Underlying Basement Membrane Research Articles

Noncanonical functions of adhesion proteins in inflammation.

Cell adhesion proteins localize to epithelial and endothelial cell membranes to form junctional complexes between neighboring cells or between cells and the underlying basement membrane. The structural and functional integrities of these junctions are critical to establish cell polarity and maintain tissue barrier function, while also facilitating leukocyte migration and adhesion to sites of inflammation. In addition to their adhesive properties, however, junctional proteins can also serve important noncanonical functions in inflammatory signaling and transcriptional regulation. Intriguingly, recent work has unveiled novel roles for cell adhesion proteins as both signaling initiators and downstream targets during inflammation. In this review, we discuss both the traditional functions of junction proteins in cell adhesion and tissue barrier function as well as their noncanonical signaling roles that have been implicated in facilitating diverse inflammatory pathologies.

Dermal stiffness governs the topography of the epidermis and the underlying basement membrane in young and old human skin.

The epidermis is a stratified epithelium that forms the outer layer of the skin. It is composed primarily of keratinocytes and is constantly renewed by the proliferation of stem cells and their progeny that undergo terminal differentiation as they leave the basal layer and migrate to the skin surface. Basal keratinocytes rest on a basement membrane composed of an extracellular matrix that controls their fate via integrin-mediated focal adhesions and hemidesmosomes which are critical elements of the epidermal barrier and promote its regenerative capabilities. The distribution of basal cells with optimal activity provides the basement membrane with its characteristic undulating shape; this configuration disappears with age, leading to epidermal weakness. In this study, we present an in-depth imaging analysis of basal keratinocyte anchorage in samples of human skin from participants across the age spectrum. Our findings reveal that skin aging is associated with the depletion of hemidesmosomes that provide crucial support for stem cell maintenance; their depletion correlates with the loss of the characteristic basement membrane structure. Atomic force microscopy studies of skin and invitro experiments revealed that the increase in tissue stiffness observed with aging triggers mechanical signals that alter the basement membrane structure and reduce the extent of basal keratinocyte anchorage, forcing them to differentiate. Genomic analysis revealed that epidermal aging was associated with mechanical induction of the transcription factor Krüppel-like factor 4. The altered mechanical properties of tissue being a new hallmark of aging, our work opens new avenues for the development of skin rejuvenation strategies.

Diagnostic Techniques in Autoimmune Blistering Diseases

Autoimmune blistering diseases (AIBD) comprise a heterogeneous group of uncommon disorders of the skin and mucous membranes, characterised by antibodies targeting structural proteins within epithelial tissue and the underlying basement membrane. There can be significant overlap in clinical presentation of these diseases and accurate diagnosis relies on the detection and characterisation of relevant autoantibodies. Immunofluorescence provides the gold-standard diagnostic tool for these diseases, identifying both tissue-bound autoantibodies in biopsy material using direct immunofluorescence and circulating antibodies in serum through indirect immunofluorescence. Following advances in the identification and subsequent characterisation of numerous antigenic targets in these diseases, the development of antigen-specific tests, in particular, enzyme-linked immunosorbent assays on serum specimens, has provided a third key tool to not only identify, but also quantify AIBD autoantibodies. This quantification has proven particularly useful in monitoring disease activity and informing clinical management decisions. Accurate diagnosis of these diseases is important since optimal treatment strategies differ between them and, prognostically, some diagnoses are associated with an increased risk of malignancy. This review outlines the molecular pathology underlying the major AIBD and describes how the three principal techniques can be used in combination, to provide best practice for diagnosis and treatment monitoring.

Levels of type XVII collagen (BP180) ectodomain are elevated in circulation from patients with multiple cancer types and is prognostic for patients with metastatic colorectal cancer

