Removal of endothelial surface-associated von villebrand factor suppresses accelerate datherosclerosis after myocardial infarction.

BACKGROUND: Thromboinflammation involving platelet adhesion to endothelial surface-associated von Willebrand factor (VWF) has been implicated in the accelerated progression of non-culprit plaques after MI. The aim of this study was to use arterial endothelial molecular imaging to mechanistically evaluate endothelial-associated VWF as a therapeutic target for reducing remote plaque activation after myocardial infarction (MI). METHODS: Hyperlipidemic mice deficient for the low-density lipoprotein receptor and Apobec-1 underwent closed-chest MI and were treated chronically with either: (i) recombinant ADAMTS13 which is responsible for proteolytic removal of VWF from the endothelial surface, (ii) N-acetylcysteine (NAC) which removes VWF by disulfide bond reduction, (iii) function-blocking anti-factor XI (FXI) antibody, or (iv) no therapy. Non-ischemic controls were also studied. At day 3 and 21, ultrasound molecular imaging was performed with probes targeted to endothelial-associated VWF A1-domain, platelet GPIbα, P-selectin and vascular cell adhesion molecule-1 (VCAM-1) at lesion-prone sites of the aorta. Histology was performed at day 21. RESULTS: Aortic signal for P-selectin, VCAM-1, VWF, and platelet-GPIbα were all increased several-fold (p < 0.01) in post-MI mice versus sham-treated animals at day 3 and 21. Treatment with NAC and ADAMTS13 significantly attenuated the post-MI increase for all four molecular targets by > 50% (p < 0.05 vs. non-treated at day 3 and 21). On aortic root histology, mice undergoing MI versus controls had 2-4 fold greater plaque size and macrophage content (p < 0.05), approximately 20-fold greater platelet adhesion (p < 0.05), and increased staining for markers of platelet transforming growth factor-ß1 signaling. Accelerated plaque growth and inflammatory activation was almost entirely prevented by ADAMTS13 and NAC. Inhibition of FXI had no significant effect on molecular imaging signal or plaque morphology. CONCLUSIONS: Plaque inflammatory activation in remote arteries after MI is strongly influenced by VWF-mediated platelet adhesion to the endothelium. These findings support investigation into new secondary preventive therapies for reducing non-culprit artery events after MI.

Adipose Tissue Analysis Toolkit (ATAT) for automated analysis of adipocyte size and extracellular matrix in white adipose tissue.

OBJECTIVE: The pathological expansion of white adipose tissue (WAT) in obesity involves adipocyte hypertrophy accompanied by expansion of the collagen-rich pericellular extracellular matrix (ECM) and development of crown-like structures (CLS). Traditionally, WAT morphology is assessed through immunohistochemical analysis of WAT sections. However, manual analysis of large histological sections is time-consuming, and the available digital tools for analyzing adipocyte size and pericellular ECM are limited. To address this gap, the authors developed the Adipose Tissue Analysis Toolkit (ATAT), an ImageJ plugin facilitating analysis of adipocyte size, WAT ECM, and CLS. METHODS AND RESULTS: ATAT utilizes local and image-level differentials in pixel intensity to independently threshold image background, distinguishing adipocyte-free tissue without user input. It accurately captures adipocytes in histological sections stained with common dyes and automates the analysis of adipocyte cross-sectional area, total-field, and localized region-of-interest ECM. ATAT allows fully automated batch analysis of histological images using default or user-defined adipocyte detection parameters. CONCLUSIONS: ATAT provides several advantages over existing WAT image analysis tools, enabling high-throughput analyses of adipocyte-specific parameters and facilitating the assessment of ECM changes associated with WAT remodeling due to weight changes and other pathophysiological alterations that affect WAT function.

Theoretical Comparison of Different Envelope Elimination and Restoration Transmitter PWM Modulator Configurations to Expand the Possible Antenna Mismatch.

