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.

Rab27a Regulates Human Perivascular Adipose Progenitor Cell Differentiation.

PURPOSE: Perivascular adipose tissue (PVAT) surrounds blood vessels and regulates vascular tone through paracrine secretion of cytokines. During conditions promoting cardiometabolic dysfunction, such as obesity, cytokine secretion is altered towards a proinflammatory and proatherogenic profile. Despite the clinical implications for cardiovascular disease, studies addressing the biology of human PVAT remain limited. We are interested in characterizing the resident adipose progenitor cells (APCs) because of their potential role in PVAT expansion during obesity. We also focused on proteins regulating paracrine interactions, including the small GTPase Rab27a, which regulates protein trafficking and secretion. METHODS: PVAT from the ascending aorta was collected from patients with severe cardiovascular disease undergoing coronary artery bypass grafting (CABG). Freshly-isolated PVAT was digested and APC expanded in culture for characterizing progenitor markers, evaluating adipogenic potential and assessing the function(s) of Rab27a. RESULTS: Using flow cytometry, RT-PCR, and immunoblot, we characterized APC from human PVAT as negative for CD45 and CD31 and expressing CD73, CD105, and CD140A. These APCs differentiate into multilocular, UCP1-producing adipocytes in vitro. Rab27a was detected in interstitial cells of human PVAT in vivo and along F-actin tracks of PVAT-APC in vitro. Knockdown of Rab27a using siRNA in PVAT-APC prior to induction resulted in a marked reduction in lipid accumulation and reduced expression of adipogenic differentiation markers. CONCLUSIONS: PVAT-APC from CABG donors express common adipocyte progenitor markers and differentiate into UCP1-containing adipocytes. Rab27a has an endogenous role in promoting the maturation of adipocytes from human PVAT-derived APC.

A receptor-specific function for Notch2 in mediating vascular smooth muscle cell growth arrest through cyclin-dependent kinase inhibitor 1B.

RATIONALE: Deregulated vascular smooth muscle cell (VSMC) proliferation contributes to multiple vascular pathologies, and Notch signaling regulates VSMC phenotype. OBJECTIVE: Previous work focused on Notch1 and Notch3 in VSMC during vascular disease; however, the role of Notch2 is unknown. Because injured murine carotid arteries display increased Notch2 in VSMC as compared with uninjured arteries, we sought to understand the impact of Notch2 signaling in VSMCs. METHODS AND RESULTS: In human primary VSMCs, Jagged-1 (Jag-1) significantly reduced proliferation through specific activation of Notch2. Increased levels of p27(kip1) were observed downstream of Jag-1/Notch2 signaling and were required for cell cycle exit. Jag-1 activation of Notch resulted in increased phosphorylation on serine 10, decreased ubiquitination, and prolonged half-life of p27(kip1). Jag-1/Notch2 signaling robustly decreased S-phase kinase-associated protein, an F-box protein that degrades p27(kip1) during G1. Overexpression of S-phase kinase-associated protein before Notch activation by Jag-1 suppressed the induction of p27(kip1). Additionally, increased Notch2 and p27(kip1) expression was colocalized to the nonproliferative zone of injured arteries as indicated by co-staining with proliferating cell nuclear antigen, whereas Notch3 was expressed throughout normal and injured arteries, suggesting Notch2 may negatively regulate lesion formation. CONCLUSIONS: We propose a receptor-specific function for Notch2 in regulating Jag-1-induced p27(kip1) expression and growth arrest in VSMCs. During vascular remodeling, colocalization of Notch2 and p27(kip1) to the nonproliferating region supports a model where Notch2 activation may negatively regulate VSMC proliferation to lessen the severity of the lesion. Thus, Notch2 is a potential target for control of VSMC hyperplasia.

The miR-143/145 cluster is a novel transcriptional target of Jagged-1/Notch signaling in vascular smooth muscle cells.

