An Expanding Role for Nuclear Factor κB in Muscle Stem Cells: Implications for the Treatment of Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is a fatal disease characterized by progressive skeletal muscle degeneration. Inhibition of the transcription factor nuclear factor-κ B (NF-κB), and more specifically the p65 subunit, significantly improves the phenotype of mdx mice, a murine DMD model. However, the ubiquity of NF-κB stands as an obstacle to clinical translation. In this dissertation, we explore the roles of NF-κB/p65 in the regenerative capacity of muscle-derived stem cells (MDSCs) with the goal of identifying alternative approaches to DMD treatment. We found that both cell proliferation and myogenic potential were increased in MDSCs lacking one allele of p65 (p65+/-). In wild type MDSCs, in vitro pharmacologic inhibition of the upstream activating kinase, IKKβ, increased myotube formation in a dose-dependent manner. When transplanted into mdx hind limb muscle, p65+/- MDSCs resulted in significantly larger engraftments. Furthermore, engraftments in cardiotoxin (CTX) injured muscle were associated with reduced local host necrosis and inflammation. Not only were p65+/- MDSCs found to be more resistant to oxidative stress, but we found that p65 depletion improved the anti-inflammatory capacity of MDSCs in vitro and in vivo via upregulation of hepatocyte growth factor (HGF). Moreover, accelerated regeneration in p65 haploinsufficient mdx mice (mdx;p65+/-) coincided with HGF upregulation. Intraperitoneal injection of a musculotropic adeno-associated virus carrying shRNA targeting HGF reversed the phenotypic improvements of mdx;p65+/- mice, increasing both muscle inflammation and necrosis. These data implicate NF-κB/p65 in muscle stem cell proliferation, differentiation, survival, and growth factor gene expression, further underlining the danger of broadly targeting such an important pathway. Finally, this research has also identified HGF as a downstream effector of NF-κB/p65 inhibition in mdx mice. Thus, delivery of HGF or activation of its receptor, MET, may represent a new approach to reduce chronic inflammation and preserve muscle fiber integrity in DMD

Inhibition of NF-κB improves the stress resistance and myogenic differentiation of MDSPCs isolated from naturally aged mice

A decline in the regenerative capacity of adult stem cells with aging is well documented. As a result of this decline, the efficacy of autologous stem cell therapies is likely to decline with increasing donor age. In these cases, strategies to restore the function of aged stem cells would have clinical utility. Globally, the transcription factor NF-κB is up-regulated in aged tissues. Given the negative role that NF-κB plays in myogenesis, we investigated whether the age-related decline in the function of muscle-derived stem/progenitor cells (MDSPCs) could be improved by inhibition of NF-κB. Herein, we demonstrate that pharmacologic or genetic inhibition of NF-κB activation increases myogenic differentiation and improves resistance to oxidative stress. Our results suggest that MDSPC “aging” may be reversible, and that pharmacologic targeting of pathways such as NF-κB may enhance the efficacy of cell-based therapies

Deficiency of AXL in Bone Marrow-Derived Cells Does Not Affect Advanced Atherosclerotic Lesion Progression

AXL, a member of the TAM (Tyro3, Axl, MerTK) family of receptors, plays important roles in cell survival, clearance of dead cells (efferocytosis), and suppression of inflammation, which are processes that critically influence atherosclerosis progression. Whereas MerTK deficiency promotes defective efferocytosis, inflammation, and plaque necrosis in advanced murine atherosclerosis, the role of Axl in advanced atherosclerosis progression is not known. Towards this end, bone marrow cells from Axl−/− or wild-type mice were transplanted into lethally irradiated Ldlr−/− mice. These chimeric mice were then fed the Western-type diet (WD) for 17 weeks. We demonstrate that lesional macrophages in WT mice express Axl but that Axl deficiency in bone marrow-derived cells does not affect lesion size, cellularity, necrosis, or inflammatory parameters in advanced atherosclerotic plaques. Moreover, apoptosis of lesional cells was unaffected, and we found no evidence of defective lesional efferocytosis. In contrast to previously reported findings with MerTK deficiency, hematopoietic cell-Axl deficiency in WD-fed Ldlr−/− mice does not affect the progression of advanced atherosclerosis or lesional processes associated with TAM receptor signaling. These findings suggest a heretofore unappreciated TAM receptor hierarchy in advanced atherosclerosis

An imbalance between specialized pro-resolving lipid mediators and pro-inflammatory leukotrienes promotes instability of atherosclerotic plaques

