Immunohistochemical staining with CD117 and PGP9.5 of excised vestibular tissue from patients with neuroproliferative vestibulodynia.

BACKGROUND: Neuroproliferative vestibulodynia (NPV), a provoked genital pain characterized by severe allodynia and hyperalgesia, is confirmed in excised vestibular tissue by immunohistochemical staining (>8 CD117-positive immunostained cells/100× microscopic field) rather than by hematoxylin and eosin staining. AIM: In this study we sought to assess immunostaining of tissue samples obtained during vestibulectomy surgery and to correlate results with patient outcomes. METHODS: Patients (n = 65) meeting criteria for NPV who underwent vestibulectomy during the period from June 2019 through December 2022 formed the study cohort. We performed assessment of pathology of vestibular tissues by use of immunohistochemical staining, including quantitation of mast cells by CD117 (mast cell marker) and nerve fibers by protein gene product (PGP) 9.5 (neuronal marker). We analyzed 725 photomicrographs of immunostained tissue sections (100× and 200×) by manual counting and computer-assisted histometry and correlated these data to clinical assessments. OUTCOMES: Outcomes included density of CD117 and PGP9.5 immunostaining in the 1:00-11:00 o'clock and 12:00 o'clock vestibular regions, and patient-reported outcomes assessing sexual function, pain, distress, and symptom improvement. RESULTS: All 65 NPV patients (median age 26 years), 45 with lifelong and 20 with acquired NPV, had severe pain documented by PROs and vulvoscopy and had >8 CD117-immunopositive cells/100× microscopic field. Median cell count values were similar in the 1:00-11:00 o'clock and 12:00 vestibular regions (28.5 and 29.5/100× field, respectively). Likewise, the marker) and nerve fibers by protein gene product (PGP) 9.5 (neuronal marker). We analyzed 725 photomicrographs of immunostained tissue sections (100× and 200×) by manual counting and computer-assisted histometry and correlated these data to clinical assessments. OUTCOMES: Outcomes included density of CD117 and PGP9.5 immunostaining in the 1:00-11:00 o'clock and 12:00 o'clock vestibular regions, and patient-reported outcomes assessing sexual function, pain, distress, and symptom improvement. RESULTS: All 65 NPV patients (median age 26 years), 45 with lifelong and 20 with acquired NPV, had severe pain documented by PROs and vulvoscopy and had >8 CD117-immunopositive cells/100× microscopic field. Median cell count values were similar in the 1:00-11:00 o'clock and 12:00 vestibular regions (28.5 and 29.5/100× field, respectively). Likewise, the median area of CD117 immunostaining was similar in both regions (0.69% and 0.73%). The median area of PGP9.5 immunostaining was 0.47% and 0.31% in these same regions. Pain scores determined with cotton-tipped swab testing were nominally higher in lifelong vs acquired NPV patients, reaching statistical significance in the 1:00-11:00 o'clock region (P < .001). The median score for the McGill Pain Questionnaire affective subscale dimension was also significantly higher in lifelong vs acquired NPV patients (P = .011). No correlations were observed between hematoxylin and eosin results and density of mast cells or neuronal markers. Of note, 63% of the patient cohort reported having additional conditions associated with aberrant mast cell activity. CLINICAL IMPLICATIONS: The pathology of NPV is primarily localized to the vestibular epithelial basement membrane and subepithelial stroma with no visible vulvoscopic findings, making clinical diagnosis challenging. STRENGTHS AND LIMITATIONS: Strengths of this study include the large number of tissues examined with what is to our knowledge the first-ever assessment of the 12:00 vestibule. Major limitations are specimens from a single timepoint within the disease state and lack of control tissues. CONCLUSIONS: Performing immunohistochemical staining of excised vestibular tissue with CD117 and PGP9.5 led to histometric confirmation of NPV, indications that NPV is a field disease involving all vestibular regions, validation for patients whose pain had been ignored and who had experienced negative psychosocial impact, and appreciation that such staining can advance knowledge.

A Metabolic-Epigenetic Mechanism Directs Cell Fate and Therapeutic Sensitivity in Breast Cancer.

