Stem Cells and Their Niches in Angiogenesis : Vascular Endothelial Stem Cells, Hematopoietic Progenitors and Hematopoietic Effector Cells

Most tissues possess tissue-specific stem cells that allow them to maintain tissue integrity. Stem cell niches provide an ideal regulatory microenvironment to support the maintenance and proliferation of adult stem cells. However, adult stem cells and stem cell niches have not been identified for all tissues. Adult stem cells that give rise to the vascular endothelium are still unknown. In this thesis work, we identified a rare population of vascular endothelial stem cells (VESC) on the vascular wall by the phenotype lin-CD31+CD105+Sca-1+c-kit+. A single c-kit expressing VESC with highly proliferative capacity generated functional blood vessels in vivo. A genetic defect in endothelial c-kit resulted in an abolished colony-forming ability of VESCs and impaired tumor angiogenesis and tumor growth. Angiogenesis, the growth of new blood vessels from pre-existing vessels, is actively involved in many physiological and pathological processes such as wound repair, female reproductive cycling, ischemic disease and tumor development. There are two major groups of cells involved in the adult vascular growth, cells that contribute directly by composing the blood vessels, such as vascular endothelial stem cells, and cells that contribute indirectly in a paracrine manner such as infiltrating hematopoietic cells. Infiltrating hematopoietic cells from the bone marrow contribute to angiogenesis in a paracrine manner by secreting angiogenic factors or by remodeling the extracellular matrix. In this thesis work, we found that transforming growth factor-β (TGF-β) recruited a massive amount of hematopoietic cells to local microenvironment. TGF-β stimulated vascular endothelial growth factor (VEGF) expression on these hematopoietic effectors and thus induced vascular growth. This stimulation was regulated by p38 and p44/p42 mitogen-activated protein kinase (MAPK) signaling pathways. These results together provided evidence for a dual action mechanism for TGF-β-induced angiogenesis in vivo. In malignant tumors, we found that the tumor expressed osteoblastic and vascular hematopoietic stem cell (HSC) niche molecules and enclosed multipotent hematopoietic progenitors. The proliferating hematopoietic progenitors generated hematopoietic effector cells and supported angiogenesis and tumor growth by secreting matrix metalloprotease 9 (MMP-9) and VEGF. HSPCs were found to be in proximity to tumor vasculature. Tumor microenvironment shared features of HSC niche in the bone marrow. Therapeutic ablation of hematopoietic cells including proliferating hematopoietic cells from tumor using AMD3100 in vivo resulted in inhibited tumor angiogenesis and growth. In conclusion, we identified and characterized a rare population of c-kit expressing VESCs that give rise to the vascular endothelium in adult. Further purification and detailed characterization of VESCs will provide a better understanding of VESCs and hierarchy of endothelial lineage. Our observations on the in vivo angiogenesis induced by TGF-β elucidated the mechanisms of action of TGF-β in promoting vascular growth. Together with current findings on the ectopic tumor hematopoietic microenvironment, the proliferating hematopoietic progenitors and angiogenic hematopoietic effector cells, these results provided potential therapeutic targets to treat cancer and other diseases.Not Availabl

Cardiovascular disease risk factors induce mesenchymal features and senescence in mouse cardiac endothelial cells

Aging, obesity, hypertension, and physical inactivity are major risk factors for endothelial dysfunction and cardiovascular disease (CVD). We applied fluorescence-activated cell sorting (FACS), RNA sequencing, and bioinformatic methods to investigate the common effects of CVD risk factors in mouse cardiac endothelial cells (ECs). Aging, obesity, and pressure overload all upregulated pathways related to TGF-beta signaling and mesenchymal gene expression, inflammation, vascular permeability, oxidative stress, collagen synthesis, and cellular senescence, whereas exercise training attenuated most of the same pathways. We identified collagen chaperone Serpinhl (also called as Hsp47) to be significantly increased by aging and obesity and repressed by exercise training. Mechanistic studies demonstrated that increased SERPINH1 in human ECs induced mesenchymal properties, while its silencing inhibited collagen deposition. Our data demonstrate that CVD risk factors significantly remodel the transcriptomic landscape of cardiac ECs inducing inflammatory, senescence, and mesenchymal features. SERPINH1 was identified as a potential therapeutic target in ECs.Peer reviewe

Decoding cancer cell death-driven immune cell recruitment: An in vivo method for site-of-vaccination analyses