The main characteristics of high-efficiency switching-mode solid-state power amplifiers with envelope elimination and restoration (EER) methods depend on all their elements. In this article, we study the influence of the types and parameters of the envelope path low-pass filters (LPFs) on the EER transmitter out-of-band emissions. This article presents for the first time an analysis of EER transmitter operation where the output impedance of the PWM modulator is not equal to zero, as usual (with a one-sided loaded LPF), but is matched with the low-pass filter and the load (with a double-sided loaded LPF). Theoretical comparisons of EER transmitters' out-of-band emissions were carried out with four envelope path LPF configurations (one-sided and double-sided loaded LPFs with a smooth and sharp transition, respectively), for both the nominal load (broadband antenna) and resonant antennas with a limited bandwidth. The analysis showed that for the case of transmitter operation on a resonant antenna with a limited bandwidth, the preferable option was the use of a sixth-order double-sided loaded LPF with a smooth transition. The use of the proposed modulator configuration allowed the transmitter to operate on an antenna with VSWR = 1.07 at the edges of the transmitted signal band with a minimum LPF bandwidth equal to 5.8 bands of the amplified signal. This could significantly expand its application capabilities and allow one to reduce the PWM clock frequency and increase efficiency.

Western-style diet in the presence of elevated circulating testosterone induces adipocyte hypertrophy without proinflammatory responses in rhesus macaques.

PROBLEM: Anovulatory infertility is commonly associated with hyperandrogenemia (elevated testosterone, T), insulin resistance, obesity, and white adipose tissue (WAT) dysfunction associated with adipocyte hypertrophy. However, whether hyperandrogenemia and adipocyte hypertrophy per se induce a proinflammatory response is unknown. METHOD OF STUDY: Young adult female rhesus macaques were exposed to an obesogenic Western-style diet (WSD) in the presence of elevated circulating testosterone (T+WSD) or a low-fat control diet with no exogenous T. Immune cells residing in visceral omental white adipose tissue (OM-WAT), corpus luteum and the contralateral ovary, endometrium, lymph nodes, bone marrow, and peripheral blood mononuclear cells were characterized by flow cytometry during the luteal phase of the reproductive cycle. RESULTS: Following one year of treatment, T+WSD animals became more insulin-resistant and exhibited increased body fat and adipocyte hypertrophy compared to controls. T+WSD treatment did not induce macrophage polarization toward a proinflammatory phenotype in the tissues examined. Additionally, T+WSD treatment did not affect TNFα production by bone marrow macrophages in response to toll-like receptor agonists. While the major lymphoid subsets were not significantly affected by T+WSD treatment, we observed a significant reduction in the frequency of effector memory CD8+ T-cells (Tem) in OM-WAT, but not in other tissues. Notably, OM-WAT Tem frequencies were negatively correlated with insulin resistance as assessed by the Homeostatic Model Assessment for Insulin Resistance (HOMA-IR). CONCLUSION: This study shows that short-term T+WSD treatment induces weight gain, insulin resistance, and adipocyte hypertrophy, but does not have a significant effect on systemic and tissue-resident proinflammatory markers, suggesting that adipocyte hypertrophy and mild hyperandrogenemia alone are not sufficient to induce a proinflammatory response.

Elevated LDL Cholesterol Increases Microvascular Endothelial VWF and Thromboinflammation After Myocardial Infarction.

BACKGROUND: In reperfused myocardial infarction, VWF (von Willebrand factor)-mediated platelet adhesion contributes to impaired microvascular reflow and possibly also to postmyocardial infarction inflammation. We hypothesized that postischemic thromboinflammatory processes are worsened by elevated LDL (low-density lipoprotein) cholesterol. METHODS: Myocardial ischemia-reperfusion or sham procedure was performed in wild-type mice and hyperlipidemic mice deficient for the LDL receptor and Apobec-1 (apolipoprotein-B mRNA editing enzyme catalytic polypeptide-1; DKO [double knockout]). DKO subgroups were treated with N-acetylcysteine, which inhibits pro-adhesive VWF multimers or with recombinant ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin motifs-13), which enzymatically cleaves endothelial surface-associated VWF. Myocardial contrast echocardiography perfusion imaging and molecular imaging for VWF, platelet glycoprotein Ibα, and leukocyte CD18 (cluster of differentiation) were performed 30 minutes post-reperfusion. Histology, infarct sizing, and echocardiography were performed at 1.5 or 72 hours; late echocardiography was performed at day 21. RESULTS: After ischemia-reperfusion, DKO compared with wild-type mice had ≈2-fold higher (P<0.05) risk area signal for microvascular platelet adhesion, VWF, and CD18; greater impairment in microvascular reflow, and 2-fold larger infarct size. Treatment of DKO mice with N-acetylcysteine and ADAMTS13 reduced molecular imaging signal for microvascular platelet adhesion, VWF, and CD18; improved early microvascular reflow; and reduced eventual infarct size. ADAMTS13 suppressed the postmyocardial infarction neutrophil and monocyte infiltration, enhanced the time-dependent recovery of left ventricular systolic function, and prevented late left ventricular remodeling. CONCLUSIONS: In reperfused myocardial infarction, elevated LDL cholesterol promotes thromboinflammation through excess microvascular endothelial VWF and platelet adhesion, resulting in less microvascular reflow and larger infarct size. In the presence of elevated LDL cholesterol, therapies that suppress endothelial-associated VWF can promote recovery of left ventricular function and protect against remodeling.

