Dario R. Lemos

Nilotinib reduces muscle fibrosis in chronic muscle injury by promoting TNF-mediated apoptosis of fibro/adipogenic progenitors

Depending on the inflammatory milieu, injury can result either in a tissues complete regeneration or in its degeneration and fibrosis, the latter of which could potentially lead to permanent organ failure. Yet how inflammatory cells regulate matrix-producing cells involved in the reparative process is unknown. Here we show that in acutely damaged skeletal muscle, sequential interactions between multipotent mesenchymal progenitors and infiltrating inflammatory cells determine the outcome of the reparative process. We found that infiltrating inflammatory macrophages, through their expression of tumor necrosis factor (TNF), directly induce apoptosis of fibro/adipogenic progenitors (FAPs). In states of chronic damage, however, such as those in mdx mice, macrophages express high levels of transforming growth factor β1 (TGF-β1), which prevents the apoptosis of FAPs and induces their differentiation into matrix-producing cells. Treatment with nilotinib, a kinase inhibitor with proposed anti-fibrotic activity, can block the effect of TGF-β1 and reduce muscle fibrosis in mdx mice. Our findings reveal an unexpected anti-fibrotic role of TNF and suggest that disruption of the precisely timed progression from a TNF-rich to a TGF-β−rich environment favors fibrotic degeneration of the muscle during chronic injury.

Fibro/adipogenic progenitors: a double-edged sword in skeletal muscle regeneration.

Comment on: Joe AWB, et al. Nat Cell Biol 2010; 12:153-163.

Functionally Convergent White Adipogenic Progenitors of Different Lineages Participate in a Diffused System Supporting Tissue Regeneration

Pathologies characterized by lipomatous infiltration of craniofacial structures as well as certain forms of lipodystrophies suggest the existence of a distinct adipogenic program in the cephalic region of mammals. Using lineage tracing, we studied the origin of craniofacial adipocytes that accumulate both in cranial fat depots and during ectopic lipomatous infiltration of craniofacial muscles. We found that unlike their counterparts in limb muscle, a significant percentage of cranial adipocytes is derived from the neural crest (NC). In addition, we identified a population of NC‐derived Lin−/α7−/CD34+/Sca‐1+ fibro/adipogenic progenitors (NC‐FAPs) that resides exclusively in the mesenchyme of cephalic fat and muscle. Comparative analysis of the adipogenic potential, impact on metabolism, and contribution to the regenerative response of NC‐FAPs and mesoderm‐derived FAPs (M‐FAPs) suggests that these cells are functionally indistinguishable. While both NC‐ and M‐FAPs express mesenchymal markers and promyogenic cytokines upon damage‐induced activation, NC‐FAPs additionally express components of the NC developmental program. Furthermore, we show that craniofacial FAP composition changes with age, with young mice containing FAPs that are almost exclusively of NC origin, while NC‐FAPs are progressively replaced by M‐FAPs as mice age. Based on these results, we propose that in the adult, ontogenetically distinct FAPs form a diffused system reminiscent of the endothelium, which can originate from multiple developmental intermediates to seed all anatomical locations. STEM CELLS2012;30:1152–1162

Role of stem/progenitor cells in reparative disorders.

Adult stem cells are activated to proliferate and differentiate during normal tissue homeostasis as well as in disease states and injury. This activation is a vital component in the restoration of function to damaged tissue via either complete or partial regeneration. When regeneration does not fully occur, reparative processes involving an overproduction of stromal components ensure the continuity of tissue at the expense of its normal structure and function, resulting in a “reparative disorder”. Adult stem cells from multiple organs have been identified as being involved in this process and their role in tissue repair is being investigated. Evidence for the participation of mesenchymal stromal cells (MSCs) in the tissue repair process across multiple tissues is overwhelming and their role in reparative disorders is clearly demonstrated, as is the involvement of a number of specific signaling pathways. Transforming growth factor beta, bone morphogenic protein and Wnt pathways interact to form a complex signaling network that is critical in regulating the fate choices of both stromal and tissue-specific resident stem cells (TSCs), determining whether functional regeneration or the formation of scar tissue follows an injury. A growing understanding of both TSCs, MSCs and the complex cascade of signals regulating both cell populations have, therefore, emerged as potential therapeutic targets to treat reparative disorders. This review focuses on recent advances on the role of these cells in skeletal muscle, heart and lung tissues.