Over the past decade, studies have increasingly shed light on a reciprocal relationship between cellular metabolism and cell fate, meaning that a cell's lineage both drives and is governed by its specific metabolic features. A recent study by Zhang and colleagues, published in Cell Metabolism, describes a novel metabolic-epigenetic regulatory axis that governs lineage identity in triple-negative breast cancer (TNBC). Among the key findings, the authors demonstrate that the metabolic enzyme pyruvate kinase M2 (PKM2) directly binds to the histone methyltransferase enhancer of zeste homolog 2 (EZH2) in the nucleus to silence expression of a set of genes that includes the mitochondrial carnitine transporter SLC16A9. Perturbation of this metabolic-epigenetic regulatory mechanism induces a metabolic shift away from glycolysis and toward fatty acid oxidation. The ensuing influx of carnitine facilitates the deposition of the activating epigenetic mark H3K27Ac onto the promoter of GATA3, driving a committed luminal lineage state. Importantly, this metabolic-epigenetic axis represents a potentially targetable vulnerability for the treatment of TNBC, a subtype that currently lacks effective therapeutic strategies. These findings lend further support for the paradigm shift underlying our understanding of cancer metabolism: that a cellular fuel source functions not only to provide energy but also to direct the epigenetic regulation of cell fate.

Rat Model of Type 2 Diabetes Mellitus Recapitulates Human Disease in the Anterior Segment of the Eye.

Changes in the anterior segment of the eye due to type 2 diabetes mellitus (T2DM) are not well-characterized, in part due to the lack of a reliable animal model. This study evaluated changes in the anterior segment, including crystalline lens health, corneal endothelial cell density, aqueous humor metabolites, and ciliary body vasculature, in a rat model of T2DM compared with human eyes. Male Sprague-Dawley rats were fed a high-fat diet (45% fat) or normal diet, and rats fed the high-fat diet were injected with streptozotocin intraperitoneally to generate a model of T2DM. Cataract formation and corneal endothelial cell density were assessed using microscopic analysis. Diabetes-related rat aqueous humor alterations were assessed using metabolomics screening. Transmission electron microscopy was used to assess qualitative ultrastructural changes ciliary process microvessels at the site of aqueous formation in the eyes of diabetic rats and humans. Eyes from the diabetic rats demonstrated cataracts, lower corneal endothelial cell densities, altered aqueous metabolites, and ciliary body ultrastructural changes, including vascular endothelial cell activation, pericyte degeneration, perivascular edema, and basement membrane reduplication. These findings recapitulated diabetic changes in human eyes. These results support the use of this model for studying ocular manifestations of T2DM and support a hypothesis postulating blood-aqueous barrier breakdown and vascular leakage at the ciliary body as a mechanism for diabetic anterior segment pathology.

Prostate lineage-specific metabolism governs luminal differentiation and response to antiandrogen treatment.

Lineage transitions are a central feature of prostate development, tumourigenesis and treatment resistance. While epigenetic changes are well known to drive prostate lineage transitions, it remains unclear how upstream metabolic signalling contributes to the regulation of prostate epithelial identity. To fill this gap, we developed an approach to perform metabolomics on primary prostate epithelial cells. Using this approach, we discovered that the basal and luminal cells of the prostate exhibit distinct metabolomes and nutrient utilization patterns. Furthermore, basal-to-luminal differentiation is accompanied by increased pyruvate oxidation. We establish the mitochondrial pyruvate carrier and subsequent lactate accumulation as regulators of prostate luminal identity. Inhibition of the mitochondrial pyruvate carrier or supplementation with exogenous lactate results in large-scale chromatin remodelling, influencing both lineage-specific transcription factors and response to antiandrogen treatment. These results establish reciprocal regulation of metabolism and prostate epithelial lineage identity.

A conserved mechanism for JNK-mediated loss of Notch function in advanced prostate cancer.

Dysregulated Notch signaling is a common feature of cancer; however, its effects on tumor initiation and progression are highly variable, with Notch having either oncogenic or tumor-suppressive functions in various cancers. To better understand the mechanisms that regulate Notch function in cancer, we studied Notch signaling in a Drosophila tumor model, prostate cancer-derived cell lines, and tissue samples from patients with advanced prostate cancer. We demonstrated that increased activity of the Src-JNK pathway in tumors inactivated Notch signaling because of JNK pathway-mediated inhibition of the expression of the gene encoding the Notch S2 cleavage protease, Kuzbanian, which is critical for Notch activity. Consequently, inactive Notch accumulated in cells, where it was unable to transcribe genes encoding its target proteins, many of which have tumor-suppressive activities. These findings suggest that Src-JNK activity in tumors predicts Notch activity status and that suppressing Src-JNK signaling could restore Notch function in tumors, offering opportunities for diagnosis and targeted therapies for a subset of patients with advanced prostate cancer.

