Generation of Cardiomyocytes From Vascular Adventitia-Resident Stem Cells.

RATIONALE: Regeneration of lost cardiomyocytes is a fundamental unresolved problem leading to heart failure. Despite several strategies developed from intensive studies performed in the past decades, endogenous regeneration of heart tissue is still limited and presents a big challenge that needs to be overcome to serve as a successful therapeutic option for myocardial infarction. OBJECTIVE: One of the essential prerequisites for cardiac regeneration is the identification of endogenous cardiomyocyte progenitors and their niche that can be targeted by new therapeutic approaches. In this context, we hypothesized that the vascular wall, which was shown to harbor different types of stem and progenitor cells, might serve as a source for cardiac progenitors. METHODS AND RESULTS: We describe generation of spontaneously beating mouse aortic wall-derived cardiomyocytes without any genetic manipulation. Using aortic wall-derived cells (AoCs) of WT (wild type), αMHC (α-myosin heavy chain), and Flk1 (fetal liver kinase 1)-reporter mice and magnetic bead-associated cell sorting sorting of Flk1+ AoCs from GFP (green fluorescent protein) mice, we identified Flk1+CD (cluster of differentiation) 34+Sca-1 (stem cell antigen-1)-CD44- AoCs as the population that gives rise to aortic wall-derived cardiomyocytes. This AoC subpopulation delivered also endothelial cells and macrophages with a particular accumulation within the aortic wall-derived cardiomyocyte containing colonies. In vivo, cardiomyocyte differentiation capacity was studied by implantation of fluorescently labeled AoCs into chick embryonic heart. These cells acquired cardiomyocyte-like phenotype as shown by αSRA (α-sarcomeric actinin) expression. Furthermore, coronary adventitial Flk1+ and CD34+ cells proliferated, migrated into the myocardium after mouse myocardial infarction, and expressed Isl-1+ (insulin gene enhancer protein-1) indicative of cardiovascular progenitor potential. CONCLUSIONS: Our data suggest Flk1+CD34+ vascular adventitia-resident stem cells, including those of coronary adventitia, as a novel endogenous source for generating cardiomyocytes. This process is essentially supported by endothelial cells and macrophages. In summary, the therapeutic manipulation of coronary adventitia-resident cardiac stem and their supportive cells may open new avenues for promoting cardiac regeneration and repair after myocardial infarction and for preventing heart failure.

Adventitial Cell Atlas of wt (Wild Type) and ApoE (Apolipoprotein E)-Deficient Mice Defined by Single-Cell RNA Sequencing.

Objective- Vascular adventitia encompasses progenitors and is getting recognized as the major site of inflammation in early stage of atherosclerosis. However, the cellular atlas of the heterogeneous adventitial cells, the intercellular communication, the cellular response of adventitia to hyperlipidemia, and its contribution to atherosclerosis have been elusive. Approach and Results- Single-cell RNA sequencing was applied to wt (wild type) and ApoE (apolipoprotein E)-deficient aortic adventitia from 12-week-old C57BL/6J mice fed on normal laboratory diet with early stage of atherosclerosis. Unbiased clustering analysis revealed that the landscape of adventitial cells encompassed adventitial mesenchyme cells, immune cells (macrophages, T cells, and B cells), and some types of rare cells, for example, neuron, lymphatic endothelial cells, and innate lymphoid cells. Seurat clustering analysis singled out 6 nonimmune clusters with distinct transcriptomic profiles, in which there predominantly were stem/progenitor cell-like and proinflammatory population (Mesen II). In ApoE-deficient adventitia, resident macrophages were activated and related to increased myeloid cell infiltration in the adventitia. Cell communication analysis further elucidated enhanced interaction between a mesenchyme cluster and inflammatory macrophages in ApoE-deficient adventitia. In vitro transwell assay confirmed the proinflammatory role of SCA1+ (stem cell antigen 1 positive) Mesen II population with increased CCL2 (chemokine [C-C motif] ligand 2) secretion and thus increased capacity to attract immune cells in ApoE-deficient adventitia. Conclusions- Cell atlas defined by single-cell RNA sequencing depicted the heterogeneous cellular landscape of the adventitia and uncovered several types of cell populations. Furthermore, resident cell interaction with immune cells appears crucial at the early stage of atherosclerosis.

