Endocardial cells form the coronary arteries by angiogenesis through myocardial-endocardial VEGF signaling.

The origins and developmental mechanisms of coronary arteries are incompletely understood. We show here by fate mapping, clonal analysis, and immunohistochemistry that endocardial cells generate the endothelium of coronary arteries. Dye tracking, live imaging, and tissue transplantation also revealed that ventricular endocardial cells are not terminally differentiated; instead, they are angiogenic and form coronary endothelial networks. Myocardial Vegf-a or endocardial Vegfr-2 deletion inhibited coronary angiogenesis and arterial formation by ventricular endocardial cells. In contrast, lineage and knockout studies showed that endocardial cells make a small contribution to the coronary veins, the formation of which is independent of myocardial-to-endocardial Vegf signaling. Thus, contrary to the current view of a common source for the coronary vessels, our findings indicate that the coronary arteries and veins have distinct origins and are formed by different mechanisms. This information may help develop better cell therapies for coronary artery disease.

Mutations in DCHS1 cause mitral valve prolapse.

Mitral valve prolapse (MVP) is a common cardiac valve disease that affects nearly 1 in 40 individuals. It can manifest as mitral regurgitation and is the leading indication for mitral valve surgery. Despite a clear heritable component, the genetic aetiology leading to non-syndromic MVP has remained elusive. Four affected individuals from a large multigenerational family segregating non-syndromic MVP underwent capture sequencing of the linked interval on chromosome 11. We report a missense mutation in the DCHS1 gene, the human homologue of the Drosophila cell polarity gene dachsous (ds), that segregates with MVP in the family. Morpholino knockdown of the zebrafish homologue dachsous1b resulted in a cardiac atrioventricular canal defect that could be rescued by wild-type human DCHS1, but not by DCHS1 messenger RNA with the familial mutation. Further genetic studies identified two additional families in which a second deleterious DCHS1 mutation segregates with MVP. Both DCHS1 mutations reduce protein stability as demonstrated in zebrafish, cultured cells and, notably, in mitral valve interstitial cells (MVICs) obtained during mitral valve repair surgery of a proband. Dchs1(+/-) mice had prolapse of thickened mitral leaflets, which could be traced back to developmental errors in valve morphogenesis. DCHS1 deficiency in MVP patient MVICs, as well as in Dchs1(+/-) mouse MVICs, result in altered migration and cellular patterning, supporting these processes as aetiological underpinnings for the disease. Understanding the role of DCHS1 in mitral valve development and MVP pathogenesis holds potential for therapeutic insights for this very common disease.

FOLFOX Therapy Induces Feedback Upregulation of CD44v6 through YB-1 to Maintain Stemness in Colon Initiating Cells.

Cancer initiating cells (CICs) drive tumor formation and drug-resistance, but how they develop drug-resistance characteristics is not well understood. In this study, we demonstrate that chemotherapeutic agent FOLFOX, commonly used for drug-resistant/metastatic colorectal cancer (CRC) treatment, induces overexpression of CD44v6, MDR1, and oncogenic transcription/translation factor Y-box-binding protein-1 (YB-1). Our study revealed that CD44v6, a receptor for hyaluronan, increased the YB-1 expression through PGE2/EP1-mTOR pathway. Deleting CD44v6, and YB-1 by the CRISPR/Cas9 system attenuates the in vitro and in vivo tumor growth of CICs from FOLFOX resistant cells. The results of DNA:CD44v6 immunoprecipitated complexes by ChIP (chromatin-immunoprecipitation) assay showed that CD44v6 maintained the stemness traits by promoting several antiapoptotic and stemness genes, including cyclin-D1, BCL2, FZD1, GINS-1, and MMP9. Further, computer-based analysis of the clones obtained from the DNA:CD44v6 complex revealed the presence of various consensus binding sites for core stemness-associated transcription factors "CTOS" (c-Myc, TWIST1, OCT4, and SOX2). Simultaneous expressions of CD44v6 and CTOS in CD44v6 knockout CICs reverted differentiated CD44v6-knockout CICs into CICs. Finally, this study for the first time describes a positive feedback loop that couples YB-1 induction and CD44 alternative splicing to sustain the MDR1 and CD44v6 expressions, and CD44v6 is required for the reversion of differentiated tumor cells into CICs.