MYC is a regulator of androgen receptor inhibition-induced metabolic requirements in prostate cancer.

Advanced prostate cancers are treated with therapies targeting the androgen receptor (AR) signaling pathway. While many tumors initially respond to AR inhibition, nearly all develop resistance. It is critical to understand how prostate tumor cells respond to AR inhibition in order to exploit therapy-induced phenotypes prior to the outgrowth of treatment-resistant disease. Here, we comprehensively characterize the effects of AR blockade on prostate cancer metabolism using transcriptomics, metabolomics, and bioenergetics approaches. The metabolic response to AR inhibition is defined by reduced glycolysis, robust elongation of mitochondria, and increased reliance on mitochondrial oxidative metabolism. We establish DRP1 activity and MYC signaling as mediators of AR-blockade-induced metabolic phenotypes. Rescuing DRP1 phosphorylation after AR inhibition restores mitochondrial fission, while rescuing MYC restores glycolytic activity and prevents sensitivity to complex I inhibition. Our study provides insight into the regulation of treatment-induced metabolic phenotypes and vulnerabilities in prostate cancer.

Prostatic proliferative inflammatory atrophy: welcome to the club†.

Single-cell RNA sequencing studies in the human prostate have defined a population of epithelial cells with transcriptional similarities to club cells in the lung. However, the localization of club-like cells in the human prostate, and their relationship to prostate cancer, is poorly understood. In a new article in The Journal of Pathology, RNA in situ hybridization was used to demonstrate that club cell markers are expressed in luminal cells adjacent to inflammation in the peripheral zone of the human prostate, where prostate cancer tends to arise. These club-like cells are commonly found in proliferative inflammatory atrophy (PIA) lesions and express markers consistent with an intermediate epithelial cell-type. Future studies will be needed to understand the functional role of club-like cells in human prostate inflammation, regeneration, and disease. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Highly multiplexed immune profiling throughout adulthood reveals kinetics of lymphocyte infiltration in the aging mouse prostate.

Aging is a significant risk factor for disease in several tissues, including the prostate. Defining the kinetics of age-related changes in these tissues is critical for identifying regulators of aging and evaluating interventions to slow the aging process and reduce disease risk. An altered immune microenvironment is characteristic of prostatic aging in mice, but whether features of aging in the prostate emerge predominantly in old age or earlier in adulthood has not previously been established. Using highly multiplexed immune profiling and time-course analysis, we tracked the abundance of 29 immune cell clusters in the aging mouse prostate. Early in adulthood, myeloid cells comprise the vast majority of immune cells in the 3-month-old mouse prostate. Between 6 and 12 months of age, there is a profound shift towards a T and B lymphocyte-dominant mouse prostate immune microenvironment. Comparing the prostate to other urogenital tissues, we found similar features of age-related inflammation in the mouse bladder but not the kidney. In summary, our study offers new insight into the kinetics of prostatic inflammaging and the window when interventions to slow down age-related changes may be most effective.

Microwell-based flow culture increases viability and restores drug response in prostate cancer spheroids.

3D cancer spheroids represent a highly promising model for study of cancer progression and therapeutic development. Wide-scale adoption of cancer spheroids, however, remains a challenge due to the lack of control over hypoxic gradients that may cloud the assessment of cell morphology and drug response. Here, we present a Microwell Flow Device (MFD) that generates in-well laminar flow around 3D tissues via repetitive tissue sedimentation. Using a prostate cancer cell line, we demonstrate the spheroids in the MFD exhibit improved cell growth, reduced necrotic core formation, enhanced structural integrity, and downregulated expression of cell stress genes. The flow-cultured spheroids also exhibit an improved sensitivity to chemotherapy with greater transcriptional response. These results demonstrate how fluidic stimuli reveal the cellular phenotype previously masked by severe necrosis. Our platform advances 3D cellular models and enables study into hypoxia modulation, cancer metabolism, and drug screening within pathophysiological conditions.

Aging of the progenitor cells that initiate prostate cancer.