FSP1 promotes the biofunctions of adventitial fibroblast through the crosstalk among RAGE, JAK2/STAT3 and Wnt3a/ß-catenin signalling pathways.

Emerging evidence indicates that fibroblast-specific protein 1 (FSP1) provides vital effects in cell biofunctions. However, whether FSP1 influences the adventitial fibroblast (AF) and vascular remodelling remains unclear. Therefore, we investigated the potential role and action mechanism of FSP1-mediated AF bioactivity. AFs were cultured and stimulated with FSP1 and siRNA-FSP1 in vitro. Viability assays demonstrated that siRNA-FSP1 counteracted AFs proliferative, migratory and adherent abilities enhanced with FSP1. Flow cytometry revealed that FSP1 increased AFs number in S phase and decreased cellular apoptosis. Contrarily, siRNA-FSP1 displayed the contrary results. RT-PCR, Western blotting and immunocytochemistry showed that FSP1 synchronously up-regulated the expression of molecules in RAGE, JAK2/STAT3 and Wnt3a/ß-catenin pathways and induced a proinflammatory cytokine profile characterized by high levels of MCP-1, ICAM-1 and VCAM-1. Conversely, FSP1 knockdown reduced the expression of these molecules and cytokines. The increased number of autophagosomes in FSP1-stimulated group and fewer autophagic corpuscles in siRNA-FSP1 group was observed by transmission electron microscope (TEM). Autophagy-related proteins (LC3B, beclin-1 and Apg7) were higher in FSP1 group than those in other groups. Conversely, the expression of p62 protein was shown an opposite trend of variation. Therefore, these pathways can promote AFs bioactivity, facilitate autophagy and induce the expression of the proinflammatory cytokines. Contrarily, siRNA-FSP1 intercepts the crosstalk of these pathways, suppresses AF functions, restrains autophagy and attenuates the expression of the inflammatory factors. Our findings indicate that crosstalk among RAGE, STAT3/JAK2 and Wnt3a/ß-catenin signalling pathways may account for the mechanism of AF functions with the stimulation of FSP1.

Differentiated Smooth Muscle Cells Generate a Subpopulation of Resident Vascular Progenitor Cells in the Adventitia Regulated by Klf4.

RATIONALE: The vascular adventitia is a complex layer of the vessel wall consisting of vasa vasorum microvessels, nerves, fibroblasts, immune cells, and resident progenitor cells. Adventitial progenitors express the stem cell markers, Sca1 and CD34 (adventitial sca1-positive progenitor cells [AdvSca1]), have the potential to differentiate in vitro into multiple lineages, and potentially contribute to intimal lesions in vivo. OBJECTIVE: Although emerging data support the existence of AdvSca1 cells, the goal of this study was to determine their origin, degree of multipotency and heterogeneity, and contribution to vessel remodeling. METHODS AND RESULTS: Using 2 in vivo fate-mapping approaches combined with a smooth muscle cell (SMC) epigenetic lineage mark, we report that a subpopulation of AdvSca1 cells is generated in situ from differentiated SMCs. Our data establish that the vascular adventitia contains phenotypically distinct subpopulations of progenitor cells expressing SMC, myeloid, and hematopoietic progenitor-like properties and that differentiated SMCs are a source to varying degrees of each subpopulation. SMC-derived AdvSca1 cells exhibit a multipotent phenotype capable of differentiating in vivo into mature SMCs, resident macrophages, and endothelial-like cells. After vascular injury, SMC-derived AdvSca1 cells expand in number and are major contributors to adventitial remodeling. Induction of the transcription factor Klf4 in differentiated SMCs is essential for SMC reprogramming in vivo, whereas in vitro approaches demonstrate that Klf4 is essential for the maintenance of the AdvSca1 progenitor phenotype. CONCLUSIONS: We propose that generation of resident vascular progenitor cells from differentiated SMCs is a normal physiological process that contributes to the vascular stem cell pool and plays important roles in arterial homeostasis and disease.

A single nucleotide polymorphism of cyclin-dependent kinase inhibitor 1B (p27Kip1) associated with human vein graft failure affects growth of human venous adventitial cells but not smooth muscle cells.

