Melanoma is an aggressive skin cancer with a high tendency for metastasis and resistance to conventional therapies. This study explores the role of preferentially expressed antigen in melanoma (PRAME), a cancer-testis antigen, in melanoma progression, focusing on its function in melanoma-derived extracellular vesicles (EVs) and its impact on benign melanocytes. We show that PRAME is highly expressed in melanoma cell lines, tissues, and patient plasma and is present in EVs. These EVs transfer PRAME protein and mRNA to benign melanocytes, leading to significant alterations in gene expression, increased cell proliferation, and a more malignant phenotype. Knockout of PRAME in melanoma cells reduces these protumorigenic effects on melanocytes, emphasizing PRAME's role in EV-mediated communication. The detection of PRAME in plasma EVs suggests its potential as a biomarker for monitoring disease progression and therapy response, including in rare melanoma subtypes. These findings highlight PRAME as a key player in melanoma progression and suggest targeting PRAME-containing EVs as a potential therapeutic strategy to inhibit melanoma progression and metastasis.

Melanoma, a deadly form of skin cancer, has seen improved survival rates due to advances in diagnosis and treatment, yet the need for further improvement remains critical. Tumor-associated antigens, such as PRAME (Preferentially Expressed Antigen in Melanoma), offer promising avenues for enhanced diagnostic precision, prognostic assessment, and targeted immunotherapy. PRAME, a cancer testis antigen, is selectively expressed in various cancers, including melanoma, and plays a key role in promoting tumorigenesis through inhibition of retinoic acid signaling, epithelial-to-mesenchymal transition, and immune evasion. This review explores the diagnostic utility of PRAME in distinguishing melanoma from benign nevi, its prognostic value in aggressive melanoma subtypes, and its potential as a therapeutic target in cancer vaccines and adoptive T-cell therapies. While PRAME-targeted therapies face challenges such as tumor heterogeneity and immune suppression, ongoing research aims to overcome these barriers, offering hope for more effective melanoma treatments.

Lipotoxicity was recently reported in several forms of kidney disease, including focal segmental glomerulosclerosis (FSGS). Susceptibility to FSGS in African Americans is associated with the presence of genetic variants of the Apolipoprotein L1 gene (APOL1) named G1 and G2. If and how endogenous APOL1 may alter mitochondrial function by the modifying cellular lipid metabolism is unknown. Using transgenic mice expressing the APOL1 variants (G0, G1 or G2) under endogenous promoter, we show that APOL1 risk variant expression in transgenic mice does not impair kidney function at baseline. However, APOL1 G1 expression worsens proteinuria and kidney function in mice characterized by the podocyte inducible expression of nuclear factor of activated T-cells (NFAT), which we have found to cause FSGS. APOL1 G1 expression in this FSGS-model also results in increased triglyceride and cholesterol ester contents in kidney cortices, where lipid accumulation correlated with loss of renal function. In vitro, we show that the expression of endogenous APOL1 G1/G2 in human urinary podocytes is associated with increased cellular triglyceride content and is accompanied by mitochondrial dysfunction in the presence of compensatory oxidative phosphorylation (OXPHOS) complexes elevation. Our findings indicate that APOL1 risk variant expression increases the susceptibility to lipid-dependent podocyte injury, ultimately leading to mitochondrial dysfunction.

