IL-33 mast cell axis is central in LL-37 induced bladder inflammation and pain in a murine interstitial cystitis model.

Interstitial cystitis (IC), also known as painful bladder syndrome (PBS), is a debilitating chronic condition that afflicts over 3 million women above the age of 18 in the U.S., and most patients fail to respond to current treatment options. Mast cells have previously been implicated as both a diagnostic and prognostic marker in IC/PBS. Patients with IC/PBS have been shown to have elevated levels of IL-33, a cytokine released in response to tissue insult, in their urine. We hypothesize that mast cell-mediated inflammation induced from IL-33 may play an important role in initiating pain and inflammation in IC/PBS. A human cathelicidin, LL-37, which is found at elevated levels in IC/PBS patients, was used to induce an IC/PBS-like state of inflammation and bladder pain in mast cell deficient C-kit (-/-) and wild type C57Bl/6 (WT) mice. Inflammation was quantified using myeloperoxidase (MPO) expression in bladder tissues measured via ELISA. Response rate to suprapubic stimulation from von Frey filaments was used to assess the relative pain and discomfort. Both types of mice increased IL-33 expression in response to LL-37 exposure. However, mast cell deficient mice demonstrated significantly lower levels of inflammation (p < 0.001) and reduced pain response (p < 0.001) compared to WT mice. These findings implicate an IL-33-mast cell dependent axis with a potential etiology of pain and inflammation in IC/PBS. Future therapeutics aimed at targeting the IL-33 - mast cell axis could potentially serve as useful targets for treating IC/PBS.

Bladder pain in an LL-37 interstitial cystitis and painful bladder syndrome model.

Our goal was to evaluate the pain response in an LL-37 induced murine model for interstitial cystitis/painful bladder syndrome (IC/PBS). In particular, we sought to characterize the dose dependence, time-course, and relationship of LL-37 induced bladder inflammation and pain. The IC/PBS model was induced in C57Bl/6 mice by instilling 50 µL of LL-37, an immunomodulatory human cathelicidin (anti-microbial peptide), in the bladder for 1 hr. Pain responses were measured using von Frey filaments (0.04 gm to 4.0 gm) before and after LL-37 instillation. Inflammation was evaluated using tissue myeloperoxidase (MPO) assay, gross inspection, and microscopic histologic examination. The dose response experiment demonstrated a graded pain response, with higher concentrations of LL-37 challenge yielding higher pain responses across all stimuli tested. Statistical significance was seen when comparing 1.0 gm von Frey filament results at 320 µM (68 ± 8% response) vs. 0 µM (38 ± 6% response). Interestingly, pain responses did not attenuate across time but increased significantly after 5 (p=0.0012) and 7 days (p=0.0096). Comparison with MPO data suggested that pain responses could be independent of inflammation. We demonstrated within our LL-37 induced IC/PBS model pain occurs in a dose-dependent fashion, pain responses persist beyond the initial point of insult, and our dose response and time course experiments demonstrated that pain was independent of inflammation.

Silk-elastinlike protein polymers enhance the efficacy of a therapeutic glycosaminoglycan for prophylactic treatment of radiation-induced proctitis.

Radiation-induced proctitis (RIP) is the most common clinical adverse effect for patients receiving radiotherapy as part of the standard course of treatment for ovarian, prostate, colon, and bladder cancers. RIP limits radiation dosage, interrupts treatment, and lowers patients' quality of life. A prophylactic treatment that protects the gastrointestinal tract from deleterious effects of radiotherapy will significantly improve patient quality of life and may allow for higher and more regular doses of radiation therapy. Semi-synthetic glycosaminoglycan (GAG), generated from the sulfation of hyaluronic acid, are anti-inflammatory but have difficulty achieving therapeutic levels in many tissues. To enhance the delivery of GAG, we created an in situ gelling rectal delivery system using silk-elastinlike protein polymers (SELPs). Using solutions of SELP 815K (which contains 6 repeats of blocks comprised of 8 silk-like units, 15 elastin-like units, and 1 lysine-substituted elastin-like unit) with GAG GM-0111, we created an injectable delivery platform that transitioned in <5min from a liquid at room temperature to a hydrogel at body temperature. The hydrogels released 50% of their payload within 30min and enhanced the accumulation of GAG in the rectum compared to traditional enema-based delivery. Using a murine model of radiation-induced proctitis, the prophylactic delivery of a single dose of GAG from a SELP matrix administered prior to irradiation significantly reduced radiation-induced pain after 3, 7, and 21days by 53±4%, 47±10%, and 12±6%, respectively. Matrix-mediated delivery of GAG by SELP represents an innovative method for more effective treatment of RIP and promises to improve quality of life of cancer patients by allowing higher radiotherapy doses with improved safety.