Role of Periostin in Cardiac Valve Development.

Although periostin plays a significant role in adult cardiac remodeling diseases, the focus of this review is on periostin as a valvulogenic gene. Periostin is expressed throughout valvular development, initially being expressed in endocardial endothelial cells that have been activated to transform into prevalvular mesenchyme termed "cushion tissues" that sustain expression of periostin throughout their morphogenesis into mature (compacted) valve leaflets. The phenotype of periostin null indicates that periostin is not required for endocardial transformation nor the proliferation of its mesenchymal progeny but rather promotes cellular behaviors that promote migration, survival (anti-apoptotic), differentiation into fibroblastic lineages, collagen secretion and postnatal remodeling/maturation. These morphogenetic activities are promoted or coordinated by periostin signaling through integrin receptors activating downstream kinases in cushion cells that activate hyaluronan synthetase II (Akt/PI3K), collagen synthesis (Erk/MapK) and changes in cytoskeletal organization (Pak1) which regulate postnatal remodeling of cells and associated collagenous matrix into a trilaminar (zonal) histoarchitecture. Pak1 binding to filamin A is proposed as one mechanism by which periostin supports remodeling. The failure to properly remodel cushions sets up a trajectory of degenerative (myxomatous-like) changes that over time reduce biomechanical properties and increase chances for prolapse, regurgitation or calcification of the leaflets. Included in the review are considerations of lineage diversity and the role of periostin as a determinant of mesenchymal cell fate.

New insights into mitral valve dystrophy: a Filamin-A genotype-phenotype and outcome study.

Aims: Filamin-A (FLNA) was identified as the first gene of non-syndromic mitral valve dystrophy (FLNA-MVD). We aimed to assess the phenotype of FLNA-MVD and its impact on prognosis. Methods and results: We investigated the disease in 246 subjects (72 mutated) from four FLNA-MVD families harbouring three different FLNA mutations. Phenotype was characterized by a comprehensive echocardiography focusing on mitral valve apparatus in comparison with control relatives. In this X-linked disease valves lesions were severe in men and moderate in women. Most men had classical features of mitral valve prolapse (MVP), but without chordal rupture. By contrast to regular MVP, mitral leaflet motion was clearly restricted in diastole and papillary muscles position was closer to mitral annulus. Valvular abnormalities were similar in the four families, in adults and young patients from early childhood suggestive of a developmental disease. In addition, mitral valve lesions worsened over time as encountered in degenerative conditions. Polyvalvular involvement was frequent in males and non-diagnostic forms frequent in females. Overall survival was moderately impaired in men (P = 0.011). Cardiac surgery rate (mainly valvular) was increased (33.3 ± 9.8 vs. 5.0 ± 4.9%, P < 0.0001; hazard ratio 10.5 [95% confidence interval: 2.9-37.9]) owing mainly to a lifetime increased risk in men (76.8 ± 14.1 vs. 9.1 ± 8.7%, P < 0.0001). Conclusion: FLNA-MVD is a developmental and degenerative disease with complex phenotypic expression which can influence patient management. FLNA-MVD has unique features with both MVP and paradoxical restricted motion in diastole, sub-valvular mitral apparatus impairment and polyvalvular lesions in males. FLNA-MVD conveys a substantial lifetime risk of valve surgery in men.

Transforming growth factor ß1 (TGFß1) regulates CD44V6 expression and activity through extracellular signal-regulated kinase (ERK)-induced EGR1 in pulmonary fibrogenic fibroblasts.

