Histologic assessments in ulcerative colitis: the evidence behind a new endpoint in clinical trials.

INTRODUCTION: Treatment goals for ulcerative colitis (UC) are evolving from the achievement of clinical remission to more rigorous goals defined by endoscopic and histologic healing. Achievement of deeper remission targets aims to reduce the risk of colectomy, hospitalizations, and colorectal cancer. AREAS COVERED: This review covers histologic assessments, histologic remission as a clinical trial endpoint, and the association between histologic disease activity and clinical outcomes. Future directions are also discussed, including the use of advanced imaging and artificial intelligence technologies, as well as potential future treatment targets beyond histologic remission. EXPERT OPINION: Histologic assessments are used for their sensitivity in measuring mucosal inflammatory changes in UC. Due to correlation with disease activity, histologic assessments may support clinical decision-making regarding treatment decisions as such assessments can be associated with rates of clinical relapse, hospitalization, colectomy, and neoplasia. While histologic remission is limited by varying definitions and multiple histologic indices, work is ongoing to create a consensus on the use of histologic assessments in clinical trials. As research advances, aspirational targets beyond histologic remission, such as molecular healing and disease clearance, are being explored.

Ulcerative colitis (UC) is the most common inflammatory bowel disease and often results in bloody diarrhea, frequent bowel movements, and bowel urgency. Patients with UC are at greater risk for hospitalization, surgery, and colorectal cancer. To reduce these risks, the goals of UC treatment are changing from mainly addressing symptoms to reducing inflammation at a deeper histologic, or microscopic, level. The inflammation in UC causes distinct microscopic changes in the colon, which can be assessed after collecting biopsies or tissue samples. This review provides an overview of histologic remission (when no signs of inflammation are seen in tissue samples viewed under a microscope) as a treatment goal in UC.Histologic remission has been shown to be associated with lower rates of relapse, hospitalization, surgical removal of the colon, and colorectal cancer. However, using histologic remission as a treatment target can be difficult due to varying definitions and the many different scoring assessments available to healthcare providers. Updated guidance from regulatory agencies and academic organizations has helped align definitions of histologic remission and how to assess histologic healing in clinical trials.The introduction of targeted advanced therapies has allowed for deeper healing with the potential for histologic resolution. This enables clinicians and researchers to aim for treatment targets that are harder to achieve but have a greater impact for patients in the course of their disease. New technologies such as artificial intelligence, high-resolution endoscopy, and digital pathology have also led to targets beyond histologic healing, aiming to restore the function of the colon's mucosal barrier and disease clearance.

Decreased Expression of Soluble Epoxide Hydrolase Suppresses Murine Choroidal Neovascularization.

Neovascular or "wet" age-related macular degeneration (nAMD) is a leading cause of blindness among older adults. Choroidal neovascularization (CNV) is a major pathological feature of nAMD, in which abnormal new blood vessel growth from the choroid leads to irreversible vision loss. There is a critical need to develop novel therapeutic strategies to address limitations of the current anti-vascular endothelial growth factor biologics. Previously, we identified soluble epoxide hydrolase (sEH) as a possible therapeutic target for CNV through a forward chemical genetic approach. The purpose of this study was to validate sEH as a target by examining retinal expression of sEH protein and mRNA by immunohistochemistry and RNAscope in situ hybridization, respectively, and to assess the efficacy of an adeno-associated virus (AAV) vector designed to knock down the sEH gene, Ephx2, in the murine laser-induced (L-) CNV model. nAMD patient postmortem eye tissue and murine L-CNV showed overexpression of sEH in photoreceptors and retinal pigment epithelial cells. Ephx2 knockdown significantly reduced CNV and normalized mRNA expression levels of CNV-related inflammatory markers. Thus, this study further establishes sEH as a promising therapeutic target against CNV associated with nAMD.

An improved method for murine laser-induced choroidal neovascularization lesion quantification from optical coherence tomography images.

Laser-induced choroidal neovascularization (L-CNV) in murine models is a standard method for assessing therapies, genetics, and mechanisms relevant to the blinding eye disease neovascular or "wet" age-related macular degeneration. The ex vivo evaluation of these lesions involves confocal microscopy analysis. In vivo evaluation via optical coherence tomography (OCT) has previously been established and allows longitudinal assessment of lesion development. However, to produce robust data, evaluation of many lesions may be required, which can be a slow, arduous process. A prior, manual method for quantifying these lesions as ellipsoids from orthogonal OCT images was effective but time consuming. We therefore developed an OCT lesion quantification that is simplified, streamlined, and less time-consuming.