Background Bone marrow-derived mesenchymal stem cells (BM-MSCs) have shown therapeutic potential in various in vitro and in vivo studies in cutaneous wound healing. Furthermore, there are ubiquitous studies highlighting the pro-regenerative effects of BM-MSC extracellular vesicles (BM-MSC EVs). The similarities and differences in BM-MSC EV cargo among potential healthy donors are not well understood. Variation in EV protein cargo is important to understand, as it may be useful in identifying potential therapeutic applications in clinical trials. We hypothesized that the donors would share both important similarities and differences in cargo relating to cell proliferation, angiogenesis, Wnt signaling, and basement membrane formation—processes shown to be critical for effective cutaneous wound healing. Methods We harvested BM-MSC EVs from four healthy human donors who underwent strict screening for whole bone marrow donation and further Good Manufacturing Practices-grade cell culture expansion for candidate usage in clinical trials. BM-MSC EV protein cargo was determined via mass spectrometry and Proteome Discoverer software. Corresponding proteomic networks were analyzed via the UniProt Consortium and STRING consortium databases. Results More than 3000 proteins were identified in each of the donors, sharing > 600 proteins among all donors. Despite inter-donor variation in protein identities, there were striking similarities in numbers of proteins per biological functional category. In terms of biologic function, the proteins were most associated with transport of ions and proteins, transcription, and the cell cycle, relating to cell proliferation. The donors shared essential cargo relating to angiogenesis, Wnt signaling, and basement membrane formation—essential processes in modulating cutaneous wound repair. Conclusions Healthy donors of BM-MSC EVs contain important similarities and differences among protein cargo that may play important roles in their pro-regenerative functions. Further studies are needed to correlate proteomic signatures to functional outcomes in cutaneous repair.

Defective cholesterol metabolism primarily linked to reduced ATP-binding cassette transporter A1 (ABCA1) expression is closely associated with the pathogenesis and progression of kidney diseases, including diabetic kidney disease and Alport Syndrome. However, whether the accumulation of free or esterified cholesterol contributes to progression in kidney disease remains unclear. Here, we demonstrate that inhibition of sterol-O-acyltransferase-1 (SOAT1), the enzyme at the endoplasmic reticulum that converts free cholesterol to cholesterol esters, which are then stored in lipid droplets, effectively reduced cholesterol ester and lipid droplet formation in human podocytes. Furthermore, we found that inhibition of SOAT1 in podocytes reduced lipotoxicity-mediated podocyte injury in diabetic kidney disease and Alport Syndrome in association with increased ABCA1 expression and ABCA1-mediated cholesterol efflux. In vivo, Soat1 deficient mice did not develop albuminuria or mesangial expansion at 10-12 months of age. However, Soat1 deficiency/inhibition in experimental models of diabetic kidney disease and Alport Syndrome reduced cholesterol ester content in kidney cortices and protected from disease progression. Thus, targeting SOAT1-mediated cholesterol metabolism may represent a new therapeutic strategy to treat kidney disease in patients with diabetic kidney disease and Alport Syndrome, like that suggested for Alzheimer's disease and cancer treatments.

BACKGROUND:Discoidin domain receptor 1 (DDR1) is a receptor tyrosine kinase that is activated by collagens that is involved in the pathogenesis of fibrotic disorders. Interestingly, de novo production of the collagen type I (Col I) has been observed in Col4a3 knockout mice, a mouse model of Alport Syndrome (AS mice). Deletion of the DDR1 in AS mice was shown to improve survival and renal function. However, the mechanisms driving DDR1-dependent fibrosis remain largely unknown.METHODS:Podocyte pDDR1 levels, Collagen and cluster of differentiation 36 (CD36) expression was analyzed by Real-time PCR and Western blot. Lipid droplet accumulation and content was determined using Bodipy staining and enzymatic analysis. CD36 and DDR1 interaction was determined by co-immunoprecipitation. Creatinine, BUN, albuminuria, lipid content, and histological and morphological assessment of kidneys harvested from AS mice treated with Ezetimibe and/or Ramipril or vehicle was performed.FINDINGS:We demonstrate that Col I-mediated DDR1 activation induces CD36-mediated podocyte lipotoxic injury. We show that Ezetimibe interferes with the CD36/DDR1 interaction in vitro and prevents lipotoxicity in AS mice thus preserving renal function similarly to ramipril.INTERPRETATION:Our study suggests that Col I/DDR1-mediated lipotoxicity contributes to renal failure in AS and that targeting this pathway may represent a new therapeutic strategy for patients with AS and with chronic kidney diseases (CKD) associated with Col4 mutations.FUNDING:This study is supported by the NIH grants R01DK117599, R01DK104753, R01CA227493, U54DK083912, UM1DK100846, U01DK116101, UL1TR000460 (Miami Clinical Translational Science Institute, National Center for Advancing Translational Sciences and the National Institute on Minority Health and Health Disparities), F32DK115109, Hoffmann-La Roche and Alport Syndrome Foundation.