The appearance of myofibroblasts is generally thought to be the underlying cause of the fibrotic changes that underlie idiopathic pulmonary fibrosis. However, the cellular/molecular mechanisms that account for the fibroblast-myofibroblast differentiation/activation in idiopathic pulmonary fibrosis remain poorly understood. We investigated the functional role of hyaluronan receptor CD44V6 (CD44 containing variable exon 6 (v6)) for differentiation of lung fibroblast to myofibroblast phenotype. Increased hyaluronan synthesis and CD44 expression have been detected in numerous fibrotic organs. Previously, we found that the TGFß1/CD44V6 pathway is important in lung myofibroblast collagen-1 and α-smooth-muscle actin synthesis. Because increased EGR1 (early growth response-1) expression has been shown to appear very early and nearly coincident with the expression of CD44V6 found after TGFß1 treatment, we investigated the mechanism(s) of regulation of CD44V6 expression in lung fibroblasts by TGFß1. TGFß1-mediated CD44V6 up-regulation was initiated through EGR1 via ERK-regulated transcriptional activation. We showed that TGFß1-induced CD44V6 expression is through EGR1-mediated AP-1 (activator protein-1) activity and that the EGR1- and AP-1-binding sites in the CD44v6 promoter account for its responsiveness to TGFß1 in lung fibroblasts. We also identified a positive-feedback loop in which ERK/EGR1 signaling promotes CD44V6 splicing and found that CD44V6 then sustains ERK signaling, which is important for AP-1 activity in lung fibroblasts. Furthermore, we identified that HAS2-produced hyaluronan is required for CD44V6 and TGFßRI co-localization and subsequent CD44V6/ERK1/EGR1 signaling. These results demonstrate a novel positive-feedback loop that links the myofibroblast phenotype to TGFß1-stimulated CD44V6/ERK/EGR1 signaling.

Transforming growth factor ß1 (TGFß1)-induced CD44V6-NOX4 signaling in pathogenesis of idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive clinical syndrome of fatal outcome. The lack of information about the signaling pathways that sustain fibrosis and the myofibroblast phenotype has prevented the development of targeted therapies for IPF. Our previous study showed that isolated fibrogenic lung fibroblasts have high endogenous levels of the hyaluronan receptor, CD44V6 (CD44 variant containing exon 6), which enhances the TGFß1 autocrine signaling and induces fibroblasts to transdifferentiate into myofibroblasts. NADPH oxidase 4 (NOX4) enzyme, which catalyzes the reduction of O2 to hydrogen peroxide (H2O2), has been implicated in the cardiac and lung myofibroblast phenotype. However, whether CD44V6 regulates NOX4 to mediate tissue repair and fibrogenesis is not well-defined. The present study assessed the mechanism of how TGF-ß-1-induced CD44V6 regulates the NOX4/reactive oxygen species (ROS) signaling that mediates the myofibroblast differentiation. Specifically, we found that NOX4/ROS regulates hyaluronan synthesis and the transcription of CD44V6 via an effect upon AP-1 activity. Further, CD44V6 is part of a positive-feedback loop with TGFß1/TGFßRI signaling that acts to increase NOX4/ROS production, which is required for myofibroblast differentiation, myofibroblast differentiation, myofibroblast extracellular matrix production, myofibroblast invasion, and myofibroblast contractility. Both NOX4 and CD44v6 are up-regulated in the lungs of mice subjected to experimental lung injury and in cases of human IPF. Genetic (CD44v6 shRNA) or a small molecule inhibitor (CD44v6 peptide) targeting of CD44v6 abrogates fibrogenesis in murine models of lung injury. These studies support a function for CD44V6 in lung fibrosis and offer proof of concept for therapeutic targeting of CD44V6 in lung fibrosis disorders.

miR-21 represses Pdcd4 during cardiac valvulogenesis.