Small-molecule inhibitors of ferrochelatase are antiangiogenic agents.

Activity of the heme synthesis enzyme ferrochelatase (FECH) is implicated in multiple diseases. In particular, it is a mediator of neovascularization in the eye and thus an appealing therapeutic target for preventing blindness. However, no drug-like direct FECH inhibitors are known. Here, we set out to identify small-molecule inhibitors of FECH as potential therapeutic leads using a high-throughput screening approach to identify potent inhibitors of FECH activity. A structure-activity relationship study of a class of triazolopyrimidinone hits yielded drug-like FECH inhibitors. These compounds inhibit FECH in cells, bind the active site in cocrystal structures, and are antiangiogenic in multiple in vitro assays. One of these promising compounds was antiangiogenic in vivo in a mouse model of choroidal neovascularization. This foundational work may be the basis for new therapeutic agents to combat not only ocular neovascularization but also other diseases characterized by FECH activity.

Measurement of mitochondrial respiration in the murine retina using a Seahorse extracellular flux analyzer.

Mitochondrial metabolism is a critical mechanism that is deregulated in numerous retinal diseases. Here, we elaborate a protocol to quantify oxygen consumption rate as a measure of mitochondrial respiration directly from mouse retinal tissue pieces. Our procedure combines the use of Seahorse extracellular flux technology and ex vivo retinal tissue isolation and is robustly reproducible under different treatment conditions. This protocol allows direct assessment of mitochondrial function in response to drug treatments or genetic manipulation in mouse models. For complete details on the use and execution of this protocol, please refer to Shetty et al. (2020), Sardar Pasha et al. (2021), Kooragayala et al. (2015), and Joyal et al. (2016).

A smartphone based method for mouse fundus imaging.

Noninvasive in vivo imaging of the mouse retina is essential for eye research. However, imaging the mouse fundus is challenging due to its small size and requires specialized equipment, maintenance, and training. These issues hinder the routine evaluation of the mouse retina. In this study, we developed a noncontact imaging system consisting of a smartphone, a 90D condensing lens, a homemade light diaphragm, a tripod, and a Bluetooth remote. With minimal training, examiners were able to capture fundus images from the mouse retina. We also found that fundus images captured using our system from wild type mice, mice with laser-induced retinal injury, and a mouse model of retinitis pigmentosa showed a quality similar to those captured using a commercial fundus camera. These images enabled us to identify normal structures and pathological changes in the mouse retina. Additionally, fluorescein angiography was possible with the smartphone system. We believe that the smartphone imaging system is low cost, simple, accessible, easy to operate, and suitable for the routine screening and examination of the mouse eye.

Pharmacological Potential of Small Molecules for Treating Corneal Neovascularization.

Under healthy conditions, the cornea is an avascular structure which allows for transparency and optimal visual acuity. Its avascular nature is maintained by a balance of proangiogenic and antiangiogenic factors. An imbalance of these factors can result in abnormal blood vessel proliferation into the cornea. This corneal neovascularization (CoNV) can stem from a variety of insults including hypoxia and ocular surface inflammation caused by trauma, infection, chemical burns, and immunological diseases. CoNV threatens corneal transparency, resulting in permanent vision loss. Mainstay treatments of CoNV have partial efficacy and associated side effects, revealing the need for novel treatments. Numerous natural products and synthetic small molecules have shown potential in preclinical studies in vivo as antiangiogenic therapies for CoNV. Such small molecules include synthetic inhibitors of the vascular endothelial growth factor (VEGF) receptor and other tyrosine kinases, plus repurposed antimicrobials, as well as natural source-derived flavonoid and non-flavonoid phytochemicals, immunosuppressants, vitamins, and histone deacetylase inhibitors. They induce antiangiogenic and anti-inflammatory effects through inhibition of VEGF, NF-κB, and other growth factor receptor pathways. Here, we review the potential of small molecules, both synthetics and natural products, targeting these and other molecular mechanisms, as antiangiogenic agents in the treatment of CoNV.

Heme Synthesis Inhibition Blocks Angiogenesis via Mitochondrial Dysfunction.

