Summary Nicotinamide Adenine Dinucleotide (NAD) levels decrease during aging, and are involved in age-related metabolic decline. To date, the mechanism responsible for the age-related reduction in NAD has not been elucidated. Here we demonstrate that expression and activity of the NADase CD38 increase with aging and that CD38 is required for the age-related NAD decline and mitochondrial dysfunction via a pathway mediated at least in part by regulation of SIRT3 activity. We also identified CD38 as the main enzyme involved in the degradation of the NAD precursor nicotinamide mononucleotide (NMN) in vivo, indicating that CD38 has a key role in the modulation of NAD-replacement therapy for aging and metabolic diseases.

Mesenchymal stem/stromal cells (MSCs) have distinct capability for renal repair, but may have safety concerns. MSC-derived extracellular vesicles emerged as a novel non-cellular alternative. Using a porcine model of metabolic syndrome and renal artery stenosis we tested whether extracellular vesicles attenuate renal inflammation, and if this capacity is mediated by their cargo of the anti-inflammatory cytokine interleukin (IL)10. Pigs with metabolic syndrome were studied after 16 weeks of renal artery stenosis untreated or treated four weeks earlier with a single intrarenal delivery of extracellular vesicles harvested from adipose tissue-derived autologous MSCs. Lean and sham metabolic syndrome animals served as controls (seven each). Five additional pigs with metabolic syndrome and renal artery stenosis received extracellular vesicles with pre-silenced IL10 (IL10 knock-down). Single-kidney renal blood flow, glomerular filtration rate, and oxygenation were studied in vivo and renal injury pathways ex vivo. Retention of extracellular vesicles in the stenotic-kidney peaked two days after delivery and decreased thereafter. Four weeks after injection, extracellular vesicle fragments co-localized with stenotic-kidney tubular cells and macrophages, indicating internalization or fusion. Extracellular vesicle delivery attenuated renal inflammation, improved medullary oxygenation and fibrosis. Renal blood flow and glomerular filtration rate fell in metabolic syndrome and renal artery stenosis compared to metabolic syndrome, but was restored in pigs treated with extracellular vesicles. These renoprotective effects were blunted in pigs treated with IL10-depleted extracellular vesicles. Thus, extracellular vesicle-based regenerative strategies might be useful for patients with metabolic syndrome and renal artery stenosis.

Decreased nicotinamide adenine dinucleotide (NAD + ) levels have been shown to contribute to metabolic dysfunction during aging. NAD + decline can be partially prevented by knockout of the enzyme CD38. However, it is not known how CD38 is regulated during aging, and how its ecto-enzymatic activity impacts NAD + homeostasis. Here we show that increases in CD38 in white adipose tissue (WAT) and liver during aging is mediated by accumulation of CD38 + immune cells. Inflammation increases CD38 and decreases NAD + . In addition, senescent cells and their secreted signals promote accumulation of CD38 + cells in WAT, and ablation of senescent cells or their secretory phenotype decrease CD38, partially reversing NAD + decline. Finally, blocking the ecto-enzymatic activity of CD38 can increase NAD + through a nicotinamide mononucleotide (NMN)-dependent process. Our findings demonstrate that senescence-induced inflammation promotes accumulation of CD38 in immune cells that through its ecto-enzymatic activity decreases levels of NMN and NAD + .

Extracellular vesicles (EVs) isolated from mesenchymal stem/stromal cells (MSCs) contribute to recovery of damaged tissue. We have previously shown that porcine MSC-derived EVs transport mRNA and miRNA capable of modulating cellular pathways in recipient cells. To identify candidate factors that contribute to the therapeutic effects of porcine MSC-derived EVs, we characterized their protein cargo using proteomics. Porcine MSCs were cultured from abdominal fat, and EVs characterized for expression of typical MSC and EV markers. LC-MS/MS proteomic analysis was performed and proteins classified. Functional pathway analysis was performed and five candidate proteins were validated by western blot. Proteomics analysis identified 5,469 distinct proteins in MSCs and 4,937 in EVs. The average protein expression was higher in MSCs vs. EVs. Differential expression analysis revealed 128 proteins that are selectively enriched in EVs versus MSCs, whereas 563 proteins were excluded from EVs. Proteins enriched in EVs are linked to a broad range of biological functions, including angiogenesis, blood coagulation, apoptosis, extracellular matrix remodeling, and regulation of inflammation. Excluded are mostly nuclear proteins, like proteins involved in nucleotide binding and RNA splicing. EVs have a selectively-enriched protein cargo with a specific biological signature that MSCs may employ for intercellular communication to facilitate tissue repair.