The discovery of small non-coding microRNAs has revealed novel mechanisms of post-translational regulation of gene expression, the implications of which are still incompletely understood. We focused on microRNA 21 (miR-21), which is expressed in cardiac valve endothelium during development, in order to better understand its mechanistic role in cardiac valve development. Using a combination of in vivo gene knockdown in zebrafish and in vitro assays in human cells, we show that miR-21 is necessary for proper development of the atrioventricular valve (AV). We identify pdcd4b as a relevant in vivo target of miR-21 and show that protection of pdcd4b from miR-21 binding results in failure of AV development. In vitro experiments using human pulmonic valve endothelial cells demonstrate that miR-21 overexpression augments endothelial cell migration. PDCD4 knockdown alone was sufficient to enhance endothelial cell migration. These results demonstrate that miR-21 plays a necessary role in cardiac valvulogenesis, in large part due to an obligatory downregulation of PDCD4.

Periostin induces intracellular cross-talk between kinases and hyaluronan in atrioventricular valvulogenesis.

Periostin (PN), a novel fasciclin-related matricellular protein, has been implicated in cardiac development and postnatal remodeling, but the mechanism remains unknown. We examined the role of PN in mediating intracellular kinase activation for atrioventricular valve morphogenesis using well defined explant cultures, gene transfection systems, and Western blotting. The results show that valve progenitor (cushion) cells secrete PN into the extracellular matrix, where it can bind to INTEGRINs and activate INTEGRIN/focal adhesion kinase signaling pathways and downstream kinases, PI3K/AKT and ERK. Functional assays with prevalvular progenitor cells showed that activating these signaling pathways promoted adhesion, migration, and anti-apoptosis. Through activation of PI3K/ERK, PN directly enhanced collagen expression. Comparing PN-null to WT mice also revealed that expression of hyaluronan (HA) and activation of hyaluronan synthase-2 (Has2) are also enhanced upon PN/INTEGRIN/focal adhesion kinase-mediated activation of PI3K and/or ERK, an effect confirmed by the reduction of HA synthase-2 in PN-null mice. We also identified in valve progenitor cells a potential autocrine signaling feedback loop between PN and HA through PI3K and/or ERK. Finally, in a three-dimensional assay to simulate normal valve maturation in vitro, PN promoted collagen compaction in a kinase-dependent fashion. In summary, this study provides the first direct evidence that PN can act to stimulate a valvulogenic signaling pathway.

Overexpression of c-Met and CD44v6 receptors contributes to autocrine TGF-ß1 signaling in interstitial lung disease.

The hepatocyte growth factor (HGF) and the HGF receptor Met pathway are important in the pathogenesis of interstitial lung disease (ILD). Alternatively spliced isoforms of CD44 containing variable exon 6 (CD44v6) and its ligand hyaluronan (HA) alter cellular function in response to interaction between CD44v6 and HGF. TGF-ß1 is the crucial cytokine that induces fibrotic action in ILD fibroblasts (ILDFbs). We have identified an autocrine TGF-ß1 signaling that up-regulates both Met and CD44v6 mRNA and protein expression. Western blot analysis, flow cytometry, and immunostaining revealed that CD44v6 and Met colocalize in fibroblasts and in tissue sections from ILD patients and in lungs of bleomycin-treated mice. Interestingly, cell proliferation induced by TGF-ß1 is mediated through Met and CD44v6. Further, cell proliferation mediated by TGF-ß1/CD44v6 is ERK-dependent. In contrast, action of Met on ILDFb proliferation does not require ERK but does require p38(MAPK). ILDFbs were sorted into CD44v6(+)/Met(+) and CD44v6(-)/Met(+) subpopulations. HGF inhibited TGF-ß1-stimulated collagen-1 and α-smooth muscle cell actin expression in both of these subpopulations by interfering with TGF-ß1 signaling. HGF alone markedly stimulated CD44v6 expression, which in turn regulated collagen-1 synthesis. Our data with primary lung fibroblast cultures with respect to collagen-1, CD44v6, and Met expressions were supported by immunostaining of lung sections from bleomycin-treated mice and from ILD patients. These results define the relationships between CD44v6, Met, and autocrine TGF-ß1 signaling and the potential modulating influence of HGF on TGF-ß1-induced CD44v6-dependent fibroblast function in ILD fibrosis.

