Vascular Stem Cells Research Articles

Inhibitory role of reactive oxygen species in the differentiation of multipotent vascular stem cells into vascular smooth muscle cells in rats: a novel aspect of traditional culture of rat aortic smooth muscle cells.

Proliferative or synthetic vascular smooth muscle cells (VSMCs) are widely accepted to be mainly derived from the dedifferentiation or phenotypic modulation of mature contractile VSMCs, i.e., a phenotype switch from a normally quiescent and contractile type into a proliferative or synthetic form. However, this theory has been challenged by recent evidence that synthetic VSMCs predominantly originate instead from media-derived multipotent vascular stem cells (MVSCs). To test these hypotheses further, we re-examine whether the conventional rat aortic SMC (RASMC) culture involves the VSMC differentiation of MVSCs or the dedifferentiation of mature VSMCs and the potential mechanism for controlling the synthetic phenotype of RASMCs. We enzymatically isolated RASMCs and cultured the cells in both a regular growth medium (RGM) and a stem cell growth medium (SCGM). Regardless of culture conditions, only a small portion of freshly isolated RASMCs attaches, survives and grows slowly during the first 7days of primary culture, while expressing both SMC- and MVSC-specific markers. RGM-cultured cells undergo a process of synthetic SMC differentiation, whereas SCGM-cultured cells can be differentiated into not only synthetic SMCs but also other somatic cells. Notably, compared with the RGM-cultured differentiated RASMCs, the SCGM-cultured undifferentiated cells exhibit the phenotype of MVSCs and generate greater amounts of reactive oxygen species (ROS) that act as a negative regulator of differentiation into synthetic VSMCs. Knockdown of phospholipase A2, group 7 (Pla2g7) suppresses ROS formation in the MVSCs while enhancing SMC differentiation of MVSCs. These results suggest that cultured synthetic VSMCs can be derived from the SMC differentiation of MVSCs with ROS as a negative regulator.

PC166. Identification and Characterization of Vascular Wall-Resident Stem Cell and Adipose-Derived Mesenchymal Stem Cell Populations in Peripheral Arterial Disease

PC166. Identification and Characterization of Vascular Wall-Resident Stem Cell and Adipose-Derived Mesenchymal Stem Cell Populations in Peripheral Arterial Disease

The effect of blocking angiogenesis on anterior cruciate ligament healing following stem cell transplantation

Ruptured human anterior cruciate ligaments (ACL) contain vascular stem cells capable of enhancing the healing of tendon grafts. In the current study we explored the role that neo-angiogenesis plays in ACL healing. ACL-derived CD34+ cells were isolated via Fluorescence Activated Cell Sorting (FACS) from the rupture sites of human ACLs. The cells were then virally transduced to express either vascular endothelial growth factor (VEGF) or soluble FLT-1 (sFLT-1), which is an antagonist of VEGF. We established five groups: CD34+VEGF(100%), where 100% of the cells were transduced with VEGF, CD34+VEGF(25%), where only 25% of the cells were transduced with VEGF, CD34+, CD34+sFLT-1, and a No cells group. The CD34+sFLT1 group had a significant reduction in biomechanical strength compared to the CD34+ group at 4 and 8 weeks; whereas the biomechanical strength of the CD34+VEGF(25%) group was significantly greater than the CD34+ group at week 4; however, no difference was observed by week 8. Immunohistochemical staining demonstrated a significantly lower number of isolectin B4 and hCD31 positive cells, markers associated with angiogenesis, in the CD34+sFLT1 group, and a higher number of isolectin B4 and hCD31 positive cells in the CD34+VEGF(100%) and CD34+VEGF(25%) groups compared to the CD34+ group. Graft maturation was significantly delayed in the CD34+sFLT1 group and accelerated in the CD34+VEGF(25%) group compared to the CD34+ group. In conclusion, blocking VEGF reduced angiogenesis, graft maturation and biomechanical strength following ACL reconstruction. Native expression of VEGF by the CD34+ cells improved tendon graft maturation and biomechanical strength; however, over-expression of VEGF impeded improvements in biomechanical strength.

