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Emerging data suggest that hypercholesterolemia has stimulatory effects on adaptive immunity and that these effects can promote atherosclerosis and perhaps other inflammatory diseases. However, research in this area has relied primarily on inbred strains of mice whose adaptive immune system can differ substantially from that of humans. Moreover, the genetically induced hypercholesterolemia in these models typically results in plasma cholesterol levels that are much higher than those in most humans. To overcome these obstacles, we studied human immune system-reconstituted mice (hu-mice) rendered hypercholesterolemic by treatment with adeno-associated virus 8-proprotein convertase subtilisin/kexin type 9 (AAV8-PCSK9) and a high-fat/high-cholesterol Western-type diet (WD). These mice had a high percentage of human T cells and moderate hypercholesterolemia. Compared with hu-mice that had lower plasma cholesterol, the PCSK9-WD mice developed a T cell-mediated inflammatory response in the lung and liver. Human CD4+ and CD8+ T cells bearing an effector memory phenotype were significantly elevated in the blood, spleen, and lungs of PCSK9-WD hu-mice, whereas splenic and circulating regulatory T cells were reduced. These data show that moderately high plasma cholesterol can disrupt human T cell homeostasis in vivo. This process may not only exacerbate atherosclerosis, but also contribute to T cell-mediated inflammatory diseases in the hypercholesterolemia setting.

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.

Duchenne muscular dystrophy (DMD) patients lack dystrophin from birth; however, muscle weakness becomes apparent only at 3–5 years of age, which happens to coincide with the depletion of the muscle progenitor cell (MPC) pools. Indeed, MPCs isolated from older DMD patients demonstrate impairments in myogenic potential. To determine whether the progression of muscular dystrophy is a consequence of the decline in functional MPCs, we investigated two animal models of DMD: (i) dystrophin-deficient mdx mice, the most commonly utilized model of DMD, which has a relatively mild dystrophic phenotype and (ii) dystrophin/utrophin double knock-out (dKO) mice, which display a similar histopathologic phenotype to DMD patients. In contrast to age-matched mdx mice, we observed that both the number and regeneration potential of dKO MPCs rapidly declines during disease progression. This occurred in MPCs at both early and late stages of myogenic commitment. In fact, early MPCs isolated from 6-week-old dKO mice have reductions in proliferation, resistance to oxidative stress and multilineage differentiation capacities compared with age-matched mdx MPCs. This effect may potentially be mediated by fibroblast growth factor overexpression and/or a reduction in telomerase activity. Our results demonstrate that the rapid disease progression in the dKO model is associated, at least in part, with MPC depletion. Therefore, alleviating MPC depletion could represent an approach to delay the onset of the histopathologies associated with DMD patients.

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.

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.

Chronic inflammation, promoted by an upregulated NF-kappa B (NF-κB) pathway, has a key role in Duchenne muscular dystrophy (DMD) patients' pathogenesis. Blocking the NF-κB pathway has been shown to be a viable approach to diminish chronic inflammation and necrosis in the dystrophin-defective mdx mouse, a murine DMD model. In this study, we used the recombinant adeno-associated virus serotype 9 (AAV9) carrying an short hairpin RNA (shRNA) specifically targeting the messenger RNA of NF-κB/p65 (p65-shRNA), the major subunit of NF-κB associated with chronic inflammation in mdx mice. We examined whether i.m. AAV9-mediated delivery of p65-shRNA could decrease NF-κB activation, allowing for amelioration of muscle pathologies in 1- and 4-month-old mdx mice. At 1 month after treatment, NF-κB/p65 levels were significantly decreased by AAV gene transfer of p65-shRNA in the two ages of treatment groups, with necrosis significantly decreased compared with controls. Quantitative analysis revealed that central nucleation (CN) of the myofibers of p65-shRNA-treated 1-month-old mdx muscles was reduced from 67 to 34%, but the level of CN was not significantly decreased in treated 4-month-old mdx mice. Moreover, delivery of the p65-shRNA enhanced the capacity of myofiber regeneration in old mdx mice treated at 4 months of age when the dystrophic myofibers were most exhausted; however, such p65 silencing diminished the myofiber regeneration in young mdx mice treated at 1 month of age. Taken together, these findings demonstrate that the AAV-mediated delivery of p65-shRNA has the capacity to ameliorate muscle pathologies in mdx mice by selectively reducing NF-κB/p65 activity.