A Modified Glycosaminoglycan, GM-0111, Inhibits Molecular Signaling Involved in Periodontitis.

BACKGROUND: Periodontitis is characterized by microbial infection, inflammation, tissue breakdown, and accelerated loss of alveolar bone matrix. Treatment targeting these multiple stages of the disease provides ways to treat or prevent periodontitis. Certain glycosaminoglycans (GAGs) block multiple inflammatory mediators as well as suppress bacterial growth, suggesting that these GAGs may be exploited as a therapeutic for periodontitis. METHODS: We investigated the effects of a synthetic GAG, GM-0111, on various molecular events associated with periodontitis: growth of Porphyromonas gingivalis (P. gingivalis) and Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) pathogenic bacteria associated with periodontitis; activation of pro-inflammatory signaling through TLR2 and TLR4 in mouse macrophage RAW 264.7 cells and heterologously expressed HEK 293 cells; osteoclast formation and bone matrix resorption in cultured mouse pre-osteoclasts. RESULTS: (1) GM-0111 suppressed the growth of P. gingivalis and A. actinomycetemcomitans even at 1% (w/v) solution. The antibacterial effects of GM-0111 were stronger than hyaluronic acid (HA) or xylitol in P. gingivalis at all concentrations and comparable to xylitol in A. actinomycetemcomitans at ≥2% (w/v) solution. We also observed that GM-0111 suppressed biofilm formation of P. gingivalis and these effects were much stronger than HA. (2) GM-0111 inhibited TLR-mediated pro-inflammatory cellular signaling both in macrophage and HEK 293 cells with higher selectivity for TLR2 than TLR4 (IC50 of 1-10 ng/mL vs. > 100 µg/mL, respectively). (3) GM-0111 blocked RANKL-induced osteoclast formation (as low as 300 ng/mL) and bone matrix resorption. While GM-0111 showed high affinity binding to RANKL, it did not interfere with RANKL/RANK/NF-κB signaling, suggesting that GM-0111 inhibits osteoclast formation by a RANKL-RANK-independent mechanism. CONCLUSIONS: We report that GM-0111 inhibits multiple molecular events involved in periodontitis, spanning from the early pro-inflammatory TLR signaling, to pathways activated at the later stage component of bone loss.

Carboxymethyl Hyaluronan-Stabilized Nanoparticles for Anticancer Drug Delivery.

Carboxymethyl hyaluronic acid (CMHA) is a semisynthetic derivative of HA that is recognized by HA binding proteins but contains an additional carboxylic acid on some of the 6-hydroxyl groups of the N-acetyl glucosamine sugar units. These studies tested the ability of CMHA to stabilize the formation of calcium phosphate nanoparticles and evaluated their potential to target therapy resistant, CD44(+)/CD24(-/low) human breast cancer cells (BT-474EMT). CMHA stabilized particles (nCaP(CMHA)) were loaded with the chemotherapy drug cis-diamminedichloroplatinum(II) (CDDP) to form nCaP(CMHA)CDDP. nCaP(CMHA)CDDP was determined to be poorly crystalline hydroxyapatite, 200 nm in diameter with a -43 mV zeta potential. nCaP(CMHA)CDDP exhibited a two-day burst release of CDDP that tapered resulting in 86% release by 7 days. Surface plasmon resonance showed that nCaP(CMHA)CDDP binds to CD44, but less effectively than CMHA or hyaluronan. nCaP(CMHA-AF488) was taken up by CD44(+)/CD24(-) BT-474EMT breast cancer cells within 18 hours. nCaP(CMHA)CDDP was as cytotoxic as free CDDP against the BT-474EMT cells. Subcutaneous BT-474EMT tumors were more reproducibly inhibited by a near tumor dose of 2.8 mg/kg CDDP than a 7 mg/kg dose nCaP(CMHA)CDDP. This was likely due to a lack of distribution of nCaP(CMHA)CDDP throughout the dense tumor tissue that limited drug diffusion.