Vascular endothelial growth factor-expressing neural stem cell for the treatment of neuropathic pain.

Previously, we determined that vascular endothelial growth factor (VEGF) improves the survival of neural stem cells (NSCs) transplanted into an ischemic environment and effectively enhances angiogenesis. Here, we applied NSCs expressing VEGF (SV-VEGF-NSCs) to treat neuropathic pain. In this study, our goal was to verify the therapeutic effect of SV-VEGF-NSCs by transplanting the cells in a sciatic nerve injury model. We compared the amount of VEGF secreted from DsRed-NSCs (control) or SV-VEGF-NSCs and observed that SV-VEGF-NSCs have a much higher expression level of VEGF. We next investigated whether transplantation with SV-VEGF-NSCs aids functional recovery and pain reduction. We confirmed that transplantation with SV-VEGF-NSCs enhances functional recovery, pain reduction, and remyelination as well as the number of blood vessels compared with the control groups. Our results show that VEGF aids functional recovery and pain reduction in a sciatic nerve injury model.

Abstract 9: Smooth Muscle Cells Are Crucial for Vascular Stem Cell Migration and Vasculogenesis via Keratinocyte Cell-derived Chemokine

Introduction: Recently, a population of stem/progenitor cells have been identified within the adventitia of vessel walls. While existing data indicate their capacity of differentiation into specific vascular lineages, their migratory roles within vascular diseases remain unknown. Interestingly, smooth muscle cells (SMCs) have been shown to reside within close proximity with the progenitors within the medial layer and thus may have a putative role on the motility of progenitor cells. Hypothesis: We hypothesize that mouse adventitia-derived resident progenitor cells will migrate in response to SMCs via their secretion of specific chemokine(s). Methods and Results: In vitro transwell and wound healing migration assays showed significant increases in Sca-1 + vascular progenitor cell migration in response to both SMCs and SMC-conditioned medium (CM). A chemokine ELISA array revealed that keratinocyte cell-derived chemokine (KC) level was markedly increased in SMC-CM. Exogenous recombinant KC significantly increased progenitor cell migration and KC knockdown in SMC-CM using siRNA markedly inhibited progenitor migration. The expression of its receptor, CXCR2, was upregulated following treatment with SMC-CM and treatment with a CXCR2 antagonist was found to significantly inhibit SMC-mediated progenitor migration. Furthermore, we found that the p38 MAP kinase pathway was involved in SMC-mediated migration; the phosphorylation of p38 in response to SMC-CM was partially reduced following knockdown with KC SiRNA. The migration of progenitor cells was also significantly decreased following treatment with a p38 inhibitor. Using a matrigel plug angiogenesis assay in vivo , we found that the KC induced Sca-1 + progenitors migration and enhanced markedly vasculogenesis after 2 weeks. The functional knockdown of KC in C57BL/6 mice using a KC morpholino system potentially reduces Sca-1 + progenitor cell migration compared to corresponding controls (n≥4 per group). Conclusion: In conclusion, SMCs can induce Sca-1 + progenitor cell migration via the release of KC and subsequent activation of p38 MAPK signalling pathway via CXCR2. The lack of functional KC inhibited progenitor cell migration and vasculogenesis in vivo .

Abstract 200: Vascular Stem Cell Migration in Response to Monocyte Chemoattractant Protein-1 Promotes Neointima Lesion Formation