Maternal diet alters long-term innate immune cell memory in fetal and juvenile hematopoietic stem and progenitor cells in nonhuman primate offspring.

Maternal overnutrition increases inflammatory and metabolic disease risk in postnatal offspring. This constitutes a major public health concern due to increasing prevalence of these diseases, yet mechanisms remain unclear. Here, using nonhuman primate models, we show that maternal Western-style diet (mWSD) exposure is associated with persistent pro-inflammatory phenotypes at the transcriptional, metabolic, and functional levels in bone marrow-derived macrophages (BMDMs) from 3-year-old juvenile offspring and in hematopoietic stem and progenitor cells (HSPCs) from fetal and juvenile bone marrow and fetal liver. mWSD exposure is also associated with increased oleic acid in fetal and juvenile bone marrow and fetal liver. Assay for transposase-accessible chromatin with sequencing (ATAC-seq) profiling of HSPCs and BMDMs from mWSD-exposed juveniles supports a model in which HSPCs transmit pro-inflammatory memory to myeloid cells beginning in utero. These findings show that maternal diet alters long-term immune cell developmental programming in HSPCs with proposed consequences for chronic diseases featuring altered immune/inflammatory activation across the lifespan.

Maternal obesity blunts antimicrobial responses in fetal monocytes.

Maternal pre-pregnancy (pregravid) obesity is associated with adverse outcomes for both mother and offspring. Amongst the complications for the offspring is increased susceptibility and severity of neonatal infections necessitating admission to the intensive care unit, notably bacterial sepsis and enterocolitis. Previous studies have reported aberrant responses to LPS and polyclonal stimulation by umbilical cord blood monocytes that were mediated by alterations in the epigenome. In this study, we show that pregravid obesity dysregulates umbilical cord blood monocyte responses to bacterial and viral pathogens. Specifically, interferon-stimulated gene expression and inflammatory responses to respiratory syncytial virus (RSV) and E. coli were significantly dampened, respectively . Although upstream signaling events were comparable, translocation of the key transcription factor NF-κB and chromatin accessibility at pro-inflammatory gene promoters following TLR stimulation was significantly attenuated. Using a rhesus macaque model of western style diet-induced obesity, we further demonstrate that this defect is detected in fetal peripheral monocytes and tissue-resident macrophages during gestation. Collectively, these data indicate that maternal obesity alters metabolic, signaling, and epigenetic profiles of fetal monocytes leading to a state of immune paralysis during late gestation and at birth.

Adipose Tissue Analysis Toolkit (ATAT) for Automated Analysis of Adipocyte Size and Extracellular Matrix in White Adipose Tissue.

Objective: The pathological expansion of white adipose tissue (WAT) in obesity involves adipocyte hypertrophy accompanied by expansion of collagen-rich pericellular extracellular matrix (ECM) and the development of crown-like structures (CLS). Traditionally, WAT morphology is assessed through immunohistochemical analysis of WAT sections. However, manual analysis of large histological sections is time-consuming, and available digital tools for analyzing adipocyte size and pericellular ECM are limited. To address this gap, we developed the Adipose Tissue Analysis Toolkit (ATAT), an ImageJ plugin facilitating analysis of adipocyte size, WAT ECM and CLS. Methods and Results: ATAT utilizes local and image-level differentials in pixel intensity to independently threshold background, distinguishing adipocyte-free tissue without user input. It accurately captures adipocytes in histological sections stained with common dyes and automates the analysis of adipocyte cross-sectional area, total-field, and localized region-of-interest ECM. ATAT allows fully automated batch analysis of histological images using default or user-defined adipocyte detection parameters. Conclusions: ATAT provides several advantages over existing WAT image analysis tools, enabling high-throughput analyses of adipocyte-specific parameters and facilitating the assessment of ECM changes associated with WAT remodeling due to weight changes and other pathophysiological alterations that affect WAT function. Study Importance Questions: What is already known about this subject?: The manual analysis of large WAT histological sections is very time-consuming, while digital tools for the analysis of WAT are limited.What are the new findings in your manuscript?: - ATAT enables fully automated analysis of batches of histological images using either default or user-defined adipocyte detection parameters- ATAT allows high-throughput analyses of adipocyte-specific parameters and pericellular extracellular matrix- ATAT enables the assessment of fibrotic changes associated with WAT remodeling and crown-like structuresHow might your results change the direction of research or the focus of clinical practice?: - ATAT is designed to work with histological sections and digital images obtained using a slide scanner or a microscope.- This tool will help basic and clinical researchers to conduct automated analyses of adipose tissue histological sections.

