Automatic Determination of Endothelial Cell Density From Donor Cornea Endothelial Cell Images.

Purpose: To determine endothelial cell density (ECD) from real-world donor cornea endothelial cell (EC) images using a self-supervised deep learning segmentation model. Methods: Two eye banks (Eversight, VisionGift) provided 15,138 single, unique EC images from 8169 donors along with their demographics, tissue characteristics, and ECD. This dataset was utilized for self-supervised training and deep learning inference. The Cornea Image Analysis Reading Center (CIARC) provided a second dataset of 174 donor EC images based on image and tissue quality. These images were used to train a supervised deep learning cell border segmentation model. Evaluation between manual and automated determination of ECD was restricted to the 1939 test EC images with at least 100 cells counted by both methods. Results: The ECD measurements from both methods were in excellent agreement with rc of 0.77 (95% confidence interval [CI], 0.75-0.79; P < 0.001) and bias of 123 cells/mm2 (95% CI, 114-131; P < 0.001); 81% of the automated ECD values were within 10% of the manual ECD values. When the analysis was further restricted to the cropped image, the rc was 0.88 (95% CI, 0.87-0.89; P < 0.001), bias was 46 cells/mm2 (95% CI, 39-53; P < 0.001), and 93% of the automated ECD values were within 10% of the manual ECD values. Conclusions: Deep learning analysis provides accurate ECDs of donor images, potentially reducing analysis time and training requirements. Translational Relevance: The approach of this study, a robust methodology for automatically evaluating donor cornea EC images, could expand the quantitative determination of endothelial health beyond ECD.

Effects of Two Soluble ACE2-Fc Variants on Blood Pressure and Albuminuria in Hypertensive Mice: Research Letter.

Background: Angiotensin-converting enzyme 2 (ACE2) hydrolyzes angiotensin (Ang) II to Ang-(1-7), promoting vasodilatation, and inhibiting oxidative stress and inflammation. Plasma membrane ACE2 is the receptor for all known SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) viral variants. In COVID-19 infection, soluble ACE2 variants may act as decoys to bind and neutralize the coronavirus, reducing its tissue infectivity. Furthermore, soluble ACE2 variants have been proposed as potential therapeutics for kidney disease and hypertensive disorders. Objective: Soluble ACE2 variants conjugated to human Fc domains and selected for high-potency viral SARS-CoV-2 neutralization were prepared and evaluated for ACE2 activity in vitro. Lead candidates were then tested for systemic ACE2 activity, stability, and effects on blood pressure and albuminuria in mice with Ang II-induced hypertension. Methods: ACE2 activity of 10 soluble ACE2 variants was first assessed in cell-free conditions using a fluorogenic substrate, or by Ang II hydrolysis to Ang-(1-7). Hypertension was induced in male or female mice by implantation of osmotic minipumps containing Ang II. Two lead ACE2 variants were injected intravenously (i.v.) into hypertensive mice, followed by measurements of blood pressure (tail-cuff plethysmography), albuminuria, and tissue ACE2 activity and protein (immunoblots). Results: Soluble ACE2-Fc variants demonstrated significant ACE2 enzymatic activity, with kinetics comparable with human recombinant ACE2. In hypertensive mice, single dose i.v. injection of ACE2-Fc variant K (10 mg/kg) significantly decreased systolic blood pressure at 24 hours, with partial lowering sustained to 48 hours, and tendency to reduce albuminuria at 72 hours. By contrast, ACE2-Fc variant I had no effect on blood pressure or albuminuria in hypertensive mice; ACE2-Fc variant K was detected by immunoblotting in plasma, kidney, heart, lung, liver, and spleen lysates 72 hours after injection, associated with significantly increased ACE2 activity in all tissues except kidney and spleen. Angiotensin-converting enzyme 2-Fc variant I had no effect on plasma ACE2 activity. Conclusions: Soluble ACE2-Fc variant K reduces blood pressure and tends to lower albuminuria in hypertensive mice. Furthermore, soluble ACE2-Fc variant K has prolonged tissue retention, associated with increased tissue ACE2 activity. The results support further studies directed at the therapeutic potential of soluble ACE2-Fc variant K for cardiovascular and kidney protection.