Fibroblasts from patients with Tangier disease carrying ATP-binding cassette A1 (ABCA1) loss-of-function mutations are characterized by cardiolipin accumulation, a mitochondrial-specific phospholipid. Suppression of ABCA1 expression occurs in glomeruli from patients with diabetic kidney disease (DKD) and in human podocytes exposed to DKD sera collected prior to the development of DKD. We demonstrated that siRNA ABCA1 knockdown in podocytes led to reduced oxygen consumption capabilities associated with alterations in the oxidative phosphorylation (OXPHOS) complexes and with cardiolipin accumulation. Podocyte-specific deletion of Abca1 (Abca1(fl/fl)) rendered mice susceptible to DKD, and pharmacological induction of ABCA1 improved established DKD. This was not mediated by free cholesterol, as genetic deletion of sterol-o-acyltransferase-1 (SOAT1) in Abca1(fl/fl) mice was sufficient to cause free cholesterol accumulation but did not cause glomerular injury. Instead, cardiolipin mediates ABCA1-dependent susceptibility to podocyte injury, as inhibition of cardiolipin peroxidation with elamipretide improved DKD in vivo and prevented ABCA1-dependent podocyte injury in vitro and in vivo. Collectively, we describe a pathway definitively linking ABCA1 deficiency to cardiolipin-driven mitochondrial dysfunction. We demonstrated that this pathway is relevant to DKD and that ABCA1 inducers or inhibitors of cardiolipin peroxidation may each represent therapeutic strategies for the treatment of established DKD.

Members of the family of serine proteinase inhibitors, such as kallistatin, have been shown to inhibit canonical Wnt-TCF/LEF-β-catenin signaling via their interactions with the Wnt co-receptor LRP6. Yet the effects of transgenic overexpression of anti-Wnt serpins on hematopoiesis and lymphopoiesis are not well known. We studied the effects of human kallistatin (SERPINA4) on Wnt reporter activity in various cell types throughout the hematopoietic system and associated impacts on circulating white blood cell profiles. Transgenic overexpression of kallistatin suppressed Wnt-TCF/LEF-β-catenin signaling in bone marrow, as demonstrated using a Wnt reporter mouse. Further, kallistatin overexpression and treatment were associated with reduced Wnt-TCF/LEF-β-catenin activity in CD34+ c-kit+ bone marrow cells and CD19+ B lymphocytes, with reduced levels of these populations in bone marrow and peripheral circulation, respectively. The presence of CD3+CD4+, CD3+CD8+, and CD3− NK1.1+ T lymphocytes were not significantly affected. Our data suggest that overexpression of kallistatin interferes with lymphopoiesis, ultimately impacting the level of circulating CD19+ B lymphocytes.

Wound healing, angiogenesis, and hair follicle maintenance are often impaired in the skin of diabetic patients, but the pathogenesis has not been well understood. Here, we report that circulation levels of kallistatin, a member of the serine proteinase inhibitor (SERPIN) superfamily with antiangiogenic activities, were elevated in type 2 diabetic patients with diabetic vascular complications. To test the hypothesis that elevated kallistatin levels could contribute to a wound-healing deficiency via the inhibition of Wnt/beta-catenin signaling, we generated kallistatin-transgenic (KS-TG) mice. KS-TG mice had reduced cutaneous hair-follicle density, microvascular density, and panniculus adiposus layer thickness, as well as altered skin microvascular hemodynamics and delayed cutaneous wound healing. Using Wnt reporter mice, our results showed that Wnt/beta-catenin signaling is suppressed in the dermal endothelium and hair follicles in KS-TG mice. Lithium, a known activator of beta-catenin via inhibition of glycogen synthase kinase-3 beta, reversed the inhibition of Wnt/beta-catenin signaling by kallistatin and rescued the wound-healing deficiency in KS-TG mice. These observations suggest that elevated circulating antiangiogenic serpins in diabetic patients may contribute to impaired wound healing through inhibition of Wnt/ beta-catenin signaling. Activation of Wnt/beta-catenin signaling, at a level downstream of Wnt receptors, may ameliorate the wound-healing deficiency in diabetic patients.

