Angiopoietin-like 4 induces a ß-catenin-mediated upregulation of ID3 in fibroblasts to reduce scar collagen expression.

In adult skin wounds, collagen expression rapidly re-establishes the skin barrier, although the resultant scar is aesthetically and functionally inferior to unwounded tissue. Although TGFß signaling and fibroblasts are known to be responsible for scar-associated collagen production, there are currently no prophylactic treatments for scar management. Fibroblasts in crosstalk with wound keratinocytes orchestrate collagen expression, although the precise paracrine pathways involved remain poorly understood. Herein, we showed that the matricellular protein, angiopoietin-like 4 (ANGPTL4), accelerated wound closure and reduced collagen expression in diabetic and ANGPTL4-knockout mice. Similar observations were made in wild-type rat wounds. Using human fibroblasts as a preclinical model for mechanistic studies, we systematically elucidated that ANGPTL4 binds to cadherin-11, releasing membrane-bound ß-catenin which translocate to the nucleus and transcriptionally upregulate the expression of Inhibitor of DNA-binding/differentiation protein 3 (ID3). ID3 interacts with scleraxis, a basic helix-loop-helix transcription factor, to inhibit scar-associated collagen types 1α2 and 3α1 production by fibroblasts. We also showed ANGPTL4 interaction with cadherin-11 in human scar tissue. Our findings highlight a central role for matricellular proteins such as ANGPTL4 in the attenuation of collagen expression and may have a broader implication for other fibrotic pathologies.

Delivery of doxorubicin and paclitaxel from double-layered microparticles: The effects of layer thickness and dual-drug vs. single-drug loading.

Double-layered microparticles composed of poly(d,l-lactic-co-glycolic acid, 50:50) (PLGA) and poly(l-lactic acid) (PLLA) were loaded with doxorubicin HCl (DOX) and paclitaxel (PCTX) through a solvent evaporation technique. DOX was localized in the PLGA shell, while PCTX was localized in the PLLA core. The aim of this study was to investigate how altering layer thickness of dual-drug, double-layered microparticles can influence drug release kinetics and their antitumor capabilities, and against single-drug microparticles. PCTX-loaded double-layered microparticles with denser shells retarded the initial release of PCTX, as compared with dual-drug-loaded microparticles. The DOX release from both DOX-loaded and dual-drug-loaded microparticles were observed to be similar with an initial burst. Through specific tailoring of layer thicknesses, a suppressed initial burst of DOX and a sustained co-delivery of two drugs can be achieved over 2months. Viability studies using spheroids of MCF-7 cells showed that controlled co-delivery of PCTX and DOX from dual-drug-loaded double-layered microparticles were better in reducing spheroid growth rate. This study provides mechanistic insights into how by tuning the layer thickness of double-layered microparticles the release kinetics of two drugs can be controlled, and how co-delivery can potentially achieve better anticancer effects. STATEMENT OF SIGNIFICANCE: While the release of multiple drugs has been reported to achieve successful apoptosis and minimize drug resistance, most conventional particulate systems can only deliver a single drug at a time. Recently, although a number of formulations (e.g. micellar nanoparticles, liposomes) have been successful in delivering two or more anticancer agents, sustained co-delivery of these agents remains inadequate due to the complex agent loading processes and rapid release of hydrophilic agents. Therefore, the present work reports the multilayered particulate system that simultaneously hosts different drugs, while being able to tune their individual release over months. We believe that our findings would be of interest to the readers of Acta Biomaterialia because the proposed system could open a new avenue on how two drugs can be released, through rate-controlling carriers, for combination chemotherapy.

How can we better predict the risk of spontaneous miscarriage among women experiencing threatened miscarriage?

This study seeks to establish progesterone and progesterone-induced blocking factor (PIBF) levels as predictors of subsequent completed miscarriage among women presenting with threatened miscarriage between 6 and 10 weeks of gestation. Our secondary objective was to assess the known maternal risk factors, toward development of a parsimonious and clinician-friendly risk assessment model for predicting completed miscarriage. In this article, we present a prospective cohort study of 119 patients presenting with threatened miscarriage from gestation weeks 6 to 10 at a tertiary women's hospital emergency unit in Singapore. Thirty (25.2%) women had a spontaneous miscarriage. Low progesterone and PIBF levels are similarly predictive of subsequent completed miscarriage. Study results (OR, 95% CI) showed that higher levels of progesterone (0.91, 95% CI 0.88-0.94) and PIBF (0.99, 95% CI 0.98-0.99) were associated with lower risk of miscarriage. Low progesterone level was a very strong predictor of miscarriage risk in our study despite previous concerns about its pulsatile secretion. Low serum progesterone and PIBF levels predicted spontaneous miscarriage among women presenting with threatened miscarriage between gestation weeks 6 to 10. Predictive models to calculate probability of spontaneous miscarriage based on serum progesterone, together with maternal BMI and fetal heart are proposed.