Many organs experience a loss of tissue mass and a decline in regenerative capacity during aging. In contrast, the prostate continues to grow in volume. In fact, age is the most important risk factor for prostate cancer. However, the age-related factors that influence the composition, morphology and molecular features of prostate epithelial progenitor cells, the cells-of-origin for prostate cancer, are poorly understood. Here, we review the evidence that prostate luminal progenitor cells are expanded with age. We explore the age-related changes to the microenvironment that may influence prostate epithelial cells and risk of transformation. Finally, we raise a series of questions about models of aging and regulators of prostate aging which need to be addressed. A fundamental understanding of aging in the prostate will yield critical insights into mechanisms that promote the development of age-related prostatic disease.

Distinct cell-types in the prostate share an aging signature suggestive of metabolic reprogramming.

Age is a significant risk factor for disease of the prostate. However, the mechanisms by which age increases disease risk have not been well described. We previously reported age-related changes within the inflammatory and luminal compartments of the mouse prostate. Old mouse prostates exhibit an expansion of the population of Trop2+ luminal progenitor cells and a reduction in the frequency and functional capacity of Trop2- luminal cells, indicating that different cell-types have distinct responses to aging. Whether distinct cell-types in the prostate share a common signature of aging has not been established. We transcriptionally profiled four distinct cell-types in young adult and old mouse prostates: stromal, basal, Trop2+ luminal progenitor and Trop2- luminal cells. Motif analysis of genes upregulated in old prostate cell-types pointed to transcriptional regulators of inflammatory and hypoxia-related signaling. Glutathione metabolism and the antioxidant response emerged as a common signature of aging across prostatic lineages. Expression of genes implicated in mouse prostate aging, including the antioxidant response gene Hmox1, correlates with age of diagnosis in primary prostate tumors from the TCGA cohort. These findings reveal a common signature shared by distinct cell-types in the old prostate reflective of age-associated metabolic reprogramming.

The effects of diabetes mellitus on the corneal endothelium: A review.

The corneal endothelium plays a critical role in maintaining corneal clarity. There is an expected decline in cell density with age and disease, and maintaining the health of this cell layer is important as corneal endothelial cells generally are amitotic in vivo. Diabetes mellitus is a highly prevalent disease that damages the corneal endothelium. Diabetes causes structural and functional impairments in the corneal endothelium that decrease cellular reserve in response to stress. These effects have implications to consider for diabetic patients undergoing anterior segment surgery, and for corneal surgeons who use diabetic donor tissue and treat diabetic patients. In this review, we discuss the specifics of how diabetes mellitus impacts the corneal endothelium including alterations in cell morphology, cell density, ultrastructure, pump and barrier function, cataract surgery outcomes, and corneal transplant outcomes with attention to the use of diabetic donor tissue and diabetic transplant recipients.

Evaluating the Differentiation Capacity of Mouse Prostate Epithelial Cells Using Organoid Culture.

The prostate epithelium is comprised predominantly of basal and luminal cells. In vivo lineage tracing has been utilized to define the differentiation capacity of mouse prostate basal and luminal cells during development, tissue-regeneration and transformation. However, evaluating cell-intrinsic and extrinsic regulators of prostate epithelial differentiation capacity using a lineage tracing approach often requires extensive breeding and can be cost-prohibitive. In the prostate organoid assay, basal and luminal cells generate prostatic epithelium ex vivo. Importantly, primary epithelial cells can be isolated from mice of any genetic background or mice treated with any number of small molecules prior to, or after, plating into three-dimensional (3D) culture. Sufficient material for evaluation of differentiation capacity is generated after 7-10 days. Collection of basal-derived and luminal-derived organoids for (1) protein analysis by Western blot and (2) immunohistochemical analysis of intact organoids by whole-mount confocal microscopy enables researchers to evaluate the ex vivo differentiation capacity of prostate epithelial cells. When used in combination, these two approaches provide complementary information about the differentiation capacity of prostate basal and luminal cells in response to genetic or pharmacological manipulation.

Targeting cellular heterogeneity with CXCR2 blockade for the treatment of therapy-resistant prostate cancer.

