Human Organ Culture Research Articles

Establishment of an Ex Vivo Human Corneal Endothelium Wound Model.

A human model able to simulate the manifestation of corneal endothelium decompensation could be advantageous for wound healing and future cell therapy assessment. The study aimed to establish an ex vivo human cornea endothelium wound model where endothelium function can be evaluated by measuring corneal thickness changes. The human cornea was maintained in an artificial anterior chamber, with a continuous culture medium infusion system designed to sustain corneal endothelium and epithelium simultaneously. The corneal thickness was used to assess corneal endothelial cell function. Immunostaining was used to evaluate cell viability and endothelial cell marker expression, ZO-1 and Na/K ATPase. Human corneas with intact corneal endothelium were maintained in the ex vivo model for 28days, showing normal corneal thickness with a clear and transparent appearance. Corneal endothelial cells were alive and expressed ZO-1 and Na/K ATPase at the end of the organ culture. The endothelium wounded corneas showed persistent corneal edema with an increase in corneal thickness at 654.6 ± 31.7 µm. Staining results showed that no cells migrated to cover the wound and no expression of ZO-1 and Na/K ATPase on the posterior surface of the cornea was found. This study provided a novel method to establish an ex vivo human cornea organ culture model, where corneal endothelium function can be evaluated by accessing the corneal thickness. The ex vivo model established in this study can provide an alternative to the animal model in studying corneal endothelium decompensation.

ZFYVE21 promotes endothelial nitric oxide signaling and vascular barrier function in the kidney during aging

ZFYVE21 is an ancient, endosome-associated protein that is highly expressed in endothelial cells (ECs) but whose function(s) invivo are undefined. Here, we identified ZFYVE21 as an essential regulator of vascular barrier function in the aging kidney. ZFYVE21 levels significantly decline in ECs in aged human and mouse kidneys. To investigate attendant effects, we generated EC-specific Zfyve21-/- reporter mice. These knockout mice developed accelerated aging phenotypes including reduced endothelial nitric oxide (ENOS) activity, failure to thrive, and kidney insufficiency. Kidneys from Zfyve21 EC-/- mice showed interstitial edema and glomerular EC injury. ZFYVE21-mediated phenotypes were not programmed developmentally as loss of ZFYVE21 in ECs during adulthood phenocopied its loss prenatally, and a nitric oxide donor normalized kidney function in adult hosts. Using live cell imaging and human kidney organ cultures, we found that in a GTPase Rab5- and protein kinase Akt-dependent manner, ZFYVE21 reduced vesicular levels of inhibitory caveolin-1 and promoted transfer of Golgi-derived ENOS to a perinuclear Rab5+ vesicular population to functionally sustain ENOS activity. Thus, our work defines a ZFYVE21- mediated trafficking mechanism sustaining ENOS activity and demonstrates the relevance of this pathway for maintaining kidney function with aging.

Lack of Elevated Expression of TGFβ3 Contributes to the Delay of Epithelial Wound Healing in Diabetic Corneas.