Vinyl sulfone analogs of lysophosphatidylcholine irreversibly inhibit autotaxin and prevent angiogenesis in melanoma.

Autotaxin (ATX) is an enzyme discovered in the conditioned medium of cultured melanoma cells and identified as a protein that strongly stimulates motility. This unique ectonucleotide pyrophosphatase and phosphodiesterase facilitates the removal of a choline headgroup from lysophosphatidylcholine (LPC) to yield lysophosphatidic acid (LPA), which is a potent lipid stimulator of tumorigenesis. Thus, ATX has received renewed attention because it has a prominent role in malignant progression with significant translational potential. Specifically, we sought to develop active site-targeted irreversible inhibitors as anti-cancer agents. Herein we describe the synthesis and biological activity of an LPC-mimetic electrophilic affinity label that targets the active site of ATX, which has a critical threonine residue that acts as a nucleophile in the lysophospholipase D reaction to liberate choline. We synthesized a set of quaternary ammonium derivative-containing vinyl sulfone analogs of LPC that function as irreversible inhibitors of ATX and inactivate the enzyme. The analogs were tested in cell viability assays using multiple cancer cell lines. The IC50 values ranged from 6.74 to 0.39 µM, consistent with a Ki of 3.50 µM for inhibition of ATX by the C16H33 vinyl sulfone analog CVS-16 (10b). A phenyl vinyl sulfone control compound, PVS-16, lacking the choline-like quaternary ammonium mimicking head group moiety, had little effect on cell viability and did not inhibit ATX. Most importantly, CVS-16 (10b) significantly inhibited melanoma progression in an in vivo tumor model by preventing angiogenesis. Taken together, this suggests that CVS-16 (10b) is a potent and irreversible ATX inhibitor with significant biological activity both in vitro and in vivo.

Why regenerative medicine needs an extracellular matrix.

Regenerative medicine is now coming of age. Many attempts at cell therapy have failed to show significant efficacy, and the umbrella term 'stem cell therapy' is perceived in some quarters as hype or just expensive and unnecessary medical tourism. Here we present a short editorial in three parts. First, we examine the importance of using a semisynthetic extracellular matrix (ECM) mimetic, or sECM, to deliver and retain therapeutic cells at the site of administration. Second, we describe one approach in which biophysical and biochemical properties are tailored to each tissue type, which we call "design for optimal functionality." Third, we describe an alternative approach to sECM design and implementation, called "design for simplicity," in which a deconstructed, minimalist sECM is employed and biology is allowed to perform the customization in situ. We opine that an sECM, whether minimal or instructive, is an essential contributor to improve the outcomes of cell-based therapies.

Targeting Glutathione S-transferase M4 in Ewing sarcoma.