Activation of Notch signaling by Jagged-1 (Jag-1) in vascular smooth muscle cells (VSMC) promotes a differentiated phenotype characterized by increased expression of contractile proteins. Recent studies show that microRNAs (miR)-143/145 regulates VSMC phenotype. The serum response factor (SRF)/myocardin complex binds to CArG sequences to activate miR-143/145 transcription, but no other regulators are known in VSMC. Using miR arrays, we found miR-143/145 induced following expression of a constitutively active Notch1 intracellular domain (N1ICD). We hypothesized that miR-143/145 is required for Jag-1/Notch-induced VSMC differentiation. Activation of Notch receptors by Jag-1 caused CBF1-dependent up-regulation of miR-143/145, increased differentiation, and decreased proliferation. Conversely, inhibiting basal Notch signaling decreased steady state levels of miR-143/145. Using SRF knockdown, we found that Jag-1/Notch induction of miR-143/145 is SRF independent, although full acquisition of contractile markers requires SRF. Using miR-143/145 promoter reporter constructs we show Jag-1/Notch increases promoter activity, and this is dependent on intact CBF1 consensus sites within the promoter. Chromatin immunoprecipitation (ChIP) assays revealed that N1ICD-containing complexes bind to CBF1 sites in the miR-143/145 promoter. We also identified N1ICD complex binding to CBF1 sites within the endogenous human miR-143/145 promoter. Using miR-143/145-interfering oligonucleotides, we demonstrate that Jag-1/Notch signaling requires induction of both miR-143 and miR-145 to promote the VSMC contractile phenotype. Thus, miR-143/145 is a novel transcriptional target of Jag-1/Notch signaling in VSMC. We propose miR-143/145 as activated independently by Jag-1/Notch and SRF in parallel pathways. Multiple pathways converging on miR-143/145 provides potential for fine-tuning or amplification of VSMC differentiation signals.

Human Perivascular Adipose Tissue as a Regulator of the Vascular Microenvironment and Diseases of the Coronary Artery and Aorta.

Perivascular adipose tissue (PVAT) is an adipose depot that surrounds blood vessels in the human body and exerts local paracrine signaling. Under physiologically healthy conditions, PVAT has an anti-contractile effect on vessels, but in obesity this effect is lost. During metabolic disease, adiponectin secretion is dysregulated, influencing nitric oxide bioavailability and macrophage infiltration and inflammation, all of which mediate PVAT signaling. However, based on the location in the body, and the type of adipocyte present, PVAT has different relationships with risk factors for disease. Imaging studies in patients with cardiovascular disease have demonstrated important associations between PVAT structure and pathology, yet insight into molecular pathways regulating human PVAT function are still lacking. This review focuses on our current understanding of human PVAT and its secretory role in the vascular microenvironment. A current area of priority is defining molecular differences in the secretome between PVAT depots, as this could inform the treatment of diseases that occur in anatomically restricted locations. In addition, understanding progressive changes in PVAT structure and function during metabolic disease is required for effective targeted therapies.

Differentiation Capacity of Human Aortic Perivascular Adipose Progenitor Cells.

Adipose tissue is a rich source of multi-potent mesenchymal stem cells (MSC) capable of differentiating into osteogenic, adipogenic, and chondrogenic lineages. Adipogenic differentiation of progenitor cells is a major mechanism driving adipose tissue expansion and dysfunction in response to obesity. Understanding changes to perivascular adipose tissue (PVAT) is thus clinically relevant in metabolic disease. However, previous studies have been predominately performed in the mouse and other animal models. This protocol uses human thoracic PVAT samples collected from patients undergoing coronary artery bypass graft surgery. Adipose tissue from the ascending aorta was collected and used for explantation of the stromal vascular fraction. We previously confirmed the presence of adipose progenitor cells in human PVAT with the capacity to differentiate into lipid-containing adipocytes. In this study, we further analyzed the differentiation potential of cells from the stromal vascular fraction, presumably containing multi-potent progenitor cells. We compared PVAT-derived cells to human bone marrow MSC for differentiation into adipogenic, osteogenic, and chondrogenic lineages. Following 14 days of differentiation, specific stains were utilized to detect lipid accumulation in adipocytes (Oil red O), calcific deposits in osteogenic cells (Alizarin Red), or glycosaminoglycans and collagen in chondrogenic cells (Masson's Trichrome). While bone marrow MSC efficiently differentiated into all three lineages, PVAT-derived cells had adipogenic and chondrogenic potential, but lacked robust osteogenic potential.