Hormonal therapy targeting androgen receptor (AR) is initially effective to treat prostate cancer (PCa), but it eventually fails. It has been hypothesized that cellular heterogeneity of PCa, consisting of AR+ luminal tumor cells and AR- neuroendocrine (NE) tumor cells, may contribute to therapy failure. Here, we describe the successful purification of NE cells from primary fresh human prostate adenocarcinoma based on the cell surface receptor C-X-C motif chemokine receptor 2 (CXCR2). Functional studies revealed CXCR2 to be a driver of the NE phenotype, including loss of AR expression, lineage plasticity, and resistance to hormonal therapy. CXCR2-driven NE cells were critical for the tumor microenvironment by providing a survival niche for the AR+ luminal cells. We demonstrate that the combination of CXCR2 inhibition and AR targeting is an effective treatment strategy in mouse xenograft models. Such a strategy has the potential to overcome therapy resistance caused by tumor cell heterogeneity.

Mass cytometry reveals species-specific differences and a new level of complexity for immune cells in the prostate.

Chronic inflammation in the benign prostate has been associated with a higher risk of developing prostate cancer. While a range of immune lineages is found in the prostate including T cells, B cells and myeloid cells, the specific subsets of immune cells with each major lineage have not been well described. In this study, we use mass cytometry (CyTOF) to comprehensively and reproducibly profile immune cells in mouse and human prostate. Using 4 myeloid markers (CD11b, CD11c, F4/80, Ly6C) in the mouse, we identified 8 phenotypically-distinct myeloid populations, demonstrating considerable heterogeneity within the immune compartment of the mouse prostate. We then profiled the prostate immune microenvironment from 9 human patients. Unlike the mouse prostate which is myeloid-dominant, the immune compartment in the benign human prostate is consistently T-lymphocyte-dominant. Using the X-shift algorithm to identify individual immune subsets based on marker expression, we found 57 phenotypically-distinct immune cell types in the human prostate. Despite similar proportions of T, B and myeloid lineage cells in the benign human prostate of all patients evaluated, we observed considerable interpatient heterogeneity in the abundance of more specific immune subsets. These findings highlight the importance of studying the immune compartment in the prostate at a granular level and will lead to future studies addressing the functional role of specific immune subsets in prostate epithelial transformation.

Expansion of Luminal Progenitor Cells in the Aging Mouse and Human Prostate.

Aging is associated with loss of tissue mass and a decline in adult stem cell function in many tissues. In contrast, aging in the prostate is associated with growth-related diseases including benign prostatic hyperplasia (BPH). Surprisingly, the effects of aging on prostate epithelial cells have not been established. Here we find that organoid-forming progenitor activity of mouse prostate basal and luminal cells is maintained with age. This is caused by an age-related expansion of progenitor-like luminal cells that share features with human prostate luminal progenitor cells. The increase in luminal progenitor cells may contribute to greater risk for growth-related disease in the aging prostate. Importantly, we demonstrate expansion of human luminal progenitor cells in BPH. In summary, we define a Trop2+ luminal progenitor subset and identify an age-related shift in the luminal compartment of the mouse and human prostate epithelium.

Assessing the Effects of Ripasudil, a Novel Rho Kinase Inhibitor, on Human Corneal Endothelial Cell Health.

PURPOSE: To determine how the Rho kinase inhibitor, ripasudil, affects metabolic function and cell viability in donor human corneal endothelial cells (HCECs). METHODS: Endothelial cell-Descemet membrane (EDM) tissues were treated with 10 µM ripasudil and assayed for mitochondrial and glycolytic activity using extracellular flux analysis and then compared to untreated controls. In addition, EDM tissues with a 24-h ripasudil treatment and control tissues were exposed to 1 µM staurosporine to induce apoptosis and then analyzed for cell viability using apoptosis and necrosis assays. RESULTS: Mitochondrial respiration metrics, specifically maximal respiration (P = 0.758) and spare respiratory capacity (P = 0.777), did not differ among the 1-h ripasudil treatment, 24-h treatment, and untreated tissues. Glycolytic activity assays showed an increase in glycolytic capacity at 1 h compared to the 24-h exposure group (P = 0.049) and controls (P = 0.009). Following exposure to staurosporine, the percentage of apoptotic HCECs was lower (P = 0.009) in ripasudil-treated tissues (2.473%, standard error of the mean [SEM] 0.477%) compared to untreated controls (3.349%, SEM 0.566%). In contrast, the percentage of necrotic HCECs decreased but did not differ statistically (P = 0.158) between ripasudil-treated (3.789%, SEM 0.487%) and untreated (4.567%, SEM 0.571%) tissues. CONCLUSIONS: Exposures to ripasudil did not result in any detectable reduction in metabolic function for HCECs in an ex vivo donor tissue model, and an increase in glycolytic activity at the 1-h time point was detected. In addition, HCECs treated with ripasudil gained a protective effect against induced apoptosis, suggesting that ripasudil may help improve the integrity of the corneal endothelium.

