Abstract
Vascular calcification and stiffening of the arterial wall is a systemic phenomenon that is associated with aging and it can be increased by several risk factors. The underlying mechanisms, especially the pathways of cellular senescence, are under current investigation. Easily manageable in vitro settings help to study the signaling pathways. The experimental setting presented here is based on an in vitro model using rat vascular smooth muscle cells and the detection of senescence and osteoblastic markers via immunofluorescence and RNAscope™. Co-staining of the senescence marker p21, the osteoblastic marker osteopontin, detection of senescence-associated heterochromatin foci, and senescence-associated β-galactosidase is possible within one test approach requiring fewer cells. The protocol is a fast and reliable evaluation method for multiplexing of calcifying and senescence markers with fluorescence microscopy detection. The experimental setting enables analysis on single cell basis and allows detection of intra-individual variances of cultured cells.
Highlights
Aging is associated with a variety of characteristic changes of the vessel wall [1]
A hallmark of vascular aging is a stiffening of the arterial wall with increasing pulse-wave velocity and the mineralization of vascular smooth muscle cells (VSMC) in the media layer of the vessel wall [1,5]
The detection of more than one marker is used for reliable detection and the senescence levels can vary between cells and tissue, respectively, within the same animal [10]
Summary
Aging is associated with a variety of characteristic changes of the vessel wall [1]. There are several disorders, in which patients show signs of premature aging of vessels that appear much older than their biological age e.g., in chronic kidney disease [2,3,4]. A hallmark of vascular aging is a stiffening of the arterial wall with increasing pulse-wave velocity and the mineralization of vascular smooth muscle cells (VSMC) in the media layer of the vessel wall [1,5]. Several hypotheses exist regarding joint or consecutive appearance of calcification and senescence in a vicious cycle in smooth muscle cells e.g., induced by uremic toxins [4,9]. The senescence level can vary within and between cells and the tissue of the same individual [10]. It has to be illuminated whether, in one cell population, cells experience senescence and calcification jointly or consecutively, or whether aged vessels contain distinctly different cell populations of aged and calcified cells
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