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.

Dynamic alterations in decoy VEGF receptor-1 stability regulate angiogenesis.

Blood vessel expansion is driven by sprouting angiogenesis of endothelial cells, and is essential for development, wound healing and disease. Membrane-localized vascular endothelial growth factor receptor-1 (mVEGFR1) is an endothelial cell-intrinsic decoy receptor that negatively modulates blood vessel morphogenesis. Here we show that dynamic regulation of mVEGFR1 stability and turnover in blood vessels impacts angiogenesis. mVEGFR1 is highly stable and constitutively internalizes from the plasma membrane. Post-translational palmitoylation of mVEGFR1 is a binary stabilization switch, and ligand engagement leads to depalmitoylation and lysosomal degradation. Trafficking of palmitoylation enzymes via Rab27a regulates mVEGFR1 stability, as reduced levels of Rab27a impaired palmitoylation of mVEGFR1, decreased its stability, and elevated blood vessel sprouting and in vivo angiogenesis. These findings identify a regulatory axis affecting blood vessel morphogenesis that highlights exquisite post-translational regulation of mVEGFR1 in its role as a molecular rheostat.

Antiangiogenic VEGF-A in peripheral artery disease.

Vascular endothelial growth factor A (VEGF-A) is a potent proangiogenic cytokine elevated in patients with peripheral artery disease (PAD). A new study links impaired vascular regrowth in PAD to increased expression of an antiangiogenic splice variant of VEGF-A.

Histone deacetylase activity selectively regulates notch-mediated smooth muscle differentiation in human vascular cells.

BACKGROUND: Histone deacetylases (HDACs) modify smooth muscle cell (SMC) proliferation and affect neointimal lesion formation by regulating cell cycle progression. HDACs might also regulate SMC differentiation, although this is not as well characterized. METHODS AND RESULTS: Notch signaling activates SMC contractile markers and the differentiated phenotype in human aortic SMCs. Using this model, we found that HDAC inhibition antagonized the ability of Notch to increase levels of smooth muscle α-actin, calponin1, smooth muscle 22α, and smooth muscle myosin heavy chain. However, inhibition of HDAC activity did not suppress Notch activation of the HRT target genes. In fact, HDAC inhibition increased activation of the canonical C-promoter binding factor-1 (CBF-1)-mediated Notch pathway, which activates HRT transcription. Although CBF-1-mediated Notch signaling was increased by HDAC inhibition in human SMCs and in a C3H10T1/2 model, SMC differentiation was inhibited in both cases. Further characterization of downstream Notch signaling pathways showed activation of the c-Jun N-terminal kinase, p38 mitogen-activated protein kinase, and PI3K/Akt pathways. The activation of these pathways was sensitive to HDAC inhibition and was positively correlated with the differentiated phenotype. CONCLUSIONS: Our studies define novel signaling pathways downstream of Notch signaling in human SMCs. In addition to the canonical CBF-1 pathway, Notch stimulates c-Jun N-terminal kinase, mitogen-activated protein kinase, and PI3K cascades. Both canonical and noncanonical pathways downstream of Notch promote a differentiated, contractile phenotype in SMCs. Although CBF-1-mediated Notch signaling is not suppressed by HDAC inhibition, HDAC activity is required for Notch differentiation signals through mitogen-activated protein kinase and PI3K pathways in SMCs. (J Am Heart Assoc. 2012;1:e000901 doi: 10.1161/JAHA.112.000901).