BackgroundCollagens are the major components of the extracellular matrix (ECM) and are known to contribute to tumor progression and metastasis. There are 28 different types of collagens each with unique functions in maintaining tissue structure and function. Type XVII collagen (BP180) is a type II transmembrane protein that provides stable adhesion between epithelial cells and the underlying basement membrane. Aberrant expression and ectodomain shedding of type XVII collagen have been associated with epithelial damage, tumor invasiveness, and metastasis in multiple tumor types and may consequently be used as a potential (non-invasive) biomarker in cancer and treatment target.MethodAn ELISA targeting the type XVII collagen ectodomain (PRO-C17) was developed for use in serum. PRO-C17 was measured in a cohort of patients with 11 different cancer types (n = 214) and compared to healthy controls (n = 23) (cohort 1). Based on the findings from cohort 1, PRO-C17 and its association with survival was explored in patients with metastatic colorectal cancer (mCRC) treated with bevacizumab in combination with chemotherapy (n = 212) (cohort 2).ResultsPRO-C17 was robust and specific towards the ectodomain of type XVII collagen. In cohort 1, PRO-C17 levels were elevated (p < 0.05) in serum from patients with CRC, kidney, ovarian, bladder, breast, and head and neck cancer compared to healthy controls. PRO-C17 was especially good at discriminating between CRC patients and healthy controls with an AUROC of 0.904. In cohort 2, patients with mCRC and high levels (tertile 3) of PRO-C17 had shorter overall survival (OS) with a median OS of 390 days compared to 539 days for patients with low levels of PRO-C17. When evaluated by multivariate Cox regression analysis, high PRO-C17 was predictive for poor OS independent of risk factors and the tumor fibrosis biomarker PRO-C3.ConclusionPRO-C17 measures the ectodomain of type XVII collagen in serum and is a promising non-invasive biomarker that can aid in understanding tumor heterogeneity as well as elaborate on the role of collagen XVII in tumor progression. Moreover, the findings in the study proposes PRO-C17 as novel biomarker of epithelial damage in specific cancer types including CRC.

Epithelial folding of alveolar cells derived from human induced pluripotent stem cells on artificial basement membrane.

Epithelial folding depends on mechanical properties of both epithelial cells and underlying basement membrane (BM). While folding is essential for tissue morphogenesis and functions, it is difficult to recapitulate features of a growing epithelial monolayer for in vitro modeling due to lack of in vivo like BM. Herein, we report a method to overcome this difficulty by culturing on an artificial basement membrane (ABM) the primordial lung progenitors (PLPs) from human induced pluripotent stem cells (hiPSCs). The ABM was achieved by self-assembling collagen IV and laminin, the two principal natural BM proteins, in the pores of a monolayer of crosslinked gelatin nanofibers deposited on a honeycomb micro-frame. The hiPSC-PLPs were seeded on the ABM for alveolar differentiation under submerged and air-liquid interface culture conditions. As results, the forces generated by the growing epithelial monolayer led to a geometry-dependent folding. Analysis of strain distribution in a clamped membrane provided instrumental insights into some of the observed phenomena. Moreover, the forces generated by the growing epithelial layer led to a high-level expression of surfactant protein C and a high percentage of aquaporin 5 positive cells compared with the results obtained with a nanofiber-covered bulk substrate. Thus, this work demonstrated the importance of recapitulating natural BM for advanced epithelial modeling. STATEMENT OF SIGNIFICANCE: The effort to develop in vitro epithelial models has not been entirely successful to date, due to lack of in vivo like basement membrane (BM). This lack has been overcome by using a microfabricated dense thin and pliable sheet like structure made of natural BM proteins. With such an artificial BM, alveolar epithelial deformation and folding could be studied and date could be correlated to numerical analyses of a plate theory. This method is simple and effective, enabling further developments in epithelial tissue modeling.

The incidence and prevalence of recurrent corneal erosion syndrome in London, UK.

