Shou-Ching Jaminet

Human endothelial colony-forming cells serve as trophic mediators for mesenchymal stem cell engraftment via paracrine signaling

Significance Endothelial colony-forming cells (ECFCs) circulate in peripheral blood and contribute to the formation of new vasculature. Here, we demonstrate that ECFCs can also function as paracrine mediators prior to the establishment of blood perfusion, modulating the regenerative potential of human mesenchymal stem cells (MSCs). We show that MSCs are highly dependable on platelet-derived growth factor BB and that ECFCs provide critical angiocrine factors needed to preserve MSCs as viable. This early angiocrine support ultimately enables extensive engraftment and long-term differentiation of transplanted MSCs. This study provides new insights into the biological attributes of ECFCs that may expand their therapeutic potential. We foresee the use of ECFCs as a means to improve the outcome of MSC transplantation. Endothelial colony-forming cells (ECFCs) are endothelial precursors that circulate in peripheral blood. Studies have demonstrated that human ECFCs have robust vasculogenic properties. However, whether ECFCs can exert trophic functions in support of specific stem cells in vivo remains largely unknown. Here, we sought to determine whether human ECFCs can function as paracrine mediators before the establishment of blood perfusion. We used two xenograft models of human mesenchymal stem cell (MSC) transplantation and studied how the presence of ECFCs modulates MSC engraftment and regenerative capacity in vivo. Human MSCs were isolated from white adipose tissue and bone marrow aspirates and were s.c. implanted into immunodeficient mice in the presence or absence of cord blood-derived ECFCs. MSC engraftment was regulated by ECFC-derived paracrine factors via platelet-derived growth factor BB (PDGF-BB)/platelet-derived growth factor receptor (PDGFR)-β signaling. Cotransplanting ECFCs significantly enhanced MSC engraftment by reducing early apoptosis and preserving stemness-related properties of PDGFR-β+ MSCs, including the ability to repopulate secondary grafts. MSC engraftment was negligible in the absence of ECFCs and completely impaired in the presence of Tyrphostin AG1296, an inhibitor of PDGFR kinase. Additionally, transplanted MSCs displayed fate-restricted potential in vivo, with adipose tissue-derived and bone marrow-derived MSCs contributing exclusive differentiation along adipogenic and osteogenic lineages, respectively. This work demonstrates that blood-derived ECFCs can serve as paracrine mediators and regulate the regenerative potential of MSCs via PDGF-BB/PDGFR-β signaling. Our data suggest the systematic use of ECFCs as a means to improve MSC transplantation.

Tumor surrogate blood vessel subtypes exhibit differential susceptibility to anti-VEGF therapy

Antivascular therapy directed against VEGF or its receptors (VEGFR) has been successful when administered at early stages of tumor vessel growth but is less effective when administered later. Tumor blood vessels are heterogeneous, so vessel subpopulations may differ in their requirements for tumor cell-secreted VEGF and in their susceptibility to anti-VEGF/VEGFR therapy. Human cancers contain several distinct blood vessel types, including mother vessels (MV), glomeruloid microvascular proliferations (GMP), vascular malformations (VM), feeding arteries (FA), and draining veins (DV), all of which can be generated in mice in the absence of tumor cells using expression vectors for VEGF-A(164). In this study, we investigated the sensitivity of each of these vessel types to anti-VEGF therapy with Aflibercept (VEGF Trap), a potent inhibitor of VEGF-A(164). Administering VEGF Trap treatment before or shortly after injection of a recombinant VEGF-A(164)-expressing adenovirus could prevent or regress tumor-free neovasculature, but it was progressively less effective if initiated at later times. Early-forming MVs and GMPs in which the lining endothelial cells expressed high levels of VEGFR-2 were highly susceptible to blockade by VEGF Trap. In contrast, late-forming VMs, FAs, and DVs that expressed low levels of VEGFR-2 were largely resistant. Together, our findings define the susceptibility of different blood vessel subtypes to anti-VEGF therapy, offering a possible explanation for the limited effectiveness of anti-VEGF-A/VEGFR treatment of human cancers, which are typically present for months to years before discovery and are largely populated by late-forming blood vessels.