Effects of age on clock gene expression in the rhesus macaque pituitary gland.

Recent studies have shown that circadian clock genes are expressed in various peripheral tissues, raising the possibility that multiple clocks regulate circadian physiology. To study clock gene expression in the rhesus macaque pituitary gland we used gene microarray data and found that the pituitary glands of young and old adult males express several components of the circadian clock (Per1, Per2, Cry1, Bmal1, Clock, Rev-erbalpha and Csnk1varepsilon). Semi-quantitative reverse-transcription polymerase chain reaction (sqRT-PCR) confirmed the presence of these core-clock genes and detected significant age-related differences in the expression of Per2. sqRT-PCR also showed differential expression of core-clock genes at two opposing time-points over the 24-h day, with greater expression of Per2 and Bmal1 (P<0.05) at 1300h as compared to 0100h. Immunohistochemistry revealed rhythmic expression of REV-ERBalpha in the pituitary glands of female macaques. These data provide evidence that the rhesus macaque pituitary gland expresses core-clock genes and their associated protein products in a 24-h rhythmic pattern, and that their expression is moderately impacted by aging processes.

Pharmacological blockage of fibro/adipogenic progenitor expansion and suppression of regenerative fibrogenesis is associated with impaired skeletal muscle regeneration

Acute skeletal muscle injury triggers an expansion of fibro/adipogenic progenitors (FAPs) and a transient stage of fibrogenesis characterized by extracellular matrix deposition. While the perpetuation of such phase can lead to permanent tissue scarring, the consequences of its suppression remain to be studied. Using a model of acute muscle damage we were able to determine that pharmacological inhibition of FAP expansion by Nilotinib, a tyrosine kinase inhibitor with potent antifibrotic activity, exerts a detrimental effect on myogenesis during regeneration. We found that Nilotinib inhibits the damage-induced expansion of satellite cells in vivo, but it does not affect in vitro proliferation, suggesting a non cell-autonomous effect. Nilotinib impairs regenerative fibrogenesis by preventing the injury-triggered expansion and differentiation of resident CD45(-):CD31(-):α7integrin(-):Sca1(+) mesenchymal FAPs. Our data support the notion that the expansion of FAPs and transient fibrogenesis observed during regeneration play an important trophic role toward tissue-specific stem cells.

Photoperiodic modulation of adrenal gland function in the rhesus macaque: effect on 24-h plasma cortisol and dehydroepiandrosterone sulfate rhythms and adrenal gland gene expression

In temperate zones, day length changes markedly across the year, and in many mammals these photoperiodic variations are associated with physiological adaptations. However, the influence of this environmental variable on human behavior and physiology is less clear, and the potential underlying mechanisms are unknown. To address this issue, we examined the effect of changing photoperiods on adrenal gland function in ovariectomized female rhesus macaques (Macaca mulatta), both in terms of steroid hormone output and in terms of gene expression. The animals were sequentially exposed to the following lighting regimens, which were designed to simulate photoperiods associated with winter, spring/autumn and summer respectively: 8 h light:16 h darkness (short days), 12 h light:12 h darkness and 16 h light:8 h darkness (long days). Remote 24-h serial blood sampling failed to disclose any effect of photoperiod on mean or peak plasma levels of cortisol or dehydroepiandrosterone sulfate. However, there was a marked phase-advancement of both hormonal rhythms in short days, which was reflected as a similar phase-advancement of the daily motor activity rhythm. Gene microarray analysis of the adrenal gland transcriptome revealed photoperiod-induced differences in the expression of genes associated with homeostatic functions, including: development, lipid synthesis and metabolism, and immune function. Taken together, the results indicate that in primates, both circadian adrenal physiology and gene expression are influenced by seasonal changes in day length, which may have implications for adrenal-regulated physiology and behavior.