Murine muscle-derived stem cells (MDSCs) have been shown capable of regenerating bone in a critical size calvarial defect model when transduced with BMP 2 or 4; however, the contribution of the donor cells and their interactions with the host cells during the bone healing process have not been fully elucidated. To address this question, C57/BL/6J mice were divided into MDSC/BMP4/GFP, MDSC/GFP, and scaffold groups. After transplanting MDSCs into the critical-size calvarial defects created in normal mice, we found that mice transplanted with BMP4GFP-transduced MDSCs healed the bone defect in 4 wk, while the control groups (MDSC-GFP and scaffold) demonstrated no bone healing. The newly formed trabecular bone displayed similar biomechanical properties as the native bone, and the donor cells directly participated in endochondral bone formation via their differentiation into chondrocytes, osteoblasts, and osteocytes via the BMP4-pSMAD5 and COX-2-PGE2 signaling pathways. In contrast to the scaffold group, the MDSC groups attracted more inflammatory cells initially and incurred faster inflammation resolution, enhanced angiogenesis, and suppressed initial immune responses in the host mice. MDSCs were shown to attract macrophages via the secretion of monocyte chemotactic protein 1 and promote endothelial cell proliferation by secreting multiple growth factors. Our findings indicated that BMP4GFP-transduced MDSCs not only regenerated bone by direct differentiation, but also positively influenced the host cells to coordinate and promote bone tissue repair through paracrine effects.—Gao, X., Usas, A., Proto, J. D., Lu, A., Cummins, J. H., Proctor, A., Chen, C.-W., Huard, J. Role of donor and host cells in muscle-derived stem cell-mediated bone repair: differentiation vs. paracrine effects.

Human microvascular pericytes (CD146+/34−/45−/56−) contain multipotent precursors and repair/regenerate defective tissues, notably skeletal muscle. However, their ability to repair the ischemic heart remains unknown. We investigated the therapeutic potential of human pericytes, purified from skeletal muscle, for treating ischemic heart disease and mediating associated repair mechanisms in mice. Echocardiography revealed that pericyte transplantation attenuated left ventricular dilatation and significantly improved cardiac contractility, superior to CD56+ myogenic progenitor transplantation, in acutely infarcted mouse hearts. Pericyte treatment substantially reduced myocardial fibrosis and significantly diminished infiltration of host inflammatory cells at the infarct site. Hypoxic pericyte-conditioned medium suppressed murine fibroblast proliferation and inhibited macrophage proliferation in vitro. High expression by pericytes of immunoregulatory molecules, including interleukin-6, leukemia inhibitory factor, cyclooxygenase-2, and heme oxygenase-1, was sustained under hypoxia, except for monocyte chemotactic protein-1. Host angiogenesis was significantly increased. Pericytes supported microvascular structures in vivo and formed capillary-like networks with/without endothelial cells in three-dimensional cocultures. Under hypoxia, pericytes dramatically increased expression of vascular endothelial growth factor-A, platelet-derived growth factor-β, transforming growth factor-β1 and corresponding receptors while expression of basic fibroblast growth factor, hepatocyte growth factor, epidermal growth factor, and angiopoietin-1 was repressed. The capacity of pericytes to differentiate into and/or fuse with cardiac cells was revealed by green fluorescence protein labeling, although to a minor extent. In conclusion, intramyocardial transplantation of purified human pericytes promotes functional and structural recovery, attributable to multiple mechanisms involving paracrine effects and cellular interactions.

Inhibition of the inhibitor of kappa B kinase (IKK)/nuclear factor-kappa B (NF-κB) pathway enhances muscle regeneration in injured and diseased skeletal muscle, but it is unclear exactly how this pathway contributes to the regeneration process. In this study, we examined the role of NF-κB in regulating the proliferation and differentiation of muscle-derived stem cells (MDSCs). MDSCs isolated from the skeletal muscles of p65(+/-) mice (haploinsufficient for the p65 subunit of NF-κB) had enhanced proliferation and myogenic differentiation compared to MDSCs isolated from wild-type (wt) littermates. In addition, selective pharmacological inhibition of IKKβ, an upstream activator of NF-κB, enhanced wt MDSC differentiation into myotubes in vitro. The p65(+/-) MDSCs also displayed a higher muscle regeneration index than wt MDSCs following implantation into adult mice with muscular dystrophy. Additionally, using a muscle injury model, we observed that p65(+/-) MDSC engraftments were associated with reduced inflammation and necrosis. These results suggest that inhibition of the IKK/NF-κB pathway represents an effective approach to improve the myogenic regenerative potential of MDSCs and possibly other adult stem cell populations. Moreover, our results suggest that the improved muscle regeneration observed following inhibition of IKK/NF-κB, is mediated, at least in part, through enhanced stem cell proliferation and myogenic potential.