Broadband and Efficient Envelope Amplifier for Envelope Elimination and Restoration/Envelope Tracking Higher-Efficiency Power Amplifiers.

Increasing the efficiency of transmitters, as the largest consumers of energy, is relevant for any wireless communication devices. For higher efficiency, a number of methods are used, including envelope tracking and envelope elimination and restoration. Increasing the bandwidth of used frequencies requires expanding envelope modulators bandwidth up to 250-500 MHz or more. The possibility of using amplifiers with input signal quantization (AISQ), as an alternative to the most common hybrid envelope tracking modulators, is considered. An approach has been developed for optimizing AISQ characteristics according to the criterion of minimum loss when amplifying modern telecommunication signals with Rayleigh envelope distribution. The optimal quantization levels are determined and the energy characteristics of AISQ are calculated. AISQ loss power is shown to decrease by 1.66 times with two-level quantization, by 2.4 times with three-level quantization, and by a factor of 3.0-3.7 for four-five quantization levels compared to a class B amplifier. With these parameters, AISQ becomes competitive with respect to hybrid envelope tracking modulators but does not have electromagnetic interference from the pulse width modulation (PWM) path.

Maternal Western-style diet remodels the transcriptional landscape of fetal hematopoietic stem and progenitor cells in rhesus macaques.

Maternal obesity adversely impacts the in utero metabolic environment, but its effect on fetal hematopoiesis remains incompletely understood. During late development, the fetal bone marrow (FBM) becomes the major site where macrophages and B lymphocytes are produced via differentiation of hematopoietic stem and progenitor cells (HSPCs). Here, we analyzed the transcriptional landscape of FBM HSPCs at single-cell resolution in fetal macaques exposed to a maternal high-fat Western-style diet (WSD) or a low-fat control diet. We demonstrate that maternal WSD induces a proinflammatory response in FBM HSPCs and fetal macrophages. In addition, maternal WSD consumption suppresses the expression of B cell development genes and decreases the frequency of FBM B cells. Finally, maternal WSD leads to poor engraftment of fetal HSPCs in nonlethally irradiated immunodeficient NOD/SCID/IL2rγ-/- mice. Collectively, these data demonstrate for the first time that maternal WSD impairs fetal HSPC differentiation and function in a translationally relevant nonhuman primate model.

Adipocyte mesenchymal transition contributes to mammary tumor progression.

Obesity is associated with increased cancer incidence and progression. However, the relationship between adiposity and cancer remains poorly understood at the mechanistic level. Here, we report that adipocytes from tumor-invasive mammary fat undergo de-differentiation to fibroblast-like precursor cells during tumor progression and integrate into the tumor microenvironment. Single-cell sequencing reveals that these de-differentiated adipocytes lose their original identities and transform into multiple cell types, including myofibroblast- and macrophage-like cells, with their characteristic features involved in immune response, inflammation, and extracellular matrix remodeling. The de-differentiated cells are metabolically distinct from tumor-associated fibroblasts but exhibit comparable effects on tumor cell proliferation. Inducing de-differentiation by Xbp1s overexpression promotes tumor progression despite lower adiposity. In contrast, promoting lipid-storage capacity in adipocytes through MitoNEET overexpression curbs tumor growth despite greater adiposity. Collectively, the metabolic interplay between tumor cells and adipocytes induces adipocyte mesenchymal transition and contributes to reconfigure the stroma into a more tumor-friendly microenvironment.

Reduced Proteolytic Cleavage of von Willebrand Factor Leads to Aortic Valve Stenosis and Load-Dependent Ventricular Remodeling.

