Ablation of hematopoietic stem cell derived adipocytes reduces tumor burden in syngeneic mouse models of high-grade serous carcinoma.

In this study we examined the influence of hematopoietic stem cell-derived adipocytes (HSCDAs) on the proliferation and metastasis of high-grade serous carcinoma (HGSC) - the most common type of ovarian cancer. HSCDAs are a subtype of adipocytes that differentiate from myeloid precursors that traffic from bone marrow to adipose tissue and accumulate therein. These are distinct from conventional mesenchymal adipocytes (CMAs), which are derived from mesenchymal precursors. We hypothesized that HSCDAs promote HGSC progression and establish a pro-tumoral niche within peritoneal adipose tissues such as the omentum. Primary human white adipose tissue samples were obtained via biopsy and then sorted into myeloid and mesenchymal populations through flow cytometry. These adipose precursors were then differentiated in vitro into mature HSCDAs and CMAs, respectively. Transcriptomic analysis showed that HSCDAs have a distinct transcriptional profile from CMAs, including downregulation of cell cycle and upregulation of multiple metabolic and adipogenic pathways. Using ELISA, we found that HSCDAs secreted greater amounts of inflammatory cytokines IL-6 and IL-8 than CMAs. Next, we incubated HGSC cells in conditioned media from HSCDAs and CMAs and performed proliferation and protein expression profiling. HGSC cells in HSCDA media, compared to those in CMA media, had elevated expression of protein markers related to epithelial to mesenchymal plasticity, including fibronectin, as well as increased serine phosphorylation of pro-survival AKT1/2. Conversely, HGSC cells in HSCDA media exhibited comparably downregulated expression of tumor suppressors including the Wnt regulator GSK3ß. Depending on the cell line and adipose donor, HGSC cells also showed altered growth rates in conditioned media. We next investigated the role of HSCDAs in HGSC progression and metastasis in vivo . We generated immunocompetent mice that were either HSCDA Proficient (can make both adipocyte subtypes) or Deficient (can only make CMAs). Using these models, we conducted two independent tumor studies using the ID8 ( Tp53-/- , Brca2-/- ) and SO ( Tp53-/- , Brca1/2 wild-type, Hras and Myc amplified) syngeneic models. Overall tumor burden was lower in HSCDA Deficient mice in both models. In the ID8 model, omental tumors from HSCDA Deficient mice showed reduced proliferation (Ki67) and apoptosis (cleaved caspase 3) relative to those from Proficient mice. Transcriptionally, omental ID8 tumors from HSCDA Deficient downregulated oxidative phosphorylation, adipogenesis, and fatty acid metabolism relative to tumors from HSCDA Proficient mice. These pathways were enriched in HSCDA cells in vitro , suggesting that ablation of HSCDAs had a significant influence on the tumor metabolic environment. Reduced inflammatory pathways in ID8 tumors from HSCDA Deficient mice were also observed leading us to interrogate immune cell infiltration into omental tumors. Compared to HSCDA Proficient mice, tumors from HSCDA Deficient mice showed reduced densities of dendritic cells (DC) and natural killer (NK) cells, as well as fewer DCs, NKs, and B-cells in proximity to tumor cells, as determined by spatial analysis. Overall, our data suggest that HSCDAs promote HGSC survival and plasticity while downregulating expression of tumor suppressors and altering the peritoneal immune and metabolic environment to promote HGSC progression.

Adipogenic differentiation of hematopoietic lineage cells isolated from adipose tissue of humans.

We previously discovered some adipocytes in the major white fat depots of mice and humans arise from bone marrow-derived cells of hematopoietic lineage rather than conventional mesenchymal precursors, termed bone marrow-derived adipocytes (BMDA). Here we aimed to determine if hematopoietic lineage cells isolated from adipose tissue and circulation of humans could undergo adipogenic differentiation in vitro, thereby establishing an in vitro model for studies of BMDA. We hypothesized that hematopoietic lineage cells isolated from adipose tissue, but not circulation, of humans would demonstrate adipogenic potential. Participants included younger (20-50 years) and older (>50-75 years) men and women, BMI 20-37 kg/m2. Subcutaneous abdominal adipose tissue biopsies were obtained and stromal cell populations identified by flow cytometry. Sorted cells underwent in vitro cultivation via traditional mesenchymal culture methodology (mesenchymal lineage) or a novel 3D-fibrin clot followed by traditional adherent culture (hematopoietic lineage) for assessment of proliferation and differentiation capacity. We found hematopoietic lineage cells isolated from the adipose tissue stroma, but not the circulation, were capable of proliferation and multilineage (adipogenic and osteogenic) differentiation in vitro. We provide a new investigative tool that can be used to perform translational studies of BMDAs and provide initial evidence that hematopoietic lineage cells isolated from the adipose tissue of humans can undergo hematopoietic-to-mesenchymal transition with multilineage differentiation potential in an in vitro environment.