BACKGROUND: Cyclin-dependent kinase inhibitor 1B (p27Kip1) is a cell-cycle inhibitor whose -838C>A single nucleotide polymorphism (rs36228499; hereafter called p27 SNP) has been associated with the clinical failure of peripheral vein grafts, but the functional effects of this SNP have not been demonstrated. METHODS: Human saphenous vein adventitial cells and intimal/medial smooth muscle cells (SMCs) were derived from explants obtained at the time of lower extremity bypass operations. We determined the following in adventitial cells and SMCs as a function of the p27 SNP genotype: (1) p27 promoter activity, (2) p27 messenger (m)RNA and protein levels, and (3) growth and collagen gel contraction. Deoxyribonuclease I footprinting was also performed in adventitial cells and SMCs. RESULTS: p27 promoter activity, deoxyribonuclease I footprinting, p27 mRNA levels, and p27 protein levels demonstrated that the p27 SNP is functional in adventitial cells and SMCs. Both cell types with the graft failure protective AA genotype had more p27 mRNA and protein. As predicted because of higher levels of p27 protein, adventitial cells with the AA genotype grew slower than those of the CC genotype. Unexpectedly, SMCs did not show this genotype-dependent growth response. CONCLUSIONS: These results support the functionality of the p27 SNP in venous SMCs and adventitial cells, but an effect of the SNP on cell proliferation is limited to only adventitial cells. These data point to a potential role for adventitial cells in human vein graft failure and also suggest that SMCs express factors that interfere with the activity of p27.

Hydroxytyrosol regulates the autophagy of vascular adventitial fibroblasts through the SIRT1-mediated signaling pathway.

Hydroxytyrosol (HT), a phenolic compound in olive oil, exerts an anti-inflammatory effect in cardiovascular diseases. Recent studies found that autophagy was a therapeutic target of diseases. However, the effect of HT on autophagy in vascular adventitial fibroblasts (VAFs) remains unknown. Thus, in this study, we aimed to determine the effect of HT on cell autophagy and related signaling pathway and whether HT regulates the inflammatory response through autophagy in VAFs. Our results showed that HT promoted cell autophagy by increasing the conversion of LC3 and Beclin1 expression and the autophagic flux in VAFs stimulated with tumor necrosis factor-α (TNF-α). HT also upregulated the expression of the deacetylase sirtuin 1 (SIRT1) protein and mRNA compared with the TNF-α group. The molecular docking studies showed the good compatibility between HT and SIRT1, indicating that HT might act through SIRT1. Further study found that HT regulated autophagy through SIRT1-mediated Akt/mTOR suppression in VAFs. In addition, HT inhibited TNF-α-induced inflammatory response in VAFs through SIRT1. Furthermore, the study showed that HT inhibited the inflammatory response of VAFs through autophagy. These findings indicate that HT regulates the autophagy of VAFs through SIRT1-mediated Akt/mTOR suppression and then inhibits the inflammatory response of VAFs.

Adipose-derived Human Perivascular Stem Cells May Improve Achilles Tendon Healing in Rats.