Angiopoietin-like 4 Increases Pulmonary Tissue Leakiness and Damage during Influenza Pneumonia.

Excessive host inflammatory responses negatively impact disease outcomes in respiratory infection. Host-pathogen interactions during the infective phase of influenza are well studied, but little is known about the host's response during the repair stage. Here, we show that influenza infection stimulated the expression of angiopoietin-like 4 (ANGPTL4) via a direct IL6-STAT3-mediated mechanism. ANGPTL4 enhanced pulmonary tissue leakiness and exacerbated inflammation-induced lung damage. Treatment of infected mice with neutralizing anti-ANGPTL4 antibodies significantly accelerated lung recovery and improved lung tissue integrity. ANGPTL4-deficient mice also showed reduced lung damage and recovered faster from influenza infection when compared to their wild-type counterparts. Retrospective examination of human lung biopsy specimens from infection-induced pneumonia with tissue damage showed elevated expression of ANGPTL4 when compared to normal lung samples. These observations underscore the important role that ANGPTL4 plays in lung infection and damage and may facilitate future therapeutic strategies for the treatment of influenza pneumonia.

Early controlled release of peroxisome proliferator-activated receptor ß/δ agonist GW501516 improves diabetic wound healing through redox modulation of wound microenvironment.

Diabetic wounds are imbued with an early excessive and protracted reactive oxygen species production. Despite the studies supporting PPARß/δ as a valuable pharmacologic wound-healing target, the therapeutic potential of PPARß/δ agonist GW501516 (GW) as a wound healing drug was never investigated. Using topical application of polymer-encapsulated GW, we revealed that different drug release profiles can significantly influence the therapeutic efficacy of GW and consequently diabetic wound closure. We showed that double-layer encapsulated GW microparticles (PLLA:PLGA:GW) provided an earlier and sustained dose of GW to the wound and reduced the oxidative wound microenvironment to accelerate healing, in contrast to single-layered PLLA:GW microparticles. The underlying mechanism involved an early GW-mediated activation of PPARß/δ that stimulated GPx1 and catalase expression in fibroblasts. GPx1 and catalase scavenged excessive H2O2 accumulation in diabetic wound beds, prevented H2O2-induced ECM modification and facilitated keratinocyte migration. The microparticles with early and sustained rate of GW release had better therapeutic wound healing activity. The present study underscores the importance of drug release kinetics on the therapeutic efficacy of the drug and warrants investigations to better appreciate the full potential of controlled drug release.

Angiopoietin-like 4 stimulates STAT3-mediated iNOS expression and enhances angiogenesis to accelerate wound healing in diabetic mice.

Impaired wound healing is a major source of morbidity in diabetic patients. Poor outcome has, in part, been related to increased inflammation, poor angiogenesis, and deficiencies in extracellular matrix components. Despite the enormous impact of these chronic wounds, effective therapies are lacking. Here, we showed that the topical application of recombinant matricellular protein angiopoietin-like 4 (ANGPTL4) accelerated wound reepithelialization in diabetic mice, in part, by improving angiogenesis. ANGPTL4 expression is markedly elevated upon normal wound injury. In contrast, ANGPTL4 expression remains low throughout the healing period in diabetic wounds. Exogenous ANGPTL4 modulated several regulatory networks involved in cell migration, angiogenesis, and inflammation, as evidenced by an altered gene expression signature. ANGPTL4 influenced the expression profile of endothelial-specific CD31 in diabetic wounds, returning its profile to that observed in wild-type wounds. We showed ANGPTL4-induced nitric oxide production through an integrin/JAK/STAT3-mediated upregulation of inducible nitric oxide synthase (iNOS) expression in wound epithelia, thus revealing a hitherto unknown mechanism by which ANGPTL4 regulated angiogenesis via keratinocyte-to-endothelial-cell communication. These data show that the replacement of ANGPTL4 may be an effective adjunctive or new therapeutic avenue for treating poor healing wounds. The present finding also confirms that therapeutic angiogenesis remains an attractive treatment modality for diabetic wound healing.