Effects of follicle-stimulating hormone on energy balance and tissue metabolic health after loss of ovarian function.

Loss of ovarian function imparts increased susceptibility to obesity and metabolic disease. These effects are largely attributed to decreased estradiol (E2), but the role of increased follicle-stimulating hormone (FSH) in modulating energy balance has not been fully investigated. Previous work that blocked FSH binding to its receptor in mice suggested this hormone may play a part in modulating body weight and energy expenditure after ovariectomy (OVX). We used an alternate approach to isolate the individual and combined contributions of FSH and E2 in mediating energy imbalance and changes in tissue-level metabolic health. Female Wistar rats were ovariectomized and given the gonadotropin releasing hormone (GnRH) antagonist degarelix to suppress FSH production. E2 and FSH were then added back individually and in combination for a period of 3 wk. Energy balance, body mass composition, and transcriptomic profiles of individual tissues were obtained. In contrast to previous studies, suppression and replacement of FSH in our paradigm had no effect on body weight, body composition, food intake, or energy expenditure. We did, however, observe organ-specific effects of FSH that produced unique transcriptomic signatures of FSH in retroperitoneal white adipose tissue. These included reductions in biological processes related to lipogenesis and carbohydrate transport. In addition, rats administered FSH had reduced liver triglyceride concentration (P < 0.001), which correlated with FSH-induced changes at the transcriptomic level. Although not appearing to modulate energy balance after loss of ovarian function in rats, FSH may still impart tissue-specific effects in the liver and white adipose tissue that might affect the metabolic health of those organs.NEW & NOTEWORTHY We find no effect of follicle-stimulating hormone (FSH) on energy balance using a novel model in which rats are ovariectomized, subjected to gonadotropin-releasing hormone antagonism, and systematically given back FSH by osmotic pump. However, tissue-specific effects of FSH on adipose tissue and liver were observed in this study. These include unique transcriptomic signatures induced by the hormone and a stark reduction in hepatic triglyceride accumulation.

Hematopoietic Stem Cell-Derived Adipocytes Modulate Adipose Tissue Cellularity, Leptin Production and Insulin Responsiveness in Female Mice.

A subpopulation of adipocytes in the major adipose depots of mice is produced from hematopoietic stem cells rather than mesenchymal progenitors that are the source of conventional white and brown/beige adipocytes. To analyze the impact of hematopoietic stem cell-derived adipocytes (HSCDAs) in the adipose niche we transplanted HSCs in which expression of a diphtheria toxin gene was under the control of the adipocyte-specific adiponectin gene promoter into irradiated wild type recipients. Thus, only adipocytes produced from HSC would be ablated while conventional white and brown adipocytes produced from mesenchymal progenitor cells would be spared. Wild type mice transplanted with HSCs from mice containing a reporter gene, but not the diphtheria toxin gene, regulated by the adiponectin gene promoter served as controls. In mice in which HSCDA production was suppressed, adipocyte size declined while adipose depot weights were unchanged and the number of conventional adipocyte progenitors significantly increased. We also measured a paradoxical increase in circulating leptin levels while physical activity was significantly decreased in the HSCDA depleted mice. Finally, insulin sensitivity was significantly reduced in HSCDA depleted mice. In contrast, loss of HSCDA production had no effect on body weight, components of energy balance, or levels of several circulating adipokines and tissue-resident inflammatory cells. These data indicate that ablation of this low-abundance subpopulation of adipocytes is associated with changes in circulating leptin levels and leptin-regulated endpoints associated with adipose tissue function. How they do so remains a mystery, but our results highlight the need for additional studies to explore the role of HSCDAs in other physiologic contexts such as obesity, metabolic dysfunction or loss of sex hormone production.