Introduction: While vascular smooth muscle cells are traditionally known for their role in neointima formation, recent studies show that a population of Sca-1 + stem/progenitor cells within the adventitia of vessel walls can also contribute to restenosis formation. To date, their migratory properties and motility in response to fundamental micro-environmental stimuli within the vasculature remain unknown. The occurrence of adventitial inflammation in injured vessels or during atherosclerosis is known to mount an increased secretion of the chemokine, monocyte chemoattractant protein-1 (MCP-1), by cells such as macrophages, endothelial and smooth muscle cells. Hypothesis: We hypothesize that the MCP-1 plays an important role on the mobilization of vascular progenitor cells. Methods and Results: Vascular progenitor cells were derived from vein graft and isolated with an anti-Sca-1 antibody microbead system. Using transwell assays, we found that recombinant MCP-1 significantly increased progenitor cell migration. The knockdown of its receptor expression, CCR2, using lentiviral shRNA significantly inhibited MCP-1 induced migration. Mechanistically, cytoskeleton related GTPase rac1 and cdc42 in progenitor cells were activated (in the GTP-bound state) by MCP-1. Furthermore, we showed that the ERK 1/2 pathway was also involved in MCP-1-mediated migration. Consistently, the migration of progenitor cells was significantly decreased following treatment with a MAPKK inhibitor. Using a femoral artery wire injury mouse model, we found that the seeding of Sca-1 + progenitors on the outer layers of the adventitia markedly enhanced neointima formation after 1 week, whereas vessels without progenitor cell seeding did not form neointima. Neointima formation in Sca-1 + progenitors-seeded vessels were found inhibited in mice that lacked functional MCP-1 (n≥8 per group). Conclusion: In conclusion, MCP-1 can induce adventitia-derived Sca-1 + progenitor cell migration by the activation of GTP-rac1/cdc42 and ERK 1/2 signalling pathway via CCR2. The lack of functional MCP-1 inhibited progenitor cell migration and neointima formation in vivo .

Do Vascular Pericytes Contribute to Neurovasculogenesis in the Central Nervous System as Multipotent Vascular Stem Cells?

Increasing evidence suggests that multipotent stem cells are harbored within a vascular niche inside various organs. Although a precise phenotype of resident vascular stem cells (VSCs) that can function as multipotent stem cells remains unclear, accumulating evidence shows that multipotent VSCs are likely vascular pericytes (PCs) that localize within blood vessels. These PCs are multipotent, possessing the ability to differentiate into various cell types, including vascular lineage cells. In addition, brain PCs are unique: They are derived from neural crest and can differentiate into neural lineage cells. Because PCs in the central nervous system (CNS) can contribute to both neurogenesis and vasculogenesis, they may mediate the reparative process of neurovascular units that are constructed by neural and vascular cells. Here, we describe the activity of PCs when viewed as multipotent VSCs, primarily regarding their neurogenic and vasculogenic potential in the CNS. We also discuss similarities between PCs and other candidates for multipotent VSCs, including perivascular mesenchymal stem cells, neural crest-derived stem cells, adventitial progenitor cells, and adipose-derived stem cells.

Vascular endothelial growth factor 165-transfected adipose-derived mesenchymal stem cells promote vascularization-assisted fat transplantation

Objective: To investigate the effect of vascular endothelial growth factor 165 (VEGF165) and adipose-derived mesenchymal stem cells (ASCs) in promoting the survival of fat grafts, and to provide new methods and theoretical evidence for increasing the survival rate of autologous fat particle grafts. Methods: The VEGF165 gene was recombined with the target fragment, and the recombinant gene was introduced into adenovirus pAdEasy-1 system; the virus was then packaged and the titer was detected. The control group received the same processing. ASCs were cultured and subcultured, and then identified with immunohistochemistry and adipogenic differentiation assay. The subsequent experiments were performed in three groups: the VEGF165 gene-virus group, blank virus group, and control group. After the viral solution was transfected into the ASCs, the viral transfection efficiency was detected using a tracing factor, EGFP. The expression of VEGF165 mRNA and protein in the transfected cells were determined. The proliferation of ASCs in each group was detected with the MTT assay. Results: (1) Recombinant adenoviral vector was constructed successfully in the two groups and the packaging was identified. The viral titer was 2.0 × 108 pfu/ml and 1.9 × 108 pfu/ml, which was in line with the requirements of the subsequent transfection experiments. (2) Immunohistochemistry and adipogenic differentiation results showed that the culture of ASCs was successful, and the cultured cells could serve as seed cells in this experiment. (3) The RT-PCR analysis showed that the relative optical density of VEGF165 mRNA expression was 0.76 ± 0.05 in the experimental group, and there were statistically significant differences compared with the values obtained for the other two groups (P < 0.05). (4) The western blot analysis showed that the relative optical density of VEGF165 protein expression in the experimental group was significantly higher than that in the other two groups (P < 0.05). (5) The proliferation of ASCs was significantly enhanced after transfection in the experimental group, relative to the other two groups (P < 0.05). This evidence indicated that VEGF165 significantly promoted the proliferation of ASCs. Conclusion: After transfection with the VEGF165-adenoviral vector, ASCs demonstrate sustained expression of the target protein and obviously promote the proliferation of ASCs, which lay the foundation for the in vitro experiments on transplantation of VEGF165 combined with ASCs, for the treatment of tissue defects.