BACKGROUND: Achilles tendon rupture is a common injury and the best treatment option remains uncertain between surgical and nonoperative methods. Biologic approaches using multipotent stem cells such as perivascular stem cells pose a possible treatment option, although there is currently a paucity of evidence regarding their clinical therapeutic use. QUESTIONS/PURPOSES: The purpose of this study was to determine whether injected perivascular stem cells (PSCs) would (1) improve histologic signs of tendon healing (such as percent area of collagen); and (2) improve biomechanical properties (peak load or stiffness) in a rat model of Achilles tendon transection. METHODS: Two subtypes of PSCs were derived from human adipose tissue: pericytes (CD146CD34CD45CD31) and adventitial cells (CD146CD34CD45CD31). Thirty-two athymic rats underwent right Achilles transection and were randomized to receive injection with saline (eight tendons), hydrogel (four tendons), pericytes in hydrogel (four tendons), or adventitial cells in hydrogel (eight tendons) 3 days postoperatively with the left serving as an uninjured control. Additionally, a subset of pericytes was labeled with CM-diI to track cell viability and localization. At 3 weeks, the rats were euthanized, and investigators blinded to treatment group allocation evaluated tendon healing by peak load and stiffness using biomechanical testing and percent area of collagen using histologic analysis with picrosirius red staining. RESULTS: Histologic analysis showed a higher mean percent area collagen for pericytes (30%) and adventitial cells (28%) than hydrogel (21%) or saline (26%). However, a nonparametric statistical analysis yielded no statistical difference. Mechanical testing demonstrated that the pericyte group had a higher peak load than the saline group (41 ± 7 N versus 26 ± 9 N; mean difference 15 N; 95% confidence interval [CI], 4-27 N; p = 0.003) and a higher peak load than the hydrogel group (41 ± 7 N versus 25 ± 3 N; mean difference 16; 95% CI, 8-24 N; p = 0.001). The pericyte group demonstrated higher stiffness than the hydrogel group (36 ± 12 N/mm versus 17 ± 6 N/mm; mean difference 19 N/mm; 95% CI, 5-34 N/mm; p = 0.005). CONCLUSIONS: Our results suggest that injection of PSCs improves mechanical but not the histologic properties of early Achilles tendon healing. CLINICAL RELEVANCE: This is a preliminary study that provides more insight into the use of adipose-derived PSCs as a percutaneous therapy in the setting of Achilles tendon rupture. Further experiments to characterize the function of these cells may serve as a pathway to development of minimally invasive intervention aimed at improving nonoperative management while avoiding the complications associated with surgical treatment down the line.

The sheath of Waldeyer is not a specific anatomical trait of the ureterovesical junction.

The function of the ureterovesical junction depends upon a peculiar structure, the adventitial fibromuscular sheath of Waldeyer, which coats the distal end of the ureter. The origin of the smooth muscle of Waldeyer's sheath (WS) is disputed. Evidence points more likely to an ureteral one. In this regard we hypothesized the WS is not specific to the distal ureter but is rather a common trait. We therefore aimed at exploring whether or not the proximal ureter is provided with a similar adventitial fibromuscular coat. We performed an immunohistochemical study on human samples of proximal ureter resulted after nephrectomies in ten patients. We applied myoid immunohistochemical markers: α-smooth muscle actin (α-SMA), desmin, and heavy chain of smooth muscle myosin (SMM) which labeled additional adventitial smooth muscle bundles, a discontinuous inner circular one applied on the muscular coat, and outer longitudinal cords specifically located on one side of the ureter, as is the case for WS. Moreover, the lamina propria myoid deep layer showed isolated smooth muscle fibers and spindle-shaped stromal cells with telocyte morphology. Our results support the idea that WS may not be a specific structure of the distal ureter, instead being just a common anatomical characteristic of the ureter.

Endothelial Cells Inhibit the Angiotensin II Induced Phenotypic Modulation of Rat Vascular Adventitial Fibroblasts.

The phenotypic modulation of vascular adventitial fibroblasts plays an important role in vascular remodeling. Evidence have shown that endothelial cells and adventitial fibroblasts interact under certain conditions. In this study, we investigated the influence of endothelial cells on the phenotypic modulation of adventitial fibroblasts. Endothelial cells and adventitial fibroblasts from rat thoracic aorta were cultivated in a co-culture system and adventitial fibroblasts were induced with angiotensin II (Ang II). Collagen I and alpha smooth muscle actin (α-SMA) expression and migration of adventitial fibroblasts were analyzed. Ang II upregulated the expression of collagen I and α-SMA and the migration of adventitial fibroblasts. Adventitial fibroblasts-endothelial cells co-culturing attenuated the effects of Ang II. Homocysteine-treated endothelial cells, which are functionally impaired, were less inhibitory of the phenotypic modulation of adventitial fibroblasts. Supplementation of endothelial cells with L-arginine (L-Arg) or 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP) enhanced the trends, while with L-NG-nitroarginine methyl ester (L-NAME) or 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) the opposite effect was observed. Under the influence of Ang II, adventitial fibroblasts were prone to undergo phenotypic modulation, which was closely related to vascular remodeling. Our study showed that endothelial cells influenced fibroblast phenotypic transformation and such effect would be mediated through the nitric oxide (NO)/cGMP signaling pathway. J. Cell. Biochem. 118: 1921-1927, 2017. © 2017 Wiley Periodicals, Inc.