Classically activated, inflammatory macrophages are generally considered to derive from circulating monocytes, which descend from hematopoietic stem and progenitor cells (HPCs) S).The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl

BackgroundMesenchymal stromal/stem cell (MSC) transplantation is a promising therapy for tissue regeneration. Extracellular vesicles (EVs) released by MSCs act as their paracrine effectors by delivering proteins and genetic material to recipient cells. To assess how their cargo mediates biological processes that drive their therapeutic effects, we integrated miRNA, mRNA, and protein expression data of EVs from porcine adipose tissue-derived MSCs.MethodsSimultaneous expression profiles of miRNAs, mRNAs, and proteins were obtained by high-throughput sequencing and LC-MS/MS proteomic analysis in porcine MSCs and their daughter EVs (n = 3 each). TargetScan and ComiR were used to predict miRNA target genes. Functional annotation analysis was performed using DAVID 6.7 database to rank primary gene ontology categories for the enriched mRNAs, miRNA target genes, and proteins. STRING was used to predict associations between mRNA and miRNA target genes.ResultsDifferential expression analysis revealed 4 miRNAs, 255 mRNAs, and 277 proteins enriched in EVs versus MSCs (fold change >2, p<0.05). EV-enriched miRNAs target transcription factors (TFs) and EV-enriched mRNAs encode TFs, but TF proteins are not enriched in EVs. Rather, EVs are enriched for proteins that support extracellular matrix remodeling, blood coagulation, inflammation, and angiogenesis.ConclusionsPorcine MSC-derived EVs contain a genetic cargo of miRNAs and mRNAs that collectively control TF activity in EVs and recipient cells, as well as proteins capable of modulating cellular pathways linked to tissue repair. These properties provide the fundamental basis for considering therapeutic use of EVs in tissue regeneration.

Renal artery stenosis (RAS) caused by narrowing of arteries is characterized by microvascular damage. Macrophages are implicated in repair and injury, but the specific populations responsible for these divergent roles have not been identified. Here, we characterized murine kidney F4/80+CD64+ macrophages in three transcriptionally unique populations. Using fate-mapping and parabiosis studies, we demonstrate that CD11b/cint are long-lived kidney-resident (KRM) while CD11chiMϕ, CD11cloMϕ are monocyte-derived macrophages. In a murine model of RAS, KRM self-renewed, while CD11chiMϕ and CD11cloMϕ increased significantly, which was associated with loss of peritubular capillaries. Replacing the native KRM with monocyte-derived KRM using liposomal clodronate and bone marrow transplantation followed by RAS, amplified loss of peritubular capillaries. To further elucidate the nature of interactions between KRM and peritubular endothelial cells, we performed RNA-sequencing on flow-sorted macrophages from Sham and RAS kidneys. KRM showed a prominent activation pattern in RAS with significant enrichment in reparative pathways, like angiogenesis and wound healing. In culture, KRM increased proliferation of renal peritubular endothelial cells implying direct pro-angiogenic properties. Human homologs of KRM identified as CD11bintCD11cintCD68+ increased in post-stenotic kidney biopsies from RAS patients compared to healthy human kidneys, and inversely correlated to kidney function. Thus, KRM may play protective roles in stenotic kidney injury through expansion and upregulation of pro-angiogenic pathways.

People in developing countries have faced multigenerational undernutrition and are currently undergoing major lifestyle changes, contributing to an epidemic of metabolic diseases, though the underlying mechanisms remain unclear. Using a Wistar rat model of undernutrition over 50 generations, we show that Undernourished rats exhibit low birth-weight, high visceral adiposity (DXA/MRI), and insulin resistance (hyperinsulinemic-euglycemic clamps), compared to age-/gender-matched control rats. Undernourished rats also have higher circulating insulin, homocysteine, endotoxin and leptin levels, lower adiponectin, vitamin B12 and folate levels, and an 8-fold increased susceptibility to Streptozotocin-induced diabetes compared to control rats. Importantly, these metabolic abnormalities are not reversed after two generations of unrestricted access to commercial chow (nutrient recuperation). Altered epigenetic signatures in insulin-2 gene promoter region of Undernourished rats are not reversed by nutrient recuperation, and may contribute to the persistent detrimental metabolic profiles in similar multigenerational undernourished human populations.