Critical Role of Astrocyte NAD+ Glycohydrolase in Myelin Injury and Regeneration.

Western-style diets cause disruptions in myelinating cells and astrocytes within the mouse CNS. Increased CD38 expression is present in the cuprizone and experimental autoimmune encephalomyelitis models of demyelination and CD38 is the main nicotinamide adenine dinucleotide (NAD+)-depleting enzyme in the CNS. Altered NAD+ metabolism is linked to both high fat consumption and multiple sclerosis (MS). Here, we identify increased CD38 expression in the male mouse spinal cord following chronic high fat consumption, after focal toxin [lysolecithin (LL)]-mediated demyelinating injury, and in reactive astrocytes within active MS lesions. We demonstrate that CD38 catalytically inactive mice are substantially protected from high fat-induced NAD+ depletion, oligodendrocyte loss, oxidative damage, and astrogliosis. A CD38 inhibitor, 78c, increased NAD+ and attenuated neuroinflammatory changes induced by saturated fat applied to astrocyte cultures. Conditioned media from saturated fat-exposed astrocytes applied to oligodendrocyte cultures impaired myelin protein production, suggesting astrocyte-driven indirect mechanisms of oligodendrogliopathy. In cerebellar organotypic slice cultures subject to LL-demyelination, saturated fat impaired signs of remyelination effects that were mitigated by concomitant 78c treatment. Significantly, oral 78c increased counts of oligodendrocytes and remyelinated axons after focal LL-induced spinal cord demyelination. Using a RiboTag approach, we identified a unique in vivo brain astrocyte translatome profile induced by 78c-mediated CD38 inhibition in mice, including decreased expression of proinflammatory astrocyte markers and increased growth factors. Our findings suggest that a high-fat diet impairs oligodendrocyte survival and differentiation through astrocyte-linked mechanisms mediated by the NAD+ase CD38 and highlights CD38 inhibitors as potential therapeutic candidates to improve myelin regeneration.SIGNIFICANCE STATEMENT Myelin disturbances and oligodendrocyte loss can leave axons vulnerable, leading to permanent neurologic deficits. The results of this study suggest that metabolic disturbances, triggered by consumption of a diet high in fat, promote oligodendrogliopathy and impair myelin regeneration through astrocyte-linked indirect nicotinamide adenine dinucleotide (NAD+)-dependent mechanisms. We demonstrate that restoring NAD+ levels via genetic inactivation of CD38 can overcome these effects. Moreover, we show that therapeutic inactivation of CD38 can enhance myelin regeneration. Together, these findings point to a new metabolic targeting strategy positioned to improve disease course in multiple sclerosis and other conditions in which the integrity of myelin is a key concern.

Characterization of a resident population of adventitial macrophage progenitor cells in postnatal vasculature.

RATIONALE: Macrophages regulate blood vessel structure and function in health and disease. The origins of tissue macrophages are diverse, with evidence for local production and circulatory renewal. OBJECTIVE: We identified a vascular adventitial population containing macrophage progenitor cells and investigated their origins and fate. METHODS AND RESULTS: Single-cell disaggregates from adult C57BL/6 mice were prepared from different tissues and tested for their capacity to form hematopoietic colony-forming units. Aorta showed a unique predilection for generating macrophage colony-forming units. Aortic macrophage colony-forming unit progenitors coexpressed stem cell antigen-1 and CD45 and were adventitially located, where they were the predominant source of proliferating cells in the aortic wall. Aortic Sca-1(+)CD45(+) cells were transcriptionally and phenotypically distinct from neighboring cells lacking stem cell antigen-1 or CD45 and contained a proliferative (Ki67(+)) Lin(-)c-Kit(+)CD135(-)CD115(+)CX3CR1(+)Ly6C(+)CD11b(-) subpopulation, consistent with the immunophenotypic profile of macrophage progenitors. Adoptive transfer studies revealed that Sca-1(+)CD45(+) adventitial macrophage progenitor cells were not replenished via the circulation from bone marrow or spleen, nor was their prevalence diminished by depletion of monocytes or macrophages by liposomal clodronate treatment or genetic deficiency of macrophage colony-stimulating factor. Rather adventitial macrophage progenitor cells were upregulated in hyperlipidemic ApoE(-/-) and LDL-R(-/-) mice, with adventitial transfer experiments demonstrating their durable contribution to macrophage progeny particularly in the adventitia, and to a lesser extent the atheroma, of atherosclerotic carotid arteries. CONCLUSIONS: The discovery and characterization of resident vascular adventitial macrophage progenitor cells provides new insight into adventitial biology and its participation in atherosclerosis and provokes consideration of the broader existence of local macrophage progenitors in other tissues.

Tissue factor pathway inhibitor blocks angiogenesis via its carboxyl terminus.