Ewing sarcoma is a malignant pediatric bone and soft tissue tumor. Although the 5-year survival rate of localized disease approaches 75%, the prognosis of metastatic and/or therapy-resistant disease remains dismal despite the wide use of aggressive therapeutic strategies. We previously reported that high expression of glutathione S-transferase M4 (GSTM4) in primary tumors correlates with poor patient outcomes. GSTM4 is required for oncogenic transformation and mediates resistance to chemotherapeutic drugs in Ewing sarcoma cells. Here, we performed RNA-sequencing analyses of Ewing sarcoma cells and combined our results with publicly available datasets to demonstrate that GSTM4 is a major GST specifically expressed in Ewing sarcoma. Pharmacological inhibition of GSTM4 activity using a pan GST inhibitor, 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio) hexanol (NBDHEX), significantly limited cellular proliferation and oncogenic transformation of Ewing sarcoma cells. Moreover, combined use of NBDHEX and etoposide synergistically increased cytotoxicity, suggesting a role for GSTM4 as an inhibitor of apoptosis. Mechanistic studies revealed that GSTM4 limits apoptosis owing to its ability to interact with Apoptosis Signal-regulating Kinase 1 (ASK1) and inhibit signaling via the c-Jun N-terminal Kinase axis. To exploit our observation that GSTM4 expression is specifically up-regulated in Ewing sarcoma, we tested the effect of a GSTM4-activated anti-cancer agent, O(2)-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate or JS-K, on tumor growth and survival. We found that JS-K robustly decreased Ewing sarcoma cell viability and xenograft tumor growth and improved overall survival of xenograft mice. Our data suggest that GSTM4 is a novel therapeutic target for the treatment of high GSTM4-expressing Ewing sarcoma. Strategies that combine standard chemotherapy with agents that inhibit GSTM4, that are activated by GSTM4, or that block GSTM4/ASK1 interactions, can potentially be more specific and/or efficacious than standard therapeutic approaches.

Augmentation of integrin-mediated mechanotransduction by hyaluronic acid.

Changes in tissue and organ stiffness occur during development and are frequently symptoms of disease. Many cell types respond to the stiffness of substrates and neighboring cells in vitro and most cell types increase adherent area on stiffer substrates that are coated with ligands for integrins or cadherins. In vivo cells engage their extracellular matrix (ECM) by multiple mechanosensitive adhesion complexes and other surface receptors that potentially modify the mechanical signals transduced at the cell/ECM interface. Here we show that hyaluronic acid (also called hyaluronan or HA), a soft polymeric glycosaminoglycan matrix component prominent in embryonic tissue and upregulated during multiple pathologic states, augments or overrides mechanical signaling by some classes of integrins to produce a cellular phenotype otherwise observed only on very rigid substrates. The spread morphology of cells on soft HA-fibronectin coated substrates, characterized by formation of large actin bundles resembling stress fibers and large focal adhesions resembles that of cells on rigid substrates, but is activated by different signals and does not require or cause activation of the transcriptional regulator YAP. The fact that HA production is tightly regulated during development and injury and frequently upregulated in cancers characterized by uncontrolled growth and cell movement suggests that the interaction of signaling between HA receptors and specific integrins might be an important element in mechanical control of development and homeostasis.

Non-invasive imaging of tumors by monitoring autotaxin activity using an enzyme-activated near-infrared fluorogenic substrate.

Autotaxin (ATX), an autocrine motility factor that is highly upregulated in metastatic cancer, is a lysophospholipase D enzyme that produces the lipid second messenger lysophosphatidic acid (LPA) from lysophosphatidylcholine (LPC). Dysregulation of the lysolipid signaling pathway is central to the pathophysiology of numerous cancers, idiopathic pulmonary fibrosis, rheumatoid arthritis, and other inflammatory diseases. Consequently, the ATX/LPA pathway has emerged as an important source of biomarkers and therapeutic targets. Herein we describe development and validation of a fluorogenic analog of LPC (AR-2) that enables visualization of ATX activity in vivo. AR-2 exhibits minimal fluorescence until it is activated by ATX, which substantially increases fluorescence in the near-infrared (NIR) region, the optimal spectral window for in vivo imaging. In mice with orthotopic ATX-expressing breast cancer tumors, ATX activated AR-2 fluorescence. Administration of AR-2 to tumor-bearing mice showed high fluorescence in the tumor and low fluorescence in most healthy tissues with tumor fluorescence correlated with ATX levels. Pretreatment of mice with an ATX inhibitor selectively decreased fluorescence in the tumor. Together these data suggest that fluorescence directly correlates with ATX activity and its tissue expression. The data show that AR-2 is a non-invasive and selective tool that enables visualization and quantitation of ATX-expressing tumors and monitoring ATX activity in vivo.

Prevention of anti-microbial peptide LL-37-induced apoptosis and ATP release in the urinary bladder by a modified glycosaminoglycan.