45 Comparative study of autologous stromal vascular fraction and adipose-derived stem cells for erectile function recovery in a rat model of cavernous nerve injury

45 Comparative study of autologous stromal vascular fraction and adipose-derived stem cells for erectile function recovery in a rat model of cavernous nerve injury

Adventitial vessel growth and progenitor cells activation in an ex vivo culture system mimicking human saphenous vein wall strain after coronary artery bypass grafting.

Saphenous vein graft disease is a timely problem in coronary artery bypass grafting. Indeed, after exposure of the vein to arterial blood flow, a progressive modification in the wall begins, due to proliferation of smooth muscle cells in the intima. As a consequence, the graft progressively occludes and this leads to recurrent ischemia. In the present study we employed a novel ex vivo culture system to assess the biological effects of arterial-like pressure on the human saphenous vein structure and physiology, and to compare the results to those achieved in the presence of a constant low pressure and flow mimicking the physiologic vein perfusion. While under both conditions we found an activation of Matrix Metallo-Proteases 2/9 and of microRNAs-21/146a/221, a specific effect of the arterial-like pressure was observed. This consisted in a marked geometrical remodeling, in the suppression of Tissue Inhibitor of Metallo-Protease-1, in the enhanced expression of TGF-β1 and BMP-2 mRNAs and, finally, in the upregulation of microRNAs-138/200b/200c. In addition, the veins exposed to arterial-like pressure showed an increase in the density of the adventitial vasa vasorum and of cells co-expressing NG2, CD44 and SM22α markers in the adventitia. Cells with nuclear expression of Sox-10, a transcription factor characterizing multipotent vascular stem cells, were finally found in adventitial vessels. Our findings suggest, for the first time, a role of arterial-like wall strain in the activation of pro-pathologic pathways resulting in adventitial vessels growth, activation of vasa vasorum cells, and upregulation of specific gene products associated to vascular remodeling and inflammation.

Hedgehog and Resident Vascular Stem Cell Fate.

The Hedgehog pathway is a pivotal morphogenic driver during embryonic development and a key regulator of adult stem cell self-renewal. The discovery of resident multipotent vascular stem cells and adventitial progenitors within the vessel wall has transformed our understanding of the origin of medial and neointimal vascular smooth muscle cells (SMCs) during vessel repair in response to injury, lesion formation, and overall disease progression. This review highlights the importance of components of the Hh and Notch signalling pathways within the medial and adventitial regions of adult vessels, their recapitulation following vascular injury and disease progression, and their putative role in the maintenance and differentiation of resident vascular stem cells to vascular lineages from discrete niches within the vessel wall.

From cellular to chemical approach for acute neural and alternative options for age-induced functional diseases.

Endogenous "stem cell niche" (SCN) accompanying vessels contains immune system components which in vivo determine differentiation of multi potent stem cells toward proper cell types in given tissue. Combinations of sex steroids may represent novel chemical approach for neuronal areas of regenerative medicine, since they cause transformation of vascular smooth muscle stem cells into differentiating neuronal cells. Circulating sex steroids are present during pregnancy and can be utilized where needed, when various embryonic/fetal tissues develop from their stem cells. Utilization of induced regeneration of tissues (regenerative medicine) is expected being more effective in sudden failures of younger individuals carrying intact SCN, as compared to established chronic disorders caused by SCN alteration. An essential component of SCN are monocyte-derived cells exhibiting tissue-specific "stop effect" (SE) preventing, for instance, an aging of neuronal cells. Its alteration causes that implantation of neuronal stem cells will also result in their differentiation toward aging cells. When we repair the SE by supply of circulating mononuclear cells from young healthy individuals, we may be able to provide novel regenerative treatments of age-induced neural diseases by sex steroid combinations. Questions regarding some age-induced body alterations are also addressed.

