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. 2018 Oct;32(5):519-530.
doi: 10.1007/s10557-018-6813-y.

Rab27a Regulates Human Perivascular Adipose Progenitor Cell Differentiation

Joshua M Boucher  1 , Michael Robich  1   2 , S Spencer Scott  1 , Xuehui Yang  1 , Larisa Ryzhova  1 , Jacqueline E Turner  1 , Ilka Pinz  1 , Lucy Liaw  3
Affiliations

Rab27a Regulates Human Perivascular Adipose Progenitor Cell Differentiation

Joshua M Boucher et al. Cardiovasc Drugs Ther. 2018 Oct.

Abstract

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.

Keywords: Adipose; Cardiovascular; Disease; Perivascular; Progenitor; Rab27a.

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Conflict of interest statement

Conflicts of interest: All authors confirm that they have no conflicts of interest to report.

Figures

Figure 1.
Figure 1.. Identification of adipocyte progenitor cells from human aortic PVAT
a) Immunohistochemical analysis of human aortic PVAT. Trichrome: blue=connective tissue, purple=nuclei. b) Phase contrast microscopy of progenitor cells explanted from the stromal vascular fraction of PVAT from a CABG patient at days 3 and 7 in culture. c) RT-PCR for the indicated transcripts from passage 1 PVAT-APC. d) Immunoblot for the indicated proteins from passage 2 PVAT-APC. e-g) Histograms of PVAT-APC analyzed by flow cytometry for CD73 (e) CD105 (f), and CD90 (g) from donor R17–1092. Blue, DAPI stained; grey, FMO negative control; green, labeled cells. Results are representative of results from 5 separate donors.
Figure 2.
Figure 2.. Human PVAT-APC differentiate to adipocytes in vitro
a) Oil red O (ORO) staining of lipids in non-induced (top) and induced (bottom) at day 7. b) Immunoblot for the indicated proteins of non-induced and induced PVAT-APC for 7 and 14 days. c) Confocal immunofluorescent analysis for the indicated proteins in non-induced and induced human PVAT-APC. Arrows, PLIN1 surrounding lipid droplets.
Figure 3.
Figure 3.. RAB27A is expressed in human PVAT and suppressed during adipogenic differentiation
a) Immunohistochemistry (top, brown Rab27a staining) and confocal immunofluorescence (bottom, green Rab27a staining) for the indicated proteins from sections of human PVAT. Nuclei were stained with DAPI. Negative controls were stained using isotype-matched IgG control antibodies. b) Immunoblot of human PVAT and subcutaneous WAT whole tissue for the indicated proteins. c) Confocal immunofluorescence microscopy of PVAT-APC. RAB27a is labeled in green, actin filaments are detected by phalloidin (red), and nuclei are stained with DAPI. Negative control is stained using fluorophore conjugated secondary antibody. d) Immunoblot for the indicated proteins from PVAT-APC as indicated.
Figure 4.
Figure 4.. RAB27A regulates adipogenic differentiation of human PVAT-APC
a-c) ORO staining (a), quantification of ORO incorporation (b) and % of ORO positive cells (c) from control (ntRNA) or RAB27A knockdown (siRAB27A) PVAT-APC induced to differentiate for 8 days. Negative control, non-induced cells from each condition stained with ORO. Statistics: Shown are means +SD. Students t-test with Tukey’s post-hoc analysis, n=8 CABG patients. d-j) Representative immunoblot (d) and quantification of protein levels (e-j) of non-induced and induced human PVAT-APC with and without RAB27A knockdown.
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