Neuregulin-1/ErbB4 signaling modulates Plasmodium falciparum HRP2-induced damage to brain cortical organoids.

Human cerebral malaria (HCM) is a severe complication of Plasmodium falciparum (P.f.) infection that is characterized by capillary occlusions, rupture of the blood-brain barrier (BBB), perivascular cellular injury, and brain swelling. P.f.histidine-rich protein 2 (HRP2), a byproduct of parasitized red blood cell (pRBC) lysis, crosses the BBB when compromised to cause brain injury. We hypothesized that HRP2-induced neuronal damage can be attenuated by Neuregulin-1 (NRG1), an anti-inflammatory neuroprotective factor. Using brain cortical organoids, we determined that HRP2 upregulated cell death and inflammatory markers and disorganized brain organoid tissue. We identified toll-like receptors (TLR1 and 2) as potential mediators of HRP2-induced cellular damage and inflammation. Exogenous acute treatment of organoids with NRG1 attenuated HRP2 effects. The results indicate that HRP2 mediates malaria-associated HRP2-induced brain injury and inflammation and that NRG1 may be an effective therapy against HRP2 effects in the brain.

Effects of Iron Supplements on Heme Scavengers in Pregnancy.

In malaria endemic countries, anemia in pregnant women occurs as a result of erythrocyte destruction by Plasmodium infections and other causes including malnutrition. Iron supplementation is recommended as treatment of iron-deficiency anemia. Erythrocyte destruction results in increased release of cytotoxic free heme that is scavenged by haptoglobin (Hp), hemopexin (Hx) and heme oxygenase-1 (HO-1). Paradoxically, iron supplementation in pregnant women has been reported to enhance parasitemia and increase levels of free heme. The relationship between free heme, heme scavengers, and birth outcomes has not been investigated, especially in women who are on iron supplementation. We hypothesized that parasite-infected pregnant women on routine iron supplementation have elevated heme and altered expression of heme scavengers. A cross-sectional study was conducted to determine the association between plasma levels of free heme, HO-1, Hp, Hx, and malaria status in pregnant women who received routine iron supplementation and their birth outcomes. Heme was quantified by colorimetric assay and scavenger protein concentration by ELISA. We demonstrated that iron-supplemented women with asymptomatic parasitemia had increased free heme (mean 75.6 µM; interquartile range [IQR] 38.8-96.5) compared with nonmalaria iron-supplemented women (mean 34.9 µM; IQR 17.4-43.8, P < 0.0001). Women with preterm delivery had lower levels of Hx (mean 656.0 µg/mL; IQR 410.9-861.3) compared with women with full-term delivery (mean: 860.9 µg/mL; IQR 715.2-1055.8, P = 0.0388). Our results indicate that iron supplementation without assessment of circulating levels of free heme and heme scavengers may increase the risk for adverse pregnancy outcomes.

Leptin Signaling Affects Survival and Chemoresistance of Estrogen Receptor Negative Breast Cancer.

Estrogen-receptor-negative breast cancer (BCER-) is mainly treated with chemotherapeutics. Leptin signaling can influence BCER- progression, but its effects on patient survival and chemoresistance are not well understood. We hypothesize that leptin signaling decreases the survival of BCER- patients by, in part, inducing the expression of chemoresistance-related genes. The correlation of expression of leptin receptor (OBR), leptin-targeted genes (CDK8, NANOG, and RBP-Jk), and breast cancer (BC) patient survival was determined from The Cancer Genome Atlas (TCGA) mRNA data. Leptin-induced expression of proliferation and chemoresistance-related molecules was investigated in triple-negative BC (TNBC) cells that respond differently to chemotherapeutics. Leptin-induced gene expression in TNBC was analyzed by RNA-Seq. The specificity of leptin effects was assessed using OBR inhibitors (shRNA and peptides). The results show that OBR and leptin-targeted gene expression are associated with lower survival of BCER- patients. Importantly, the co-expression of these genes was also associated with chemotherapy failure. Leptin signaling increased the expression of tumorigenesis and chemoresistance-related genes (ABCB1, WNT4, ADHFE1, TBC1D3, LL22NC03, RDH5, and ITGB3) and impaired chemotherapeutic effects in TNBC cells. OBR inhibition re-sensitized TNBC to chemotherapeutics. In conclusion, the co-expression of OBR and leptin-targeted genes may be used as a predictor of survival and drug resistance of BCER- patients. Targeting OBR signaling could improve chemotherapeutic efficacy.