To investigate the mechanisms underlying the differential roles of TGFβ1 and TGFβ3 in accelerating corneal epithelial wound healing (CEWH) in diabetic (DM) corneas, with normoglycemia (NL) corneas as the control. Two types of diabetic mice, human corneal organ cultures, mouse corneal epithelial progenitor cell lines, and bone marrow-derived macrophages (BMDMs) were employed to assess the effects of TGFβ1 and TGFβ3 on CEWH, utilizing quantitative PCR, western blotting, ELISA, and whole-mount confocal microscopy. Epithelial debridement led to an increased expression of TGFβ1 and TGFβ3 in cultured human NL corneas, but only TGFβ1 in DM corneas. TGFβ1 and TGFβ3 inhibition was significantly impeded, but exogenous TGFβ1 and, more potently, TGFβ3 promoted CEWH in cultured TKE2 cells and in NL and DM C57BL6 mouse corneas. Wounding induced similar levels of p-SMAD2/SMAD3 in NL and DM corneas but weaker ERK1/2, Akt, and EGFR phosphorylation in DM corneas compared to NL corneas. Whereas TGFβ1 augmented SMAD2/SMAD3 phosphorylation, TGFβ3 preferentially activated ERK, PI3K, and EGFR in healing DM corneas. Furthermore, TGFβ1 and TGFβ3 differentially regulated the expression of S100a9, PAI-1, uPA/tPA, and CCL3 in healing NL and DM corneas. Finally, TGFβ1 induced the expression of M1 macrophage markers iNOS, CD86, and CTGF, whereas TGFβ3 promoted the expression of M2 markers CD206 and NGF in BMDMs from db/db or db/+ mice. Hyperglycemia disrupts the balanced expression of TGFβ3/TGFβ1, resulting in delayed CEWH, including impaired sensory nerve regeneration in the cornea. Supplementing TGFβ3 in DM wounds may hold therapeutic potential for accelerating delayed wound healing in diabetic patients.

Dickkopf-related Protein 2 Promotes Hair Growth by Upregulating the Wnt/β-catenin Signaling Pathway in Human Dermal Papilla Cells.

The Wnt/β-catenin signaling pathway is crucial for the development, initiation, and growth of hair follicles (HFs). The Dickkopf-related protein (DKK) gene family encodes secreted proteins modulating the Wnt/β-catenin signaling pathway. Studies have reported that DKK1 promotes the regression of HFs and serves as a pathogenic mediator in male pattern baldness. However, the role of DKK2 on human hair growth has not yet been explored. This study investigates direct effect of DKK2 on hair growth using human dermal papilla cell (DPC) cultures and ex vivo human HF organ cultures. To elucidate the effect of DKK2 on hair growth, we examined the effect of recombinant human DKK2 (rhDKK2) treatment on cell viability, expression of mRNA and protein related to the Wnt/β-catenin signaling pathway, and cell growth in cultured human DPCs. We also performed ex vivo organ culture of HFs with rhDKK2 and measured changes in hair shaft length for 8 days. Treatment with rhDKK2 led to a dose-dependent rise in the proliferation of human DPCs (p<0.05), reaching levels comparable to those induced by 1 μM minoxidil. Moreover, rhDKK2 increased the expression of Wnt/β-catenin target genes, phosphorylated extracellular signal-regulated kinase, and cyclin-D1; it also increased the BAX-to-Bcl-2 ratio and downregulated the bone morphogenetic protein 2 gene. In human HF organ cultures, relative to the control treatment, rhDKK2 treatment significantly increased hair shaft elongation (p<0.01). Our results indicate that rhDKK2 could promote hair growth by facilitating the proliferation of human DPCs through activation of the Wnt/β-catenin signaling pathway.

Human Colonic Organ Culture Reveals XPO1-Independent Ability of Salvianolic Acid to Reduce Ulcerative Colitis–Related Inflammation

Human Colonic Organ Culture Reveals XPO1-Independent Ability of Salvianolic Acid to Reduce Ulcerative Colitis–Related Inflammation

Injectable hydrogel induces regeneration of naturally degenerate human intervertebral discs in a loaded organ culture model