SIRT1 Regulates the Inflammatory Response of Vascular Adventitial Fibroblasts through Autophagy and Related Signaling Pathway.

BACKGROUND/AIMS: Autophagy is a lysosomal degradation pathway that is essential for cellular survival, differentiation, and homeostasis. Sirtuin 1 (SIRT1), a NAD+-dependent deacetylase, plays a pivotal role in modulation of autophagy. Recent studies found that autophagy was involved in the regulation of inflammatory response. In this study, we aimed to determine the effect of SIRT1 on autophagy and inflammation, and whether autophagy can regulate the inflammatory response in vascular adventitial fibroblasts (VAFs). METHODS: Cell autophagy was evaluated by fluorescence microscope and transmission electron microscopy. The expression of protein and mRNA were determined by Western blot analysis and real time-PCR. The production of cytokine was detected by ELISA. RESULTS: TNF-α induced autophagy and increased SIRT1 expression in VAFs. SIRT1 activator resveratrol enhanced TNF-α-induced VAF autophagy. In contrast, SIRT1 knockdown attenuated VAF autophagy. Both the Akt inhibitor MK2206 and mTOR inhibitor rapamycin further increased TNF-α-induced VAF autophagy. Furthermore, SIRT1 knockdown increased Akt phosphorylation and inhibited the autophagy in VAFs. However, MK2206 attenuated the effect of SIRT1 knockdown on VAF autophagy. In addition, ingenuity pathway analysis showed that there is a relationship between cell autophagy and inflammation. We found that SIRT1 knockdown increased the expression of NLRP3 and interleukin (IL)-6 and promoted the production of IL-1ß in VAFs. Further study showed that autophagy activation decreased the expression of NLRP3 and IL-6 and inhibited the production of IL-1ß, whereas autophagy inhibition increased the inflammatory response of VAFs. More importantly, our study showed that autophagy was involved in the degradation of NLRP3 through the autophagy-lysosome pathway. CONCLUSION: SIRT1 not only regulates VAF autophagy through the Akt/mTOR signaling pathway but also suppresses the inflammatory response of VAFs through autophagy.

"String theory" of c-kit(pos) cardiac cells: a new paradigm regarding the nature of these cells that may reconcile apparently discrepant results.

Although numerous preclinical investigations have consistently demonstrated salubrious effects of c-kit(pos) cardiac cells administered after myocardial infarction, the mechanism of action remains highly controversial. We and others have found little or no evidence that these cells differentiate into mature functional cardiomyocytes, suggesting paracrine effects. In this review, we propose a new paradigm predicated on a comprehensive analysis of the literature, including studies of cardiac development; we have (facetiously) dubbed this conceptual construct "string theory" of c-kit(pos) cardiac cells because it reconciles multifarious and sometimes apparently discrepant results. There is strong evidence that, during development, the c-kit receptor is expressed in different pools of cardiac progenitors (some capable of robust cardiomyogenesis and others with little or no contribution to myocytes). Accordingly, c-kit positivity, in itself, does not define the embryonic origins, lineage capabilities, or differentiation capacities of specific cardiac progenitors. C-kit(pos) cells derived from the first heart field exhibit cardiomyogenic potential during development, but these cells are likely depleted shortly before or after birth. The residual c-kit(pos) cells found in the adult heart are probably of proepicardial origin, possess a mesenchymal phenotype (resembling bone marrow mesenchymal stem/stromal cells), and are capable of contributing significantly only to nonmyocytic lineages (fibroblasts, smooth muscle cells, and endothelial cells). If these 2 populations (first heart field and proepicardium) express different levels of c-kit, the cardiomyogenic potential of first heart field progenitors might be reconciled with recent results of c-kit(pos) cell lineage tracing studies. The concept that c-kit expression in the adult heart identifies epicardium-derived, noncardiomyogenic precursors with a mesenchymal phenotype helps to explain the beneficial effects of c-kit(pos) cell administration to ischemically damaged hearts despite the observed paucity of cardiomyogenic differentiation of these cells.