CD38 is methylated in prostate cancer and regulates extracellular NAD.

BACKGROUND: Cancer cell metabolism requires sustained pools of intracellular nicotinamide adenine dinucleotide (NAD+) which is maintained by a balance of NAD+ hydrolase activity and NAD+ salvage activity. We recently reported that human prostate cancer can be initiated following oncogene expression in progenitor-like luminal cells marked by low expression of the NAD+-consuming enzyme CD38. CD38 expression is reduced in prostate cancer compared to benign prostate, suggesting that tumor cells may reduce CD38 expression in order to enhance pools of NAD+. However, little is known about how CD38 expression is repressed in advanced prostate cancer and whether CD38 plays a role in regulating NAD+ levels in prostate epithelial cells. METHODS: CD38 expression, its association with recurrence after prostatectomy for clinically localized prostate cancer, and DNA methylation of the CD38 promoter were evaluated in human prostate tissues representing various stages of disease progression. CD38 was inducibly over-expressed in benign and malignant human prostate cell lines in order to determine the effects on cell proliferation and levels of NAD+ and NADH. NAD+ and NADH were also measured in urogenital tissues from wild-type and CD38 knockout mice. RESULTS: CD38 mRNA expression was reduced in metastatic castration-resistant prostate cancer compared to localized prostate cancer. In a large cohort of men undergoing radical prostatectomy, CD38 protein expression was inversely correlated with recurrence. We identified methylation of the CD38 promoter in primary and metastatic prostate cancer. Over-expression of wild-type CD38, but not an NAD+ hydrolase-deficient mutant, depleted extracellular NAD+ levels in benign and malignant prostate cell lines. However, expression of CD38 did not significantly alter intracellular NAD+ levels in human prostate cell lines grown in vitro and in urogenital tissues isolated from wild-type and CD38 knockout mice. CONCLUSIONS: CD38 protein expression in prostate cancer is associated with risk of recurrence. Methylation results suggest that CD38 is epigenetically regulated in localized and metastatic prostate cancer tissues. Our study provides support for CD38 as a regulator of extracellular, but not intracellular, NAD+ in epithelial cells. These findings suggest that repression of CD38 by methylation may serve to increase the availability of extracellular NAD+ in prostate cancer tissues.

Proteomic analysis of corneal endothelial cell-descemet membrane tissues reveals influence of insulin dependence and disease severity in type 2 diabetes mellitus.

The objective of this study was to characterize the proteome of the corneal endothelial cell layer and its basement membrane (Descemet membrane) in humans with various severities of type II diabetes mellitus compared to controls, and identify differentially expressed proteins across a range of diabetic disease severities that may influence corneal endothelial cell health. Endothelium-Descemet membrane complex tissues were peeled from transplant suitable donor corneas. Protein fractions were isolated from each sample and subjected to multidimensional liquid chromatography and tandem mass spectrometry. Peptide spectra were matched to the human proteome, assigned gene ontology, and grouped into protein signaling pathways unique to each of the disease states. We identified an average of 12,472 unique proteins in each of the endothelium-Descemet membrane complex tissue samples. There were 2,409 differentially expressed protein isoforms that included previously known risk factors for type II diabetes mellitus related to metabolic processes, oxidative stress, and inflammation. Gene ontology analysis demonstrated that diabetes progression has many protein footprints related to metabolic processes, binding, and catalysis. The most represented pathways involved in diabetes progression included mitochondrial dysfunction, cell-cell junction structure, and protein synthesis regulation. This proteomic dataset identifies novel corneal endothelial cell and Descemet membrane protein expression in various stages of diabetic disease. These findings give insight into the mechanisms involved in diabetes progression relevant to the corneal endothelium and its basement membrane, prioritize new pathways for therapeutic targeting, and provide insight into potential biomarkers for determining the health of this tissue.