Alexander Birbrair

Role of Pericytes in Skeletal Muscle Regeneration and Fat Accumulation

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.

Type-2 pericytes participate in normal and tumoral angiogenesis.

Tissue growth and function depend on vascularization, and vascular insufficiency or excess exacerbates many human diseases. Identification of the biological processes involved in angiogenesis will dictate strategies to modulate reduced or excessive vessel formation. We examine the essential role of pericytes. Their heterogeneous morphology, distribution, origins, and physiology have been described. Using double-transgenic Nestin-GFP/NG2-DsRed mice, we identified two pericyte subsets. We found that Nestin-GFP(-)/NG2-DsRed(+) (type-1) and Nestin-GFP(+)/NG2-DsRed(+) (type-2) pericytes attach to the walls of small and large blood vessels in vivo; in vitro, type-2, but not type-1, pericytes spark endothelial cells to form new vessels. Matrigel assay showed that only type-2 pericytes participate in normal angiogenesis. Moreover, when cancer cells were transplanted into Nestin-GFP/NG2-DsRed mice, type-1 pericytes did not penetrate the tumor, while type-2 pericytes were recruited during its angiogenesis. As inhibition of angiogenesis is a promising strategy in cancer therapy, type-2 pericytes may provide a cellular target susceptible to signaling and pharmacological manipulation in treating malignancy. This work also reports the potential of type-2 pericytes to improve blood perfusion in ischemic hindlimbs, indicating their potential for treating ischemic illnesses.

Type-1 pericytes accumulate after tissue injury and produce collagen in an organ-dependent manner

IntroductionFibrosis, or scar formation, is a pathological condition characterized by excessive production and accumulation of collagen, loss of tissue architecture, and organ failure in response to uncontrolled wound healing. Several cellular populations have been implicated, including bone marrow-derived circulating fibrocytes, endothelial cells, resident fibroblasts, epithelial cells, and recently, perivascular cells called pericytes. We previously demonstrated pericyte functional heterogeneity in skeletal muscle. Whether pericyte subtypes are present in other tissues and whether a specific pericyte subset contributes to organ fibrosis are unknown.MethodsHere, we report the presence of two pericyte subtypes, type-1 (Nestin-GFP-/NG2-DsRed+) and type-2 (Nestin-GFP+/NG2-DsRed+), surrounding blood vessels in lungs, kidneys, heart, spinal cord, and brain. Using Nestin-GFP/NG2-DsRed transgenic mice, we induced pulmonary, renal, cardiac, spinal cord, and cortical injuries to investigate the contributions of pericyte subtypes to fibrous tissue formation in vivo.ResultsA fraction of the lung’s collagen-producing cells corresponds to type-1 pericytes and kidney and heart pericytes do not produce collagen in pathological fibrosis. Note that type-1, but not type-2, pericytes increase and accumulate near the fibrotic tissue in all organs analyzed. Surprisingly, after CNS injury, type-1 pericytes differ from scar-forming PDGFRβ + cells.ConclusionsPericyte subpopulations respond differentially to tissue injury, and the production of collagen by type-1 pericytes is organ-dependent. Characterization of the mechanisms underlying scar formation generates cellular targets for future anti-fibrotic therapeutics.

Pericytes at the intersection between tissue regeneration and pathology

Perivascular multipotent cells, pericytes, contribute to the generation and repair of various tissues in response to injury. They are heterogeneous in their morphology, distribution, origin and markers, and elucidating their molecular and cellular differences may inform novel treatments for disorders in which tissue regeneration is either impaired or excessive. Moreover, these discoveries offer novel cellular targets for therapeutic approaches to many diseases. This review discusses recent studies that support the concept that pericyte subtypes play a distinctive role in myogenesis, neurogenesis, adipogenesis, fibrogenesis and angiogenesis.

Type-1 pericytes participate in fibrous tissue deposition in aged skeletal muscle.