We hypothesized that excess endothelial-associated von Willebrand factor (vWF) and secondary platelet adhesion contribute to aortic valve stenosis (AS). We studied hyperlipidemic mice lacking ADAMTS13 (LDLR -/- AD13 -/- ), which cleaves endothelial-associated vWF multimers. On echocardiography and molecular imaging, LDLR -/- AD13 -/- compared with control strains had increased aortic endothelial vWF and platelet adhesion and developed hemodynamically significant AS, arterial stiffening, high valvulo-aortic impedance, and secondary load-dependent reduction in LV systolic function. Histology revealed leaflet thickening and calcification with valve interstitial cell myofibroblastic and osteogenic transformation, and evidence for TGFß1 pathway activation. We conclude that valve leaflet endothelial vWF-platelet interactions promote AS through juxtacrine platelet signaling.

Deficient Caveolin-1 Synthesis in Adipocytes Stimulates Systemic Insulin-Independent Glucose Uptake via Extracellular Vesicles.

Caveolin-1 (cav1) is an important structural and signaling component of plasma membrane invaginations called caveolae and is abundant in adipocytes. As previously reported, adipocyte-specific ablation of the cav1 gene (ad-cav1 knockout [KO] mouse) does not result in elimination of the protein, as cav1 protein traffics to adipocytes from neighboring endothelial cells. However, this mouse is a functional KO because adipocyte caveolar structures are depleted. Compared with controls, ad-cav1KO mice on a high-fat diet (HFD) display improved whole-body glucose clearance despite complete loss of glucose-stimulated insulin secretion, blunted insulin-stimulated AKT activation in metabolic tissues, and partial lipodystrophy. The cause is increased insulin-independent glucose uptake by white adipose tissue (AT) and reduced hepatic gluconeogenesis. Furthermore, HFD-fed ad-cav1KO mice display significant AT inflammation, fibrosis, mitochondrial dysfunction, and dysregulated lipid metabolism. The glucose clearance phenotype of the ad-cav1KO mice is at least partially mediated by AT small extracellular vesicles (AT-sEVs). Injection of control mice with AT-sEVs from ad-cav1KO mice phenocopies ad-cav1KO characteristics. Interestingly, AT-sEVs from ad-cav1KO mice propagate the phenotype of the AT to the liver. These data indicate that ad-cav1 is essential for healthy adaptation of the AT to overnutrition and prevents aberrant propagation of negative phenotypes to other organs by EVs.

Single-Cell Sorting of Non-human Primate Adipocytes with Large-particle Flow Cytometry.

Fluorescence-activated cell sorting enables separation and analysis of heterogeneous cell populations based on size, granularity, and fluorescence intensity. Cell sorting has been widely used for isolation of cells that are ∼10 to 25 µm in diameter. By contrast, cell sorting of unilocular adipocytes isolated from white adipose tissue imposes a significant technological challenge. The combination of their large size (up to 200 µm) and the fragile nature of lipid-laden adipocytes requires the use of specialized flow cytometers equipped with a large nozzle and capable of using low pressure to reduce shear forces during the cell sorting process. Furthermore, isolation of single adipocytes is rarely performed due to the lack of specialized cell sorters that can dispense single adipocytes into individual wells. Conducting cell sorting on single adipocytes would enable analyses of the cell-autonomous heterogeneity in nutrient uptake and metabolism observed in white adipose tissue. In this protocol, we describe single-cell sorting of rhesus macaque adipocytes labeled with fluorescent fatty acid and live-cell indicators using large-particle flow cytometry. This methodology represents a valuable resource for basic and translational studies aimed at understanding the development and function of adipocytes. © 2021 Wiley Periodicals LLC. Basic Protocol: Single-cell flow sorting of adipocytes.

Proteolysis of Von Willebrand Factor Influences Inflammatory Endothelial Activation and Vascular Compliance in Atherosclerosis.

This study used in vivo molecular imaging to characterize endotheliall activation attributable to von Willebrand factor (vWF)-mediated platelet adhesion in atherosclerosis. In atherosclerotic mice lacking the low-density lipoprotein receptor on Western diet, the additional genetic deletion of the ADAMTS13, which cleaves endothelial-associated vWF, produced greater aortic molecular imaging signal for not only vWF and platelets, but also for endothelial adhesion molecules VCAM1 and P-selectin, larger plaque size, and lower aortic distensibility. Sustained ADAMTS13 therapy reduced signal for all 4 molecular targets and plaque size. We conclude that excess endothelial-associated vWF contributes to not only platelet adhesion, but also to up-regulation of endothelial cell adhesion molecules.