Hematopoietic stem cells produce intermediate lineage adipocyte progenitors that simultaneously express both myeloid and mesenchymal lineage markers in adipose tissue.

Some adipocytes are produced from bone marrow hematopoietic stem cells. In vitro studies previously indicated that these bone marrow-derived adipocytes (BMDAs) were generated from adipose tissue macrophage (ATM) that lose their hematopoietic markers and acquire mesenchymal markers prior to terminal adipogenic differentiation. Here we interrogated whether this hematopoietic-to-mesenchymal transition drives BMDA production In vitro. We generated transgenic mice in which the lysozyme gene promoter (LysM) indelibly labeled ATM with green fluorescent protein (GFP). We discovered that adipose stroma contained a population of LysM-positive myeloid cells that simultaneously expressed hematopoietic/myeloid markers (CD45 and CD11b), and mesenchymal markers (CD29, PDGFRa and Sca-1) typically found on conventional adipocyte progenitors. These cells were capable of adipogenic differentiation In vitro and In vitro, while other stromal populations deficient in PDGFRa and Sca-1 were non-adipogenic. BMDAs and conventional adipocytes expressed common fat cell markers but exhibited little or no expression of hematopoietic and mesenchymal progenitor cell markers. The data indicate that BMDAs are produced from ATM simultaneously expressing hematopoietic and mesenchymal markers rather than via a stepwise hematopoietic-to-mesenchymal transition. Because BMDA production is stimulated by high fat feeding, their production from hematopoietic progenitors may maintain adipocyte production when conventional adipocyte precursors are diminished.

Resident mesenchymal vascular progenitors modulate adaptive angiogenesis and pulmonary remodeling via regulation of canonical Wnt signaling.

Adaptive angiogenesis is necessary for tissue repair, however, it may also be associated with the exacerbation of injury and development of chronic disease. In these studies, we demonstrate that lung mesenchymal vascular progenitor cells (MVPC) modulate adaptive angiogenesis via lineage trace, depletion of MVPC, and modulation of ß-catenin expression. Single cell sequencing confirmed MVPC as multipotential vascular progenitors, thus, genetic depletion resulted in alveolar simplification with reduced adaptive angiogenesis. Following vascular endothelial injury, Wnt activation in MVPC was sufficient to elicit an emphysema-like phenotype characterized by increased MLI, fibrosis, and MVPC driven adaptive angiogenesis. Lastly, activation of Wnt/ß-catenin signaling skewed the profile of human and murine MVPC toward an adaptive phenotype. These data suggest that lung MVPC drive angiogenesis in response to injury and regulate the microvascular niche as well as subsequent distal lung tissue architecture via Wnt signaling.

Inactivation of Tsc2 in Abcg2 lineage-derived cells drives the appearance of polycystic lesions and fibrosis in the adult kidney.

Tuberous sclerosis complex 2 (TSC2), or tuberin, is a pivotal regulator of the mechanistic target of rapamycin signaling pathway that controls cell survival, proliferation, growth, and migration. Loss of Tsc2 function manifests in organ-specific consequences, the mechanisms of which remain incompletely understood. Recent single cell analysis of the kidney has identified ATP-binding cassette G2 (Abcg2) expression in renal proximal tubules of adult mice as well as a in a novel cell population. The impact in adult kidney of Tsc2 knockdown in the Abcg2-expressing lineage has not been evaluated. We engineered an inducible system in which expression of truncated Tsc2, lacking exons 36-37 with an intact 3' region and polycystin 1, is driven by Abcg2. Here, we demonstrate that selective expression of Tsc2fl36-37 in the Abcg2pos lineage drives recombination in proximal tubule epithelial and rare perivascular mesenchymal cells, which results in progressive proximal tubule injury, impaired kidney function, formation of cystic lesions, and fibrosis in adult mice. These data illustrate the critical importance of Tsc2 function in the Abcg2-expressing proximal tubule epithelium and mesenchyme during the development of cystic lesions and remodeling of kidney parenchyma.

Modulation of Energy Expenditure by Estrogens and Exercise in Women.