Interstitial cystitis (IC), often referred to in combination with painful bladder syndrome, is a chronic inflammatory disease of the bladder. Current therapies primarily focus on replenishing urothelial glycosaminoglycan (GAG) layer using GAG analogs and managing pain with supportive therapies. However, the elusive etiology of IC and the lack of animal models to study the disease have been major hurdles developing more effective therapeutics. Previously, we showed an increased urinary concentration of antimicrobial peptide LL-37 in spina bifida patients and used LL-37 to develop a mouse model of cystitis that mimics important clinical findings of IC. Here we investigate (1) the molecular mechanism of LL-37 induced cystitis in cultured human urothelial cells and in mice, (2) the protective effects of GM-0111, a modified GAG, within the context of this mechanism, (3) the physiological and molecular markers that correlate with the severity of the inflammation, and (4) the protective effects of several GAGs using these biomarkers in our LL-37 induced cystitis model. We find that LL-37 quickly induces release of ATP and apoptosis in the urothelium. These changes can be inhibited by a chemically-modified GAG, GM-0111. Furthermore, we also find that GAG analogs provide varying degrees of protection against LL-37 challenge in mice. These findings suggest that GM-0111 and possibly GAG molecules prevent the development of cystitis by blocking the apoptosis and the concurrent release of ATP from the urothelium.

The influence of collagen and hyaluronan matrices on the delivery and bioactivity of bone morphogenetic protein-2 and ectopic bone formation.

Bone morphogenetic protein-2 (BMP-2) is known to enhance fracture healing when delivered via a bovine collagen sponge. However, collagen rapidly releases BMP-2 with a high burst phase that is followed by a low sustained phase. As a result, supra-physiological doses of BMP-2 are often required to successfully treat bone defects. High BMP-2 dosing can introduce serious side effects that include edema, bone overgrowth, cyst-like bone formation and significant inflammation. As the release behavior of BMP-2 carriers significantly affects the efficacy of fracture healing, we sought to compare the influence of two BMP-2 delivery matrices with contrasting release profiles on BMP-2 bioactivity and ectopic bone formation. We compared a thiol-modified hyaluronan (Glycosil™) hydrogel that exhibits a low burst followed by a sustained release of BMP-2 to a collagen sponge for the delivery of three different doses of BMP-2, the bioactivities of released BMP-2 and ectopic bone formation. Analysis of bone formation by micro-computed tomography revealed that low burst followed by sustained release of BMP-2 from a hyaluronan hydrogel induced up to 456% more bone compared to a BMP-2 dose-matched collagen sponge that has a high burst and sustained release. This study demonstrates that BMP-2 released with a low burst followed by a sustained release of BMP-2 is more desirable for bone formation. This highlights the therapeutic potential of hydrogels, particularly hyaluronan-based, for the delivery of BMP-2 for the treatment of bone defects and may help abrogate the adverse clinical effects associated with high dose growth factor use.

Physiological relevance of LL-37 induced bladder inflammation and mast cells.

PURPOSE: We established the physiological relevance of LL-37 induced bladder inflammation. We hypothesized that 1) human urinary LL-37 is increased in pediatric patients with spina bifida, 2) LL-37 induced inflammation occurs in our mouse model via urothelial binding and is dose dependent and 3) LL-37 induced inflammation involves mast cells. MATERIALS AND METHODS: To test our first hypothesis, we obtained urine samples from 56 pediatric patients with spina bifida and 22 normal patients. LL-37 was measured by enzyme-linked immunosorbent assay. Our second hypothesis was tested in C57Bl/6 mice challenged with 7 LL-37 concentrations intravesically for 1 hour. At 24 hours tissues were examined histologically and myeloperoxidase assay was done to quantitate inflammation. In separate experiments fluorescent LL-37 was instilled and tissues were obtained immediately (time = 0) and at 24 hours (time = 24). To test our final hypothesis, we performed immunohistochemistry for mast cell tryptase and evaluated 5 high power fields per bladder to determine the mean number of mast cells per mm(2). RESULTS: Urinary LL-37 was 89-fold higher in patients with spina bifida. Mouse LL-37 dose escalation experiments revealed increased inflammation at higher LL-37 concentrations. Fluorescent LL-37 demonstrated global urothelial binding at time = 0 but was not visible at time = 24. Immunohistochemistry for tryptase revealed mast cell infiltration in all tissue layers. At higher concentrations the LL-37 challenge led to significantly greater mast cell infiltration. CONCLUSIONS: Urinary LL-37 was significantly increased in pediatric patients with spina bifida. To our knowledge we report for the first time that LL-37 can elicit profound, dose dependent bladder inflammation involving the urothelium. Finally, inflammation propagation involves mast cells.