Recurrent corneal erosion syndrome (RCES) is caused by repeated episodes of corneal epithelial breakdown due to improper adherence of the corneal epithelium to the underlying basement membrane. The most common aetiologies are corneal dystrophy or previous superficial ocular trauma. The incidence and prevalence of the condition is currently unknown. This study aimed to determine the incidence and prevalence of RCES within the London population over a 5-year period in order to better inform clinicians and evaluate how this condition affects ophthalmic service provision. A retrospective cohort study over a 5-year period reviewed 487,690 emergency room patient attendances at Moorfields Eye Hospital (MEH) London between 1 January 2015 and 31 December 2019. MEH caters for a local population comprising of around ten regional clinical commissioning groups (CCGs). The data for this study were collected using OpenEyesTM electronic medical records including demographics and comorbidities. The CCGs encompass 41% (3,689,000) of London's total 8,980,000 inhabitants. Using these data the crude incidence and prevalence rates of disease were estimated with results reported per 100,000 population. Out of 330,684 patients, 3623 patients were given a new diagnosis of RCES by the emergency ophthalmology services, and from these, 1056 patients attended outpatient follow-up. The crude annual incidence of RCES was estimated at 25.4 per 100,000, with a crude prevalence rate of 0.96%. There was no statistical difference in annual incidence across the 5-year period. The period prevalence of 0.96% shows that RCES is not uncommon. There was also a stable annual incidence over the 5-year period, showing no changing trend over the study period. However, identifying the true incidence and period prevalence is a challenging task, as minor cases may heal prior to examination by an ophthalmologist. It is highly likely that RCES is underdiagnosed and therefore underreported.

Epithelial HVEM maintains intraepithelial T cell survival and contributes to host protection.

Intraepithelial T cells (IETs) are in close contact with intestinal epithelial cells and the underlying basement membrane, and they detect invasive pathogens. How intestinal epithelial cells and basement membrane influence IET survival and function, at steady state or after infection, is unclear. The herpes virus entry mediator (HVEM), a member of the TNF receptor superfamily, is constitutively expressed by intestinal epithelial cells and is important for protection from pathogenic bacteria. Here, we showed that at steady-state LIGHT, an HVEM ligand, binding to epithelial HVEM promoted the survival of small intestine IETs. RNA-seq and addition of HVEM ligands to epithelial organoids indicated that HVEM increased epithelial synthesis of basement membrane proteins, including collagen IV, which bound to β1 integrins expressed by IETs. Therefore, we proposed that IET survival depended on β1 integrin binding to collagen IV and showed that β1 integrin-collagen IV interactions supported IET survival in vitro. Moreover, the absence of β1 integrin expression by T lymphocytes decreased TCR αβ+ IETs in vivo. Intravital microscopy showed that the patrolling movement of IETs was reduced without epithelial HVEM. As likely consequences of decreased number and movement, protective responses to Salmonella enterica were reduced in mice lacking either epithelial HVEM, HVEM ligands, or β1 integrins. Therefore, IETs, at steady state and after infection, depended on HVEM expressed by epithelial cells for the synthesis of collagen IV by epithelial cells. Collagen IV engaged β1 integrins on IETs that were important for their maintenance and for their protective function in mucosal immunity.

Phototherapeutische Keratektomie bei rezidivierenden Hornhauterosionen verschiedener epithelialer Genese: Einfluss der Ablationstiefe auf Pachymetrie und Refraktion

Phototherapeutic keratectomy (PTK) is an established treatment method for patients suffering from either genetic corneal dystrophy or recurrent corneal erosion (RCE) without underlying basement membrane dystrophy, often caused by trauma. This study aimed to describe the changes in manifest subjective refraction and pachymetry after PTK treatment in patients suffering from epithelial basement membrane dystrophy (EBMD) and traumatic or atraumatic RCE without underlying EBMD. This was aretrospective, single-center study performed at the Department of Ophthalmology of the Ludwig-Maximilians University (Munich). Patient data were retrospective collected using the smart-eye database of the Department of Ophthalmology including diagnostic data from an autorefractometer and from the Pentacam HR. All laser treatments were performed with an ablation depth of 10 μm for EBMD patients and 6 μm for RCE patients without EBMD. Both collectives showed adecrease in pachymetry larger than the calculated ablation depths after afollow-up interval of 126days (95% CI 104-147days). While the EBMD collective receiving an ablation of 10 μm showed adecrease of 25.8 μm (N = 74; 95% CI 21.2-30.3; p < 0.001), the non-EBMD collective receiving an ablation of 6 μm showed a decrease of 12.3 μm (N = 44, 95% CI 7.0-17.7; p < 0.001). Both, total corneal refractive power (TCRP) as well as spherical equivalent (SE) offered no significant change in preoperative and postoperative comparison for the EBMD collective. On the other hand, patients without underlying EBMD showed asignificant myopic decrease in SE of 0.4 dpt (±0.7 dpt SD, p < 0.05). The mean follow-up interval was 126 days (95% CI 104-147 days). PTK treatment offers an effective method for patients suffering from either EBMD dystrophy or RCE syndrome without underlying EBMD. The final ablation based on pachymetry at the apex can be estimated at 2.3 to 2.6 times higher compared to the original ablation depth. The reasons for this are on the one hand the laser ablation itself and the influence of the reactive wound healing of the corneal epithelium.