Reducing estrogen in women results in decreases in energy expenditure, but the mechanism(s) remain largely unknown. We postulate that the loss of estrogens in women is associated with increased accumulation of bone marrow-derived adipocytes in white adipose tissue, decreased activity of brown adipose tissue, and reduced levels of physical activity. Regular exercise may counteract the effects of estrogen deficiency.

Ovarian Hormones Regulate the Production of Adipocytes From Bone Marrow-Derived Cells.

Sex differences in body fat distribution and menopause-associated shifts in regional adiposity suggest that sex hormones play an important role in regulating the differentiation and distribution of adipocytes, but the underlying mechanisms have not been fully explained. The aim of this study was to determine whether ovarian hormone status influences the production and distribution of adipocytes in adipose tissue arising from bone marrow-derived cells. Nine- to ten-week-old ovariectomized (OVX), surgery naïve (WT), and estrogen receptor alpha knockout (αERKO) mice underwent bone marrow transplantation from luciferase or green fluorescent protein expressing donors. A subset of OVX animals had estradiol (E2) added back. Eight-weeks posttransplant, whole body and gonadal fat BM-derived adipocyte production was highest in OVX and αERKO mice, which was attenuated in OVX mice by E2 add-back. All groups demonstrated the highest bone marrow derived adipocyte (BMDA) production in the gonadal adipose depot, a visceral fat depot in mice. Taken together, the loss of ovarian hormones increases the production of BMDAs. If translatable across species, production of BMDA may be a mechanism by which visceral adiposity increases in estrogen-deficient postmenopausal women.

Disruption of lineage specification in adult pulmonary mesenchymal progenitor cells promotes microvascular dysfunction.

Pulmonary vascular disease is characterized by remodeling and loss of microvessels and is typically attributed to pathological responses in vascular endothelium or abnormal smooth muscle cell phenotypes. We have challenged this understanding by defining an adult pulmonary mesenchymal progenitor cell (MPC) that regulates both microvascular function and angiogenesis. The current understanding of adult MPCs and their roles in homeostasis versus disease has been limited by a lack of genetic markers with which to lineage label multipotent mesenchyme and trace the differentiation of these MPCs into vascular lineages. Here, we have shown that lineage-labeled lung MPCs expressing the ATP-binding cassette protein ABCG2 (ABCG2+) are pericyte progenitors that participate in microvascular homeostasis as well as adaptive angiogenesis. Activation of Wnt/ß-catenin signaling, either autonomously or downstream of decreased BMP receptor signaling, enhanced ABCG2+ MPC proliferation but suppressed MPC differentiation into a functional pericyte lineage. Thus, enhanced Wnt/ß-catenin signaling in ABCG2+ MPCs drives a phenotype of persistent microvascular dysfunction, abnormal angiogenesis, and subsequent exacerbation of bleomycin-induced fibrosis. ABCG2+ MPCs may, therefore, account in part for the aberrant microvessel function and remodeling that are associated with chronic lung diseases.

Hematopoietic-to-mesenchymal transition of adipose tissue macrophages is regulated by integrin ß1 and fabricated fibrin matrices.

Some bona fide adult adipocytes arise de novo from a bone marrow-derived myeloid lineage. These studies further demonstrate that adipose tissue stroma contains a resident population of myeloid cells capable of adipocyte and multilineage mesenchymal differentiation. These resident myeloid cells lack hematopoietic markers and express mesenchymal and progenitor cell markers. Because bone marrow mesenchymal progenitor cells have not been shown to enter the circulation, we hypothesized that myeloid cells acquire mesenchymal differentiation capacity in adipose tissue. We fabricated a 3-dimensional fibrin matrix culture system to define the adipose differentiation potential of adipose tissue-resident myeloid subpopulations, including macrophages, granulocytes and dendritic cells. Our data show that multilineage mesenchymal potential was limited to adipose tissue macrophages, characterized by the acquisition of adipocyte, osteoblast, chondrocyte and skeletal muscle myocyte phenotypes. Fibrin hydrogel matrices stimulated macrophage loss of hematopoietic cell lineage determinants and the expression of mesenchymal and progenitor cell markers, including integrin ß1. Ablation of integrin ß1 in macrophages inhibited adipocyte specification. Therefore, some bona fide adipocytes are specifically derived from adipose tissue-resident macrophages via an integrin ß1-dependent hematopoietic-to-mesenchymal transition, whereby they become capable of multipotent mesenchymal differentiation. The requirement for integrin ß1 highlights this molecule as a potential target for controlling the production of marrow-derived adipocytes and their contribution to adipose tissue development and function.