LL-37 induced cystitis and the receptor for advanced glycation end-products (RAGE) pathway.

To elucidate pathways in bladder inflammation, we employed our physiologically relevant LL-37 induced cystitis model. Based on inflammatory studies involving other organ systems implicating the receptor for advanced glycation end-products (RAGE), we first hypothesized that RAGE is critically involved in LL-37 induced cystitis. We further hypothesized a common RAGE ligand - high mobility group box 1 (HMGB1) is up-regulated in bladders challenged with LL-37. Finally, we hypothesized NF-κB dependent inflammatory genes are activated in LL-37 induced cystitis. Testing our first hypothesis, C57Bl/6 mice were challenged with either saline (control) or 320 µM of LL-37 intravesically for 1 hr. After 12 or 24 hours, tissues were examined with immunohistochemistry (IHC) for RAGE, and both mRNA and protein isolation for respective qRT-PCR and Western Blot analysis. Our second hypothesis was tested by employing HMGB1 IHC. Testing our final hypothesis, qRT-PCR was performed investigating five genes: TNFα, IL-6, IL-1ß, GM-CSF, COX-2. In control and LL-37 challenged tissues, IHC for RAGE revealed similar qualitative expression. Evaluation with qRT-PCR and Western Blot for RAGE revealed diminished expression at the mRNA and protein level within LL-37 challenged bladders. IHC for HMGB1 revealed a moderate qualitative increase within LL-37 challenged tissues. Finally, with the exception of TNF α, all NF- κB dependent inflammatory genes yielded substantial up-regulation. We have employed our LL-37 induced cystitis model to gain insight towards a possible mechanistic pathway involved in bladder inflammation. This work provides data for future studies involving the inflammatory ligand HMGB1, RAGE, and receptor pathways that activate NF-κB.

Silk-hyaluronan-based composite hydrogels: a novel, securable vehicle for drug delivery.

A new, biocompatible hyaluronic acid (HA)-silk hydrogel composite was fabricated and tested for use as a securable drug delivery vehicle. The composite consisted of a hydrogel formed by cross-linking thiol-modified HA with poly(ethylene glycol)-diacrylate, within which was embedded a reinforcing mat composed of electrospun silk fibroin protein. Both HA and silk are biocompatible, selectively degradable biomaterials with independently controllable material properties. Mechanical characterization showed the composite tensile strength as fabricated to be 4.43 ± 2.87 kPa, two orders of magnitude above estimated tensions found around potential target organs. In the presence of hyaluronidase (HAse) in vitro, the rate of gel degradation increased with enzyme concentration although the reinforcing silk mesh was not digested. Composite gels demonstrated the ability to store and sustainably deliver therapeutic agents. Time constants for in vitro release of selected representative antibacterial and anti-inflammatory drugs varied from 46.7 min for cortisone to 418 min for hydrocortisone. This biocomposite showed promising mechanical characteristics for direct fastening to tissue and organs, as well as controllable degradation properties suitable for storage and release of therapeutically relevant drugs.

The translational imperative: making cell therapy simple and effective.

The current practice of cell therapy, in which multipotent or terminally differentiated cells are injected into tissues or intravenously, is inefficient. Few therapeutic cells are retained at the site of administration and engraftment is low. An injectable and biologically appropriate vehicle for delivery, retention, growth and differentiation of therapeutic cells is needed to improve the efficacy of cell therapy. We focus on a hyaluronan-based semi-synthetic extracellular matrix (sECM), HyStem®, which is a manufacturable, approvable and affordable clinical product. The composition of this sECM can be customized for use with mesenchymal stem cells as well as cells derived from embryonic or induced pluripotent sources. In addition, it can support therapeutic uses of progenitor and mature cell populations obtained from skin, fat, liver, heart, muscle, bone, cartilage, nerves and other tissues. This overview presents four pre-clinical uses of HyStem® for cell therapy to repair injured vocal folds, improve post-myocardial infarct heart function, regenerate damaged liver tissue and restore brain function following ischemic stroke. Finally, we address the real-world limitations - manufacture, regulation, market acceptance and financing - surrounding cell therapy and the development of clinical combination products.