PURPOSE Endothelial progenitor cells (EPC) have been shown to participate in ischemia-induced retinal neovascularization (NV). Overactivation of Wnt signaling has a pathogenic role in ischemia-induced retinal NV. The purpose of this study is to determine whether Wnt signaling regulates EPC release. METHODS Oxygen-induced retinopathy (OIR) was used as a model of retinal NV and Wnt pathway activation. The EPC, marked as c-Kit(+)/Tie-2(+) cells in the peripheral blood and bone marrow, were quantified using flow cytometry following immunolabeling. The Wnt signaling activity was evaluated by measuring nonphosphorylated β-catenin levels and X-gal staining in the Wnt reporter mice (Bat-gal mice). RESULTS The c-Kit(+)/Tie-2(+) cells were increased significantly in the peripheral blood and bone marrow of mice with OIR, compared to non-OIR mice. Overexpression of kallistatin, an endogenous inhibitor of the Wnt pathway, in kallistatin transgenic (kallistatin-TG) mice with OIR attenuated the increases of c-Kit(+)/Tie-2(+) cells in the peripheral blood and bone marrow, compared to WT mice with OIR. When the Bat-gal mice were crossed with kallistatin-TG mice, kallistatin overexpression suppressed the OIR-induced increases of X-gal-positive cells in the retinas and bone marrow, suggesting inhibition of Wnt signaling in these tissues. Furthermore, intraperitoneal injection of LiCl, a Wnt signaling activator, increased c-Kit(+)/Tie-2(+) cells in the peripheral blood of normal mice. Consistently, LiCl activated Wnt signaling in the retina and bone marrow cells in Bat-gal mice. CONCLUSIONS The Wnt signaling pathway has an important role in EPC release during retinal NV in OIR.

Kallistatin is a member of the serine proteinase inhibitor superfamily. Kallistatin levels have been shown to be decreased in the vitreous while increased in the circulation of patients with diabetic retinopathy (DR). Overactivation of the Wnt pathway is known to play pathogenic roles in DR. To investigate the role of kallistatin in DR and in Wnt pathway activation, we generated kallistatin transgenic (kallistatin-TG) mice overexpressing kallistatin in multiple tissues including the retina. In the oxygen-induced retinopathy (OIR) model, kallistatin overexpression attenuated ischemia-induced retinal neovascularization. In diabetic kallistatin-TG mice, kallistatin overexpression ameliorated retinal vascular leakage, leukostasis, and overexpression of vascular endothelial growth factor and intracellular adhesion molecule. Furthermore, kallistatin overexpression also suppressed Wnt pathway activation in the retinas of the OIR and diabetic models. In diabetic Wnt reporter (BAT-gal) mice, kallistatin overexpression suppressed retinal Wnt reporter activity. In cultured retinal cells, kallistatin blocked Wnt pathway activation induced by high glucose and by Wnt ligand. Coprecipitation and ligand-binding assays both showed that kallistatin binds to a Wnt coreceptor LRP6 with high affinity (Kd = 4.5 nmol/L). These observations suggest that kallistatin is an endogenous antagonist of LRP6 and inhibitor of Wnt signaling. The blockade of Wnt signaling may represent a mechanism for its antiangiogenic and antineuroinflammatory effects.