Leukemic Stem Cells Evade Chemotherapy by Metabolic Adaptation to an Adipose Tissue Niche.

Adipose tissue (AT) has previously been identified as an extra-medullary reservoir for normal hematopoietic stem cells (HSCs) and may promote tumor development. Here, we show that a subpopulation of leukemic stem cells (LSCs) can utilize gonadal adipose tissue (GAT) as a niche to support their metabolism and evade chemotherapy. In a mouse model of blast crisis chronic myeloid leukemia (CML), adipose-resident LSCs exhibit a pro-inflammatory phenotype and induce lipolysis in GAT. GAT lipolysis fuels fatty acid oxidation in LSCs, especially within a subpopulation expressing the fatty acid transporter CD36. CD36(+) LSCs have unique metabolic properties, are strikingly enriched in AT, and are protected from chemotherapy by the GAT microenvironment. CD36 also marks a fraction of human blast crisis CML and acute myeloid leukemia (AML) cells with similar biological properties. These findings suggest striking interplay between leukemic cells and AT to create a unique microenvironment that supports the metabolic demands and survival of a distinct LSC subpopulation.

De novo generation of adipocytes from circulating progenitor cells in mouse and human adipose tissue.

White adipocytes in adults are typically derived from tissue resident mesenchymal progenitors. The recent identification of de novo production of adipocytes from bone marrow progenitor-derived cells in mice challenges this paradigm and indicates an alternative lineage specification that adipocytes exist. We hypothesized that alternative lineage specification of white adipocytes is also present in human adipose tissue. Bone marrow from transgenic mice in which luciferase expression is governed by the adipocyte-restricted adiponectin gene promoter was adoptively transferred to wild-type recipient mice. Light emission was quantitated in recipients by in vivo imaging and direct enzyme assay. Adipocytes were also obtained from human recipients of hematopoietic stem cell transplantation. DNA was isolated, and microsatellite polymorphisms were exploited to quantify donor/recipient chimerism. Luciferase emission was detected from major fat depots of transplanted mice. No light emission was observed from intestines, liver, or lungs. Up to 35% of adipocytes in humans were generated from donor marrow cells in the absence of cell fusion. Nontransplanted mice and stromal-vascular fraction samples were used as negative and positive controls for the mouse and human experiments, respectively. This study provides evidence for a nontissue resident origin of an adipocyte subpopulation in both mice and humans.

Shared gene expression patterns in mesenchymal progenitors derived from lung and epidermis in pulmonary arterial hypertension: identifying key pathways in pulmonary vascular disease.

Rapid access to lung-derived cells from stable subjects is a major challenge in the pulmonary hypertension field, given the relative contraindication of lung biopsy. In these studies, we sought to demonstrate the importance of evaluating a cell type that actively participates in disease processes, as well as the potential to translate these findings to vascular beds in other nonlung tissues, in this instance perivascular skin mesenchymal cells (MCs). We utilized posttransplant or autopsy lung explant-derived cells (ABCG2-expressing mesenchymal progenitor cells [MPCs], fibroblasts) and skin-derived MCs to test the hypothesis that perivascular ABCG2 MPCs derived from pulmonary arterial hypertension (PAH) patient lung and skin would express a gene profile reflective of ongoing vascular dysfunction. By analyzing the genetic signatures and pathways associated with abnormal ABCG2 lung MPC phenotypes during PAH and evaluating them in lung- and skin-derived MCs, we have identified potential predictor genes for detection of PAH as well as a targetable mechanism to restore MPCs and microvascular function. These studies are the first to explore the utility of expanding the study of ABCG2 MPC regulation of the pulmonary microvasculature to the epidermis, in order to identify potential markers for adult lung vascular disease, such as PAH.

ABCG2pos lung mesenchymal stem cells are a novel pericyte subpopulation that contributes to fibrotic remodeling.