Chronic unresolved inflammation plays a causal role in the development of advanced atherosclerosis, but the mechanisms that prevent resolution in atherosclerosis remain unclear. Here, we use targeted mass spectrometry to identify specialized pro-resolving lipid mediators (SPM) in histologically-defined stable and vulnerable regions of human carotid atherosclerotic plaques. The levels of SPMs, particularly resolvin D1 (RvD1), and the ratio of SPMs to pro-inflammatory leukotriene B4 (LTB₄), are significantly decreased in the vulnerable regions. SPMs are also decreased in advanced plaques of fat-fed Ldlr⁻/⁻ mice. Administration of RvD1 to these mice during plaque progression restores the RvD1:LTB₄ ratio to that of less advanced lesions and promotes plaque stability, including decreased lesional oxidative stress and necrosis, improved lesional efferocytosis, and thicker fibrous caps. These findings provide molecular support for the concept that defective inflammation resolution contributes to the formation of clinically dangerous plaques and offer a mechanistic rationale for SPM therapy to promote plaque stability

Beneficial effect of mechanical stimulation on the regenerative potential of muscle-derived stem cells is lost by inhibiting vascular endothelial growth factor

OBJECTIVE - : We previously reported that mechanical stimulation increased the effectiveness of muscle-derived stem cells (MDSCs) for tissue repair. The objective of this study was to determine the importance of vascular endothelial growth factor (VEGF) on mechanically stimulated MDSCs in a murine model of muscle regeneration. APPROACH AND RESULTS - : MDSCs were transduced with retroviral vectors encoding the LacZ reporter gene (lacZ-MDSCs), the soluble VEGF receptor Flt1 (sFlt1-MDSCs), or a short hairpin RNA (shRNA) targeting messenger RNA of VEGF (shRNA-VEGF MDSCs). Cells were subjected to 24 hours of mechanical cyclic strain and immediately transplanted into the gastrocnemius muscles of mdx/scid mice. Two weeks after transplantation, angiogenesis, fibrosis, and regeneration were analyzed. There was an increase in angiogenesis in the muscles transplanted with mechanically stimulated lacZ-MDSCs compared with nonstimulated lacZ-MDSCs, sFlt1-MDSCs, and shRNA -VEGF MDSCs. Dystrophin-positive myofiber regeneration was significantly lower in the shRNA-VEGF-MDSC group compared with the lacZ-MDSC and sFlt1-MDSC groups. In vitro proliferation of MDSCs was not decreased by inhibition of VEGF; however, differentiation into myotubes and adhesion to collagen were significantly lower in the shRNA-VEGF-MDSC group compared with the lacZ-MDSC and sFlt1-MDSC groups. CONCLUSIONS - : The beneficial effects of mechanical stimulation on MDSC-mediated muscle repair are lost by inhibiting VEGF. © 2013 American Heart Association, Inc

Multiple compounds improve aged MDSPC proliferation (self-renewal).

<p>(A) Aged MDSPCs were cultured in medium containing aspirin, nordihydroguaiaretic acid (NDGA), or rapamycin, and proliferation was monitored by live cell imaging for up to 60 hours, or (B) cells were treated in fusion medium and assessed for myogenic differentiation after 5 days (*p≤0.05 Aged WT vs Treated; ANOVA followed by Tukey post-hoc analysis).</p

NF-κB inhibition improves the differentiation of MDSPCs isolated from old mice.

<p>(A) Brightfield images demonstrate a dose dependent increase in myotube formation following IKKi administration (10x images). (B) Immunofluorescent staining for MyHC demonstrated significantly improved differentiation following treatment at the 5μM dose (20x magnification), quantified in (C). (D) The total number of nuclei per field of view for experiment in B, C (*p≤0.05; ANOVA followed by Tukey post-hoc analysis). (E) IKKi treatment of young WT MDSPCs did not significantly improve differentiation (*p≤0.05, T-test).</p

NF-κB inhibition improves the oxidative stress resistance of MDSPCs isolated from old mice.

<p>MDSPCs were exposed to 250 μM H₂O₂ and monitored by live cell imaging for up to 24 hours. IKKi treated aged WT MDSPCs and aged <i>p65</i>^+/- MDSPCs demonstrated improved survival compared to vehicle treated aged WT MDSPCs (*p≤0.05 Aged WT vs +IKKi; +p≤0.05 Aged WT vs Aged <i>p65</i>^+/-; ANOVA followed by Tukey post-hoc analysis).</p