Regenerating airway epithelium using fibrous biomimetic basement membranes.

There are reciprocal interactions between epithelial cells and underlying basement membrane. The resemblance of biomaterials to native basement membrane is thus critical for their success when used to regenerate epithelium-containing organs. Particularly, the use of nanofibers and the incorporation of basement membrane proteins may mimic both biophysical and biochemical properties of basement membrane, respectively. Herein we tested how electrospun polycaprolactone/heparin fibers with and without adsorbed laminin and collagen IV proteins affect epithelial cell functions. We found that airway epithelial cells attached, migrated, and proliferated on all scaffolds but protein-functionalized fibers promoted higher attachment, quicker migration, and increased proliferation. Fibers were then integrated on polyethylene scaffolds and cultured at an air-liquid interface. The detection of secretory and ciliated cell markers was higher in cells on polyethylene with fibers. These findings demonstrate that electrospun fibers incite beneficial epithelial cell responses and can be used in the fabrication of bioengineered functional epithelia.

The pathologic diagnosis of carcinoma in various tissues.

Carcinoma is defined as cancer arising from the epithelial cells that line an organ or tissue. The most common carcinoma in males arises in the prostate and breast in females; while the most significant cause of cancer related mortality in the United States is carcinoma of the lung. Cancers typically begin as a clonal proliferation of cells that have acquired distinct mutations, which then progress to invasive carcinoma as the cells breach the underlying basement membrane associated with the tissue of origin. This transition to invasive carcinoma carries with it the potential to invade blood vessels or lymphatic channels and metastasize to lymph nodes or distant tissues resulting in increased morbidity and mortality. The histologic diagnosis of carcinoma is rendered based on both the cytologic and architectural features of the tumor, as well as the location of the proliferating cells and the interaction with the surrounding stromal elements.

The Effects of Early Diabetes on Duodenal Alterations in the Rats

Diabetes mellitus (DM) mainly affects functional changes in the duodenum, which plays an important role in the digestion and absorption of food. The impairment of duodenal function contributes to malnutrition, abdominal bloating and pain in diabetic patients. Thus, this study aimed to investigate the histological alterations and quantitative measurements of duodenal structures in the early stage of streptozotocin (STZ)-induced diabetic rats. Eight male Sprague-Dawley rats were divided into three control and five diabetic rats. Diabetes was induced by a single intraperitoneal dose of 60 mg/kg STZ. After four weeks of diabetic induction, the duodenum was prepared for histological study and morphometric analysis. In diabetic rats, there were deformed villi with disrupted surface epithelium and mildly distorted shapes of crypts, together with an increase in villus height and crypt depth. The epithelial cells detached from their underlying basement membrane. The goblet cells decreased in number, whereas an increased number of Cellula panethensis (Paneth cells) with pale-stained eosinophilic granules occurred in the DM group. A diabetic thickened submucosal layer was observed as enhanced duodenal glands (Brunner’s glands) hypertrophy and collagen accumulation. These findings indicated that histopathologic lesions of the duodenum developed in the early stage of diabetes. The destruction of villi, crypts, and epithelium may affect digestion and absorption. The structural changes in goblet and Cellula panethensis and duodenal glands may be associated with malfunction to protect duodenal mucosa from bacteria and stomach acid. These conditions can worsen the quality of life in diabetic individuals, leading to complications such as maldigestion, malabsorption, and duodenal ulcer.

Alcohol-associated capillarization of sinusoids: A critique since the discovery by Schaffner and Popper in 1963.