The relationship between heme metabolism and angiogenesis is poorly understood. The final synthesis of heme occurs in mitochondria, where ferrochelatase (FECH) inserts Fe2+ into protoporphyrin IX to produce proto-heme IX. We previously showed that FECH inhibition is antiangiogenic in human retinal microvascular endothelial cells (HRECs) and in animal models of ocular neovascularization. In the present study, we sought to understand the mechanism of how FECH and thus heme is involved in endothelial cell function. Mitochondria in endothelial cells had several defects in function after heme inhibition. FECH loss changed the shape and mass of mitochondria and led to significant oxidative stress. Oxidative phosphorylation and mitochondrial Complex IV were decreased in HRECs and in murine retina ex vivo after heme depletion. Supplementation with heme partially rescued phenotypes of FECH blockade. These findings provide an unexpected link between mitochondrial heme metabolism and angiogenesis.

Calcitriol and non-calcemic vitamin D analogue, 22-oxacalcitriol, attenuate developmental and pathological choroidal vasculature angiogenesis ex vivo and in vivo.

Aberrant ocular angiogenesis can underpin vision loss in leading causes of blindness, including neovascular age-related macular degeneration and proliferative diabetic retinopathy. Current pharmacological interventions require repeated invasive administrations, may lack efficacy and are associated with poor patient compliance and tachyphylaxis. Vitamin D has de novo anti-angiogenic properties. Here, our aim was to validate the ocular anti-angiogenic activity of biologically active vitamin D, calcitriol, and selected vitamin D analogue, 22-oxacalcitriol. Calcitriol induced a significant reduction in ex vivo mouse choroidal fragment sprouting. Viability studies in a human RPE cell line suggested non-calcemic vitamin D analogues including 22-oxacalcitriol have less off-target anti-proliferative activity compared to calcitriol and other analogues. Thereafter, the anti-angiogenic activity of 22-oxacalcitriol was demonstrated in an ex vivo mouse choroidal fragment sprouting assay. In zebrafish larvae, 22-oxacalcitriol was found to be anti-angiogenic, inducing a dose-dependent reduction in choriocapillaris development. Subcutaneously administered calcitriol failed to attenuate mouse retinal vasculature development. However, calcitriol and 22-oxacalcitriol administered intraperitoneally, significantly attenuated lesion volume in the laser-induced choroidal neovascularisation mouse model. In summary, calcitriol and 22-oxacalcitriol attenuate ex vivo and in vivo choroidal vasculature angiogenesis. Therefore, vitamin D may have potential as an interventional treatment for ophthalmic neovascular indications.

Soluble Epoxide Hydrolase Inhibition for Ocular Diseases: Vision for the Future.

Ocular diseases cause visual impairment and blindness, imposing a devastating impact on quality of life and a substantial societal economic burden. Many such diseases lack universally effective pharmacotherapies. Therefore, understanding the mediators involved in their pathophysiology is necessary for the development of therapeutic strategies. To this end, the hydrolase activity of soluble epoxide hydrolase (sEH) has been explored in the context of several eye diseases, due to its implications in vascular diseases through metabolism of bioactive epoxygenated fatty acids. In this mini-review, we discuss the mounting evidence associating sEH with ocular diseases and its therapeutic value as a target. Substantial data link sEH with the retinal and choroidal neovascularization underlying diseases such as wet age-related macular degeneration, retinopathy of prematurity, and proliferative diabetic retinopathy, although some conflicting results pose challenges for the synthesis of a common mechanism. sEH also shows therapeutic relevance in non-proliferative diabetic retinopathy and diabetic keratopathy, and sEH inhibition has been tested in a uveitis model. Various approaches have been implemented to assess sEH function in the eye, including expression analyses, genetic manipulation, pharmacological targeting of sEH, and modulation of certain lipid metabolites that are upstream and downstream of sEH. On balance, sEH inhibition shows considerable promise for treating multiple eye diseases. The possibility of local delivery of inhibitors makes the eye an appealing target for future sEH drug development initiatives.

Ref-1/APE1 Inhibition with Novel Small Molecules Blocks Ocular Neovascularization.