OBJECTIVE: Tissue factor pathway inhibitor (TFPI) is the primary regulator of the tissue factor (TF) coagulation pathway. As such, TFPI may regulate the proangiogenic effects of TF. TFPI may also affect angiogenesis independently of TF, through sequences within its polybasic carboxyl terminus (TFPI C terminus [TFPIct]). We aimed to determine the effects of TFPI on angiogenesis and the role of TFPIct. METHODS AND RESULTS: Transgenic overexpression of TFPI attenuated angiogenesis in the murine hindlimb ischemia model and an aortic sprout assay. In vitro, TFPI inhibited endothelial cell migration. Peptides within the human TFPIct inhibited endothelial cell cord formation and migration in response to vascular endothelial growth factor (VEGF) 165 but not VEGF121. Furthermore, exposure to human TFPIct inhibited the phosphorylation of VEGF receptor 2 at residue Lys951, a residue known to be critical for endothelial cell migration. Finally, systemic delivery of a murine TFPIct peptide inhibited angiogenesis in the hindlimb model. CONCLUSION: These data demonstrate an inhibitory role for TFPI in angiogenesis that is, in part, mediated through peptides within its carboxyl terminus. In addition to its known role as a TF antagonist, TFPI, via its carboxyl terminus, may regulate angiogenesis by directly blocking VEGF receptor 2 activation and attenuating the migratory capacity of endothelial cells.

Identification of a monocyte-predisposed hierarchy of hematopoietic progenitor cells in the adventitia of postnatal murine aorta.

BACKGROUND: Hematopoiesis originates from the dorsal aorta during embryogenesis. Although adult blood vessels harbor progenitor populations for endothelial and smooth muscle cells, it is not known if they contain hematopoietic progenitor or stem cells. Here, we hypothesized that the arterial wall is a source of hematopoietic progenitor and stem cells in postnatal life. METHODS AND RESULTS: Single-cell aortic disaggregates were prepared from adult chow-fed C57BL/6 and apolipoprotein E-null (ApoE(-/-)) mice. In short- and long-term methylcellulose-based culture, aortic cells generated a broad spectrum of multipotent and lineage-specific hematopoietic colony-forming units, with a preponderance of macrophage colony-forming units. This clonogenicity was higher in lesion-free ApoE(-/-) mice and localized primarily to stem cell antigen-1-positive cells in the adventitia. Expression of stem cell antigen-1 in the aorta colocalized with canonical hematopoietic stem cell markers, as well as CD45 and mature leukocyte antigens. Adoptive transfer of labeled aortic cells from green fluorescent protein transgenic donors to irradiated C57BL/6 recipients confirmed the content of rare hematopoietic stem cells (1 per 4 000 000 cells) capable of self-renewal and durable, low-level reconstitution of leukocytes. Moreover, the predominance of long-term macrophage precursors was evident by late recovery of green fluorescent protein-positive colonies from recipient bone marrow and spleen that were exclusively macrophage colony-forming units. Although trafficking from bone marrow was shown to replenish some of the hematopoietic potential of the aorta after irradiation, the majority of macrophage precursors appeared to arise locally, suggesting long-term residence in the vessel wall. CONCLUSIONS: The postnatal murine aorta contains rare multipotent hematopoietic progenitor/stem cells and is selectively enriched with stem cell antigen-1-positive monocyte/macrophage precursors. These populations may represent novel, local vascular sources of inflammatory cells.

Biodesign of a renal-protective peptide based on alternative splicing of B-type natriuretic peptide.

Alternative RNA splicing may provide unique opportunities to identify drug targets and therapeutics. We identified an alternative spliced transcript for B-type natriuretic peptide (BNP) resulting from intronic retention. This transcript is present in failing human hearts and is reduced following mechanical unloading. The intron-retained transcript would generate a unique 34 amino acid (aa) carboxyl terminus while maintaining the remaining structure of native BNP. We generated antisera to this carboxyl terminus and identified immunoreactivity in failing human heart tissue. The alternatively spliced peptide (ASBNP) was synthesized and unlike BNP, failed to stimulate cGMP in vascular cells or vasorelax preconstricted arterial rings. This suggests that ASBNP may lack the dose-limiting effects of recombinant BNP. Given structural considerations, a carboxyl-terminal truncated form of ASBNP was generated (ASBNP.1) and was determined to retain the ability of BNP to stimulate cGMP in canine glomerular isolates and cultured human mesangial cells but lacked similar effects in vascular cells. In a canine-pacing model of heart failure, systemic infusion of ASBNP.1 did not alter mean arterial pressure but increased the glomerular filtration rate (GFR), suppressed plasma renin and angiotensin, while inducing natriuresis and diuresis. Consistent with its distinct in vivo effects, the activity of ASBNP.1 may not be explained through binding and activation of NPR-A or NPR-B. Thus, the biodesigner peptide ASBNP.1 enhances GFR associated with heart failure while lacking the vasoactive properties of BNP. These findings demonstrate that peptides with unique properties may be designed based on products of alternatively splicing.

Distribution of circulation-derived endothelial progenitors following systemic delivery.

