Role of pericytes in skeletal muscle regeneration and fat accumulation

Abstract

Stem cells ensure tissue regeneration, while overgrowth of adipogenic cells may compromise organ recovery and impair function. In myopathies and muscle atrophy associated with aging, fat accumulation increases dysfunction, and after chronic injury, the process of fatty degeneration, in which muscle is replaced by white adipocytes, further compromises tissue function and environment. Some studies suggest that pericytes may contribute to muscle regeneration as well as fat formation. This work reports the presence of two pericyte subpopulations in the skeletal muscle and characterizes their specific roles. Skeletal muscle from Nestin-GFP/NG2-DsRed mice show two types of pericytes, Nestin-GFP-/NG2-DsRed+ (type-1) and Nestin-GFP+/NG2-DsRed+ (type-2), in close proximity to endothelial cells. We also found that both Nestin-GFP-/NG2-DsRed+ and Nestin-GFP+/NG2-DsRed+ cells colocalize with staining of two pericyte markers, PDGFRβ and CD146, but only type-1 pericyte express the adipogenic progenitor marker PDGFRα. Type-2 pericytes participate in muscle regeneration, while type-1 contribute to fat accumulation. Transplantation studies indicate that type-1 pericytes do not form muscle in vivo, but contribute to fat deposition in the skeletal muscle, while type-2 pericytes contribute only to the new muscle formation after injury, but not to the fat accumulation. Our results suggest that type-1 and type-2 pericytes contribute to successful muscle regeneration which results from a balance of myogenic and nonmyogenic cells activation.

FIG. 1.

Two bona fide pericyte subtypes in skeletal muscle. Histological analysis of pericyte subtypes in the skeletal muscle from Nestin-GFP/NG2-DsRed mice. (A) Pericytes surround the endothelial cell layers of the capillary network in skeletal muscle. Muscle section showing small blood vessels with CD31+ endothelial cells, characteristically surrounded by NG2-DsRed+ pericytes. Nestin-GFP−/NG2-DsRed+ (type-1) (red arrow) and Nestin-GFP+/NG2-DsRed+ (type-2) (green arrow) pericytes and the blood vessels' CD31+ (orange) labeled contour. All panels show the same area for different channels (Nestin-GFP, NG2-DsRed, Hoechst, CD31 staining, brightfield, merged fluorescence images, and all the images merged with brightfield). (B) Pericyte markers PDGFRβ and CD146 colocalize with skeletal muscle interstitial Nestin-GFP−/NG2-DsRed+ and Nestin-GFP+/NG2-DsRed+ cells. The top and bottom six panels show identical muscle areas from left to right: CD146 (top) or PDGFRβ (bottom) (orange), NG2-DsRed (red), Nestin-GFP+ (green), Hoechst (blue), brightfield, and merged images. The red arrow indicates type-1 pericytes (Nestin-GFP−/NG2-DsRed+ cells), and the green arrow, type-2 (Nestin-GFP+/NG2-DsRed+ cells). Scale bar=20 μm. Color images available online at www.liebertpub.com/scd

FIG. 2.