Pulmonary autotaxin expression contributes to the pathogenesis of pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, fibrotic form of diffuse lung disease occurring mainly in older adults. Increased lysophosphatidic acid (LPA) concentrations have been reported in the alveolar space of both idiopathic pulmonary fibrosis patients and a corresponding animal model, whereas the genetic deletion or pharmacological inhibition of LPA receptor 1 attenuated the development of the modeled disease, suggesting a direct involvement of LPA in disease pathogenesis. In this report, increased concentrations of autotaxin (ATX; ENPP2), the enzyme largely responsible for extracellular LPA production, were detected in both murine and human fibrotic lungs. The genetic deletion of ATX from bronchial epithelial cells or macrophages attenuated disease severity, establishing ATX as a novel player in IPF pathogenesis. Furthermore, the pharmacological inhibition of ATX attenuated the development of the modeled disease, suggesting that ATX is a possible therapeutic target in IPF.

Functional performance of human cardiosphere-derived cells delivered in an in situ polymerizable hyaluronan-gelatin hydrogel.

The vast majority of cells delivered into the heart by conventional means are lost within the first 24 h. Methods are needed to enhance cell retention, so as to minimize loss of precious material and maximize effectiveness of the therapy. We tested a cell-hydrogel delivery strategy. Cardiosphere-derived cells (CDCs) were grown from adult human cardiac biopsy specimens. In situ polymerizable hydrogels made of hyaluronan and porcine gelatin (Hystem(®)-C™) were formulated as a liquid at room temperature so as to gel within 20 min at 37 °C. CDC viability and migration were not compromised in Hystem-C™. Myocardial infarction was created in SCID mice and CDCs were injected intramyocardially in the infarct border zone. Real-time PCR revealed engraftment of CDCs delivered in Hystem-C™ was increased by nearly an order of magnitude. LVEF (left ventricular ejection fraction) deteriorated in the control (PBS only) group over the 3-week time course. Hystem-C™ alone or CDCs alone preserved LVEF relative to baseline, while CDCs delivered in Hystem-C™ resulted in a sizable boost in LVEF. Heart morphometry revealed the greatest attenuation of LV remodeling in the CDC + Hystem-C™ group. Histological analysis suggested cardiovascular differentiation of the CDCs in Hystem-C™. However, the majority of functional benefit is likely from paracrine mechanisms such as tissue preservation and neovascularization. A CDC/hydrogel formulation suitable for catheter-based intramyocardial injection exhibits superior engraftment and functional benefits relative to naked CDCs.

Metabolically stabilized derivatives of phosphatidylinositol 4-phosphate: synthesis and applications.

Phosphatidylinositol 4-phosphate (PtdIns(4)P) lipid is an essential component of eukaryotic membranes and a marker of the Golgi complex. Here, we developed metabolically stabilized (ms) analogs of PtdIns(4)P and the inositol 1,4-bisphosphate (IP(2)) head group derivative and demonstrated that these compounds can substitute the natural lipid fully retaining its physiological activities. The methylenephosphonate (MP) and phosphorothioate (PT) analogs of PtdIns(4)P and the aminohexyl (AH)-IP(2) probe are recognized by the PtdIns(4)P-specific PH domain of four phosphate adaptor protein 1 (FAPP1). Binding of FAPP1 to the PtdIns(4)P derivatives stimulates insertion of the PH domain into the lipid layers and induces tubulation of membranes. Both ms analogs and IP(2) probes could be invaluable for identifying protein effectors and characterizing PtdIns(4)P-dependent signaling cascades within the trans-Golgi network (TGN).