Cells with an endothelial phenotype can be cultured from peripheral blood. These cells include cells of a monocytic origin with endothelial features (culture-modified mononuclear cells, CMMCs) and, at later time points, blood outgrowth endothelial cells (BOECs). Both are promising candidates for systemic cell-based cardiovascular therapies and each may have unique capabilities. Indeed, the combined use of both cell types has been shown to have synergistic therapeutic features requiring simultaneous delivery. However, the majority of preclinical studies of cell delivery have used splenectomized animals to increase systemic distribution. The goal of this study was to directly compare the distribution of these two cell types following systemic delivery in an intact animal model. A similar pattern of delivery was seen following delivery of both cell types with detection in the lung, liver, bone marrow, and spleen. Taken together, the data suggest that strategies using systemic delivery of circulation-derived cells must consider the distribution and efficiency of delivery in intact animals.

Use of blood outgrowth endothelial cells as virus-producing vectors for gene delivery to tumors.

Cell-based delivery of therapeutic viruses has potential advantages over systemic viral administration, including attenuated neutralization and improved viral targeting. One of the exciting new areas of investigation is the potential ability of endothelial-lineage cells to deliver genes to the areas of neovascularization. In the present study, we compared two types of endothelial-lineage cells [outgrowth endothelial cells (OECs) and culture-modified mononuclear cells (CMMCs), also known as "endothelial progenitor cells"] for their ability to be infected with adenovirus and to home to the areas of neovascularization. Both cell types were isolated from peripheral blood of healthy human donors and expanded in culture. We demonstrate that OECs are more infectable and home better to tumors expressing VEGF on systemic administration. Furthermore, we used an adenoviral/retroviral chimeric system to convert OECs to retrovirus-producing cells. When injected systemically into tumor-bearing mice, OECs retain their ability to produce retrovirus and infect surrounding tumor cells. Our data demonstrate that OECs could be efficient carriers for viral delivery to areas of tumor neovascularization.

Modulation of the vascular response to injury by autologous blood-derived outgrowth endothelial cells.

Delivery of a heterogeneous population of cells with endothelial phenotype derived from peripheral blood has been shown to improve vascular responses after balloon arterial injury in an endothelium-dependent manner. Refinement of culture techniques has enabled the generation of outgrowth endothelial cells (OECs), a homogeneous population of distinctly endothelial cells expanded from circulating progenitor cells. The present study tested the hypothesis that OEC delivery would confer vascular protection after balloon arterial injury in a rabbit model. Rabbit peripheral blood mononuclear cells (PBMCs) were cultured in endothelial growth medium for 4-5 wk, yielding proliferative OECs with distinct endothelial phenotype (morphology, incorporation of acetylated LDL, and expression of endothelial nitric oxide synthase and caveolin-1 but not CD14). Animals underwent balloon carotid injury immediately followed by local delivery of autologous OECs for 20 min. Fluorescent-labeled OECs were detected in all layers at 4 wk, with immunostaining revealing maintenance of endothelial phenotype (von Willebrand factor-positive and RAM-11-negative) by luminal and nonluminal cells. To evaluate functional effects, additional animals received autologous OECs, saline, or freshly harvested PBMCs as noncultured cell controls by local dwell after balloon injury. Local OEC delivery improved endothelium-dependent vasoreactivity (P < 0.05 vs. saline and PBMC) and similarly reduced neointimal formation (P < 0.05 vs. saline and PBMC). These data suggest that OECs can be detected in injured arterial segments at 4 wk. Moreover, delivery of OECs confers greater vascular protection than PBMCs or saline controls and may thus offer a novel, autologous strategy to limit the response to mechanical injury.

Caveolin-1 can regulate vascular smooth muscle cell fate by switching platelet-derived growth factor signaling from a proliferative to an apoptotic pathway.

BACKGROUND: Caveolin-1 is a regulator of signaling events originating from plasma membrane microdomains termed caveolae. This study was performed to determine the regulatory role of caveolin-1 on the proliferative events induced by platelet-derived growth factor (PDGF) in vascular smooth muscle cells (VSMCs). METHODS AND RESULTS: Treatment of VSMCs with PDGF for 24 hours resulted in a loss of caveolin-1 protein expression and plasma membrane-associated caveolae, despite a 3-fold increase in caveolin-1 mRNA. Pretreatment of VSMCs with chloroquine, an inhibitor of lysosomal function, inhibited the PDGF-induced loss of caveolin-1. These studies demonstrated that caveolin-1 was a target of PDGF signaling events. Adenoviral overexpression of caveolin-1 was associated with a switch in PDGF-induced signaling events from a proliferative response to an apoptotic response. This overexpression inhibited PDGF-induced expression of cyclin D1 in the presence of unaffected mitogen-activated protein kinase activation. CONCLUSIONS: Taken together, these studies suggest that caveolin-1 is an inhibitor of PDGF proliferative responses and might be capable of transforming PDGF-induced proliferative signals into death signals.