The adventitia: essential regulator of vascular wall structure and function.

The vascular adventitia acts as a biological processing center for the retrieval, integration, storage, and release of key regulators of vessel wall function. It is the most complex compartment of the vessel wall and is composed of a variety of cells, including fibroblasts, immunomodulatory cells (dendritic cells and macrophages), progenitor cells, vasa vasorum endothelial cells and pericytes, and adrenergic nerves. In response to vascular stress or injury, resident adventitial cells are often the first to be activated and reprogrammed to influence the tone and structure of the vessel wall; to initiate and perpetuate chronic vascular inflammation; and to stimulate expansion of the vasa vasorum, which can act as a conduit for continued inflammatory and progenitor cell delivery to the vessel wall. This review presents the current evidence demonstrating that the adventitia acts as a key regulator of vascular wall function and structure from the outside in.

CYP2J2-Derived EETs Attenuated Angiotensin II-Induced Adventitial Remodeling via Reduced Inflammatory Response.

BACKGROUND: Cytochrome P450 (CYP) epoxygenases metabolize arachidonic acids (AA) to form epoxyeicosatrienoic acids (EETs), which exert beneficial roles in the treatment of cardiovascular diseases, but little is known about its role on adventitial remodeling. METHODS: We used C57BL/6J mice in vivo and primary rat adventitial fibroblasts (AFs) in vitro treated with Angiotensin II to investigate the effects of CYP2J2 gene delivery and exogenous EETs administration on adventitial remodeling. RESULTS: CYP/sEH system was found to exist in human adventitia, and involved in adventitial remodeling process. Exogenous EETs administration significantly inhibited Ang II-induced AFs activation, characterized by differentiation, proliferation, migration, and collagen synthesis. These protective effects were partially reversed by PPARx03B3; antagonist GW9662 pretreatment or SOCS3 siRNA transfection. EETs suppressed Ang II-induced Ix03BA;Bα phosphorylation, subsequent NF-x03BA;B nuclear translocation via PPARx03B3; dependent signaling pathway in AFs. Additionally, EETs reduced Ang II-induced JAK2, STAT3 phosphorylation and subsequent phosphor-STAT3 nuclear translocation, which were mediated by SOCS3 induction but independent of PPARx03B3; activation. Furthermore, rAAV-CYP2J2 gene delivery reduced vessel wall thickening, AFs differentiation, proliferation and collagen deposition in aortic adventitia induced by Ang II infusion, which were mediated by NF-x03BA;B and SOCS3/JAK/STAT signaling pathways in blood pressure dependent and independent manner, respectively. CONCLUSION: We concluded that CYP2J2 overexpression attenuated Ang II-induced adventitial remodeling via PPARx03B3;-dependent NF-x03BA;B and PPARx03B3;-independent SOCS3/JAK/STAT inflammatory signaling pathways.

Macrophages activate iNOS signaling in adventitial fibroblasts and contribute to adventitia fibrosis.

A large amount of NO is generated through the inducible nitric oxide synthase (iNOS) pathway from the vascular adventitia in various vascular diseases. However, it is currently not fully understood how the iNOS signaling pathway is activated. In the present study, this question was addressed in the context of adventitial cellular interactions. A rat model of acute hypertension in the contralateral carotid arteries was established through transverse aortic constriction (TAC) surgery. In this model, activated macrophages were found surrounded by a large quantity of iNOS-expressing adventitial fibroblasts (AFs), suggesting a possible causal relationship between macrophages and iNOS activation of the neighboring AFs. In an in vitro model, a macrophage-like cell line RAW 264.7 was first activated by LPS treatment. The supernatant was then harvested and applied to treat primary rat AFs. iNOS in AFs was activated robustly by the supernatant treatment but not by LPS itself. Treating AFs with interleukin-1ß (IL-1ß) also activated iNOS signaling, suggesting that the IL-1ß pathway might be a possible mediator. As a consequence of the iNOS activation, total protein nitration and S-nitrosylation significantly increased in those AFs. Additionally, increased deposition of type I and type III collagens was observed in both in vitro and in vivo models. The collagen deposition was partially restored by an iNOS inhibitor, 1400 W. These findings highlight the importance of iNOS signaling during vascular inflammation, and advance our understanding of its activation through a cellular interaction perspective.