SIRT1 and stem cells: In the forefront with cardiovascular disease, neurodegeneration and cancer.

Cardiovascular disease, nervous system disorders, and cancer in association with other diseases such as diabetes mellitus result in greater than sixty percent of the global annual deaths. These noncommunicable diseases also affect at least one-third of the population in low and middle-income countries and lead to hypertension, elevated cholesterol, malignancy, and neurodegenerative disorders such as Alzheimer's disease and stroke. With the climbing lifespan of the world's population, increased prevalence of these disorders is expected requiring the development of new therapeutic strategies against these disabling disease entities. Targeting stem cell proliferation for cardiac disease, vascular disorders, cancer, and neurodegenerative disorders is receiving great enthusiasm, especially those that focus upon SIRT1, a mammalian homologue of the yeast silent information regulator-2. Modulation of the cellular activity of SIRT1 can involve oversight by nicotinamide/nicotinic acid mononucleotide adenylyltransferase, mammalian forkhead transcription factors, mechanistic of rapamycin pathways, and cysteine-rich protein 61, connective tissue growth factor, and nephroblastoma over-expressed gene family members that can impact cytoprotective outcomes. Ultimately, the ability of SIRT1 to control the programmed cell death pathways of apoptosis and autophagy can determine not only cardiac, vascular, and neuronal stem cell development and longevity, but also the onset of tumorigenesis and the resistance against chemotherapy. SIRT1 therefore has a critical role and holds exciting prospects for new therapeutic strategies that can offer reparative processes for cardiac, vascular, and nervous system degenerative disorders as well as targeted control of aberrant cell growth during cancer.

The vascular stem cell niche: roadmap for transplanted neural progenitor cells during environmental enrichment?

One of the challenges of cell transplantation into the brain is poor graft survival. Graft survival may be affected by an immunological response of the host towards transplanted cells, shear injury to cells during transplantation or an unsuitable micro-environment for the transplanted cell type. Neural progenitor cells have an affinity to laminin, which is commonly used to maintain neural progenitors as monolayer cultures in vitro. The affinity of neural progenitor cells to laminin is based on the high expression of α6β1 integrin, which is required for binding of neural progenitors to endothelial cells (Hall et al., 2006; Shen et al., 2008). Mice with targeted disruption of β1 integrin have substantial layering deficits in developing neocortex (Loulier et al., 2009). Laminin is an important constituent of the basal membrane of brain capillaries. We also found abundant expression of laminin in the basal cistern along the apical surface of the molecular layer of the dentate gyrus (Jamal et al., 2015). Transplanted dentate progenitor cells did not survive in the transplant core within the dentate gyrus, but initially rather exclusively survived along the laminin scaffold of the basal cistern (Figure 1A). The laminin of the basal cistern is connected to the laminin of the capillary network of the dentate gyrus. It appeared as if transplanted dentate progenitor cells initially settled on the dense laminin scaffold of the basal cistern and then migrated into the hippocampal formation using laminin of dentate capillaries as tram tracks (Figure ​Figure1B1B, ​,C).C). Even though we expanded neural progenitor cells on laminin-coated tissue culture flasks, which may have selected for neural progenitor cells with a strong affinity to laminin, we observed the same in vivo affinity to laminin after growing neural progenitor cells as neurospheres for 5 days before transplantation. Also, previous experiments with neural progenitor cells grown exclusively as neurospheres before transplantation showed the greatest survival of cells along the laminin-rich basal cistern adjacent to the dentate gyrus instead of in the transplant core within the parenchyma of the hippocampal formation (Waldau et al., 2010).

Adult vascular wall resident multipotent vascular stem cells, matrix metalloproteinases, and arterial aneurysms.