FoxO1-Mediated Activation of Akt Plays a Critical Role in Vascular Homeostasis

Rationale: Forkhead box-O transcription factors (FOXOs) transduce a wide range of extracellular signals, resulting in changes in cell survival, cell cycle progression, and several cell type-specific responses. FOXO1 is expressed in many cell types, including endothelial cells (ECs). Previous studies have shown that Foxo1 knockout in mice results in embryonic lethality at E11 because of impaired vascular development. In contrast, somatic deletion of Foxo1 is associated with hyperproliferation of ECs. Thus, the precise role of FOXO1 in the endothelium remains enigmatic. Objective: To determine the effect of endothelial-specific knockout and overexpression of FOXO1 on vascular homeostasis. Methods and Results: We show that EC-specific disruption of Foxo1 in mice phenocopies the full knockout. Although endothelial expression of FOXO1 rescued otherwise Foxo1-null animals, overexpression of constitutively active FOXO1 resulted in increased EC size, occlusion of capillaries, elevated peripheral resistance, heart failure, and death. Knockdown of FOXO1 in ECs resulted in marked inhibition of basal and vascular endothelial growth factor–induced Akt-mammalian target of rapamycin complex 1 (mTORC1) signaling. Conclusions: Our findings suggest that in mice, endothelial expression of FOXO1 is both necessary and sufficient for embryonic development. Moreover, FOXO1-mediated feedback activation of Akt maintains growth factor responsive Akt/mTORC1 activity within a homeostatic range.

TM4SF1: a new vascular therapeutic target in cancer

AbstractTransmembrane-4 L-six family member-1 (TM4SF1) is a small plasma membrane glycoprotein that regulates cell motility and proliferation. TM4SF1 is an attractive cancer target because of its high expression in both tumor cells and on the vascular endothelial cells lining tumor blood vessels. We generated mouse monoclonal antibodies against human TM4SF1 in order to evaluate their therapeutic potential; 13 of the antibodies we generated reacted with extracellular loop-2 (EL2), TM4SF1’s larger extracellular, lumen-facing domain. However, none of these antibodies reacted with mouse TM4SF1, likely because the EL2 of mouse TM4SF1 differs significantly from that of its human counterpart. Therefore, to test our antibodies in vivo, we employed an established model of engineered human vessels in which human endothelial colony-forming cells (ECFC) and human mesenchymal stem cells (MSC) are incorporated into Matrigel plugs that are implanted subcutaneously in immunodeficient nude mice. We modified the original protocol by (1) preculturing human ECFC on laminin, fibronectin, and collagen-coated plates, and (2) increasing the ECFC/MSC ratio. These modifications significantly increased the human vascular network in Matrigel implants. Two injections of one of our anti-TM4SF1 EL2 monoclonal antibodies, 8G4, effectively eliminated the human vascular component present in these plugs; they also abrogated human PC3 prostate cancer cells that were incorporated into the ECFC/MSC Matrigel mix. Together, these studies provide a mouse model for assessing tumor xenografts that are supplied by a human vascular network and demonstrate that anti-TM4SF1 antibodies such as 8G4 hold promise for cancer therapy.

Human white adipose tissue vasculature contains endothelial colony-forming cells with robust in vivo vasculogenic potential

Blood-derived endothelial colony-forming cells (ECFCs) have robust vasculogenic potential that can be exploited to bioengineer long-lasting human vascular networks in vivo. However, circulating ECFCs are exceedingly rare in adult peripheral blood. Because the mechanism by which ECFCs are mobilized into circulation is currently unknown, the reliability of peripheral blood as a clinical source of ECFCs remains a concern. Thus, there is a need to find alternative sources of autologous ECFCs. Here we aimed to determine whether ECFCs reside in the vasculature of human white adipose tissue (WAT) and to evaluate if WAT-derived ECFCs have equal clinical potential to blood-derived ECFCs. We isolated the complete endothelial cell (EC) population from intact biopsies of normal human subcutaneous WAT by enzymatic digestion and selection of CD31+ cells. Subsequently, we extensively compared WAT-derived EC phenotype and functionality to bonafide ECFCs derived from both umbilical cord blood and adult peripheral blood. We demonstrated that human WAT is indeed a dependable source of ECFCs with indistinguishable properties to adult peripheral blood ECFCs, including hierarchical clonogenic ability, large expansion potential, stable endothelial phenotype, and robust in vivo blood vessel-forming capacity. Considering the unreliability and low rate of occurrence of ECFCs in adult blood and that biopsies of WAT can be obtained with minimal intervention in an ambulatory setting, our results indicate WAT as a more practical alternative to obtain large amounts of readily available autologous ECFCs for future vascular cell therapies.