Ocular neovascular diseases like wet age-related macular degeneration are a major cause of blindness. Novel therapies are greatly needed for these diseases. One appealing antiangiogenic target is reduction-oxidation factor 1-apurinic/apyrimidinic endonuclease 1 (Ref-1/APE1). This protein can act as a redox-sensitive transcriptional activator for nuclear factor (NF)-κB and other proangiogenic transcription factors. An existing inhibitor of Ref-1's function, APX3330, previously showed antiangiogenic effects. Here, we developed improved APX3330 derivatives and assessed their antiangiogenic activity. We synthesized APX2009 and APX2014 and demonstrated enhanced inhibition of Ref-1 function in a DNA-binding assay compared with APX3330. Both compounds were antiproliferative against human retinal microvascular endothelial cells (HRECs; GI50 APX2009: 1.1 µM, APX2014: 110 nM) and macaque choroidal endothelial cells (Rf/6a; GI50 APX2009: 26 µM, APX2014: 5.0 µM). Both compounds significantly reduced the ability of HRECs and Rf/6a cells to form tubes at mid-nanomolar concentrations compared with control, and both significantly inhibited HREC and Rf/6a cell migration in a scratch wound assay, reducing NF-κB activation and downstream targets. Ex vivo, APX2009 and APX2014 inhibited choroidal sprouting at low micromolar and high nanomolar concentrations, respectively. In the laser-induced choroidal neovascularization mouse model, intraperitoneal APX2009 treatment significantly decreased lesion volume by 4-fold compared with vehicle (P < 0.0001, ANOVA with Dunnett's post-hoc tests), without obvious intraocular or systemic toxicity. Thus, Ref-1 inhibition with APX2009 and APX2014 blocks ocular angiogenesis in vitro and ex vivo, and APX2009 is an effective systemic therapy for choroidal neovascularization in vivo, establishing Ref-1 inhibition as a promising therapeutic approach for ocular neovascularization.

Potential roles of inorganic phosphate on the progression of initially bound glucopyranose toward the nonenzymatic glycation of human hemoglobin: mechanistic diversity and impacts on site selectivity.

Nonenzymatic glycation (NEG) begins with the non-covalent binding of a glucopyranose to a protein. The bound glucopyranose must then undergo structural modification to generate a bound electrophile that can reversibly form a Schiff base, which can then lead to Amadori intermediates, and ultimately to glycated proteins. Inorganic phosphate (Pi) is known to accelerate the glycation of human hemoglobin (HbA), although the specific mechanism(s) of Pi as an effector reagent have not been determined. The aim of this study was to determine whether Pi and a glucopyranose can concomitantly bind to HbA and react while bound within the early, noncovalent stages to generate electrophilic species capable of progress in NEG. 31P and 1HNMR of model reactions confirm that bimolecular reactions between Pi and glucopyranose occur generating modified glucose electrophiles. Computations of protein/substrate interactions predict that Pi can concomitantly bind with a glucopyranose in HbA pockets with geometries suitable for multiple acid/base mechanisms that can generate any of four transient electrophiles. Pi-facilitated mechanisms in the noncovalent stages predict that the glycation of ß-Val1 of HbA to HbA1c is a "hot spot" because the ß-Val1 pocket facilitates many more mechanisms than any other site. The mechanistic diversity of the Pi effect within the early noncovalent stages of NEG predicts well the overall site selectivity observed from the in vivo glycation of HbA in the presence of Pi. These insights extend our basic understanding of the NEG process and may have clinical implications for diabetes mellitus and even normal aging.

Chemical Proteomics Reveals Soluble Epoxide Hydrolase as a Therapeutic Target for Ocular Neovascularization.

The standard-of-care therapeutics for the treatment of ocular neovascular diseases like wet age-related macular degeneration (AMD) are biologics targeting vascular endothelial growth factor signaling. There are currently no FDA approved small molecules for treating these blinding eye diseases. Therefore, therapeutic agents with novel mechanisms are critical to complement or combine with existing approaches. Here, we identified soluble epoxide hydrolase (sEH), a key enzyme for epoxy fatty acid metabolism, as a target of an antiangiogenic homoisoflavonoid, SH-11037. SH-11037 inhibits sEH in vitro and in vivo and docks to the substrate binding cleft in the sEH hydrolase domain. sEH levels and activity are up-regulated in the eyes of a choroidal neovascularization (CNV) mouse model. sEH is overexpressed in human wet AMD eyes, suggesting that sEH is relevant to neovascularization. Known sEH inhibitors delivered intraocularly suppressed CNV. Thus, by dissecting a bioactive compound's mechanism, we identified a new chemotype for sEH inhibition and characterized sEH as a target for blocking the CNV that underlies wet AMD.