Evidences have shown the presence of multipotent stem cells (SCs) at sites of arterial aneurysms: they can differentiate into smooth muscle cells (SMCs) and are activated after residing in a quiescent state in the vascular wall. Recent studies have implicated the role of matrix metalloproteinases in the pathogenesis of arterial aneurysms: in fact the increased synthesis of MMPs by arterial SMCs is thought to be a pivotal mechanism in aneurysm formation. The factors and signaling pathways involved in regulating wall resident SC recruitment, survival, proliferation, growth factor production, and differentiation may be also related to selective expression of different MMPs. This review explores the relationship between adult vascular wall resident multipotent vascular SCs, MMPs, and arterial aneurysms.

Role of Mesenchymal Stem Cells Transfected With Vascular Endothelial Growth Factor in Maintaining Renal Structure and Function in Rats with Unilateral Ureteral Obstruction.

Mesenchymal stem cells hold promise for renal disease treatment. Vascular endothelial growth factor may heal tubule-interstitial fibrosis in unilateral ureteral obstruction by inhibiting epithelial-mesenchymal transition. We investigated the protective effect of vascular endothelial growth factor in transfected mesenchymal stem cells in unilateral ureteral obstruction-induced renal injury in rats. Male Wistar Albino rats (32 rats; weight, 250-300 g) were divided into 4 equal groups: group 1, control; group 2, unilateral ureteral obstruction; group 3, unilateral ureteral obstruction and mesenchymal stem cells; and group 4, unilateral ureteral obstruction and vascular endothelial growth factor-transfected mesenchymal stem cells. Vascular endothelial growth factor-transfected mesenchymal stem cells were administered intravenously before onset of unilateral ureteral obstruction. On day 14, the rats were killed and kidneys were retrieved. Tubular necrosis, mononuclear cell infiltration, and interstitial fibrosis were evaluated in paraffin blocks. We evaluated green fluorescent protein-positive and vascular endothelial growth factor-positive cells; anti-inflammatory (Prostaglandin E2 receptor) and interleukin 1 receptor antagonist), proinflammatory/anti-inflammatory (interleukin 6), and proinflammatory (MPO) cytokine expression levels; and levels of nitric oxide; transforming growth factor β1, E-cadherin, and hydroxyproline. Green fluorescent protein-positive cells were negative in the renal parenchyma in groups 1 and 2 and positive in groups 3 and 4. Vascular endothelial growth factor levels were significantly higher in group 4. Transforming growth factor β1, nitric oxide, and E-cadherin levels were significantly higher in the unilateral ureteral obstruction than control group; however, in the study groups, these values were not significantly different from the unilateral ureteral obstruction group. In stem cell-transplanted tissue samples, EP3, interleukin 1 receptor antagonist, and interleukin 6 levels were elevated, but MPO expression levels were low. Although there were significant differences for tubular necrosis and fibrosis in group 2, there were significant reductions in tubular injury and fibrosis in groups 3 and 4. Systemic stem cells transplanted into the kidney protected against unilateral ureteral obstruction-induced renal epithelial-mesenchymal transition and renal fibrosis.

The Vascular Niche Is Involved in Regulating Leukemic Stem Cells in Murine Chronic Myelogenous Leukemia