PDGFRα expression in a subpopulation of type-1 but not type-2 pericytes. (A) Isolation of skeletal muscle cells for flow cytometry. Representative dot plot showing GFP versus DsRed fluorescence with the gate set using cells isolated from wild-type mice. The cells were divided into four populations: Nestin-GFP+/NG2-DsRed− (purple), Nestin-GFP−/NG2-DsRed+ (red), Nestin-GFP+/NG2-DsRed+ (green), and Nestin-GFP−/NG2-DsRed− (blue). (B) Flow cytometry analysis of PDGFRα expressed by skeletal muscle-derived cells. Histograms show PDGFRα expression in each population. Left histogram (unlabeled cells) shows control staining of all cells with secondary antibody APC anti-rabbit to set the gate without using primary antibody rabbit anti-PDGFRα. Right histograms (PDGFRα-labeled cells) show the surface expression of PDGFRα on each skeletal muscle-derived cell subset. Data represent three independent experiments in cells dissociated from the hindlimb muscles of Nestin-GFP/NG2-DsRed mice. Note that only Nestin-GFP−/NG2-DsRed− and Nestin-GFP−/NG2-DsRed+ cells express PDGFRα. (C) Representative transverse cross-section of a tibialis anterior muscle from a double-transgenic Nestin-GFP/NG2-DsRed mouse. A green arrow indicates a Nestin-GFP+/NG2-DsRed+ cell, and a red arrow, a Nestin-GFP−/NG2-DsRed+ cell. Expression of PDGFRα, GFP, DsRed, and their corresponding, Hoechst 33342, brightfield, and merge images are illustrated. PDGFRα staining colocalizes with skeletal muscle interstitial Nestin-GFP−/NG2-DsRed+ but not Nestin-GFP+/NG2-DsRed+ cells. Color images available online at www.liebertpub.com/scd

FIG. 3.

Type-1 but not type-2 pericytes are adipogenic in vitro. Adipogenic induction of freshly isolated muscle-derived pericyte subtypes. (A) Protocol: freshly isolated pericytes were cultured in adipogenic medium for 14 days. (B) Representative dot plots showing DsRed versus GFP fluorescence of cells isolated from skeletal muscle of Nestin-GFP/NG2-DsRed mice. Gate was set using cells derived from wild-type skeletal muscle. Morphologic analysis is shown after type-1 and 2 pericytes were cultured for 2 weeks under adipogenic conditions; they were then stained with anti-perilipin A antibody (C, D) or Oil Red O/hematoxylin (E, F). (D) Percent of cells positive for perilipin in type-1 or type-2 pericytes (n=5 preparations). (F) Percent of type-1 or type-2 pericytes positive for Oil Red O (n=5 preparations from separate cell isolation experiments). Data are mean±standard error of the mean (SEM). Scale bars=20 μm. Color images available online at www.liebertpub.com/scd

FIG. 4.

Type-2 but not type-1 pericytes are myogenic in vitro. Myogenic induction of freshly isolated muscle-derived pericyte subtypes. (A) Protocol: freshly isolated pericytes were cultured for 3 days in growth medium followed by 2 weeks in myogenic differentiation medium. (B) Representative dot plots showing DsRed versus GFP fluorescence of cells isolated from the skeletal muscle of Nestin-GFP/NG2-DsRed mice. Gate was set using cells derived from wild-type skeletal muscle. Morphologic analysis is shown after type-1 and 2 pericytes were cultured for 2 weeks in myogenic conditions. (C, D) After 3 days in differentiation medium, Nestin-GFP−/NG2-DsRed+ and Nestin-GFP+/NG2-DsRed+ cells were stained with anti-myogenin antibody. (D) The percent of myogenin+ cells derived from each pericyte population was counted and normalized to the number of nuclei. (n=3 preparations from separate cell isolation experiments). (E, F) After 14 days in differentiation medium, both cell types were stained with anti-MHC antibody. (F) The percent of MHC+ nuclei derived from each pericyte subpopulation was counted and normalized to the total number of nuclei (n=3 preparations from separate cell isolation experiments). Data are mean±SEM. Scale bar=100 μm. Color images available online at www.liebertpub.com/scd

FIG. 5.