Inhibition of 3-D tumor spheroids by timed-released hydrophilic and hydrophobic drugs from multilayered polymeric microparticles.

First-line cancer chemotherapy necessitates high parenteral dosage and repeated dosing of a combination of drugs over a prolonged period. Current commercially available chemotherapeutic agents, such as Doxil and Taxol, are only capable of delivering single drug in a bolus dose. The aim of this study is to develop dual-drug-loaded, multilayered microparticles and to investigate their antitumor efficacy compared with single-drug-loaded particles. Results show hydrophilic doxorubicin HCl (DOX) and hydrophobic paclitaxel (PTX) localized in the poly(dl-lactic-co-glycolic acid, 50:50) (PLGA) shell and in the poly(l-lactic acid) (PLLA) core, respectively. The introduction of poly[(1,6-bis-carboxyphenoxy) hexane] (PCPH) into PLGA/PLLA microparticles causes PTX to be localized in the PLLA and PCPH mid-layers, whereas DOX is found in both the PLGA shell and core. PLGA/PLLA/PCPH microparticles with denser shells allow better control of DOX release. A delayed release of PTX is observed with the addition of PCPH. Three-dimensional MCF-7 spheroid studies demonstrate that controlled co-delivery of DOX and PTX from multilayered microparticles produces a greater reduction in spheroid growth rate compared with single-drug-loaded particles. This study provides mechanistic insights into how distinctive structure of multilayered microparticles can be designed to modulate the release profiles of anticancer drugs, and how co-delivery can potentially provide better antitumor response.

Klf2 is an essential factor that sustains ground state pluripotency.

The maintenance of mouse embryonic stem cells (mESCs) requires LIF and serum. However, a pluripotent "ground state," bearing resemblance to preimplantation mouse epiblasts, can be established through dual inhibition (2i) of both prodifferentiation Mek/Erk and Gsk3/Tcf3 pathways. While Gsk3 inhibition has been attributed to the transcriptional derepression of Esrrb, the molecular mechanism mediated by Mek inhibition remains unclear. In this study, we show that Krüppel-like factor 2 (Klf2) is phosphorylated by Erk2 and that phospho-Klf2 is proteosomally degraded. Mek inhibition hence prevents Klf2 protein phosphodegradation to sustain pluripotency. Indeed, while Klf2-null mESCs can survive under LIF/Serum, they are not viable under 2i, demonstrating that Klf2 is essential for ground state pluripotency. Importantly, we also show that ectopic Klf2 expression can replace Mek inhibition in mESCs, allowing the culture of Klf2-null mESCs under Gsk3 inhibition alone. Collectively, our study defines the Mek/Erk/Klf2 axis that cooperates with the Gsk3/Tcf3/Esrrb pathway in mediating ground state pluripotency.

Fabrication and drug release study of double-layered microparticles of various sizes.

Double-layered microparticles, composed of poly(D,L-lactide-co-glycolide) (50:50) (PLGA) core and poly(L-lactide) (PLLA) shell, of controllable sizes ranging from several hundred microns to few microns were fabricated using a one-step solvent evaporation method. Metoclopramide monohydrochloride monohydrate (MCA), a hydrophilic drug, was selectively localized in the PLGA core. To achieve the double-layered particles of size approximately 2 µm, the process parameters were carefully manipulated to extend the phase separation time by increasing oil-to-water ratio and saturating the surrounding aqueous phase with solvent. Subsequently, the drug release profiles of the double-layered particles of various sizes were studied. Increased particle size resulted in faster degradation of polymers because of autocatalysis, accelerating the release rate of MCA. Interestingly, the effect of degradation rates, affected by particle sizes, on drug release was insignificant when the particle size was drastically reduced to 2-20 µm in the investigated double-layered particles. This understanding would provide critical insights into how the controllable formation and unique drug release profiles of double-layered particles of various sizes can be achieved.

Matricellular proteins: a sticky affair with cancers.