Genesis of myofibroblasts is obligatory for the development of pathology in many adult lung diseases. Adult lung tissue contains a population of perivascular ABCG2(pos) mesenchymal stem cells (MSC) that are precursors of myofibroblasts and distinct from NG2 pericytes. We hypothesized that these MSC participate in deleterious remodeling associated with pulmonary fibrosis (PF) and associated hypertension (PH). To test this hypothesis, resident lung MSC were quantified in lung samples from control subjects and PF patients. ABCG2(pos) cell numbers were decreased in human PF and interstitial lung disease compared with control samples. Genetic labeling of lung MSC in mice enabled determination of terminal lineage and localization of ABCG2 cells following intratracheal administration of bleomycin to elicit fibrotic lung injury. Fourteen days following bleomycin injury enhanced green fluorescent protein (eGFP)-labeled lung MSC-derived cells were increased in number and localized to interstitial areas of fibrotic and microvessel remodeling. Finally, gene expression analysis was evaluated to define the response of MSC to bleomycin injury in vivo using ABCG2(pos) MSC isolated during the inflammatory phase postinjury and in vitro bleomycin or transforming growth factor-ß1 (TGF-ß1)-treated cells. MSC responded to bleomycin treatment in vivo with a profibrotic gene program that was not recapitulated in vitro with bleomycin treatment. However, TGF-ß1 treatment induced the appearance of a profibrotic myofibroblast phenotype in vitro. Additionally, when exposed to the profibrotic stimulus, TGF-ß1, ABCG2, and NG2 pericytes demonstrated distinct responses. Our data highlight ABCG2(pos) lung MSC as a novel cell population that contributes to detrimental myofibroblast-mediated remodeling during PF.

Obesity-related pulmonary arterial hypertension in rats correlates with increased circulating inflammatory cytokines and lipids and with oxidant damage in the arterial wall but not with hypoxia.

Obesity is causally linked to a number of comorbidities, including cardiovascular disease, diabetes, renal dysfunction, and cancer. Obesity has also been linked to pulmonary disorders, including pulmonary arterial hypertension (PAH). It was long believed that obesity-related PAH was the result of hypoventilation and hypoxia due to the increased mechanical load of excess body fat. However, in recent years it has been proposed that the metabolic and inflammatory disturbances of obesity may also play a role in the development of PAH. To determine whether PAH develops in obese rats in the absence of hypoxia, we assessed pulmonary hemodynamics and pulmonary artery (PA) structure in the diet-resistant/diet-induced obesity (DR/DIO) and Zucker lean/fatty rat models. We found that high-fat feeding (DR/DIO) or overfeeding (Zucker) elicited PA remodeling, neomuscularization of distal arterioles, and elevated PA pressure, accompanied by right ventricular (RV) hypertrophy. PA thickening and distal neomuscularization were also observed in DIO rats on a low-fat diet. No evidence of hypoventilation or chronic hypoxia was detected in either model, nor was there a correlation between blood glucose or insulin levels and PAH. However, circulating inflammatory cytokine levels were increased with high-fat feeding or calorie overload, and hyperlipidemia and oxidant damage in the PA wall correlated with PAH in the DR/DIO model. We conclude that hyperlipidemia and peripheral inflammation correlate with the development of PAH in obese subjects. Obesity-related inflammation may predispose to PAH even in the absence of hypoxia.

Inhibition of phosphatidylinositol 3-kinase/Akt signaling attenuates hypoxia-induced pulmonary artery remodeling and suppresses CREB depletion in arterial smooth muscle cells.

Hypoxia-induced pulmonary hypertension is characterized by progressive remodeling of the pulmonary artery (PA) system and loss of the transcription factor, cAMP response element binding protein (CREB) in PA smooth muscle cells (SMCs). Previous in vitro studies suggested that platelet-derived growth factor, a mitogen produced in the hypoxic arterial wall, elicits loss of CREB in medial SMCs via the PI3K/Akt pathway. These events trigger switching of SMCs from a quiescent, contractile phenotype to a proliferative, migratory, dedifferentiated, and synthetic phenotype, which contributes to PA thickening. Here, we investigated whether inhibition of PI3K or Akt could attenuate arterial remodeling in the lung and prevent CREB loss in PA medial SMCs in rats subjected to chronic hypoxia. Inhibition of either enzyme-blunted hypoxia-induced PA remodeling and SMC CREB depletion and diminished SMC proliferation and collagen deposition. Inhibition of Akt, but not PI3K, suppressed muscularization of distal arterioles and blunted right ventricular hypertrophy. Interestingly, mean PA pressure was elevated equally by hypoxia in untreated and inhibitor-treated groups but was normalized acutely by the Rho kinase inhibitor, Fasudil. We conclude that PI3K and Akt inhibitors can attenuate hypoxia-induced PA remodeling and SMC CREB depletion but fail to block the development of pulmonary hypertension because of their inability to repress Rho kinase-mediated vasoconstriction.