Modelling heme-mediated brain injury associated with cerebral malaria in human brain cortical organoids.

Human cerebral malaria (HCM), a severe encephalopathy associated with Plasmodium falciparum infection, has a 20-30% mortality rate and predominantly affects African children. The mechanisms mediating HCM-associated brain injury are difficult to study in human subjects, highlighting the urgent need for non-invasive ex vivo human models. HCM elevates the systemic levels of free heme, which damages the blood-brain barrier and neurons in distinct regions of the brain. We determined the effects of heme on induced pluripotent stem cells (iPSCs) and a three-dimensional cortical organoid system and assessed apoptosis and differentiation. We evaluated biomarkers associated with heme-induced brain injury, including a pro-inflammatory chemokine, CXCL-10, and its receptor, CXCR3, brain-derived neurotrophic factor (BDNF) and a receptor tyrosine-protein kinase, ERBB4, in the organoids. We then tested the neuroprotective effect of neuregulin-1 (NRG-1) against heme treatment in organoids. Neural stem and mature cells differentially expressed CXCL-10, CXCR3, BDNF and ERBB4 in the developing organoids and in response to heme-induced neuronal injury. The organoids underwent apoptosis and structural changes that were attenuated by NRG-1. Thus, cortical organoids can be used to model heme-induced cortical brain injury associated with HCM pathogenesis as well as for testing agents that reduce brain injury and neurological sequelae.

Leptin-induced Notch and IL-1 signaling crosstalk in endometrial adenocarcinoma is associated with invasiveness and chemoresistance.

BACKGROUND: Obesity is a recognized risk factor for endometrial cancer (EmCa) and other cancer types. Leptin levels are significantly increased in obese individuals. Leptin-induced signaling crosstalk [Notch, Interleukin-1 (IL-1) and leptin outcome, NILCO] has been associated with breast cancer progression. This complex signaling crosstalk affects cancer cell proliferation, migration, invasion, angiogenesis, apoptosis and chemoresistance. NILCO expression was previously detected in human EmCa. However, it is unknown whether leptin regulates NILCO and alters EmCa's response to chemotherapeutics. It is hypothesized that leptin induces NILCO and increases aggressiveness and chemoresistance in EmCa cells. AIM: To determine whether leptin induces NILCO molecules in EmCa affecting cell proliferation, aggressiveness and chemoresistance. METHODS: Leptin's effects on the expression of NILCO molecules [mRNAs and proteins for Notch receptors (Notch1-4), ligands (JAG1 and DLL4) and downstream effectors (survivin, Hey2), and leptin (OB-R) and IL-1 (IL-1R tI) receptors] was examined in EmCa cells (type I: Ishikawa, and HEC-1A, and type II: An3Ca and KLE) using Real-time PCR and Western blot analysis, respectively. In addition, the effects of leptin on cell cycle, proliferation and cell invasion were determined using cytometric analysis (Cellometer Vision CBA system), MTT cell proliferation and Matrigel-based invasion assays, respectively. Inhibitors of leptin (nanoparticle-bound leptin peptide receptor antagonist-2, IONP-LPrA2), IL-1 (anti-IL-1R tI antibody) and Notch (siRNA interference RNA) were used to investigate NILCO's effects on cell proliferation and invasion. Leptin's effects on Paclitaxel cytotoxicity in EmCa cells was determined by the CCK8 and Cellometer-based Annexin V assays. RESULTS: For the first time it was shown that leptin is an inducer of Notch in EmCa. Experimental data suggest that leptin induced the expression of NILCO molecules, promoted proliferation and S- phase progression, and reduced Paclitaxel cytotoxicity on EmCa cells. Leptin's effects were higher in type II EmCa cells. The progression of this more aggressive form of the disease is associated with obesity. Remarkably, the use of the leptin signaling antagonist, IONP-LPrA2, re-sensitized EmCa cells to Paclitaxel. CONCLUSION: Present data suggest the notion that leptin-induced NILCO could be a link between obesity and EmCa progression and chemoresistance. Most aggressive type II EmCa cells were higher sensitive to leptin, which appears to increase proliferation, cell cycle progression, aggressiveness, and chemoresistance to Paclitaxel. Therefore, leptin and NILCO could be novel therapeutic targets for type II EmCa, which does not have targeted therapy. Overall, IONP-LPrA2 has a potential as a novel adjuvant drug to enhance the effectiveness of type II EmCa chemotherapy.