Contexte: L'enzyme de conversion de l'angiotensine 2 (ACE2) hydrolyse l'angiotensine (Ang) II en angiotensine (Ang)-(1-7), ce qui favorise la vasodilatation et inhibe le stress oxydatif et l'inflammation. L'ACE2 de la membrane plasmique est le récepteur de tous les variants connus du SARS-COV-2. Dans les cas d'infection à la COVID-19, les variants solubles de l'ACE2 peuvent agir comme leurres pour lier et neutraliser le coronavirus, et réduire ainsi son infectiosité dans les tissus. Des variants solubles de l'ACE2 ont également été proposés comme agents thérapeutiques potentiels pour l'insuffisance rénale et les troubles liés à l'hypertension. Objectif: Des variants solubles de l'ACE2 conjugués au domaine Fc humain ont été sélectionnés pour leur fort potentiel neutralisant du virus SARS-COV-2, puis préparés et évalués pour la mesure de l'activité de l'ACE2 in vitro. Les meilleurs candidats ont ensuite été testés chez des souris souffrant d'hypertension induite par l'Ang II afin de mesurer l'activité d'ACE2, ainsi que leur stabilité et leurs effets sur la pression artérielle et l'albuminurie. Méthodologie: L'activité de 10 variants solubles de l'ACE2 a d'abord été évaluée en conditions acellulaires à l'aide d'un substrat fluorogène, ou par hydrolyse de l'Ang II en Ang-(1-7). L'hypertension a été induite chez des souris mâles ou femelles par l'implantation de minipompes osmotiques contenant de l'Ang II. Deux des meilleurs variants de l'ACE2 ont été injectés par voie intraveineuse (i.v.) à des souris hypertendues, puis des mesures de la pression artérielle (pléthysmographie par manchon caudal), de l'albuminurie, de l'activité de l'ACE2 dans les tissus et des protéines (immunobuvardage) ont été effectuées. Résultats: Les variants solubles ACE2-Fc ont montré une activité enzymatique significative, avec une cinétique comparable à celle de l'ACE2 recombinante humaine. Chez les souris hypertendues, l'injection i.v. d'une dose unique (10 mg/kg) du variant K ACE2-Fc a abaissé significativement la pression artérielle systolique après 24 heures­une réduction partielle s'étant prolongée jusqu'à 48 heures­et a montré une tendance à réduire l'albuminurie après 72 heures. En revanche, le variant I ACE2-Fc n'a eu aucun effet sur la pression artérielle ou l'albuminurie des souris hypertendues. Après 72 heures, le variant K ACE2-Fc a été détecté par immunobuvardage dans le plasma, ainsi que dans des lysats de reins, de cœur, de poumon, de foie et de rate, ce qui a été associé à une augmentation significative de l'activité de l'ACE2 dans tous les tissus sauf dans les reins et la rate. Le variant I ACE2-Fc n'a montré aucun effet sur l'activité de l'ACE2 dans le plasma. Conclusion: Le variant soluble K ACE2-Fc abaisse la pression artérielle et tend à diminuer l'albuminurie chez les souris hypertendues. Il présente en outre une rétention tissulaire prolongée, laquelle est associée à une plus grande activité de l'ACE2 dans les tissus. Ces résultats appuient d'autres études portant sur le potentiel thérapeutique du variant soluble K ACE2-Fc dans la protection cardiovasculaire et rénale.

Pharmacokinetics and Pharmacodynamic Effect of a Blood-Brain Barrier-Crossing Fusion Protein Therapeutic for Alzheimer's Disease in Rat and Dog.