The role of periostin in tissue remodeling across health and disease.

Periostin, also termed osteoblast-specific factor 2, is a matricellular protein with known functions in osteology, tissue repair, oncology, cardiovascular and respiratory systems, and in various inflammatory settings. However, most of the research to date has been conducted in divergent and circumscribed areas meaning that the overall understanding of this intriguing molecule remains fragmented. Here, we integrate the available evidence on periostin expression, its normal role in development, and whether it plays a similar function during pathologic repair, regeneration, and disease in order to bring together the different research fields in which periostin investigations are ongoing. In spite of the seemingly disparate roles of periostin in health and disease, tissue remodeling as a response to insult/injury is emerging as a common functional denominator of this matricellular molecule. Periostin is transiently upregulated during cell fate changes, either physiologic or pathologic. Combining observations from various conditions, a common pattern of events can be suggested, including periostin localization during development, insult and injury, epithelial-mesenchymal transition, extracellular matrix restructuring, and remodeling. We propose mesenchymal remodeling as an overarching role for the matricellular protein periostin, across physiology and disease. Periostin may be seen as an important structural mediator, balancing appropriate versus inappropriate tissue adaption in response to insult/injury.

Study of droplet formation process during drop-on-demand inkjetting of living cell-laden bioink.

Biofabrication offers a great potential for the fabrication of three-dimensional living tissues and organs by precisely layer-by-layer placing various tissue spheroids as anatomically designed. Inkjet printing of living cell-laden bioink is one of the most promising technologies enabling biofabrication, and the bioink printability must be carefully examined for it to be a viable biofabrication technology. In this study, the cell-laden bioink droplet formation process has been studied in terms of the breakup time, droplet size and velocity, and satellite formation using a time-resolved imaging approach. The bioink has been prepared using fibroblasts and sodium alginate with four different cell concentrations: without cells, 1 × 10(6), 5 × 10(6), and 1 × 10(7) cells/mL to appreciate the effect of cell concentration on the droplet formation process. Furthermore, the bioink droplet formation process is compared with that during the inkjetting of a comparable polystyrene microbead-laden suspension under the identical operating conditions to understand the effect of particle physical properties on the droplet formation process. It is found that (1) as the cell concentration of bioink increases, the droplet size and velocity decrease, the formation of satellite droplets is suppressed, and the breakup time increases, and (2) compared to the hard bead-laden suspension, the bioink tends to have a less ejected fluid volume, lower droplet velocity, and longer breakup time.

Novel di-tertiary-butyl phenylhydrazones as dual cyclooxygenase-2/5-lipoxygenase inhibitors: synthesis, COX/LOX inhibition, molecular modeling, and insights into their cytotoxicities.

Although dual inhibition of Cyclooxygenase-2 (COX-2) and 5-Lipoxygenase (5-LOX) enzymes is highly effective than targeting COX or LOX alone, there are only a few reports of examining such compounds in case of colorectal cancers (CRC). In the present work we report that the novel di-tert-butyl phenol-based dual inhibitors DTPSAL, DTPBHZ, DTPINH, and DTPNHZ exhibit significant cytotoxicity against human CRC cell lines. Molecular docking studies revealed a good fit of these compounds in the COX-2 and 5-LOX protein cavities. The inhibitors show significant inhibition of COX-2 and 5-LOX activities and are effective against a panel of human colon cancer cell lines including HCA-7, HT-29, SW480 and intestinal Apc10.1 cells as well as the hyaluronan synthase-2 (Has2) enzyme over-expressing colon cancer cells, through inhibition of the Hyaluronan/CD44v6 cell survival pathway. Western blot analysis and qRT-PCR analyses indicated that the di-tert-butyl phenol-based dual inhibitors reduce the expression of COX-2, 5-LOX, and CD44v6 in human colon cancer HCA-7 cells, while the combination of CD44v6shRNA and DTPSAL has an additional inhibitory effect on CD44v6 mRNA expression. The synergistic inhibitory effect of Celecoxib and Licofelone on CD44v6 mRNA expression suggests that the present dual inhibitors down-regulate cyclooxygenase and lipoxygenase enzymes through CD44v6. The compounds also exhibited enhanced antiproliferative potency compared to standard dual COX/LOX inhibitor, viz. Licofelone. Importantly, the HA/CD44v6 antagonist CD44v6shRNA in combination with synthetic compounds had a sensitizing effect on the cancer cells which enhanced their antiproliferative potency, a finding which is crucial for the anti-proliferative potency of the novel synthetic di-tert-butyl phenol based dual COX-LOX inhibitors in colon cancer cells.