Anticancer vaccines have recently received renewed attention for immunotherapy of at least a subset of cancer-types. Such vaccines mostly involve either killed cancer or tumor cells alone, or combinations thereof with specific (co-incubated) innate immune cells. In recent years, the immunogenic characteristics of the dead or dying cancer cells have emerged as decisive factors behind the success of anticancer vaccines. This has amplified the importance of accounting for immunology of cell death while preparing anticancer vaccines. This, in turn, has increased the emphasis on the immune reactions at the site-of-vaccination since the therapeutic efficacy of the killed cancer/tumor cell vaccines is contingent upon the nature and characteristics of these reactions at the site-of-injection. In this article, we present a systematic methodology that exploits the murine ear pinna model to study differential immune cell recruitment by dead/dying cancer cells injected in vivo, thereby modeling the site-of-injection relevant for anticancer vaccines.keywords: Apoptosis, Necrosis, Immunotherapy, Dendritic cells, Immunogenic cell death (ICD), Macrophages, Neutrophils, Innate immunity, Danger signals, Necroptosisstatus: publishe

Generation of Functional Blood Vessels from a Single c-kit+ Adult Vascular Endothelial Stem Cell

<div><p>In adults, the growth of blood vessels, a process known as angiogenesis, is essential for organ growth and repair. In many disorders including cancer, angiogenesis becomes excessive. The cellular origin of new vascular endothelial cells (ECs) during blood vessel growth in angiogenic situations has remained unknown. Here, we provide evidence for adult vascular endothelial stem cells (VESCs) that reside in the blood vessel wall endothelium. VESCs constitute a small subpopulation within CD117+ (c-kit+) ECs capable of undergoing clonal expansion while other ECs have a very limited proliferative capacity. Isolated VESCs can produce tens of millions of endothelial daughter cells in vitro. A single transplanted c-kit-expressing VESC by the phenotype lin−CD31+CD105+Sca1+CD117+ can generate in vivo functional blood vessels that connect to host circulation. VESCs also have long-term self-renewal capacity, a defining functional property of adult stem cells. To provide functional verification on the role of c-kit in VESCs, we show that a genetic deficit in endothelial c-kit expression markedly decreases total colony-forming VESCs. In vivo, c-kit expression deficit resulted in impaired EC proliferation and angiogenesis and retardation of tumor growth. Isolated VESCs could be used in cell-based therapies for cardiovascular repair to restore tissue vascularization after ischemic events. VESCs also provide a novel cellular target to block pathological angiogenesis and cancer growth.</p> </div

A genetic deficit in endothelial c-kit expression decreases colony-forming VESCs and results in impaired EC proliferation and angiogenesis, and retardation of tumor growth in vivo.

<p>(A) Equivalent lin−CD31+CD105+ EC populations were detected in mutant C57BL/6J mice with a genetic c-kit expression deficit (C57BL/6J-Kit^W-sh mice) and in the wt C57BL/6J controls. However, the Kit^W-sh mutant mice have very low numbers of CD117+ ECs (here 1% of total lin−CD31+CD105+ ECs). Typical results from FACS analysis of lung ECs are shown. Histograms indicate the percentage of CD117+ cells, control IgG labeling and gatings are also shown. (B) lin−CD31+CD105+ ECs from kit deficient Kit^W-sh mutant mice contain abnormally low levels of endothelial CFCs in comparison to wt mice (<i>p</i><0.0001, the Mann-Whitney test). The horizontal lines indicate 10th, 25th, 50th (median), 75th, and 90th percentiles. The results of 12 independent experiments, each performed in duplicate, are shown. The Mann-Whitney test was used to compare the groups. (C) When syngeneic B16 melanoma tumors were implanted to kit deficient Kit^W-sh mutant mice, a highly significant impairment of tumor angiogenesis was observed (<i>p</i> = 0.0006; <i>n</i> = 7 for each group). vWF/DAPI stains are also shown. The tumor vasculature in kit deficient Kit^W-sh mutant mice contained a significantly diminished number of proliferating ECs (<i>p</i> = 0.01; <i>n</i> = 7 for each group). The percentiles of mean percentages of proliferating (ki-67+) ECs are shown. ki-67/CD31/DAPI stains are also shown. The Mann-Whitney test was used to compare the groups. Scale bars, 100 µm. (D) A highly significant retardation of tumor growth was observed in the kit deficient Kit^W-sh mice. *<i>p</i><0.01; **<i>p</i><0.001; ***<i>p</i><0.0001; <i>n</i> = 17 for each group.</p

The CD117+ EC population greatly enriches for ECs capable of generating functional blood vessels in vivo.