This article reviews the literature on capillarization of hepatic sinusoids since its discovery in 1963. Liver sinusoidal endothelial cells are uniquely fenestrated and lack an underlying basement membrane. In chronic liver disease, the sinusoids capillarize and transform into systemic capillaries, a process termed capillarization of sinusoids. The histopathology is marked by defenestration, basement membrane formation, and space of Disse fibrogenesis. Capillarized sinusoids compromise the bidirectional exchange of materials between sinusoids and hepatocytes, leading to hepatocellular dysfunction. Sinusoidal capillarization was first described in active cirrhosis of alcoholics in 1963. Since then, it has been found in early and progressive stages of alcoholic hepatic fibrosis before the onset of cirrhosis. The sinusoidal structure is not altered in alcoholic steatosis without fibrosis. Defenestration impairs the ability of the endothelium to filter chylomicron remnants from sinusoids into the Disse's space, contributing to alcohol-induced postprandial hyperlipidemia and possibly atherosclerosis. Ethanol also modulates the fenestration dynamics in animals. In baboons, chronic alcohol consumption diminishes endothelial porosity in concomitance with hepatic fibrogenesis and in rats defenestrates the endothelium in the absence of fibrosis, and sometimes capillarizes the sinusoids. Acute ethanol ingestion enlarges fenestrations in rats and contracts fenestrations in rabbits. In sinusoidal endothelial cell culture, ethanol elicits fenestration dilation, which is likely related to its interaction with fenestration-associated cytoskeleton. Ethanol potentiates sinusoidal injury caused by cocaine, acetaminophen or lipopolysaccharide in mice and rats. Understanding ethanol's mechanisms on pathogenesis of sinusoidal capillarization and fenestration dynamics will lead to development of methods to prevent risks for atherosclerosis in alcoholism.

Regulation of hemidesmosome dynamics and cell signaling by integrin α6β4.

Hemidesmosomes (HDs) are specialized multiprotein complexes that connect the keratin cytoskeleton of epithelial cells to the extracellular matrix (ECM). In the skin, these complexes provide stable adhesion of basal keratinocytes to the underlying basement membrane. Integrin α6β4 is a receptor for laminins and plays a vital role in mediating cell adhesion by initiating the assembly of HDs. In addition, α6β4 has been implicated in signal transduction events that regulate diverse cellular processes, including proliferation and survival. In this Review, we detail the role of α6β4 in HD assembly and beyond, and we discuss the molecular mechanisms that regulate its function.

Fibronectin‐Mediated Inflammatory Signaling Through Integrin α5 in Vascular Remodeling

BackgroundAdhesion of vascular endothelial cells to the underlying basement membrane potently modulates endothelial cells to cells' inflammatory activation. The normal basement membrane proteins laminin and collagen IV attenuate inflammatory signaling in part through integrin α2β1. In contrast, fibronectin, the provisional matrix protein found in injured, remodeling or inflamed vessels, sensitizes endothelial cells to inflammatory stimuli through integrins α5β1and and αvβ3. A chimeric integrin in which the cytoplasmic domain of α5 is replaced with that of α2 pairs with β1 and binds fibronectin but signals like α2β1.Methods and ResultsHere, we examined mice in which integrin α5 is replaced with the α5/2 chimera, using the transverse aortic constriction and partial carotid ligation models of vessel remodeling. Following transverse aortic constriction and partial carotid ligation surgery, wild‐type mice showed increased fibronectin deposition and expression of inflammatory markers, which were strongly attenuated in a5/2 mice. α5/2 mice also showed reduced artery wall hypertrophy in the transverse aortic constriction model and diminished inward remodeling in the partial carotid ligation model. Acute atherosclerosis after partial carotid ligation in hyperlipidemic ApoE−/− mice on a high fat diet was dramatically decreased in α5/2 mice.ConclusionsFibronectin and integrin α5 signaling is a key element of pathological vascular remodeling in acute models of both hypertension and disturbed flow. These results underscore the key role for integrin α5 signaling in pathological vascular remodeling associated with hypertension and atherosclerosis and support its potential as a therapeutic target.

The transcription factor Rreb1 regulates epithelial architecture, invasiveness, and vasculogenesis in early mouse embryos.