Isothiocyanate analogs targeting CD44 receptor as an effective strategy against colon cancer.

Inflammatory pathway plays an important role in tumor cell progression of colorectal cancers. Although colon cancer is considered as one of the leading causes of death worldwide, very few drugs are available for its effective treatment. Many studies have examined the effects of specific COX-2 and 5-LOX inhibitors on human colorectal cancer, but the role of isothiocyanates (ITSCs) as COX-LOX dual inhibitors engaged in hyaluronan-CD44 interaction has not been studied. In the present work, we report series of ITSC analogs incorporating bioisosteric thiosemicarbazone moiety. These inhibitors are effective against panel of human colon cancer cell lines including COX-2 positive HCA-7, HT-29 cells lines, and hyaluronan synthase-2 (Has2) enzyme over-expressing transformed intestinal epithelial Apc10.1Has2 cells. Specifically, our findings indicate that HA-CD44v6-mediated COX-2/5-LOX signaling mediate survivin production, which in turn, supports anti-apoptosis and chemo-resistance leading to colon cancer cell survival. The over-expression of CD44v6shRNA as well as ITSC treatment significantly decreases the survival of colon cancer cells. The present results thus offer an opportunity to evolve potent inhibitors of HA synthesis and CD44v6 pathway and thus underscoring the importance of the ITSC analogs as chemopreventive agents for targeting HA/CD44v6 pathway.

Atrioventricular valve development: new perspectives on an old theme.

Atrioventricular valve development commences with an EMT event whereby endocardial cells transform into mesenchyme. The molecular events that induce this phenotypic change are well understood and include many growth factors, signaling components, and transcription factors. Besides their clear importance in valve development, the role of these transformed mesenchyme and the function they serve in the developing prevalve leaflets is less understood. Indeed, we know that these cells migrate, but how and why do they migrate? We also know that they undergo a transition to a mature, committed cell, largely defined as an interstitial fibroblast due to their ability to secrete various matrix components including collagen type I. However, we have yet to uncover mechanisms by which the matrix is synthesized, how it is secreted, and how it is organized. As valve disease is largely characterized by altered cell number, cell activation, and matrix disorganization, answering questions of how the valves are built will likely provide us with information of real clinical relevance. Although expression profiling and descriptive or correlative analyses are insightful, to advance the field, we must now move past the simplicity of these assays and ask fundamental, mechanistic based questions aimed at understanding how valves are "built". Herein we review current understandings of atrioventricular valve development and present what is known and what isn't known. In most cases, basic, biological questions and hypotheses that were presented decades ago on valve development still are yet to be answered but likely hold keys to uncovering new discoveries with relevance to both embryonic development and the developmental basis of adult heart valve diseases. Thus, the goal of this review is to remind us of these questions and provide new perspectives on an old theme of valve development.

Methodology for the Evaluation of Double-Layered Microcapsule Formability Zone in Compound Nozzle Jetting Based on Growth Rate Ratio.