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.

Epigenetic profile of human adventitial progenitor cells correlates with therapeutic outcomes in a mouse model of limb ischemia.

OBJECTIVE: We investigated the association between the functional, epigenetic, and expressional profile of human adventitial progenitor cells (APCs) and therapeutic activity in a model of limb ischemia. APPROACH AND RESULTS: Antigenic and functional features were analyzed throughout passaging in 15 saphenous vein (SV)-derived APC lines, of which 10 from SV leftovers of coronary artery bypass graft surgery and 5 from varicose SV removal. Moreover, 5 SV-APC lines were transplanted (8×10(5) cells, IM) in mice with limb ischemia. Blood flow and capillary and arteriole density were correlated with functional characteristics and DNA methylation/expressional markers of transplanted cells. We report successful expansion of tested lines, which reached the therapeutic target of 30 to 50 million cells in ≈10 weeks. Typical antigenic profile, viability, and migratory and proangiogenic activities were conserved through passaging, with low levels of replicative senescence. In vivo, SV-APC transplantation improved blood flow recovery and revascularization of ischemic limbs. Whole genome screening showed an association between DNA methylation at the promoter or gene body level and microvascular density and to a lesser extent with blood flow recovery. Expressional studies highlighted the implication of an angiogenic network centered on the vascular endothelial growth factor receptor as a predictor of microvascular outcomes. FLT-1 gene silencing in SV-APCs remarkably reduced their ability to form tubes in vitro and support tube formation by human umbilical vein endothelial cells, thus confirming the importance of this signaling in SV-APC angiogenic function. CONCLUSIONS: DNA methylation landscape illustrates different therapeutic activities of human APCs. Epigenetic screening may help identify determinants of therapeutic vasculogenesis in ischemic disease.

Responses of adventitial CD34+ vascular wall-resident stem/progenitor cells and medial smooth muscle cells to carotid injury in rats.

Cell culture and carotid injury studies with SD rats were performed to investigate the roles of CD34+ vascular wall-resident stem/progenitor cells (VRS/Pcs) and vascular smooth muscle cells (SMCs) in neointimal formation. In vitro, the media-isolated SM MHC+ SMCs occupied 93.92±8.62% of total BrdU+ cells, whereas the CD34+ cells, only 2.61±0.82%, indicating that the cell expansion in SMC culture was attributed to SM MHC+ SMCs. The adventitia-isolated CD34+ VRS/Pcs responded to PDGF-BB by differentiating into SMC-like cells which expressed SM22α (an early stage SMC marker), but seldom SM MHC (a late stage SMC marker). In carotid injury model, the CD34+ VRS/Pcs differentiated SMC-like cells migrated in very few numbers into only the outer layer of the media, and this was further confirmed by a cell tracking analysis. While the neointimal cells were consistently SM MHC+ and CD34- SMCs during whole course of the post-injury remodeling. Thus it is speculated that the adventitial CD34+ VRS/Pcs, at least in rat model, do not directly participate in neointimal formation, but function to maintain homeostasis of the media during injury-induced vascular wall remodeling.

PKCε is a negative regulator of PVAT-derived vessel formation.

RATIONALE: Vessel formation is a crucial event in tissue repair after injury. Thus, one assumption of innovative therapeutic approaches is the understanding of its molecular mechanisms. Notwithstanding our knowledge of the role of Protein Kinase C epsilon (PKCε) in cardio-protection and vascular restenosis, its role in vessel progenitor differentiation remains elusive. OBJECTIVE: Given the availability of PKCε pharmacological modulators already tested in clinical trials, the specific aim of this study is to unravel the role of PKCε in vessel progenitor differentiation, with implications in vascular pathology and vasculogenesis. METHODS AND RESULTS: Mouse Peri-Vascular Adipose Tissue (PVAT) was used as source of mesenchymal vessel progenitors. VEGF-induced differentiation of PVAT cells down-regulates both PKCε and p-PAK1 protein expression levels. PKCε overexpression and activation: i) reduced the expression levels of SMA and PECAM in endothelial differentiation of PVAT cells; ii) completely abrogated tubules formation in collagen gel assays; iii) increased the expression of p-PAK1. CONCLUSION: PKCε negatively interferes with vessel progenitor differentiation via interaction with PAK-1.

