/J mice were fed a 60% high-fat diet for 12 weeks or a cardioprotective 30% calorie-restricted diet for 5 weeks. Proteomic analysis identified PVAT as a molecularly distinct adipose depot, and novel markers for thermogenic adipocytes, such as GRP75 (stress-70 protein, mitochondrial), were identified. High-fat diet increased the similarity of protein signatures in PVAT and brown adipose, suggesting activation of a conserved whitening pathway. The whitening phenotype was characterized by suppression of UCP1 (uncoupling protein 1) and increased lipid deposition, leptin, and inflammation, and specifically in PVAT, elevated Notch signaling. Conversely, PVAT from calorie-restricted mice had decreased Notch signaling and less lipid. Using the Adipoq-Cre strain, we constitutively activated Notch1 signaling in adipocytes, which phenocopied the changes in PVAT caused by a high-fat diet, even on a standard diet. Preadipocytes from mouse PVAT expressed Sca1, CD140a, Notch1, and Notch2, but not CD105, showing differences compared with preadipocytes from other depots. Inhibition of Notch signaling during differentiation of PVAT-derived preadipocytes reduced lipid deposition and adipocyte marker expression. CONCLUSIONS: PVAT shares features with other adipose depots, but has a unique protein signature that is regulated by dietary stress. Increased Notch signaling in PVAT is sufficient to initiate the pathological conversion of PVAT by promoting adipogenesis and lipid accumulation and may thus prime the microenvironment for vascular disease.

Exploring mechanisms of increased cardiovascular disease risk with antipsychotic medications: Risperidone alters the cardiac proteomic signature in mice.

Atypical antipsychotic (AA) medications including risperidone (RIS) and olanzapine (OLAN) are FDA approved for the treatment of psychiatric disorders including schizophrenia, bipolar disorder and depression. Clinical side effects of AA medications include obesity, insulin resistance, dyslipidemia, hypertension and increased cardiovascular disease risk. Despite the known pharmacology of these AA medications, the mechanisms contributing to adverse metabolic side-effects are not well understood. To evaluate drug-associated effects on the heart, we assessed changes in the cardiac proteomic signature in mice administered for 4 weeks with clinically relevant exposure of RIS or OLAN. Using proteomic and gene enrichment analysis, we identified differentially expressed (DE) proteins in both RIS- and OLAN-treated mouse hearts (p < 0.05), including proteins comprising mitochondrial respiratory complex I and pathways involved in mitochondrial function and oxidative phosphorylation. A subset of DE proteins identified were further validated by both western blotting and quantitative real-time PCR. Histological evaluation of hearts indicated that AA-associated aberrant cardiac gene expression occurs prior to the onset of gross pathomorphological changes. Additionally, RIS treatment altered cardiac mitochondrial oxygen consumption and whole body energy expenditure. Our study provides insight into the mechanisms underlying increased patient risk for adverse cardiac outcomes with chronic treatment of AA medications.

The antipsychotic medication, risperidone, causes global immunosuppression in healthy mice.

Atypical antipsychotic medications such as risperidone are widely prescribed for diverse psychiatric indications including schizophrenia, bipolar disorder and depression. These medications have complex pharmacology and are associated with significant endocrine and metabolic side effects. This class of medications also carries FDA black box warnings due to increased risk of death in elderly patients. Clinical reports indicate that patients treated with these medications are more susceptible to infections; however, the underlying mechanisms/pharmacology are unclear. We have previously reported that risperidone and it's active metabolite distributes to the bone marrow in clinically relevant concentrations in preclinical species, leading us to hypothesize that the hematopoietic system may be impacted by these medications. To test this hypothesis, using proteomic and cytokine array technology, we evaluated the expression of genes involved in inflammatory and immune function following short term (5 days) and longer term (4 weeks) treatment in healthy animals. We report that low-dose risperidone treatment results in global immunosuppression in mice, observed following 5 days of dosing and exacerbated with longer term drug treatment (4 weeks). These data are consistent with increased susceptibility to infection in patients administered these medications and have profound implications for the increasing off-label prescribing to vulnerable patient populations including children and the elderly.

Interactions with Muscle Cells Boost Fusion, Stemness, and Drug Resistance of Prostate Cancer Cells.

Poorly understood interactions with nonmalignant cells within the tumor microenvironment play an important role in cancer progression. Here, we explored interactions between prostate cancer and muscle cells that surround the prostate. We found that coculturing of prostate cancer cells with skeletal or smooth muscle cells expands the subpopulations of cancer cells with features characteristic of cancer stem-like cells, including anchorage-independent growth, elevated CD133 expression, and drug resistance. These changes in the properties of cancer cells depend on: (i) the muscle cell-induced increases in the concentrations of interleukins 4 and 13; (ii) the cytokine-induced upregulation of the expression of syncytin 1 and annexin A5; and (iii) cancer cell fusion. In human prostate cancer tissues, expression of syncytin 1 and annexin A5, proteins that we found to be required for the cell fusion, positively correlated with the cancer development suggesting that these proteins can be used as biomarkers to evaluate cancer progression and potential therapeutic targets. IMPLICATIONS: The discovered effects of muscle cells on prostate cancer cells reveal a novel and specific pathway by which muscle cells in the microenvironment of prostate cancer cells promote cell fusion and cancer progression.

Development of a 3D bone marrow adipose tissue model.

Over the past twenty years, evidence has accumulated that biochemically and spatially defined networks of extracellular matrix, cellular components, and interactions dictate cellular differentiation, proliferation, and function in a variety of tissue and diseases. Modeling in vivo systems in vitro has been undeniably necessary, but when simplified 2D conditions rather than 3D in vitro models are used, the reliability and usefulness of the data derived from these models decreases. Thus, there is a pressing need to develop and validate reliable in vitro models to reproduce specific tissue-like structures and mimic functions and responses of cells in a more realistic manner for both drug screening/disease modeling and tissue regeneration applications. In adipose biology and cancer research, these models serve as physiologically relevant 3D platforms to bridge the divide between 2D cultures and in vivo models, bringing about more reliable and translationally useful data to accelerate benchtop to bedside research. Currently, no model has been developed for bone marrow adipose tissue (BMAT), a novel adipose depot that has previously been overlooked as "filler tissue" but has more recently been recognized as endocrine-signaling and systemically relevant. Herein we describe the development of the first 3D, BMAT model derived from either human or mouse bone marrow (BM) mesenchymal stromal cells (MSCs). We found that BMAT models can be stably cultured for at least 3 months in vitro, and that myeloma cells (5TGM1, OPM2 and MM1S cells) can be cultured on these for at least 2 weeks. Upon tumor cell co-culture, delipidation occurred in BMAT adipocytes, suggesting a bidirectional relationship between these two important cell types in the malignant BM niche. Overall, our studies suggest that 3D BMAT represents a "healthier," more realistic tissue model that may be useful for elucidating the effects of MAT on tumor cells, and tumor cells on MAT, to identify novel therapeutic targets. In addition, proteomic characterization as well as microarray data (expression of >22,000 genes) coupled with KEGG pathway analysis and gene set expression analysis (GSEA) supported our development of less-inflammatory 3D BMAT compared to 2D culture. In sum, we developed the first 3D, tissue-engineered bone marrow adipose tissue model, which is a versatile, novel model that can be used to study numerous diseases and biological processes involved with the bone marrow.