The Role of Notch Signaling and Leptin-Notch Crosstalk in Pancreatic Cancer.

There is accumulating evidence that deregulated Notch signaling affects cancer development, and specifically pancreatic cancer (PC) progression. Notch canonical and non-canonical signaling has diverse impact on PC. Moreover, the actions of RBP-Jk (nuclear partner of activated Notch) independent of Notch signaling pathway seem to affect differently cancer progression. Recent data show that in PC and other cancer types the adipokine leptin can modulate Notch/RBP-Jk signaling, thereby, linking the pandemic obesity with cancer and chemoresistance. The potential pivotal role of leptin on PC, and its connection with Notch signaling and chemoresistance are still not completely understood. In this review, we will describe the most important aspects of Notch-RBP-Jk signaling in PC. Further, we will discuss on studies related to RBP-Jk-independent Notch and Notch-independent RPB-Jk signaling. We will also discuss on the novel crosstalk between leptin and Notch in PC and its implications in chemoresistance. The effects of leptin-Notch/RBP-Jk signaling on cancer cell proliferation, apoptosis, and drug resistance require more investigation. Data from these investigations could help to open unexplored ways to improve PC treatment success that has shown little progress for many years.

Leptin-Notch axis impairs 5-fluorouracil effects on pancreatic cancer.

5-FU chemotherapy is a current strategy to treat pancreatic cancer (PC), but unfortunately chemoresistance is eventually developed in most patients. Obesity is a risk factor for PC that could affect 5-FU effectiveness through the adipokine leptin, which is a known proliferation, survival factor and Notch inducer. We investigated whether leptin signaling affects 5-FU cytotoxicity on PC. To this end, tumorspheres developed from BxPC-3 and MiaPaCa-2 PC cells were treated with 5-FU, leptin, inhibitors for Notch (DAPT) and leptin signaling (IONP-LPrA2) and ATP-binding cassette of proteins (Probenecid). Leptin treatment decreased 5-FU cytotoxicity, and increased cell proliferation, colony forming ability, stem cell, pluripotency, EMT markers, drug efflux proteins (ABCC5, ABCC11) and Notch. In addition, leptin reduced the 5-FU effects on apoptosis by decreasing pro-apoptotic (Bax, Caspase-3 activation and PARP degradation) and increasing anti-apoptotic factors (RIP and Bcl-XL). Leptin's effects on PC tumorspheres treated with 5-FU were reduced by IONP-LPrA2 and were mainly Notch signaling- dependent and more evident in MiaPaCa-2-derived tumorspheres. Present results suggest that leptin could impair 5-FU cytotoxicity and promote chemoresistance. Therefore, targeting the leptin-Notch axis could be a novel way to improve 5-FU therapy for PC patients, especially in obesity context.

Nanoparticle-Mediated Cell Capture Enables Rapid Endothelialization of a Novel Bare Metal Stent.