Activation of PPAR alpha by fenofibrate inhibits apoptosis in vascular adventitial fibroblasts partly through SIRT1-mediated deacetylation of FoxO1.

Recent studies demonstrated that the ligand-activated transcription factor peroxisome proliferator-activated receptorα (PPARα) acts in association with histone deacetylase sirtuin 1 (SIRT1) in the regulation of metabolism and inflammation involved in cardiovascular diseases. PPARα activation also participates in the modulation of cell apoptosis. Our previous study found that SIRT1 inhibits the apoptosis of vascular adventitial fibroblasts (VAFs). However, whether the role of PPARα in apoptosis of VAFs is mediated by SIRT1 remains unknown. In this study, we aimed to determine the effect of PPARα agonist fenofibrate on cell apoptosis and SIRT1 expression and related mechanisms in ApoE(-/-) mice and VAFs in vitro. We found that fenofibrate inhibited cell apoptosis in vascular adventitia and up-regulated SIRT1 expression in aorta of ApoE(-/-) mice. Moreover, SIRT1 activator resveratrol (RSV) further enhanced these effects of fenofibrate. In vitro study showed that activation of PPARα by fenofibrate inhibited TNF-α-induced cell apoptosis and cell cycle arrest in VAFs. Meanwhile, fenofibrate up-regulated SIRT1 expression and inhibited SIRT1 translocation from nucleus to cytoplasm in VAFs stimulated with TNF-α. Moreover, the effects of fenofibrate on cell apoptosis and SIRT1 expression in VAFs were reversed by PPARα antagonist GW6471. Importantly, treatment of VAFs with SIRT1 siRNA or pcDNA3.1(+)-SIRT1 showed that the inhibitory effect of fenofibrate on cell apoptosis in VAFs through SIRT1. On the other hand, knockdown of FoxO1 decreased cell apoptosis of VAFs compared with fenofibrate group. Overexpression of FoxO1 increased cell apoptosis of VAFs compared with fenofibrate group. Further study found that fenofibrate decreased the expression of acetylated-FoxO1 in TNF-α-stimulated VAFs, which was abolished by SIRT1 knockdown. Taken together, these findings indicate that activation of PPARα by fenofibrate inhibits cell apoptosis in VAFs partly through the SIRT1-mediated deacetylation of FoxO1.

TGF-ß1/FGF-2 signaling mediates the 15-HETE-induced differentiation of adventitial fibroblasts into myofibroblasts.

BACKGROUND: Pulmonary adventitial fibroblasts (PAFs) are activated under stress stimuli leading to their differentiation into myofibroblasts, which is involved in vessel remodeling. 15-HETE is known as an important factor in vessel remodeling under hypoxia; however, the role of 15-HETE in PAF phenotypic alteration is not clear. RESULTS: The effect of 15-HETE on PAF phenotypic alterations was investigated in the present study. PAFs were treated with 15-HETE (0.5 µM) for 24 h, and the myofibroblast marker α-smooth muscle actin (α-SMA) was analyzed. The 15-HETE induced α-SMA expression and cell morphology. 15-HETE upregulated FGF-2 levels in PAFs, and knockdown FGF-2 by siRNAs blocked the enhanced α-SMA expression induced by 15-HETE. p38 kinase was activated, and blocked depressed 15-HETE-induced FGF-2 expression. The downstream of p38 pathway, Egr-1 activation, was also raised by 15-HETE treatment, and silenced Egr-1 suppressed the 15-HETE-induced upregulation of FGF-2. TGF-ß1 was upregulated with FGF-2 treatment, and α-SMA expression induced by FGF-2 was inhibited after the cell was transferred with TGF-ß1 siRNA. Meanwhile, FGF-2 increased α-SMA expression and improved proliferation, which was associated with p27(kip1) and cyclin E variation. CONCLUSIONS: The above results suggest that p38/Egr-1 pathway-mediated FGF-2 is involved in 15-HETE-induced differentiation of PAFs into myofibroblasts and cell proliferation.