PURPOSE:To develop a mouse model of limbal stem cell deficiency (LSCD) by topical administration of benzalkonium chloride (BAC).METHODS:BAC solutions (0%-0.5%) were applied to the mouse ocular surface for 4 weeks. Corneal neovascularization, inflammation, and epithelial status were observed under slit-lamp microscope. The eyeball and ocular surface tissues were collected at 4 and 12 weeks and labeled with a series of antibodies. Limbal structure was evaluated by light and transmission electron microscopy (TEM). Corneal impression cytology was performed at 12 weeks, and specimens were labeled with periodic acid Schiff (PAS) reagents.RESULTS:BAC (0.5%) four times per day for 28 days successfully induced the typical manifestations of LSCD, including corneal neovascularization, severe inflammation in the stroma, and diffuse epithelial defect (P < 0.001). Conjunctival epithelium markers K19 and K13 were positive on the corneal surface. Expression of the putative limbal stem cell markers P63 and ABCG2 was abolished in the limbal epithelium. β-catenin was negative in the basal layer. TEM revealed the irregular basement membrane and the loss of stem cell-specific ultrastructure in the limbal basal epithelium. In the 0.5% BAC group, goblet cells could not be observed on day 28 but emerged after the cessation of BAC, and remained over the cornea after 8 weeks. K13-positive cells were still present over the cornea with the loss of K12.CONCLUSIONS:Topical administration of BAC at high concentration and frequency in mouse induces ocular surface changes resembling those of LSCD in humans, representing a novel model of LSCD.

OBJECTIVE:Randomized controlled experimental study to investigate the therapeutic effect and the possible mechanism of Pranoprofen on the recovery of dry eye induced by topical medication of Benzalkonium Chloride (BAC) in mouse.METHODS:It was an experimental study. Seventy BALB/c mice were treated with topical administration of 0.25% BAC to establish the dry eye condition. Based on the consistency of break-up time of tear-film (BUT), corneal fluorescein staining scores and inflammation index, the eyes were re-selected and randomly divided into four groups on day (D) 21 after the BAC treatment. Group A was set up as blank control, while group B, C and D were treated respectively with 0.1% sodium hyaluronate eye drops, 0.1% fluorometholone eye drops plus 0.1% sodium hyaluronate eye drops, 0.1% pyranoprofen eye drops plus 0.1% sodium hyaluronate eye drops. BUTs, tear volumes, corneal fluorescein staining scores and inflammation index were evaluated in each group on D0, 1, 3 and 5 after the therapeutic treatment. Global specimens were collected on D6. Sections were stained with hematoxylin and eosin (HE) or by periodic acid-schiff (PAS) assay, and labeled with cytokeratin 10 (K10) antibody. The expression of tumor necrosis factor-α (TNF-α) in the cornea and conjunctiva was quantified by western blot.RESULTS:72 eyes were included in the sequential experiment, 18 eyes for each group. On D0, 1 and 3, no clinical differences were observed among the groups. On D5, the BUT was (2.933 ± 0.320), (2.900 ± 0.280), (3.464 ± 0.498) and (3.643 ± 0.413) s in group A, B, C and D respectively; the BUTs in group C and D were significant longer than those of group A and B (F = 13.774, P = 0.000). The corneal fluorescein staining score was (11.640 ± 1.008), (11.790 ± 1.188), (10.330 ± 1.371) and (10.270 ± 1.104)s in group A, B, C and D respectively; the scores in group C and D were significant lower than those of group A and B (F = 6.145, P = 0.001). The corneal inflammatory index was (0.232 ± 0.059), (0.229 ± 0.078), (0.151 ± 0.055) and (0.154 ± 0.056) in group A, B, C and D respectively; the index in group C and D were significant lower than those of group A and B (F = 6.703, P = 0.001). No significant difference was found in tear volume among groups. No significant difference was found between Pyranoprofen and Fluorometholone treatment in BUT, corneal fluorescein score or inflammatory index. Corneal morphology showed the feature of thicker corneal epithelial layer in group A and uniformity in group C and D. PAS assay revealed similar goblet cell numbers in group C and D, but less goblet cells in group A and B. Cytokeratin 10 was almost negatively expressed in Pranoprofen or Fluorometholone treated groups, and remained positive in the corneal epithelium with other treatments. The level of TNF-α in the cornea was down-regulated in Pranoprofen or Fluorometholone treated groups.CONCLUSIONS:Pranoprofen or Fluorometholone combined with sodium hyaluronate treatment presented similar therapeutic effects on BAC-induced mouse dry eye, with the more stable tear film, the better regularity of epithelium recovery, the down-regulation of inflammatory TNF-α, the increased number of goblet cells, and the elimination of squamous metaplasia, when compared with the treatment of sodium hyaluronate eye drops only. Our results showed the great potentialities of Pranoprofen in the clinical treatment of ocular surface inflammation in the mild and severity dry eye.