Incomplete endothelialization of intracoronary stents has been associated with stent thrombosis and recurrent symptoms, whereas prolonged use of dual antiplatelet therapy increases bleeding-related adverse events. Facilitated endothelialization has the potential to improve clinical outcomes in patients who are unable to tolerate dual antiplatelet therapy. The objective of this study was to demonstrate the feasibility of magnetic cell capture to rapidly endothelialize intracoronary stents in a large animal model. A novel stent was developed from a magnetizable duplex stainless steel (2205 SS). Polylactic-co-glycolic acid and magnetite (Fe3O4) were used to synthesize biodegradable superparamagnetic iron oxide nanoparticles, and these were used to label autologous blood outgrowth endothelial cells. Magnetic 2205 SS and nonmagnetic 316L SS control stents were implanted in the coronary arteries of pigs (n = 11), followed by intracoronary delivery of magnetically labeled cells to 2205 SS stents. In this study, we show extensive endothelialization of magnetic 2205 SS stents (median 98.4% cell coverage) within 3 days, whereas the control 316L SS stents exhibited significantly less coverage (median 48.9% cell coverage, p < 0.0001). This demonstrates the ability of intracoronary delivery of magnetic nanoparticle labeled autologous endothelial cells to improve endothelialization of magnetized coronary stents within 3 days of implantation.

Histone deacetylases, microRNA and leptin crosstalk in pancreatic cancer.

Because pancreatic cancer (PC) historically has had poor prognosis and five year survival rates, it has been intensely investigated. Analysis of PC incidence and biology has shown a link between different risk factors such as smoking, alcoholism, and obesity and disease progression. Important factors affecting PC include the epigenomic changes driven by DNA methylation and histone acetylation, and actions of microRNA inducing oncogenic or tumor suppressor effects. Studies have identified markers whose dysregulation seem to play important roles in PC progression. PC markers involve classical histone deacetylases (HDAC), PC stem cell (PCSC), and leptin. In this review, we discuss the role of several PC biomarkers, and the potential crosstalk between HDAC, microRNA, and leptin in PC progression. Dysregulated expression of these molecules can increase proliferation, survival, PCSC, resistance to chemotherapy and tumor angiogenesis. The potential relationships between these molecules are further analyzed using data from The Cancer Genome Atlas and crosstalk pathways generated by the Pathway Studio Platform (Ariadne Genomics, Inc.). Oncogenic miRNA21 and tumor suppressor miRNA200 have been previously linked to leptin signaling. Preliminary analysis of PC biopsies and signaling crosstalk suggests that the main adipokine leptin could affect the expression of microRNA and HDAC in PC. Data analysis suggests that HDAC-microRNA-leptin signaling crosstalk may be a new target for PC therapy.

Leptin signaling and cancer chemoresistance: Perspectives.

Obesity is a major health problem and currently is endemic around the world. Obesity is a risk factor for several different types of cancer, significantly promoting cancer incidence, progression, poor prognosis and resistance to anti-cancer therapies. The study of this resistance is critical as development of chemoresistance is a serious drawback for the successful and effective drug-based treatments of cancer. There is increasing evidence that augmented adiposity can impact on chemotherapeutic treatment of cancer and the development of resistance to these treatments, particularly through one of its signature mediators, the adipokine leptin. Leptin is a pro-inflammatory, pro-angiogenic and pro-tumorigenic adipokine that has been implicated in many cancers promoting processes such as angiogenesis, metastasis, tumorigenesis and survival/resistance to apoptosis. Several possible mechanisms that could potentially be developed by cancer cells to elicit drug resistance have been suggested in the literature. Here, we summarize and discuss the current state of the literature on the role of obesity and leptin on chemoresistance, particularly as it relates to breast and pancreatic cancers. We focus on the role of leptin and its significance in possibly driving these proposed chemoresistance mechanisms, and examine its effects on cancer cell survival signals and expansion of the cancer stem cell sub-populations.

Nanoparticle-linked antagonist for leptin signaling inhibition in breast cancer.