Chronic myelogenous leukemia (CML) is effectively controlled by tyrosine kinase inhibitors (TKIs) such as imatinib mesylate, leading to a hematologic remission in >90% of patients. However, the majority of patients relapse once TKI therapy is discontinued, suggesting that CML leukemia stem cells (LSC) are not eradicated. We recently showed that modulation of the osteoblastic niche can lead to reduction of LSC in CML (Krause et al., Nat. Med. 2013;19:1513), but the role of the vascular hematopoietic stem cell niche in CML has not been well defined.E-selectin is expressed on bone marrow (BM) endothelium within the vascular niche, whereas loss of E-selectin expression or treatment with GMI-1271, an E-selectin small molecule antagonist, enhances HSC quiescence and self-renewal (Winkler et al., Nat. Med. 2012;18:1651). E-selectin also plays a critical role in the homing and engraftment of CML LSCs (Krause et al., Blood 2014;123:1361) through E-selectin ligands expressed on the LSCs, including CD44 (Krause et al., Nat Med. 2006;12:1175). We, therefore, hypothesized that E-selectin blockade with GMI-1271 may overcome niche-mediated resistance to TKIs and eradicate CML LSC.Using the well-described murine retroviral transduction/transplantation model of CML we showed that the white blood cell count (WBC) of mice with BCR-ABL1-induced CML-like leukemia was significantly reduced by treatment with imatinib plus GMI-1271 (or imatinib alone) and there was a trend towards WBC reduction by treatment with GMI-1271 alone (P=0.07). The percentage of GFP+ Mac-1+ cells in peripheral blood on day 16 post-transplant was decreased by imatinib or GMI-1271 alone or by combined treatment with imatinib and GMI-1271. Spleen weights were significantly reduced by combined treatment with imatinib plus GMI 1271. Furthermore, the BM GFP+ (BCR-ABL1+) Lin– c-Kit+ Sca-1+ population, which contains the LSCs in this model, was significantly reduced in animals treated with GMI-1271 compared to vehicle controls. As expected, treatment with imatinib alone had no effect on BM LSC frequency, and there was no added benefit in the reduction of LSC when imatinib and GMI-1271 were combined. In addition, the survival of mice treated with imatinib plus GMI-1271 was significantly prolonged compared to vehicle-treated animals, with ~20% of mice treated with GMI-1271 alone or the combination of imatinib and GMI-1271 exhibiting long-term low-burden disease despite discontinuation of treatment on day 28 post-transplant. In these primary recipients neither BCR-ABL1+ myeloid cells nor BCR-ABL1+ LSC were mobilized to peripheral organs. However, fewer BCR-ABL1+ LSC were found in the spleen of mice treated with GMI-1271 compared to imatinib-treated mice. There was a significant reduction in the frequency of cycling BCR-ABL1+ LSC in mice treated with GMI-1271 and imatinib.To assess directly the effect of E-selectin inhibition on LSC frequency and function, we transplanted BM from primary leukemic mice treated with vehicle, imatinib, GMI-1271 or the combination of imatinib and GMI-1271 into irradiated secondary recipient mice. There was a significant reduction of WBC and a trend towards reduction of BCR-ABL1+ myeloid cells in secondary recipients of BM from donors treated with GMI-1271 alone or in combination with imatinib, but not by imatinib alone.These data suggest that modulation of the vascular niche and, specifically, inhibition of E-selectin may be a possible strategy to target LSC in CML, possibly via a reduction in S-G2/M as cells arrest prior to apoptosis, even when imatinib is discontinued. Further studies on the effects of GMI-1271 on homing of LSC and the more exact mechanism of LSC reduction by GMI-1271 are being performed. DisclosuresMagnani:GlycoMimetics Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees. Krause:Glycomimetics. Inc.: Research Funding.

Recent developments in vascular biology.

The field of vascular biology has transformed dramatically in recent years as new research has elucidated complex roles of novel proteins, stem/progenitor/hematopoietic cells, and miRNAs as well as redox signaling in vascular development, vascular tone regulation and disease in preclinical models and clinical settings. Review of this body of work highlights seminal findings and new developments that emphasize and illuminate underlying principles and historical connections. In the 1980s, there was the frenzied search for the identity of the Endothelium-Derived Relaxing Factor (EDRF; ie, NO).1,2 In the 1990s, the elucidation of myriad EDHFs (hyperpolarizing factors, eg, H2O2, K+, arachidonic acid metabolites) expanded endothelial cell complexity beyond NO.3–5 In the 2000s, the endothelial cell generation and overall cardiovascular effects of H2S were hotly pursued.6,7 In the 2010s, the role of the perivascular adipose tissue (PVAT) derived relaxing and contractile factors in vascular regulation under physiological and disease promoting conditions (eg, diet-induced obesity, metabolic syndrome) has emerged as a new integrative vascular issue of import because of the obesity epidemic.8 Critically important mechanisms continue to be sources of interest and renewed research. Indeed, the panoply of endothelium-derived factors that regulate vascular tone continues to grow, and identifying new ones will only enhance our understanding of the diverse factors that regulate vascular tone and blood flow in context-dependent ways. As elucidated in the article by Zhang et al, entitled “H2O2-induced dilation in human coronary arterioles: role of protein kinase G dimerization and large-conductance Ca2+-activated K+ channel activation,” redox-mediated VSMC K+-channel opening ultimately leads to relaxation and dilation in coronary blood vessels.9 Similarly, PPAR-γ stimulated Regulatory G-protein S 5 (RGS5)-control of Protein Kinase C-mediated opening of calcium-activated …