Activation of the Nrf2 Cell Defense Pathway by Ancient Foods: Disease Prevention by Important Molecules and Microbes Lost from the Modern Western Diet

The Nrf2 (NFE2L2) cell defense pathway protects against oxidative stress and disorders including cancer and neurodegeneration. Although activated modestly by oxidative stress alone, robust activation of the Nrf2 defense mechanism requires the additional presence of co-factors that facilitate electron exchange. Various molecules exhibit this co-factor function, including sulforaphane from cruciferous vegetables. However, natural co-factors that are potent and widely available from dietary sources have not been identified previously. The objectives of this study were to investigate support of the Nrf2 cell defense pathway by the alkyl catechols: 4-methylcatechol, 4-vinylcatechol, and 4-ethylcatechol. These small electrochemicals are naturally available from numerous sources but have not received attention. Findings reported here illustrate that these compounds are indeed potent co-factors for activation of the Nrf2 pathway both in vitro and in vivo. Each strongly supports expression of Nrf2 target genes in a variety of human cell types; and, in addition, 4-ethylcatechol is orally active in mice. Furthermore, findings reported here identify important and previously unrecognized sources of these compounds, arising from biotransformation of common plant compounds by lactobacilli that express phenolic acid decarboxylase. Thus, for example, Lactobacillus plantarum, Lactobacillus brevis, and Lactobacillus collinoides, which are consumed from a diet rich in traditionally fermented foods and beverages, convert common phenolic acids found in fruits and vegetables to 4-vinylcatechol and/or 4-ethylcatechol. In addition, all of the alkyl catechols are found in wood smoke that was used widely for food preservation. Thus, the potentially numerous sources of alkyl catechols in traditional foods suggest that these co-factors were common in ancient diets. However, with radical changes in food preservation, alkyl catechols have been lost from modern foods. The absence of alkyl catechols from the modern Western diet suggests serious negative consequences for Nrf2 cell defense, resulting in reduced protection against multiple chronic diseases associated with oxidative stress.

Novel Anti-TM4SF1 Antibody–Drug Conjugates with Activity against Tumor Cells and Tumor Vasculature

Antibody–drug conjugates (ADC) represent a promising therapeutic modality for managing cancer. Here, we report a novel humanized ADC that targets the tetraspanin-like protein TM4SF1. TM4SF1 is highly expressed on the plasma membranes of many human cancer cells and also on the endothelial cells lining tumor blood vessels. TM4SF1 is internalized upon interaction with antibodies. We hypothesized that an ADC against TM4SF1 would inhibit cancer growth directly by killing cancer cells and indirectly by attacking the tumor vasculature. We generated a humanized anti-human TM4SF1 monoclonal antibody, v1.10, and armed it with an auristatin cytotoxic agent LP2 (chemical name mc-3377). v1.10-LP2 selectively killed cultured human tumor cell lines and human endothelial cells that express TM4SF1. Acting as a single agent, v1.10-LP2 induced complete regression of several TM4SF1-expressing tumor xenografts in nude mice, including non–small cell lung cancer and pancreas, prostate, and colon cancers. As v1.10 did not react with mouse TM4SF1, it could not target the mouse tumor vasculature. Therefore, we generated a surrogate anti-mouse TM4SF1 antibody, 2A7A, and conjugated it to LP2. At 3 mpk, 2A7A-LP2 regressed several tumor xenografts without noticeable toxicity. Combination therapy with v1.10-LP2 and 2A7A-LP2 together was more effective than either ADC alone. These data provide proof-of-concept that TM4SF1-targeting ADCs have potential as anticancer agents with dual action against tumor cells and the tumor vasculature. Such agents could offer exceptional therapeutic value and warrant further investigation. Mol Cancer Ther; 14(8); 1868–76. ©2015 AACR.