Adipose tissue insulin sensitivity and macrophage recruitment: Does PI3K pick the pathway?

In the United States, obesity is a burgeoning health crisis, with over 30% of adults and nearly 20% of children classified as obese. Insulin resistance, a common metabolic complication associated with obesity, significantly increases the risk of developing metabolic diseases such as hypertension, coronary heart disease, stroke, type 2 diabetes, and certain cancers. With the seminal finding that obese adipose tissue harbors cytokine secreting immune cells, obesity-related research over the past decade has focused on understanding adipocyte-macrophage crosstalk and its impact on systemic insulin sensitivity. Indeed, adipose tissue has emerged as a central mediator of obesity- and diet-induced insulin resistance. In this mini-review, we focus on a potential role of adipose tissue phosphoinositide 3-kinase (PI3K) as a point of convergence of cellular signaling pathways that integrates nutrient sensing and inflammatory signaling to regulate tissue insulin sensitivity.

Attenuated Pik3r1 expression prevents insulin resistance and adipose tissue macrophage accumulation in diet-induced obese mice.

Obese white adipose tissue (AT) is characterized by large-scale infiltration of proinflammatory macrophages, in parallel with systemic insulin resistance; however, the cellular stimulus that initiates this signaling cascade and chemokine release is still unknown. The objective of this study was to determine the role of the phosphoinositide 3-kinase (PI3K) regulatory subunits on AT macrophage (ATM) infiltration in obesity. Here, we find that the Pik3r1 regulatory subunits (i.e., p85α/p55α/p50α) are highly induced in AT from high-fat diet-fed obese mice, concurrent with insulin resistance. Global heterozygous deletion of the Pik3r1 regulatory subunits (αHZ), but not knockout of Pik3r2 (p85ß), preserves whole-body, AT, and skeletal muscle insulin sensitivity, despite severe obesity. Moreover, ATM accumulation, proinflammatory gene expression, and ex vivo chemokine secretion in obese αHZ mice are markedly reduced despite endoplasmic reticulum (ER) stress, hypoxia, adipocyte hypertrophy, and Jun NH(2)-terminal kinase activation. Furthermore, bone marrow transplant studies reveal that these improvements in obese αHZ mice are independent of reduced Pik3r1 expression in the hematopoietic compartment. Taken together, these studies demonstrate that Pik3r1 expression plays a critical role in mediating AT insulin sensitivity and, more so, suggest that reduced PI3K activity is a key step in the initiation and propagation of the inflammatory response in obese AT.

Adipose lineage specification of bone marrow-derived myeloid cells.

We have reported the production of white adipocytes in adipose tissue from hematopoietic progenitors arising from bone marrow. However, technical challenges have hindered detection of this adipocyte population by certain other laboratories. These disparate results highlight the need for sensitive and definitive techniques to identify bone marrow progenitor (BMP)-derived adipocytes. In these studies we exploited new models and methods to enhance detection of this adipocyte population. Here we showed that confocal microscopy with spectrum acquisition could effectively identify green fluorescent protein (GFP) positive BMP-derived adipocytes by matching their fluorescence spectrum to that of native GFP. Likewise, imaging flow cytometry made it possible to visualize intact unilocular and multilocular GFP-positive BMP-derived adipocytes and distinguished them from non-fluorescent adipocytes and cell debris in the cytometer flow stream. We also devised a strategy to detect marker genes in flow-enriched adipocytes from which stromal cells were excluded. This technique also proved to be an efficient means for detecting genetically labeled adipocytes and should be applicable to models in which marker gene expression is low or absent. Finally, in vivo imaging of mice transplanted with BM from adipocyte-targeted luciferase donors showed a time-dependent increase in luciferase activity, with the bulk of luciferase activity confined to adipocytes rather than stromal cells. These results confirmed and extended our previous reports and provided proof-of-principle for sensitive techniques and models for detection and study of these unique cells.