Ras-responsive element-binding protein 1 (Rreb1) is a zinc-finger transcription factor acting downstream of RAS signaling. Rreb1 has been implicated in cancer and Noonan-like RASopathies. However, little is known about its role in mammalian non-disease states. Here, we show that Rreb1 is essential for mouse embryonic development. Loss of Rreb1 led to a reduction in the expression of vasculogenic factors, cardiovascular defects, and embryonic lethality. During gastrulation, the absence of Rreb1 also resulted in the upregulation of cytoskeleton-associated genes, a change in the organization of F-ACTIN and adherens junctions within the pluripotent epiblast, and perturbed epithelial architecture. Moreover, Rreb1 mutant cells ectopically exited the epiblast epithelium through the underlying basement membrane, paralleling cell behaviors observed during metastasis. Thus, disentangling the function of Rreb1 in development should shed light on its role in cancer and other diseases involving loss of epithelial integrity.

Endothelial Basement Membrane Laminins as an Environmental Cue in Monocyte Differentiation to Macrophages.

Monocyte differentiation to macrophages is triggered by migration across the endothelial barrier, which is constituted by both endothelial cells and their underlying basement membrane. We address here the role of the endothelial basement membrane laminins (laminins 411 and 511) in this monocyte to macrophage switch. Chimeric mice carrying CX3CR1-GFP bone marrow were employed to track CCL2-induced monocyte extravasation in a cremaster muscle model using intravital microscopy, revealing faster extravasation in mice lacking endothelial laminin 511 (Tek-cre::Lama5−/−) and slower extravasation in mice lacking laminin 411 (Lama4−/−). CX3CR1-GFPlow extravasating monocytes were found to have a higher motility at laminin 511 low sites and to preferentially exit vessels at these sites. However, in vitro experiments reveal that this is not due to effects of laminin 511 on monocyte migration mode nor on the tightness of the endothelial barrier. Rather, using an intestinal macrophage replenishment model and in vitro differentiation studies, we demonstrate that laminin 511, together with the attached endothelium, promote monocyte differentiation to macrophages. Macrophage differentiation is associated with a change in integrin profile, permitting differentiating macrophages to distinguish between laminin 511 high and low areas and to preferentially migrate across laminin 511 low sites. These studies highlight the endothelial basement membrane as a critical site for monocyte differentiation to macrophages, which may be relevant to the differentiation of other cells at vascular niches.

Endothelial to mesenchymal transition: a precursor to post-COVID-19 interstitial pulmonary fibrosis and vascular obliteration?

Endothelial to mesenchymal transition (EndMT) could lead to post-COVID-19 pulmonary fibrosis and vascular remodelling https://bit.ly/2QqSKxT

Abstract 5029: Targeting fatty acid oxidation to promote anoikis and inhibit ovarian cancer progression

Abstract Purpose: Epithelial-derived high-grade serous ovarian cancer (HGSOC) is the deadliest gynecologic malignancy and in the US accounts for over 14,000 deaths per year. Roughly 80% of patients are diagnosed with late-stage disease, which is defined by wide-spread cancer dissemination throughout the pelvic and peritoneal cavities. HGSOC dissemination is dependent on tumor cells acquiring the ability to resist anoikis (apoptosis triggered by the loss of attachment). Epithelial cell detachment from the underlying basement membrane or extracellular matrix leads to cellular stress, including nutrient-deprivation. Reports describe metabolic reprogramming that promotes fatty acid oxidation (FAO) potentially supports anoikis resistance. Experimental Design: Examination of a panel of HGSOC cell lines cultured in adherent and suspension conditions. Evaluated anoikis by caspase 3/7 activity, cleaved caspase 3 immunofluorescence, and flow cytometry. Using the Seahorse Bioanalyzer examined mitochondria activity in suspension. Tested the role of exogenous free fatty acids in anoikis resistance and metabolism. In a patient-derived xenograft model, administered an FAO inhibitor and/or platinum-based chemotherapy. Results: HGSOC cultured in suspension upregulated the FAO rate-limiting enzyme, Carnitine Palmitoyltransferase 1A (CPT1A). CPT1A is overexpressed in HGSOC and correlates with poor overall survival. The knockdown of CPT1A promoted anoikis. HGSOC cells in suspension culture have decreased oxygen consumption that is rescued by exogenous fatty acids. In a patient-derived xenograft of HGSOC, a CPT1A inhibitor, etomoxir, significantly inhibited tumor growth and reduced tumor distribution. Conclusions: Targeting FAO in HGSOC to promote anoikis and attenuate dissemination is a potential approach to promote a more durable anti-tumor response and improve patient outcomes. Citation Format: Benjamin Guy Bitler, Brandon Sawyer, Lubna Qamar, Jennifer K. Richer, Kian Behbakht, Isabel R. Schlaepfer. Targeting fatty acid oxidation to promote anoikis and inhibit ovarian cancer progression [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5029.