AIM: To develop a leptin peptide receptor antagonist linked to nanoparticles and determine its effect on viability of breast cancer cells. METHODS: The leptin antagonist, LPrA2, was coupled via EDAC [1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide] to iron oxide nanoparticles (IONP-LPrA2) to increase its efficacy. IONP-LPrA2 conjugation was confirmed by Western blot and nanoparticle tracking analysis. Human triple negative breast cancer (TNBC) MDA-MB-231, HCC1806 and estrogen receptor positive (ER+) MCF-7 cells were analyzed for the expression of the leptin receptor, Ob-R. The effects of leptin and antagonist on levels of leptin-induced STAT3 phosphorylation and cyclin D1, cell cycle progression, cell proliferation, and tumorsphere formation in breast cancer cells were determined. Doses of the chemotherapeutics [cisplatin (Cis), cyclophosphamide (CTX), doxorubicin (Dox) and paclitaxel (PTX)] to effectively reduce cell viability were calculated. The effects of combination treatments of IONP-LPrA2 and chemotherapeutics on cell viability were determined. RESULTS: Western blot analysis of coupling reaction products identified IONP-LPrA2 at approximately 100 kD. IONP-LPrA2 significantly decreased leptin-induced pSTAT3 levels in HCC1806 cells and drastically decreased cyclin D1 levels in all cell lines. IONP-LPrA2 significantly reduced leptin-induced S phase progression and cell proliferation in all breast cancer cell lines and the formation of tumorspheres in MDA-MB-231 cells. Also, IONP-LPrA2 showed an additive effect on the reduction of breast cancer cell survival with chemotherapeutics. Cis plus IONP-LPrA2 produced a significant reduction in the survival of MDA-MB-231 and HCC1806 cells. CTX plus IONP-LPrA2 caused a significant decrease in the survival of MDA-MB-231 cells. Dox plus IONP-LPrA2 caused a marked reduction in the survival of HCC1806 cells. Although, PTX plus IONP-LPrA2 did not have a major effect on the viability of the breast cancer cells when compared to PTX alone. CONCLUSION: Present data indicate that IONP-LPrA2 may be a useful adjuvant for chemotherapeutic treatment of breast cancer, particularly for TNBC which lacks targeted therapeutic options.

Leptin-Notch signaling axis is involved in pancreatic cancer progression.

Pancreatic cancer (PC) shows a high death rate. PC incidence and prognosis are affected by obesity, a pandemic characterized by high levels of leptin. Notch is upregulated by leptin in breast cancer. Thus, leptin and Notch crosstalk could influence PC progression. Here we investigated in PC cell lines (BxPC-3, MiaPaCa-2, Panc-1, AsPC-1), derived tumorspheres and xenografts whether a functional leptin-Notch axis affects PC progression and expansion of pancreatic cancer stem cells (PCSC). PC cells and tumorspheres were treated with leptin and inhibitors of Notch (gamma-secretase inhibitor, DAPT) and leptin (iron oxide nanoparticle-leptin peptide receptor antagonist 2, IONP-LPrA2). Leptin treatment increased cell cycle progression and proliferation, and the expression of Notch receptors, ligands and targeted molecules (Notch1-4, DLL4, JAG1, Survivin and Hey2), PCSC markers (CD24/CD44/ESA, ALDH, CD133, Oct-4), ABCB1 protein, as well as tumorsphere formation. Leptin-induced effects on PC and tumorspheres were decreased by IONP-LPrA2 and DAPT. PC cells secreted leptin and expressed the leptin receptor, OB-R, which indicates a leptin autocrine/paracrine signaling loop could also affect tumor progression. IONP-LPrA2 treatment delayed the onset of MiaPaCa-2 xenografts, and decreased tumor growth and the expression of proliferation and PCSC markers. Present data suggest that leptin-Notch axis is involved in PC. PC has no targeted therapy and is mainly treated with chemotherapy, whose efficiency could be decreased by leptin and Notch activities. Thus, the leptin-Notch axis could be a novel therapeutic target, particularly for obese PC patients.

Oncogenic role of leptin and Notch interleukin-1 leptin crosstalk outcome in cancer.