PURPOSE: We have recently demonstrated the brain-delivery of an Amyloid-ß oligomer (Aßo)-binding peptide-therapeutic fused to the BBB-crossing single domain antibody FC5. The bi-functional fusion protein, FC5-mFc-ABP (KG207-M) lowered both CSF and brain Aß levels after systemic dosing in transgenic mouse and rat models of Alzheimer's disease (AD). For development as a human therapeutic, we have humanized and further engineered the fusion protein named KG207-H. The purpose of the present study was to carry out comparative PK/PD studies of KG207-H in wild type rat and beagle dogs (middle-aged and older) to determine comparability of systemic PK and CSF exposure between rodent species and larger animals with more complex brain structure such as dogs. METHOD: Beagle dogs were used in this study as they accumulate cerebral Aß with age, as seen in human AD patients, and can serve as a model of sporadic AD. KG207-H (5 to 50 mg/kg) was administered intravenously and serum and CSF samples were serially collected for PK studies and to assess target engagement. KG207-H and Aß levels were quantified using multiplexed selected reaction monitoring mass spectrometry. RESULTS: After systemic dosing, KG207-H demonstrated similar serum pharmacokinetics in rats and dogs. KG207-H appeared in the CSF in a time- and dose-dependent manner with similar kinetics, indicating CNS exposure. Further analyses revealed a dose-dependent inverse relationship between CSF KG207-H and Aß levels in both species indicating target engagement. CONCLUSION: This study demonstrates translational attributes of BBB-crossing Aß-targeting biotherapeutic KG207-H in eliciting a pharmacodynamic response, from rodents to larger animal species.

Preclinical in vivo longitudinal assessment of KG207-M as a disease-modifying Alzheimer's disease therapeutic.

In vivo biomarker abnormalities provide measures to monitor therapeutic interventions targeting amyloid-ß pathology as well as its effects on downstream processes associated with Alzheimer's disease pathophysiology. Here, we applied an in vivo longitudinal study design combined with imaging and cerebrospinal fluid biomarkers, mirroring those used in human clinical trials to assess the efficacy of a novel brain-penetrating anti-amyloid fusion protein treatment in the McGill-R-Thy1-APP transgenic rat model. The bi-functional fusion protein consisted of a blood-brain barrier crossing single domain antibody (FC5) fused to an amyloid-ß oligomer-binding peptide (ABP) via Fc fragment of mouse IgG (FC5-mFc2a-ABP). A five-week treatment with FC5-mFc2a-ABP (loading dose of 30 mg/Kg/iv followed by 15 mg/Kg/week/iv for four weeks) substantially reduced brain amyloid-ß levels as measured by positron emission tomography and increased the cerebrospinal fluid amyloid-ß42/40 ratio. In addition, the 5-week treatment rectified the cerebrospinal fluid neurofilament light chain concentrations, resting-state functional connectivity, and hippocampal atrophy measured using magnetic resonance imaging. Finally, FC5-mFc2a-ABP (referred to as KG207-M) treatment did not induce amyloid-related imaging abnormalities such as microhemorrhage. Together, this study demonstrates the translational values of the designed preclinical studies for the assessment of novel therapies based on the clinical biomarkers providing tangible metrics for designing early-stage clinical trials.

Inhibition of carbonic anhydrase IX in glioblastoma multiforme.

Carbonic anhydrase IX (CAIX) is a transmembrane enzyme upregulated in several types of tumors including glioblastoma multiforme (GBM). GBM is among the most aggressive tumors among gliomas. Temozolomide (TMZ) therapy combined with surgical or radiation approaches is the standard treatment but not effective in long term. In this study we tested the treatment with acetazolamide (ATZ), an inhibitor of CAIX, alone or combined with TMZ. The experiments were performed in 2D and 3D cultures (spheroids) using glioblastoma U251N and human brain tumor stem cells (BTSCs). Several proteins implicated in tumor cell death were also investigated. The key results from these studies suggest the following: (1) Cell death of human glioblastoma spheroids and BTSC is significantly increased with combined treatment after 7 days, and (2) the effectiveness of ATZ is significantly enhanced against BTSC and U251N when incorporated into nano-carriers. Collectively, these results point toward the usefulness of nano-delivery of CAIX inhibitors and their combination with chemotherapeutics for glioblastoma treatment.