Satellite cells do not express NG2 proteoglycan. (A) Representative tibialis anterior (TA) muscle section from a Nestin-GFP/NG2-DsRed transgenic mouse showing laminin (basal lamina), Nestin-GFP, NG2-DsRed expression, and Hoechst positive nuclei in the same region. Brightfield and merged images are also shown. White arrow shows a satellite cell (Nestin-GFP+ located beneath the basal lamina) that does not express NG2-DsRed; the yellow arrow indicates a type-2 pericyte (Nestin-GFP+/NG2-DsRed+) located outside the basal lamina. (B) We counted 120 Nestin-GFP+ cells (NG2+ or NG2−) beneath the basal lamina. (C) Representative Nestin-GFP, NG2-DsRed, brightfield, and merged images of the same region in a dish containing freshly dissociated flexor digitorum brevis (FDB) muscle fibers from Nestin-GFP/NG2-DsRed mice. The white arrow indicates a satellite cell (Nestin-GFP+) attached to the myofiber, while the yellow arrow shows both types of pericytes (Nestin-GFP−/NG2-DsRed+ and Nestin-GFP+/NG2-DsRed+ cells) in dissociated connective tissue. Scale bar=50 μm. (D) Number of Nestin-GFP+ cells (NG2+ or NG2−) in freshly dissociated single FDB muscle fibers from Nestin-GFP/NG2-DsRed transgenic mice; n=4 preparations, more than 1,000 cells counted. (E) Representative TA muscle section from a NG2-DsRed transgenic mouse showing Pax7 staining. The white arrow shows a typical satellite cell (Pax7+/NG2-DsRed−). Color images available online at www.liebertpub.com/scd

FIG. 6.

Diagram of transplantation procedures used to track the fate of type-1 and type-2 pericytes in vivo. (A) Obtaining single cells from Nestin-GFP/β-actin-DsRed double-transgenic mouse skeletal muscle, in which all cells are DsRed+. Representative dot plots showing GFP fluorescence versus NG2+ cells with the gate set using unlabeled cells. Protocol for cell transplantation in models of injury (B) and fatty degeneration (C) in skeletal muscle. Color images available online at www.liebertpub.com/scd

FIG. 7.

Type-1 pericyte adipogenic potential in vivo. (A) Fourteen days after type-1 or type-2 pericytes were transplanted into glycerol-injured muscle, muscle sections were analyzed for perilipin expression. Representative perilipin expression (green), DsRed fluorescence, Hoechst, brightfield, and merge images of the same region. Colocalization of DsRed+ cells with perilipin, shown by the yellow arrow in the merged image, supports adipogenic differentiation of transplanted type-1 pericytes. Note that DsRed+ type-2 pericytes do not differentiate into adipocytes (perilipin−) in vivo; all perilipin+ (green) adipocytes are DsRed− as indicated by the white arrow in the merged image. (B) Quantitative analysis of transplantation experiments. Data are mean±SEM. (n=5 replicates). (C) Section of the muscle represented in (A), incubated with the fluorescent secondary but not the primary antiperilipin antibody. (D) Control section of a regenerated muscle stained with perilipin. Notice that perilipin does not stain myofibers. Scale bars=50 μm. Color images available online at www.liebertpub.com/scd

FIG. 8.

Only type-2 pericytes generate myofibers after transplantation into injured skeletal muscle. (A) DsRed fluorescence in whole TA muscles 2 weeks after injection with type-1 or type-2 pericytes. DsRed+ fibers can be detected only in muscle injected with type-2 pericytes. (B) Quantitative analysis of newly formed DsRed+ myofibers with characteristic central nuclei derived from type-1 or type-2 pericytes. Data are mean±SEM (n=5 replicates). (C) At day 14 after transplantation, clusters of DsRed myofibers (red) are present throughout the muscle of mice injected with type-2 pericytes. Type-1 pericytes stay in the interstitial space and do not differentiate into muscle cells. (D) Representative TA muscle section from a transplanted mouse (as in C), showing that type-1 pericytes (DsRed+) retain the expression of the pericyte marker NG2 proteoglycan. Scale bar=20 μm. Color images available online at www.liebertpub.com/scd

FIG. 9.

Schematic representation of normal regenerating and fatty degenerating skeletal muscle. Two pericyte subtypes are associated with blood vessels: type-1 (yellow) and type-2 (green). We suggest that type-1 pericytes contribute to the adipose infiltration observed in various disorders, such as obesity, dystrophies, and aging, while type-2 pericytes cooperate with myogenesis after healing in normal adult skeletal muscle. Color images available online at www.liebertpub.com/scd