Gelatin Methacrylate as an Enzyme-Controlled Release Vehicle of Hyaluronic Acid for the Treatment of Recurrent Corneal Erosion.

Recurrent corneal erosion (RCE) is a condition where the superficial corneal epithelial cells sporadically shed because of poor attachment to the underlying basement membrane, resulting in pain and discomfort. RCE-afflicted corneas exhibit elevated biological factors and enzymes, such as matrix metalloproteinase (MMP)-9. Soft bandage contact lenses (BCLs) are a commonly recommended treatment as they permit continued vision during recovery. However, they do not outperform alternative treatments as they currently lack active ingredients for ocular surface repair. Development of BCLs with a sustained release of active ingredients to promote RCE wound healing over an extended period of time would be clinically valuable. We fabricated gelatin methacrylate (GelMA) hydrogels with MMP-9-triggered controlled release for further BCL development. GelMA disks were characterized to determine the optimal concentration and methacrylation degree for sustained release in the presence of MMP-9. The chosen active ingredient, 60 kDa hyaluronic acid (HA), was verified to promote wound healing in rabbit corneal epithelial cell and human corneal epithelial cell wound assays. The active ingredient was loaded into GelMA hydrogels, and the bioavailability of the vehicle was determined. The GelMA hydrogels revealed a tunable profile for diffusion and enzyme-mediated controlled release of a model molecule. The 60 kDa HA-loaded GelMA disks showed improvement in wound healing at HA loadings of 150 and 250 μg by improving the wound healing rates by 28% (68.7% wound closure) and 26% (67.9% wound closure), respectively, compared to the control (50.5% wound closure) at 24 h. The HA-loaded GelMA disks with 150 and 250 μg HA also exhibited shortened wound closure time of 75 h compared to >147 h with a bolus daily delivery of HA for wound closure. Overall, we demonstrated that the fabricated GelMA hydrogels offered an enzyme-triggered option as a sustained release material for future RCE treatments.

Targeting Fatty Acid Oxidation to Promote Anoikis and Inhibit Ovarian Cancer Progression

Epithelial-derived high-grade serous ovarian cancer (HGSOC) is the deadliest gynecologic malignancy. Roughly 80% of patients are diagnosed with late-stage disease, which is defined by wide-spread cancer dissemination throughout the pelvic and peritoneal cavities. HGSOC dissemination is dependent on tumor cells acquiring the ability to resist anoikis (apoptosis triggered by cell detachment). Epithelial cell detachment from the underlying basement membrane or extracellular matrix leads to cellular stress, including nutrient deprivation. In this report, we examined the contribution of fatty acid oxidation (FAO) in supporting anoikis resistance. We examined expression Carnitine Palmitoyltransferase 1A (CPT1A) in a panel of HGSOC cell lines cultured in adherent and suspension conditions. With CPT1A knockdown cells, we evaluated anoikis by caspase 3/7 activity, cleaved caspase 3 immunofluorescence, flow cytometry, and colony formation. We assessed CPT1A-dependent mitochondrial activity and tested the effect of exogenous oleic acid on anoikis and mitochondrial activity. In a patient-derived xenograft model, we administered etomoxir, an FAO inhibitor, and/or platinum-based chemotherapy. CPT1A is overexpressed in HGSOC, correlates with poor overall survival, and is upregulated in HGSOC cells cultured in suspension. CPT1A knockdown promoted anoikis and reduced viability of cells cultured in suspension. HGSOC cells in suspension culture are dependent on CPT1A for mitochondrial activity. In a patient-derived xenograft model of HGSOC, etomoxir significantly inhibited tumor progression. IMPLICATIONS: Targeting FAO in HGSOC to promote anoikis and attenuate dissemination is a potential approach to promote a more durable antitumor response and improve patient outcomes.