Oxidized high-density lipoprotein impairs endothelial progenitor cells' function by activation of CD36-MAPK-TSP-1 pathways.

High-density lipoprotein (HDL) levels inversely correlate with cardiovascular events due to the protective effects on vascular wall and stem cells, which are susceptible to oxidative modifications and then lead to potential pro-atherosclerotic effects. We proposed that oxidized HDL (ox-HDL) might lead to endothelial progenitor cells (EPCs) dysfunction and investigated underlying mechanisms. ox-HDL was shown to increase apoptosis and intracellular reactive oxygen species levels, but to reduce migration, angiogenesis, and cholesterol efflux of EPCs in a dose-dependent manner. p38 mitogen-activated protein kinase (MAPK) and NF-κB were activated after ox-HDL stimulation, which also upregulated thrombospondin-1 (TSP-1) expression without affecting vascular endothelial growth factor. Effects caused by ox-HDL could be significantly attenuated by pretreatment with short hairpin RNA-mediated CD36 knockdown or probucol. Data of in vivo experiments and the inverse correlation of ox-HDL and circulating EPC numbers among patients with coronary artery diseases (CAD) or CAD and type 2 diabetes also supported it. Meanwhile, HDL separated from such patients could significantly increase cultured EPC's caspase 3 activity, further supporting our proposal. This is the most complete study to date of how ox-HDL would impair EPCs function, which was involved with activation of CD36-p38 MAPK-TSP-1 pathways and proved by not only the inverse relationship between ox-HDL and circulating EPCs in clinic but also pro-apoptotic effects of HDL separated from patients' serum. Activation of CD36-p38 MAPK-TSP-1 pathways contributes to the pathological effects of ox-HDL on EPCs' dysfunction, which might be one of the potential etiological factors responsible for the disturbed neovascularization in chronic ischemic disease.

Vascular and neural stem cells in the gut: do they need each other?

Enteric neurons and blood vessels form intricate networks throughout the gastrointestinal tract. To support the hypothesis of a possible interaction of both networks, we investigated whether primary mesenteric vascular cells (MVCs) and enteric nervous system (ENS)-derived cells (ENSc) depend on each other using two- and three-dimensional in vitro assays. In a confrontation assay, both cell types migrated in a target-oriented manner towards each other. The migration of MVCs was significantly increased when cultured in ENSc-conditioned medium. Co-cultures of ENSc with MVCs resulted in an improved ENSc proliferation and differentiation. Moreover, we analysed the formation of the vascular and nervous system in developing mice guts. It was found that the patterning of newly formed microvessels and neural stem cells, as confirmed by nestin and SOX2 stainings, is highly correlated in all parts of the developing gut. In particular in the distal colon, nestin/SOX2-positive cells were found in the tissues adjacent to the capillaries and in the capillaries themselves. Finally, in order to provide evidences for a mutual interaction between endothelial and neural cells, the vascular patterns of a RET((-/-)) knockout mouse model as well as human Hirschsprung's cases were analysed. In the distal colon of postnatal RET((-/-)) knockout mice, the vascular and neural networks were similarly disrupted. In aganglionic zones of Hirschsprung's patients, the microvascular density was significantly increased compared with the ganglionic zone within the submucosa. Taken together, these findings indicate a strong interaction between the enteric nervous and vascular system.