Obesity is a global pandemic characterized by high levels of body fat (adiposity) and derived-cytokines (i.e., leptin). Research shows that adiposity and leptin provide insight on the link between obesity and cancer progression. Leptin's main function is to regulate energy balance. However, obese individuals routinely develop leptin resistance, which is the consequence of the breakdown in the signaling mechanism controlling satiety resulting in the accumulation of leptin. Therefore, leptin levels are often chronically elevated in human obesity. Elevated leptin levels are related to higher incidence, increased progression and poor prognosis of several human cancers. In addition to adipose tissue, cancer cells can also secrete leptin and overexpress leptin receptors. Leptin is known to act as a mitogen, inflammatory and pro-angiogenic factor that induces cancer cell proliferation and tumor angiogenesis. Moreover, leptin signaling induces cancer stem cells, which are involved in cancer recurrence and drug resistance. A novel and complex signaling crosstalk between leptin, Notch and interleukin-1 (IL-1) [Notch, IL-1 and leptin crosstalk outcome (NILCO)] seems to be an important driver of leptin-induced oncogenic actions. Leptin and NILCO signaling mediate the activation of cancer stem cells that can affect drug resistance. Thus, leptin and NILCO signaling are key links between obesity and cancer progression. This review presents updated data suggesting that adiposity affects cancer incidence, progression, and response to treatment. Here we show data supporting the oncogenic role of leptin in breast, endometrial, and pancreatic cancers.

CD34 affinity pheresis attenuates a surge among circulating progenitor cells following vascular injury.

BACKGROUND: Intimal hyperplasia (restenosis) is an exaggerated healing response leading to failure of half of vascular interventions. Increasing evidence suggests that circulating progenitor cells contribute to intimal pathology, and clinical studies have demonstrated a correlation between progenitor cells and the incidence of restenosis after cardiovascular interventions. The aims of this study were to characterize the temporal response of CD34+ progenitors following vascular injury in an ovine model and to evaluate an affinity pheresis approach to attenuate this response. METHODS: An ovine model underwent either operative vascular injury or a nonvascular surgery (n = 3 per group). Blood was examined perioperatively over 2 weeks by flow cytometry. Next, an affinity pheresis approach to mediate systemic depletion of CD34 progenitors was designed. Custom agarose pheresis matrix with antibody affinity toward CD34 or an isotype control was evaluated in vitro. Next, following vascular injury, sheep underwent perioperative whole blood volume pheresis toward either the progenitor cell marker CD34 (n = 3) or an isotype control (n = 4) for 14 days. Animals were monitored by physical exam as well as complete blood counts. Cells recovered by pheresis were eluted and examined by flow cytometry. RESULTS: Flow cytometry revealed a focal surge of circulating CD34 cells after vascular injury but not among surgical controls (P = .05). Toward the goal of an approach to attenuate the surge of CD34 progenitors, an evaluation of high-flow affinity matrix revealed efficacy in removal of progenitors from ovine blood in vitro. Next, a separate group of animals undergoing affinity pheresis after vascular injury was evaluated to mediate systemic depletion of CD34+ cells. Again, a surge of CD34+ cells was observed among isotype pheresis animals following vascular intervention but was attenuated over 20-fold by a CD34 pheresis approach (P = .029). Furthermore, an average of 77 million CD34-positive cells were eluted from the CD34 pheresis matrix. Despite multiple sessions of pheresis, complete blood counts remained essentially unchanged over 2 weeks. CONCLUSIONS: Despite evidence suggesting a role for CD34+ circulating progenitor cells in restenotic pathology, the temporal pattern of CD34 progenitors after vascular injury has not been previously defined. We have demonstrated a surge among circulating CD34+ cells that appears confined to procedures involving vascular injury and that this event seems to occur early after vascular injury. We further conclude that CD34 affinity pheresis attenuates the surge. This approach for direct depletion of progenitors may have important implications for the study of progenitors in vascular restenosis.

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.

Resident vascular progenitor cells--diverse origins, phenotype, and function.