Purpose: To develop a dry eye model of mouse induced by topical administration of benzalkonium chloride (BAC) and investigate the possible mechanisms.Methods: BAC at concentration of 0.2% was applied to the mouse ocular surface for 7 days. Phenol red thread tear test, tear break-up time (BUT) test, corneal inflammatory index scoring, fluorescein and rose bengal test were performed to evaluate the toxic effects of BAC on the ocular surface. Global specimens were collected on day (D) 7 and labeled with a series of antibodies including cytokeratin 10 (K10) and mucin 5AC (MUC5AC). Apoptosis of ocular surface epithelium was evaluated by in situ terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Histologic analysis and transmission electron microscopy (TEM) were performed on D7.Results: BAC at a concentration of 0.2% successfully induced a dry eye condition with decreased tear volume and BUTs, increased corneal fluorescein and rose bengal scores. The Inflammatory index was increased in accompanyment with higher tumor necrosis factor-alpha (TNF-alpha) expression and more inflammatory infiltration in the cornea. Immunolabeling revealed positive K10 expression in BAC-treated corneal epithelium and fewer MUC5AC-positive cells in the BAC-treated conjunctival fornix. TUNEL assay showed more apoptotic cells in the corneal basal epithelium. TEM showed that the size and intervals of the microvillis were both reduced in the corneal epithelium.Conclusions: Topical administration of 0.2% BAC in mouse induces changes resembling that of dry eye syndrome in humans, and thus, represents a novel model of dry eye.

SERPINA3K is a member of the serine proteinase inhibitor (SERPIN) family. Here we evaluated the therapeutic effects of SERPINA3K on neovascularization and inflammation in a rat cornea alkali burn model that is commonly employed to study corneal wounding. Topical treatment of the injured rat cornea with SERPINA3K (20 µg/eye/day) for 7 days significantly decreased the neovascular area, compared with the groups treated with BSA or PBS. The SERPINA3K treatment also ameliorated the corneal inflammation as evaluated by the inflammatory index. Furthermore, SERPINA3K enhanced the recovery of corneal epithelium after the alkali injury. Toward the mechanism of action, SERPINA3K down-regulated the expression of the pro-angiogenic and pro-inflammatory factors, vascular endothelial growth factor and tumor necrosis factor-α and up-regulated the expression of the anti-angiogenic factor, pigment epithelium-derived factor. SERPINA3K specifically inhibited growth of vascular endothelial cells. Meanwhile, SERPINA3K significantly up-regulated the expression of EGFR in the corneal epithelium. These findings suggest that SERPINA3K has therapeutic potential for corneal inflammation and NV.

Benzalkonium Chloride (BAC) is commonly used in eyedrops. Although the cytotoxicity of BAC has been reported, the mechanism underlying its toxic effect has not been elucidated. The present study investigated the role of the Wnt signaling pathway in the cytotoxicity of BAC in corneal epithelial cells and in the rat cornea. We demonstrated that phosphorylation of β-catenin, a downstream effector of the canonical Wnt pathway, was down-regulated by a short exposure to BAC in both cultured human corneal epithelial cells (HCE) and a cultured mouse corneal epithelial progenitor cell line (TKE2), suggesting an activation of the Wnt pathway. The activation of Wnt pathway is correlated with the decrease of cell viability induced by BAC. On the other hand, a specific Wnt pathway inhibitor, secreted frizzled-related protein-1 (sFRP1), reversed BAC-induced down-regulating effects on the level of phosphorylation of β-catenin and ameliorated cell viability in cells treated with BAC. In the rat cornea, the levels of total β-catenin were significantly up-regulated 8 h after the topical administration of BAC. Taken together, these results provided novel evidence suggesting that the cytotoxicity of BAC may be mediated through modulation of the Wnt pathway.