The multistep process of metastasis is a major hallmark of cancer progression involving the cointeraction and coevolution of the tumor and its microenvironment. In the tumor microenvironment, tumor cells and the surrounding stromal cells aberrantly secrete matricellular proteins, which are a family of nonstructural proteins in the extracellular matrix (ECM) that exert regulatory roles via a variety of molecular mechanisms. Matricellular proteins provide signals that support tumorigenic activities characteristic of the metastastic cascade such as epithelial-to-mesenchymal (EMT) transition, angiogenesis, tumor cell motility, proliferation, invasion, evasion from immune surveillance, and survival of anoikis. Herein, we review the current understanding of the following matricellular proteins and highlight their pivotal and multifacted roles in metastatic progression: angiopoietin-like protein 4 (ANGPTL4), CCN family members cysteine-rich angiogenic inducer 61 (Cyr61/CCN1) and CCN6, osteopontin (OPN), secreted protein acidic and rich in cysteine (SPARC), tenascin C (TNC), and thrombospondin-1 and -2 (TSP1, TSP2). Insights into the signaling mechanisms resulting from the interaction of these matricellular proteins and their respective molecular partner(s), as well as their subsequent contribution to tumor metastasis, are discussed. In addition, emerging evidences of their promising potential as therapeutic options and/or targets in the treatment of cancer are also highlighted.

ANGPTL4 modulates vascular junction integrity by integrin signaling and disruption of intercellular VE-cadherin and claudin-5 clusters.

Vascular disruption induced by interactions between tumor-secreted permeability factors and adhesive proteins on endothelial cells facilitates metastasis. The role of tumor-secreted C-terminal fibrinogen-like domain of angiopoietin-like 4 (cANGPTL4) in vascular leakiness and metastasis is controversial because of the lack of understanding of how cANGPTL4 modulates vascular integrity. Here, we show that cANGPTL4 instigated the disruption of endothelial continuity by directly interacting with 3 novel binding partners, integrin α5ß1, VE-cadherin, and claudin-5, in a temporally sequential manner, thus facilitating metastasis. We showed that cANGPTL4 binds and activates integrin α5ß1-mediated Rac1/PAK signaling to weaken cell-cell contacts. cANGPTL4 subsequently associated with and declustered VE-cadherin and claudin-5, leading to endothelial disruption. Interfering with the formation of these cANGPTL4 complexes delayed vascular disruption. In vivo vascular permeability and metastatic assays performed using ANGPTL4-knockout and wild-type mice injected with either control or ANGPTL4-knockdown tumors confirmed that cANGPTL4 induced vascular leakiness and facilitated lung metastasis in mice. Thus, our findings elucidate how cANGPTL4 induces endothelial disruption. Our findings have direct implications for targeting cANGPTL4 to treat cancer and other vascular pathologies.

Angiopoietin-like 4 protein elevates the prosurvival intracellular O2(-):H2O2 ratio and confers anoikis resistance to tumors.

Cancer is a leading cause of death worldwide. Tumor cells exploit various signaling pathways to promote their growth and metastasis. To our knowledge, the role of angiopoietin-like 4 protein (ANGPTL4) in cancer remains undefined. Here, we found that elevated ANGPTL4 expression is widespread in tumors, and its suppression impairs tumor growth associated with enhanced apoptosis. Tumor-derived ANGPTL4 interacts with integrins to stimulate NADPH oxidase-dependent production of O(2)(-). A high ratio of O(2)(-):H(2)O(2) oxidizes/activates Src, triggering the PI3K/PKBα and ERK prosurvival pathways to confer anoikis resistance, thus promoting tumor growth. ANGPTL4 deficiency results in diminished O(2)(-) production and a reduced O(2)(-):H(2)O(2) ratio, creating a cellular environment conducive to apoptosis. ANGPTL4 is an important redox player in cancer and a potential therapeutic target.

Angiopoietin-like 4 interacts with integrins beta1 and beta5 to modulate keratinocyte migration.

Adipose tissue secretes adipocytokines for energy homeostasis, but recent evidence indicates that some adipocytokines also have a profound local impact on wound healing. Upon skin injury, keratinocytes use various signaling molecules to promote reepithelialization for efficient wound closure. In this study, we identify a novel function of adipocytokine angiopoietin-like 4 (ANGPTL4) in keratinocytes during wound healing through the control of both integrin-mediated signaling and internalization. Using two different in vivo models based on topical immuno-neutralization of ANGPTL4 as well as ablation of the ANGPTL4 gene, we show that ANGPTL4-deficient mice exhibit delayed wound reepithelialization with impaired keratinocyte migration. Human keratinocytes in which endogenous ANGPTL4 expression was suppressed by either siRNA or a neutralizing antibody show impaired migration associated with diminished integrin-mediated signaling. Importantly, we identify integrins ß1 and ß5, but not ß3, as novel binding partners of ANGPTL4. ANGPTL4-bound integrin ß1 activated the FAK-Src-PAK1 signaling pathway, which is important for cell migration. The findings presented herein reveal an unpredicted role of ANGPTL4 during wound healing and demonstrate how ANGPTL4 stimulates intracellular signaling mechanisms to coordinate cellular behavior. Our findings provide insight into a novel cell migration control mechanism and underscore the physiological importance of the modulation of integrin activity in cancer metastasis.

