ABSTRACT
Multipotent mesenchymal stromal cells (MSCs) integrate hormone and neuromediator signaling to coordinate tissue homeostasis, tissue renewal and regeneration. To facilitate the investigation of MSC biology, stable immortalized cell lines are created (e.g., commercially available ASC52telo). However, the ASC52telo cell line has an impaired adipogenic ability and a depressed response to hormones, including 5-HT, GABA, glutamate, noradrenaline, PTH and insulin compared to primary cells. This markedly reduces the potential of the ASC52telo cell line in studying the mechanisms of hormonal control of MSC's physiology. Here, we have established a novel immortalized culture of adipose tissue-derived MSCs via forced telomerase expression after lentiviral transduction. These immortalized cell cultures demonstrate high proliferative potential (up to 40 passages), delayed senescence, as well as preserved primary culture-like functional activity (sensitivity to hormones, ability to hormonal sensitization and differentiation) and immunophenotype up to 17-26 passages. Meanwhile, primary adipose tissue-derived MSCs usually irreversibly lose their properties by 8-10 passages. Observed characteristics of reported immortalized human MSC cultures make them a feasible model for studying molecular mechanisms, which regulate the functional activities of these cells, especially when primary cultures or commercially available cell lines are not appropriate.
Subject(s)
Mesenchymal Stem Cells , Humans , Mesenchymal Stem Cells/metabolism , Cell Line , Cell Culture Techniques , Cell Differentiation , Cells, Cultured , Hormones/metabolism , Cell ProliferationABSTRACT
Hypertension is one of the major life-threatening complications of obesity. Recently adipose multipotent mesenchymal stromal cells (MSCs) were implicated to the pathogenesis of obesity-associated hypertension. These cells amplify noradrenaline-induced vascular cell contraction via cAMP-mediated signaling pathway. In this study we tested the ability of several cAMP-mediated hormones to affect the adrenergic sensitivity of MSCs and their associated contractility. Despite that adipose MSCs express a plethora of receptors capable of cAMP signaling activation, only 5-HT was able to elevate α1A-adrenoceptor-induced Ca2+ signaling in MSCs. Furthermore, 5-HT markedly enhanced noradrenaline-induced MSCs contractility. Using HTR isoform-specific antagonists followed by CRISPRi-mediated knockdown, we identified that the observed 5-HT effect on MSCs was mediated by the HTR6 isoform. This receptor was previously associated exclusively with 5-HT central nervous system activity. Discovered effect of HTR6 on MSCs contractility points to it as a potential therapeutic target for the prevention and treatment of obesity-associated hypertension.
Subject(s)
Hypertension , Serotonin , Humans , Norepinephrine/pharmacology , Hypertension/etiology , Obesity/complications , Protein IsoformsABSTRACT
OBJECTIVE: Transforming growth factor-beta (TGF-beta) has been implicated in the pathogenesis of human atherosclerosis but its actions during lesion progression are poorly understood. Smad2, Smad3, and Smad4 proteins are signaling molecules by which TGF-beta modulates gene transcription. Our objective was to define the actions of TGF-beta during lesion progression in humans by examining the expression of Smads in relation to TGF-beta-mediated responses. METHODS AND RESULTS: Immunohistochemistry and reverse-transcription polymerase chain reaction demonstrated Smad2, Smad3, and Smad4 expression in macrophages of fibrofatty lesions and their upregulation after differentiation of monocytes to macrophages. The major Smad splice variants expressed by the macrophages were those that are transcriptionally most active. Macrophages also expressed cyclin inhibitors whose expression is induced via Smad proteins. The cytoplasmic location of p21(Waf1) suggests it may protect macrophages from apoptosis. Smooth muscle cells (SMCs) within the fibrofatty lesions did not express the Smad proteins or the cyclin inhibitors. SMCs of fibrous plaques expressed all 3 Smad proteins. CONCLUSIONS: In human atherosclerotic lesions, the actions of TGF-beta appear restricted to SMCs in fibrous plaques and macrophages in fatty streaks/fibrofatty lesions. The lack of key TGF-beta signaling components in SMCs of fibrofatty lesions indicates impaired ability of these cells to initiate TGF-beta-mediated Smad-dependent transcriptional responses.
Subject(s)
Aorta, Thoracic/metabolism , Aortic Diseases/metabolism , Arteriosclerosis/metabolism , DNA-Binding Proteins/biosynthesis , Macrophages/metabolism , Myocytes, Smooth Muscle/metabolism , Trans-Activators/biosynthesis , Transforming Growth Factor beta/physiology , Adult , Aged , Aorta, Thoracic/pathology , Aortic Diseases/pathology , Arteriosclerosis/pathology , Cell Cycle Proteins/biosynthesis , Cell Cycle Proteins/genetics , Cell Differentiation/drug effects , Cyclin-Dependent Kinase Inhibitor p15 , Cyclin-Dependent Kinase Inhibitor p21 , DNA-Binding Proteins/genetics , DNA-Binding Proteins/physiology , Death, Sudden , Female , Fibrosis , Humans , Lipids , Male , Middle Aged , Monocytes/metabolism , Muscle, Smooth, Vascular/metabolism , Muscle, Smooth, Vascular/pathology , RNA Splicing , Signal Transduction , Smad2 Protein , Smad3 Protein , Smad4 Protein , Trans-Activators/genetics , Trans-Activators/physiology , Transforming Growth Factor beta/pharmacology , Transforming Growth Factor beta1 , Tumor Suppressor Proteins/biosynthesis , Tumor Suppressor Proteins/geneticsABSTRACT
OBJECTIVE: Despite studies implicating superoxide anion-producing oxidases in atherosclerosis, their characteristics, expression, and regulation in cells of lesions are poorly understood. We examined the following: (1) whether cytochrome b558-dependent NAD(P)H oxidase-phox peptides are expressed by intimal smooth muscle cells (iSMCs) and macrophages of human aortic atherosclerotic lesions and their regulation and (2) whether cytochrome b558-dependent NAD(P)H oxidase represents a major NAD(P)H oxidase in iSMCs. METHODS AND RESULTS: Using a combination of immunochemical and reverse transcription-polymerase chain reaction procedures, we demonstrate that p22(phox) and gp91(phox) (cytochrome b558) expression in normal intima was restricted to a quarter of the iSMCs. In fatty streaks, a similar fraction of iSMCs expressed cytochrome b558, whereas macrophages also expressed low levels of p47(phox) and p67(phox). In fibrofatty lesions, the majority of iSMCs expressed the cytochrome b558 subunits; p67(phox) was also detected. Macrophages and macrophage-derived foam cells expressed the 4 phox subunits that constitute superoxide-producing cytochrome b558-dependent NAD(P)H oxidase. These were upregulated by transforming growth factor-beta1 and interferon-gamma. Aortic lesions also expressed Thox1 and Nox4, and although their expression also increases with lesion severity, their expression is less frequent than that of gp91(phox). CONCLUSIONS: In human aortic fibrofatty lesions, a cytochrome b558-dependent NAD(P)H oxidase appears to be a major iSMC and macrophage oxidase whose expression is upregulated by cytokines.