In older adults, changes in skeletal muscle composition are associated with increased fibrosis, loss of mass, and decreased force, which can lead to dependency, morbidity, and mortality. Understanding the biological mechanisms responsible is essential to sustaining and improving their quality of life. Compared with young mice, aged mice take longer to recover from muscle injury; their tissue fibrosis is more extensive, and regenerated myofibers are smaller. Strong evidence indicates that cells called pericytes, embedded in the basement membrane of capillaries, contribute to the satellite-cell pool and muscle growth. In addition to their role in skeletal muscle repair, after tissue damage, they detach from capillaries and migrate to the interstitial space to participate in fibrosis formation. Here we distinguish two bona fide pericyte subtypes in the skeletal muscle interstitium, type-1 (Nestin-GFP(-)/NG2-DsRed(+)) and type-2 (Nestin-GFP(+)/NG2-DsRed(+)), and characterize their heretofore unknown specific roles in the aging environment. Our in vitro results show that type-1 and type-2 pericytes are either fibrogenic or myogenic, respectively. Transplantation studies in young animals indicate that type-2 pericytes are myogenic, while type-1 pericytes remain in the interstitial space. In older mice, however, the muscular regenerative capacity of type-2 pericytes is limited, and type-1 pericytes produce collagen, contributing to fibrous tissue deposition. We conclude that in injured muscles from aging mice, the pericytes involved in skeletal muscle repair differ from those associated with scar formation.

Niche heterogeneity in the bone marrow

In adult mammals, hematopoietic stem cells (HSCs) are defined by their abilities to self‐renew and to differentiate to form all blood cell lineages. These rare multipotent cells occupy specific locations in the bone marrow (BM) microenvironment. The specific microenvironment regulating HSCs, commonly referred to as the niche, comprises multiple cell types whose exact contributions are under active investigation. Understanding cellular cross talk involving HSCs in the BM microenvironment is of fundamental importance for harnessing therapies against benign and malignant blood diseases. In this review, we summarize and evaluate recent advances in our understanding of niche heterogeneity and its influence on HSC function.

Nestin-GFP Transgene Reveals Neural Precursor Cells in Adult Skeletal Muscle

Background Therapy for neural lesions or degenerative diseases relies mainly on finding transplantable active precursor cells. Identifying them in peripheral tissues accessible for biopsy, outside the central nervous system, would circumvent the serious immunological and ethical concerns impeding cell therapy. Methodology/Principal Findings In this study, we isolated neural progenitor cells in cultured adult skeletal muscle from transgenic mice in which nestin regulatory elements control GFP expression. These cells also expressed the early neural marker Tuj1 and light and heavy neurofilament but not S100β, indicating that they express typical neural but not Schwann cell markers. GFP+/Tuj1+ cells were also negative for the endothelial and pericyte markers CD31 and α-smooth muscle actin, respectively. We established their a) functional response to glutamate in patch-clamp recordings; b) interstitial mesenchymal origin; c) replicative capacity; and d) the environment necessary for their survival after fluorescence-activated cell sorting. Conclusions/Significance We propose that the decline in nestin-GFP expression in muscle progenitor cells and its persistence in neural precursor cells in muscle cultures provide an invaluable tool for isolating a population of predifferentiated neural cells with therapeutic potential.

Skeletal muscle neural progenitor cells exhibit properties of NG2-glia.

Reversing brain degeneration and trauma lesions will depend on cell therapy. Our previous work identified neural precursor cells derived from the skeletal muscle of Nestin-GFP transgenic mice, but their identity, origin, and potential survival in the brain are only vaguely understood. In this work, we show that Nestin-GFP+ progenitor cells share morphological and molecular markers with NG2-glia, including NG2, PDGFRα, O4, NGF receptor (p75), glutamate receptor-1(AMPA), and A2B5 expression. Although these cells exhibit NG2, they do not express other pericyte markers, such as α-SMA or connexin-43, and do not differentiate into the muscle lineage. Patch-clamp studies displayed outward potassium currents, probably carried through Kir6.1 channels. Given their potential therapeutic application, we compared their abundance in tissues and concluded that skeletal muscle is the richest source of predifferentiated neural precursor cells. We found that these cells migrate toward the neurogenic subventricular zone displaying their typical morphology and nestin-GFP expression two weeks after brain injection. For translational purposes, we sought to identify these neural progenitor cells in wild-type species by developing a DsRed expression vector under Nestin-Intron II control. This approach revealed them in nonhuman primates and aging rodents throughout the lifespan.