<p>(A) CD117-depleted GFP-tagged lin−CD31+CD105+Sca-1+ ECs were transplanted in matrigel plugs (here 10,000 ECs per plug) into wt C57BL/6 mice. 14 d later, none of the plugs (<i>n</i> = 20) contained GFP+ blood vessels. Note that occasional GFP+ ECs from the CD117-depleted GFP+ transplant can be seen within the plug, but they are infrequent (corresponding to the transplanted cell density of 50 ECs per 1 µl) and do not form complete blood vessels. A confocal scan of an area with occasional GFP+ cells is also shown. (B) An equal number (here 10,000) of CD117-enriched GFP+ lin−CD31+CD105+Sca-1+ ECs formed GFP+ blood vessels in all the matrigel plugs (<i>n</i> = 12) in an identical control experiment. Overview fluorescence micrographs of the plugs and confocal scans of the sectioned pugs are shown for both groups. Scale bars, 50 µm. Note that in addition to donor GFP+ ECs from the EC transplant the matrigel plugs also contain various host-derived non-EC types such as perivascular pericytes, other mesenchymal/stromal cells, and numerous infiltrating inflammatory cells. Therefore, many cells within a plug do not express endothelial cell markers such as CD31 or CD105. Additionally, the plugs also contain numerous GFP-negative wt ECs and blood vessels from the wt host (arrow).</p

The present results provide evidence for adult endothelial stem cell hierarchy.

<p>The results provide evidence for the existence of a rare self-renewing adult VESC that resides at the blood vessel wall endothelium. VESCs are a small subpopulation within vessel wall CD117+ ECs capable of undergoing clonal expansion while other ECs have a very limited proliferative capacity.</p

Isolated colony-forming ECs produce tens of millions of endothelial daughter cells in vitro.

<p>(A) Growth kinetics of five separate EC monolayer cultures originating from individual lin−CD31+CD105+ CFCs that were picked up from colony assays and propagated in 2-D cultures. The cultures were split when 90% confluent and no cells were discarded during the experiment. A summary growth curve (mean ± SD) of the five cultures is also shown. Cell number is in log scale. (B) The long term 2-D EC cultures were analyzed at passage 24 by immunofluorescence microscopy. The cells express the endothelial markers CD105, VEGFR-2, and the stem/progenitor cell marker CD117. The nuclei are stained with DAPI (blue) to recognize individual cells. Scale bars, 50 µm; 5 µm (insert).</p

A single c-kit-expressing adult VESC by the phenotype lin−CD31+CD105+Sca-1+CD117+ can generate functional, perfused blood vessels in vivo.

<p>(A) Flow diagram of the FACS sorting procedure used to obtain lin−CD31+CD105+Sca1+ CD117+ cells is shown. A single clonal colony originating from a single GFP-tagged lin−CD31+CD105+Sca1+CD117+ CFC was expanded for 12 d in adherent culture to amplify the cell number, manually picked up using a micropipette, mixed with 200 µl of matrigel supplemented with VEGF and bFGF, and injected subcutaneously into wt C57BL/6J mice. An eGFP channel inverted microscope micrograph of a single colony prior that was picked up and transplanted to a wt host is also shown. Scale bar, 150 µm. (B) Functional, perfused GFP+ blood vessels generated by the transplanted descendants of a single c-kit-expressing colony-forming EC by the phenotype lin− CD31+CD105+Sca1+CD117+ cell (14 d after transplantation). The mouse was perfused with fluorescent 0.2 µm microspheres (red) to stain endothelia in functional blood vessels that are connected to the blood circulation. ECs were stained for CD31 or CD105. Scale bars, 100 µm. 1-µm thick confocal optical slices and a 3-D orthogonal projection (x–z and y–z axes) are also shown. Note the blood vessel lumina (*) and the red endothelial signal from the microsphere perfusion of functional vasculature. Scale bars, 10 µm. Six independent experiments with similar results were performed. (C) Self-renewal capacity, a defining characteristic of stem cells, was evaluated by inoculating mice with syngeneic B16 melanomas (2 million cells per mice) together with 15 CFUs of GFP-tagged isolated CD31+CD105+ ECs. After 2 wk of tumor growth, repeated isolations and serial transplantations of lineage depleted single cell suspensions containing the GFP+ tagged ECs and the B16 cells were performed. The figure shows GFP+ blood vessels in the quaternary transplant. ECs were stained for VEGFR-2 (red), vWF (white), and CD31 and CD105 (red). Scale bar, 10 µm. A 3-D reconstitution of a GFP+ blood vessel in the quaternary transplant is also shown (right; a 34-µm thick stack of 34 x–y slices from a confocal scan). Six independent experiments with similar results were performed.</p