The fundamental contributions that blood vessels make toward organogenesis and tissue homeostasis are reflected by the considerable ramifications that loss of vascular wall integrity has on pre- and postnatal health. During both neovascularization and vessel wall remodeling after insult, the dynamic nature of vascular cell growth and replacement vitiates traditional impressions that blood vessels contain predominantly mature, terminally differentiated cell populations. Recent discoveries have verified the presence of diverse stem/progenitor cells for both vascular and non-vascular progeny within the mural layers of the vasculature. During embryogenesis, this encompasses the emergence of definitive hematopoietic stem cells and multipotent mesoangioblasts from the developing dorsal aorta. Ancestral cells have also been identified and isolated from mature, adult blood vessels, showing variable capacity for endothelial, smooth muscle, and mesenchymal differentiation. At present, the characterization of these different vascular wall progenitors remains somewhat rudimentary, but there is evidence for their constitutive residence within organized compartments in the vessel wall, most compellingly in the tunica adventitia. This review overviews the spectrum of resident stem/progenitor cells that have been documented in macro- and micro-vessels during developmental and adult life and considers the implications for a local, vascular wall stem cell niche(s) in the pathogenesis and treatment of cardiovascular and other diseases.

Regulation of circulating progenitor cells in left ventricular dysfunction.

BACKGROUND: Reductions in numbers of circulating progenitor cells (CD34+ cell subsets) have been demonstrated in patients at risk for, or in the presence of, cardiovascular disease. The mediators of these reductions remain undefined. To determine whether neurohumoral factors might regulate circulating CD34+ cell subsets in vivo, we studied complementary canine models of left ventricular (LV) dysfunction. METHODS AND RESULTS: A pacing model of severe LV dysfunction and a hypertensive renal wrap model in which dogs were randomized to receive deoxycorticosterone acetate (DOCA) were studied. Circulating CD34+ cell subsets including hematopoietic precursor cells (HPCs: CD34+/CD45(dim)/VEGFR2-) and endothelial progenitor cells (EPCs: CD34+/CD45-/VEGFR2+) were quantified. Additionally, the effect of mineralocorticoid excess on circulating progenitor cells in normal dogs was studied. The majority of circulating CD34+ cells expressed CD45dimly and did not express VEGFR2, consistent with an HPC phenotype. HPCs were decreased in response to pacing, and this decrease correlated with plasma aldosterone levels (Spearman rank correlation=-0.67, P=0.03). In the hypertensive renal wrap model, administration of DOCA resulted in decreased HPCs. No changes were seen in EPCs in either model. Normal dogs treated with DOCA exhibited a decrease in HPCs in peripheral blood but not bone marrow associated with decreased telomerase activity. CONCLUSIONS: This is the first study to demonstrate that mineralocorticoid excess, either endogenous or exogenous, results in reduction in HPCs. These data suggest that mineralocorticoids may induce accelerated senescence of progenitor cells, leading to their reduced survival and decline in numbers.

Carotid repair using autologous adipose-derived endothelial cells.

BACKGROUND AND PURPOSE: Adipose tissue is an abundant source of endothelial cells as well as stem and progenitor cells which can develop an endothelial phenotype. It has been demonstrated that these cells have distinct angiogenic properties in vitro and in vivo. However, whether these cells have the capacity to directly improve large vessel form and function after vascular injury remains unknown. To define whether delivery of adipose-derived endothelial cells (ADECs) would improve healing of injured carotid arteries, a rabbit model of acute arterial injury was used. METHODS: Autologous rabbit ADECs were generated using defined culture conditions. To test the ability of ADECs to enhance carotid artery repair, cells were delivered intraarterially after acute balloon injury. Additional delivery studies were performed after functional selection of cells before delivery. RESULTS: After rabbit omental fat harvest and digestion, a proliferative, homogenous, and distinctly endothelial population of ADECs was identified. Direct delivery of autologous ADECs resulted in marked reendothelialization 48 hours after acute vascular injury as compared to saline controls (82.2+/-26.9% versus 4.2+/-3.0% P<0.001). Delivery of ADECs that were selected for their ability to take up acetylated LDL significantly improved vasoreactivity and decreased intimal formation after vascular injury. CONCLUSIONS: Taken together, these data suggest that ADECs represent an autologous source of proliferative endothelial cells, which demonstrate the capacity to rapidly improve reendothelialization, improve vascular reactivity, and decrease intimal formation in a carotid artery injury model.