Daily Ethanol Drinking Followed by an Abstinence Period Impairs Bone Marrow Niche and Mitochondrial Function of Hematopoietic Stem/Progenitor Cells in Rhesus Macaques.

BACKGROUND: Unhealthy consumption of alcohol is a major public health crisis with strong associations between immunological dysfunctions, high vulnerability to infectious disease, anemia, and an increase in the risk of hematological malignancies. However, there is a lack of studies addressing alcohol-induced changes in bone marrow (BM) and hematopoiesis as fundamental aspects of immune system function. METHODS: To address the effect of chronic alcohol consumption on hematopoietic stem and progenitor cells (HSPCs) and the BM niche, we used an established rhesus macaque model of voluntary alcohol drinking. A cohort of young adult male rhesus macaques underwent a standard ethanol self-administration protocol that allowed a choice of drinking alcohol or water 22 hours/day with periods of forced abstinence that elevated subsequent intakes when alcohol availability resumed. Following the last month of forced abstinence, the monkeys were euthanized. HSPCs and bone samples were collected and analyzed in functional assays and by confocal microscopy. RESULTS: HSPCs from alcohol animals exhibited reduced ability to form granulocyte-monocyte and erythroid colonies in vitro. HSPCs also displayed a decrease in mitochondrial oxygen consumption linked to ATP production and basal respiratory capacity. Chronic alcohol use led to vascular remodeling of the BM niche, a reduction in the number of primitive HSPCs, and a shift in localization of HSPCs from an adipose to a perivascular niche. CONCLUSIONS: Our study demonstrates, for the first time, that chronic voluntary alcohol drinking in rhesus macaque monkeys leads to the long-term impairment of HSPC function, a reduction in mitochondrial respiratory activity, and alterations in the BM microenvironment. Further studies are needed to determine whether these changes in hematopoiesis are persistent or adaptive during the abstinent period and whether an initial imprinting to alcohol primes BM to become more vulnerable to future exposure to alcohol.

Short-Term Caloric Restriction Attenuates Obesity-Induced Pro-Inflammatory Response in Male Rhesus Macaques.

White adipose tissue (WAT) hypertrophy is an essential hallmark of obesity and is associated with the activation of resident immune cells. While the benefits of caloric restriction (CR) on health span are generally accepted, its effects on WAT physiology are not well understood. We previously demonstrated that short-term CR reverses obesity in male rhesus macaques exposed to a high-fat Western-style diet (WSD). Here, we analyzed subcutaneous WAT biopsies collected from this cohort of animals before and after WSD and following CR. This analysis showed that WSD induced adipocyte hypertrophy and inhibited ß-adrenergic-simulated lipolysis. CR reversed adipocyte hypertrophy, but WAT remained insensitive to ß-adrenergic agonist stimulation. Whole-genome transcriptional analysis revealed that ß3-adrenergic receptor and de novo lipogenesis genes were downregulated by WSD and remained downregulated after CR. In contrast, WSD-induced pro-inflammatory gene expression was effectively reversed by CR. Furthermore, peripheral blood monocytes isolated during the CR period exhibited a significant reduction in the production of pro-inflammatory cytokines compared to those obtained after WSD. Collectively, this study demonstrates that short-term CR eliminates an obesity-induced pro-inflammatory response in WAT and peripheral monocytes.

Spatial and biochemical interactions between bone marrow adipose tissue and hematopoietic stem and progenitor cells in rhesus macaques.