Pericytes are heterogeneous in their origin within the same tissue

Pericytes heterogeneity is based on their morphology, distribution, and markers. It is well known that pericytes from different organs may have distinct embryonic sources. Yamazaki et al. (2017) using several transgenic mouse model reveal by cell-lineage tracing that pericytes are even more heterogeneous than previously appreciated. This study shows that pericytes from within the same tissue may be heterogeneous in their origin. Remarkably, a subpopulation of embryonic dermal pericytes derives from the hematopoietic lineage, an unexpected source. Reconstructing the lineage of pericytes is central to understanding development, and also for the diagnosis and treatment of diseases in which pericytes play important roles.

Pericytes are Essential for Skeletal Muscle Formation

Various cells in skeletal muscle have known myogenic potential [1–4], but their physiological roles in its formation are unclear. The general consensus holds that satellite cells are required for muscle regeneration [5–7]. However, in a recent article in Development, Kostallari and colleagues demonstrated that pericytes are also indispensable for postnatal growth of skeletal muscle [8]. Using a transgenic mouse model for selective diphtheria toxin-induced depletion of NG2+ pericytes, they found that pericyte ablation led to myofiber hypotrophy. Their report is the first to show that skeletal muscle formation in vivo depends on myogenic cells other than satellite cells. Future studies should test whether depleting the skeletal muscle of other interstitial cells with myogenic potential will affect muscle formation; for example, CD133+ [9]; PW1+ interstitial cells [10]; and muscle side population (SP) cells [11]. The skeletal muscle microenvironment is very heterogeneous, with such distinct cell types as fibroblasts, adipocytes, Schwann cells, and blood cells, all of which may influence and, in turn, be influenced by local structural and biochemical cues. Studies should determine how depleting each specific cell population affects skeletal muscle function and regenerative capacity. For instance, Kardon’s group showed that connective tissue fibroblasts without myogenic potential are necessary for muscle regeneration [5]. Kostallari and colleagues also found that pericytes form a niche for satellite cells, similar to the niche in the bone marrow of mesenchymal stem cells for hematopoietic stem cells [12, 13]. During postnatal skeletal muscle development, the distance between pericytes and satellite cells became progressively shorter. After pericyte depletion, the previously quiescent satellite cells were activated. When indirect co-cultures were used to assess the functional interactions between pericytes and satellite cells, the authors found that pericytes both promoted myogenic differentiation and induced satellite cell quiescence. Furthermore, using tissue-specific mouse mutants, they demonstrated that pericytes stimulate muscle growth through IGF1 and control satellite-cell quiescence through ANGPT1 [8], but whether the reverse occurs has not been shown. Do satellite cells form a niche for pericytes? How are pericyte numbers and functions affected after satellite-cell depletion?What signaling molecules/cytokines influence the communication between these cell types? Do satellite cells produce signals that inform such pericyte stem-cell functions as angiogenesis/myogenesis? Skeletal muscle pericytes are heterogeneous, and two major subpopulations have been distinguished: type-1 (Nestin-GFP-/ NG2+/PDGFRβ+) and type-2 (Nestin-GFP+/NG2+/ PDGFRβ+) [1, 3, 14–17]. Kostallari et al. propose that the pericytes in the satellite-cell niche are type-2. However, most of their experiments were done in mice and affected both pericyte subtypes, which both express NG2 proteoglycan. Pericyte subtypes have distinct functions, and only type-2 pericytes contribute to muscle formation [2]. Recombinationbased lineage tracing and ablation of a specific pericyte subpopulation, rather than all NG2+ pericytes, may better explain * Osvaldo Delbono odelbono@wakehealth.edu