Angiopoietin-like 4 interacts with matrix proteins to modulate wound healing.

A dynamic cell-matrix interaction is crucial for a rapid cellular response to changes in the environment. Appropriate cell behavior in response to the changing wound environment is required for efficient wound closure. However, the way in which wound keratinocytes modify the wound environment to coordinate with such cellular responses remains less studied. We demonstrated that angiopoietin-like 4 (ANGPTL4) produced by wound keratinocytes coordinates cell-matrix communication. ANGPTL4 interacts with vitronectin and fibronectin in the wound bed, delaying their proteolytic degradation by metalloproteinases. This interaction does not interfere with integrin-matrix protein recognition and directly affects cell-matrix communication by altering the availability of intact matrix proteins. These interactions stimulate integrin- focal adhesion kinase, 14-3-3, and PKC-mediated signaling pathways essential for effective wound healing. The deficiency of ANGPTL4 in mice delays wound re-epithelialization. Further analysis revealed that cell migration was impaired in the ANGPTL4-deficient keratinocytes. Altogether, the findings provide molecular insight into a novel control of wound healing via ANGPTL4-dependent regulation of cell-matrix communication. Given the known role of ANGPTL4 in glucose and lipid homeostasis, it is a prime therapeutic candidate for the treatment of diabetic wounds. It also underscores the importance of cell-matrix communication during angiogenesis and cancer metastasis.

Regulation of cell proliferation and migration by TAK1 via transcriptional control of von Hippel-Lindau tumor suppressor.

Skin maintenance and healing after wounding requires complex epithelial-mesenchymal interactions purportedly mediated by growth factors and cytokines. We show here that, for wound healing, transforming growth factor-beta-activated kinase 1 (TAK1) in keratinocytes activates von Hippel-Lindau tumor suppressor expression, which in turn represses the expression of platelet-derived growth factor-B (PDGF-B), integrin beta1, and integrin beta5 via inhibition of the Sp1-mediated signaling pathway in the keratinocytes. The reduced production of PDGF-B leads to a paracrine-decreased expression of hepatocyte growth factor in the underlying fibroblasts. This TAK1 regulation of the double paracrine PDGF/hepatocyte growth factor signaling can regulate keratinocyte cell proliferation and is required for proper wound healing. Strikingly, TAK1 deficiency enhances cell migration. TAK1-deficient keratinocytes displayed lamellipodia formation with distinct microspike protrusion, associated with an elevated expression of integrins beta1 and beta5 and sustained activation of cdc42, Rac1, and RhoA. Our findings provide evidence for a novel homeostatic control of keratinocyte proliferation and migration mediated via TAK1 regulation of von Hippel-Lindau tumor suppressor. Dysfunctional regulation of TAK1 may contribute to the pathology of non-healing chronic inflammatory wounds and psoriasis.

Regulation of epithelial-mesenchymal IL-1 signaling by PPARbeta/delta is essential for skin homeostasis and wound healing.

Skin morphogenesis, maintenance, and healing after wounding require complex epithelial-mesenchymal interactions. In this study, we show that for skin homeostasis, interleukin-1 (IL-1) produced by keratinocytes activates peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) expression in underlying fibroblasts, which in turn inhibits the mitotic activity of keratinocytes via inhibition of the IL-1 signaling pathway. In fact, PPARbeta/delta stimulates production of the secreted IL-1 receptor antagonist, which leads to an autocrine decrease in IL-1 signaling pathways and consequently decreases production of secreted mitogenic factors by the fibroblasts. This fibroblast PPARbeta/delta regulation of the IL-1 signaling is required for proper wound healing and can regulate tumor as well as normal human keratinocyte cell proliferation. Together, these findings provide evidence for a novel homeostatic control of keratinocyte proliferation and differentiation mediated via PPARbeta/delta regulation in dermal fibroblasts of IL-1 signaling. Given the ubiquitous expression of PPARbeta/delta, other epithelial-mesenchymal interactions may also be regulated in a similar manner.