Recent developments in in situ microscopy have enabled unparalleled resolution of the architecture of the bone marrow (BM) niche for murine hematopoietic stem and progenitor cells (HSPCs). However, the extent to which these observations can be extrapolated to human BM remains unknown. In humans, adipose tissue occupies a significant portion of the BM medullary cavity, making quantitative immunofluorescent analysis difficult due to lipid-mediated light scattering. In this study, we employed optical clearing, confocal microscopy and nearest neighbor analysis to determine the spatial distribution of CD34+ HSPCs in the BM in a translationally relevant rhesus macaque model. Immunofluorescent analysis revealed that femoral BM adipocytes are associated with the branches of vascular sinusoids, with half of HSPCs localizing in close proximity of the nearest BM adipocyte. Immunofluorescent microscopy and flow cytometric analysis demonstrate that BM adipose tissue exists as a multicellular niche consisted of adipocytes, endothelial cells, granulocytes, and macrophages. Analysis of BM adipose tissue conditioned media using liquid chromatography-tandem mass spectrometry revealed the presence of multiple bioactive proteins involved in regulation of hematopoiesis, inflammation, and bone development, with many predicted to reside inside microvesicles. Pretreatment of purified HSPCs with BM adipose tissue conditioned media, comprising soluble and exosomal/microvesicle-derived factors, led to enhanced proliferation and an increase in granulocyte-monocyte differentiation potential ex vivo. Our work extends extensive studies in murine models, indicating that BM adipose tissue is a central paracrine regulator of hematopoiesis in nonhuman primates and possibly in humans.

Synergistic Effects of Hyperandrogenemia and Obesogenic Western-style Diet on Transcription and DNA Methylation in Visceral Adipose Tissue of Nonhuman Primates.

Polycystic ovary syndrome (PCOS) is a major reproductive disorder that is responsible for 80% of anovulatory infertility and that is associated with hyperandrogenemia, increased risk of obesity, and white adipose tissue (WAT) dysfunction. We have previously demonstrated that the combination of chronic testosterone (T) treatment and an obesogenic Western-style diet (WSD) exerts synergistic functional effects on WAT, leading to increased lipid accumulation in visceral adipocytes by an unknown mechanism. In this study, we examined the whole-genome transcriptional response in visceral WAT to T and WSD, alone and in combination. We observed a synergistic effect of T and WSD on gene expression, resulting in upregulation of lipid storage genes concomitant with adipocyte hypertrophy. Because DNA methylation is known to be associated with body fat distribution and the etiology of PCOS, we conducted whole-genome DNA methylation analysis of visceral WAT. While only a fraction of differentially expressed genes also exhibited differential DNA methylation, in silico analysis showed that differentially methylated regions were enriched in transcription factor binding motifs, suggesting a potential gene regulatory role for these regions. In summary, this study demonstrates that hyperandrogenemia alone does not induce global transcriptional and epigenetic response in young female macaques unless combined with an obesogenic diet.

Molecular Imaging of VWF (von Willebrand Factor) and Platelet Adhesion in Postischemic Impaired Microvascular Reflow.

BACKGROUND: Complete mechanistic understanding of impaired microvascular reflow after myocardial infarction will likely lead to new therapies for reducing infarct size. Myocardial contrast echocardiography perfusion imaging and molecular imaging were used to evaluate the contribution of microvascular endothelial-associated VWF (von Willebrand factor) and platelet adhesion to microvascular no-reflow. METHODS AND RESULTS: Myocardial infarction was produced by transient LAD ligation in WT (wild type) mice, WT mice treated with the VWF proteolytic enzyme ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 motif, member 13), and ADAMTS13-deficient (ADAMTS13-/-) mice. Myocardial contrast echocardiography perfusion imaging and molecular imaging of VWF and platelet GP (glycoprotein) Ibα were performed 30 minutes after ischemia-reperfusion. Infarct size was measured at 3 days. Mortality during ischemia-reperfusion incrementally increased in WT+ADAMTS13, WT, and ADAMTS13-/- mice (14%, 43%, and 63%, respectively; P<0.05). For WT mice, molecular imaging signal for platelets and VWF in the postischemic risk area was 4- to 5-fold higher ( P<0.05) compared with both the remote nonischemic regions or to sham-treated mice. Signal enhancement in the risk area was completely abolished by ADAMTS13 treatment for both platelets (12.8±3.3 versus -1.0±4.4 IU; P<0.05) and VWF (13.9±4.0 versus -1.0±3.0 IU; P<0.05). ADAMTS13-/- compared with WT mice had 2- to 3-fold higher risk area signal for platelets (33.1±8.5 IU) and VWF (30.9±1.9 IU). Microvascular reflow in the risk area incrementally decreased for WT+ADAMTS13, WT, and ADAMTS13-/- mice ( P<0.05), whereas infarct size incrementally increased ( P<0.05). CONCLUSIONS: Mechanistic information on microvascular no-reflow is possible by combining perfusion and molecular imaging. In reperfused myocardial infarction, excess endothelial-associated VWF and secondary platelet adhesion in the risk area microcirculation contribute to impaired reflow and are modifiable.