Maintenance of vascular integrity by pericytes is essential for normal kidney function

Pericytes are tissue-resident mesenchymal progenitor cells anatomically associated with the vasculature that have been shown to participate in tissue regeneration. Here, we tested the hypothesis that kidney pericytes, derived from FoxD1+ mesodermal progenitors during embryogenesis, are necessary for postnatal kidney homeostasis. Diphtheria toxin delivery to FoxD1Cre::RsDTR transgenic mice resulted in selective ablation of >90% of kidney pericytes but not other cell lineages. Abrupt increases in plasma creatinine, blood urea nitrogen, and albuminuria within 96 h indicated acute kidney injury in pericyte-ablated mice. Loss of pericytes led to a rapid accumulation of neutral lipid vacuoles, swollen mitochondria, and apoptosis in tubular epithelial cells. Pericyte ablation led to endothelial cell swelling, reduced expression of vascular homeostasis markers, and peritubular capillary loss. Despite the observed injury, no signs of the acute inflammatory response were observed. Pathway enrichment analysis of genes expressed in kidney pericytes in vivo identified basement membrane proteins, angiogenic factors, and factors regulating vascular tone as major regulators of vascular function. Using novel microphysiological devices, we recapitulated human kidney peritubular capillaries coated with pericytes and showed that pericytes regulate permeability, basement membrane deposition, and microvascular tone. These findings suggest that through the active support of the microvasculature, pericytes are essential to adult kidney homeostasis.

Gene expression profiling in the rhesus macaque: Experimental design considerations

The development of species-specific gene microarrays has greatly facilitated gene expression profiling in nonhuman primates. However, to obtain accurate and physiologically meaningful data from these microarrays, one needs to consider several factors when designing the studies. This article focuses on effective experimental design while the companion article focuses on methodology and data analysis. Biological cycles have a major influence on gene expression, and at least 10% of the expressed genes are likely to show a 24-h expression pattern. Consequently, the time of day when RNA samples are collected can influence detection of significant changes in gene expression levels. Similarly, when photoperiodic species such as the rhesus macaque are housed outdoors, some of their genes show differential expression according to the time of year. In addition, the sex-steroid environment of humans and many nonhuman primates changes markedly across the menstrual cycle, and so phase of the cycle needs to be considered when studying gene expression in adult females.

Expression profiling of genes encoding glutamate and GABA receptor subunits in three immortalized gnrh cell lines

Gonadotropin-releasing hormone (GnRH) plays a central role in regulating development and function of the reproductive axis, and its secretion is known to be influenced by glutamate and GABA. In the present study, we used gene microarrays and RT-PCR to compare the expression profiles of glutamate and GABA receptor subunits in three immortalized GnRH cell lines: GT1-1, GT1-7, and Gn10. All of these cell lines expressed the AMPA glutamate receptor subunit genes GluR2 and GluR4, but only the GT1-1 and GT1-7 cells expressed the kainate glutamate receptor subunit gene KA2. Additionally, GluRdelta2, a subunit that can form heteromeric receptors with kainate and AMPA subunits, was present in GT1-1 and Gn10 cells but not in GT1-7 cells. Genes encoding the GABA(A) receptor alpha3, beta2, beta3, epsilon, and pi subunits, as well as the GABA(B) receptor 1 subunit, were evident in all three cell lines. However, the gene encoding the expression of GABA(A) receptor gamma subunit was noticeably absent. Taken together, these data demonstrate comprehensive screening of neurotransmitter receptor genes in a controlled neuronal culture system, and reveal novel features.