NFAT1 promotes intratumoral neutrophil infiltration by regulating IL8 expression in breast cancer

NFAT transcription factors are key regulators of gene expression in immune cells. In addition, NFAT1‐induced genes play diverse roles in mediating the progression of various solid tumors. Here we show that NFAT1 induces the expression of the IL8 gene by binding to its promoter and leading to IL8 secretion. Thapsigargin stimulation of breast cancer cells induces IL8 expression in an NFAT‐dependent manner. Moreover, we show that NFAT1‐mediated IL8 production promotes the migration of primary human neutrophils in vitro and also promotes neutrophil infiltration in tumor xenografts. Furthermore, expression of active NFAT1 effectively suppresses the growth of nascent and established tumors by a non cell‐autonomous mechanism. Evaluation of breast tumor tissue reveals that while the levels of NFAT1 are similar in tumor cells and normal breast epithelium, cells in the tumor stroma express higher levels of NFAT1 compared to normal stroma. Elevated levels of NFAT1 also correlate with increased neutrophil infiltrate in breast tumors. These data point to a mechanism by which NFAT1 orchestrates the communication between breast cancer cells and host neutrophils during breast cancer progression.

Role of RNA splicing in mediating lineage-specific expression of the von Willebrand factor gene in the endothelium

We previously demonstrated that the first intron of the human von Willebrand factor (vWF) is required for gene expression in the endothelium of transgenic mice. Based on this finding, we hypothesized that RNA splicing plays a role in mediating vWF expression in the vasculature. To address this question, we used transient transfection assays in human endothelial cells and megakaryocytes with intron-containing and intronless human vWF promoter-luciferase constructs. Next, we generated knockin mice in which LacZ was targeted to the endogenous mouse vWF locus in the absence or presence of the native first intron or heterologous introns from the human β-globin, mouse Down syndrome critical region 1, or hagfish coagulation factor X genes. In both the in vitro assays and the knockin mice, the loss of the first intron of vWF resulted in a significant reduction of reporter gene expression in endothelial cells but not megakaryocytes. This effect was rescued to varying degrees by the introduction of a heterologous intron. Intron-mediated enhancement of expression was mediated at a posttranscriptional level. Together, these findings implicate a role for intronic splicing in mediating lineage-specific expression of vWF in the endothelium.

Diverse Functions of Retinoic Acid in Brain Vascular Development

As neural structures grow in size and increase metabolic demand, the CNS vasculature undergoes extensive growth, remodeling, and maturation. Signals from neural tissue act on endothelial cells to stimulate blood vessel ingression, vessel patterning, and acquisition of mature brain vascular traits, most notably the blood–brain barrier. Using mouse genetic and in vitro approaches, we identified retinoic acid (RA) as an important regulator of brain vascular development via non-cell-autonomous and cell-autonomous regulation of endothelial WNT signaling. Our analysis of globally RA-deficient embryos (Rdh10 mutants) points to an important, non-cell-autonomous function for RA in the development of the vasculature in the neocortex. We demonstrate that Rdh10 mutants have severe defects in cerebrovascular development and that this phenotype correlates with near absence of endothelial WNT signaling, specifically in the cerebrovasculature, and substantially elevated expression of WNT inhibitors in the neocortex. We show that RA can suppress the expression of WNT inhibitors in neocortical progenitors. Analysis of vasculature in non-neocortical brain regions suggested that RA may have a separate, cell-autonomous function in brain endothelial cells to inhibit WNT signaling. Using both gain and loss of RA signaling approaches, we show that RA signaling in brain endothelial cells can inhibit WNT-β-catenin transcriptional activity and that this is required to moderate the expression of WNT target Sox17. From this, a model emerges in which RA acts upstream of the WNT pathway via non-cell-autonomous and cell-autonomous mechanisms to ensure the formation of an adequate and stable brain vascular plexus. SIGNIFICANCE STATEMENT Work presented here provides novel insight into important yet little understood aspects of brain vascular development, implicating for the first time a factor upstream of endothelial WNT signaling. We show that RA is permissive for cerebrovascular growth via suppression of WNT inhibitor expression in the neocortex. RA also functions cell-autonomously in brain endothelial cells to modulate WNT signaling and its downstream target, Sox17. The significance of this is although endothelial WNT signaling is required for neurovascular development, too much endothelial WNT signaling, as well as overexpression of its target Sox17, are detrimental. Therefore, RA may act as a “brake” on endothelial WNT signaling and Sox17 to ensure normal brain vascular development.