Double-layered microcapsules, which usually consist of a core (polymeric) matrix surrounded by a (polymeric) shell, have been used in many industrial and scientific applications, such as microencapsulation of drugs and living cells. Concentric compound nozzle-based jetting has been favored due to its efficiency and precise control of the core-shell compound structure. Thus far, little is known about the underlying formation mechanism of double-layered microcapsules in compound nozzle jetting. This study aims to understand the formability of double-layered microcapsules in compound nozzle jetting by combining a theoretical analysis and numerical simulations. A linear temporal instability analysis is used to define the perturbation growth rates of stretching and squeezing modes and a growth ratio as a function of the wave number, and a computational fluid dynamics (CFD) method is implemented to model the microcapsule formation process in order to determine the good microcapsule forming range based on the growth ratio curve. Using a pseudobisection method, the lower and upper bounds of the good formability range have been determined for a given materials-nozzle system. The proposed formability prediction methodology has been implemented to model a water-poly (lactide-co-glycolide) (PLGA)-air compound jetting system.

Scaffold-free inkjet printing of three-dimensional zigzag cellular tubes.

The capability to print three-dimensional (3D) cellular tubes is not only a logical first step towards successful organ printing but also a critical indicator of the feasibility of the envisioned organ printing technology. A platform-assisted 3D inkjet bioprinting system has been proposed to fabricate 3D complex constructs such as zigzag tubes. Fibroblast (3T3 cell)-based tubes with an overhang structure have been successfully fabricated using the proposed bioprinting system. The post-printing 3T3 cell viability of printed cellular tubes has been found above 82% (or 93% with the control effect considered) even after a 72-h incubation period using the identified printing conditions for good droplet formation, indicating the promising application of the proposed bioprinting system. Particularly, it is proved that the tubular overhang structure can be scaffold-free fabricated using inkjetting, and the maximum achievable height depends on the inclination angle of the overhang structure. As a proof-of-concept study, the resulting fabrication knowledge helps print tissue-engineered blood vessels with complex geometry.

Interplay Between Chemotherapy-Activated Cancer Associated Fibroblasts and Cancer Initiating Cells Expressing CD44v6 Promotes Colon Cancer Resistance.

Cancer-initiating cells (CICs) drive colorectal tumor growth by their supportive niches where CICs interact with multiple cell types within the microenvironment, including cancer-associated fibroblasts (CAFs). We investigated the interplay between the CICs and the clinically relevant chemotherapeutic FOLFOX that creates the persistent tumorigenic properties of colorectal CICs, and stimulates the microenvironmental factors derived from the CAFs. We found that the CICs expressing an immunophenotype (CD44v6[+]) promote FOLFOX-resistance and that the CIC-immunophenotype was enhanced by factors secreted by CAFs after FOLFOX treatment These secreted factors included periostin, IL17A and WNT3A, which induced CD44v6 expression by activating WNT3A/ß-catenin signaling. Blocking the interaction between CICs with any of these CAF-derived factors through tissue-specific conditional silencing of CD44v6 significantly reduced colorectal tumorigenic potential. To achieve this, we generated two unique vectors (floxed-pSico-CD44v6 shRNA plus Fabpl-Cre) that were encapsulated into transferrin coated PEG-PEI/(nanoparticles), which when introduced in vivo reduced tumor growth more effectively than using CD44v6-blocking antibodies. Notably, this tissue-specific conditional silencing of CD44v6 resulted in long lasting effects on self-renewal and tumor growth associated with a positive feedback loop linking WNT3A signaling and alternative-splicing of CD44. These findings have crucial clinical implications suggesting that therapeutic approaches for modulating tumor growth that currently focus on cell-autonomous mechanisms may be too limited and need to be broadened to include mechanisms that recognize the interplay between the stromal factors and the subsequent CIC-immunophenotype enrichment. Thus, more specific therapeutic approaches may be required to block a chemotherapy induced remodeling of a microenvironment that acts as a paracrine regulator to enrich CD44v6 (+) in colorectal CICs.

Chemotherapy induces feedback up-regulation of CD44v6 in colorectal cancer initiating cells through ß-catenin/MDR1 signaling to sustain chemoresistance.

Chemoresistance in colorectal cancer initiating cells (CICs) involves the sustained activation of multiple drug resistance (MDR) and WNT/ß-catenin signaling pathways, as well as of alternatively spliced-isoforms of CD44 containing variable exon-6 (CD44v6). In spite of its importance, mechanisms underlying the sustained activity of WNT/ß-catenin signaling have remained elusive. The presence of binding elements of the ß-catenin-interacting transcription factor TCF4 in the MDR1 and CD44 promoters suggests that crosstalk between WNT/ß-catenin/TCF4-activation and the expression of the CD44v6 isoform mediated by FOLFOX, a first-line chemotherapeutic agent for colorectal cancer, could be a fundamental mechanism of FOLFOX resistance. Our results identify that FOLFOX treatment induced WNT3A secretion, which stimulated a positive feedback loop coupling ß-catenin signaling and CD44v6 splicing. In conjunction with FOLFOX induced WNT3A signal, specific CD44v6 variants produced by alternative splicing subsequently enhance the late wave of WNT/ß-catenin activation to facilitate cell cycle progression. Moreover, we revealed that FOLFOX-mediated sustained WNT signal requires the formation of a CD44v6-LRP6-signalosome in caveolin microdomains, which leads to increased FOLFOX efflux. FOLFOX-resistance in colorectal CICs occurs in the absence of tumor-suppressor disabled-2 (DAB2), an inhibitor of WNT/ß-catenin signaling. Conversely, in sensitive cells, DAB2 inhibition of WNT-signaling requires interaction with a clathrin containing CD44v6-LRP6-signalosome. Furthermore, full-length CD44v6, once internalized through the caveolin-signalosome, is translocated to the nucleus where in complex with TCF4, it binds to ß-catenin/TCF4-regulated MDR1, or to CD44 promoters, which leads to FOLFOX-resistance and CD44v6 transcription through transcriptional-reprogramming. These findings provide evidence that targeting CD44v6-mediated LRP6/ß-catenin-signaling and drug efflux may represent a novel approach to overcome FOLFOX resistance and inhibit tumor progression in colorectal CICs. Thus, sustained drug resistance in colorectal CICs is mediated by overexpression of CD44v6, which is both a functional biomarker and a therapeutic target in colorectal cancer.

Recruitment of bone marrow-derived valve interstitial cells is a normal homeostatic process.

Advances in understanding of the maintenance of the cardiac valves during normal cardiac function and response to injury have led to several novel findings, including that there is contribution of extra-cardiac cells to the major cellular population of the valve: the valve interstitial cell (VIC). While suggested to occur in human heart studies, we have been able to experimentally demonstrate, using a mouse model, that cells of bone marrow hematopoietic stem cell origin engraft into the valves and synthesize collagen type I. Based on these initial findings, we sought to further characterize this cell population in terms of its similarity to VICs and begin to elucidate its contribution to valve homeostasis. To accomplish this, chimeric mice whose bone marrow was repopulated with enhanced green fluorescent protein (EGFP) expressing total nucleated bone marrow cells were used to establish a profile of EGFP(+) valve cells in terms of their expression of hematopoietic antigens, progenitor markers, fibroblast- and myofibroblast-related molecules, as well as their distribution within the valves. Using this profile, we show that normal (non-irradiated, non-transplanted) mice have BM-derived cell populations that exhibit identical morphology and phenotype to those observed in transplanted mice. Collectively, our findings establish that the engraftment of bone marrow-derived cells occurs as part of normal valve homeostasis. Further, our efforts demonstrate that the use of myeloablative irradiation, which is commonly employed in studies involving bone marrow transplantation, does not elicit changes in the bone marrow-derived VIC phenotype in recipient mice.