Low back pain resulting from disc degeneration is a leading cause of disability worldwide. However, to date few therapies target the cause and fail to repair the intervertebral disc (IVD). This study investigates the ability of an injectable hydrogel (NPgel), to inhibit catabolic protein expression and promote matrix expression in human nucleus pulposus (NP) cells within a tissue explant culture model isolated from degenerate discs. Furthermore, the injection capacity of NPgel into naturally degenerate whole human discs, effects on mechanical function, and resistance to extrusion during loading were investigated. Finally, the induction of potential regenerative effects in a physiologically loaded human organ culture system was investigated following injection of NPgel with or without bone marrow progenitor cells. Injection of NPgel into naturally degenerate human IVDs increased disc height and Young's modulus, and was retained during extrusion testing. Injection into cadaveric discs followed by culture under physiological loading increased MRI signal intensity, restored natural biomechanical properties and showed evidence of increased anabolism and decreased catabolism with tissue integration observed. These results provide essential proof of concept data supporting the use of NPgel as an injectable therapy for disc regeneration. Statement of significanceLow back pain resulting from disc degeneration is a leading cause of disability worldwide. However, to date few therapies target the cause and fail to repair the intervertebral disc. This study investigated the potential regenerative properties of an injectable hydrogel system (NPgel) within human tissue samples. To mimic the human in vivo conditions and the unique IVD niche, a dynamically loaded intact human disc culture system was utilised. NPgel improved the biomechanical properties, increased MRI intensity and decreased degree of degeneration. Furthermore, NPgel induced matrix production and decreased catabolic factors by the native cells of the disc. This manuscript provides evidence for the potential use of NPgel as a regenerative biomaterial for intervertebral disc degeneration.

Mammalian Animal and Human Retinal Organ Culture as Pre-Clinical Model to Evaluate Oxidative Stress and Antioxidant Intraocular Therapeutics.

Oxidative stress (OS) is involved in the pathogenesis of retinal neurodegenerative diseases such as age-related macular degeneration (AMD) and diabetic retinopathy (DR) and an important target of therapeutic treatments. New therapeutics are tested in vivo despite limits in terms of transferability and ethical concerns. Retina cultures using human tissue can deliver critical information and significantly reduce the number of animal experiments along with increased transferability. We cultured up to 32 retina samples derived from one eye, analyzed the model's quality, induced OS, and tested the efficiency of antioxidative therapeutics. Bovine, porcine, rat, and human retinae were cultured in different experimental settings for 3-14 d. OS was induced by a high amount of glucose or hydrogen peroxide (H2O2) and treated with scutellarin, pigment epithelium-derived factor (PEDF), and/or granulocyte macrophage colony-stimulating factor (GM-CSF). The tissue morphology, cell viability, inflammation, and glutathione level were determined. The retina samples showed only moderate necrosis (23.83 ± 5.05 increased to 27.00 ± 1.66 AU PI-staining over 14 d) after 14 days in culture. OS was successfully induced (reduced ATP content of 288.3 ± 59.9 vs. 435.7 ± 166.8 nM ATP in the controls) and the antioxidants reduced OS-induced apoptosis (from 124.20 ± 51.09 to 60.80 ± 319.66 cells/image after the scutellarin treatment). Enhanced mammalian animal and human retina cultures enable reliable, highly transferable research on OS-triggered age-related diseases and pre-clinical testing during drug development.

Effect of andrographolide and deep eutectic solvent extracts of Andrographis paniculata on human coronavirus organ culture 43 (HCoV-OC43)

BackgroundAndrographis paniculata (Burm. f.) Nees has demonstrated potential for treating infections caused by coronaviruses. However, no antiviral activity of andrographolide or A. paniculata extracts against human coronavirus organ culture 43 (HCoV-OC43) has been reported. PurposeThis study aimed to evaluate the anti-HCoV-OC43 effect of andrographolide and A. paniculata as well as the correlation between andrographolide concentration and the anti-HCoV-OC43 activity of A. paniculata extracts. MethodsThis study evaluated and compared the in vitro anti-HCoV-OC43 activities of various A. paniculata extracts and andrographolide. To obtain A. paniculata extracts with different concentrations of andrographolide and its components, methanol and deep eutectic solvents (DES) were used to extract the aerial parts of A. paniculata. Andrographolide content was determined using UV-HPLC, and antiviral activity was assessed in HCT-8 colon cells. ResultsThe methanol and five acidic DES (containing malic acid or citric acid) extracts of A. paniculata exerted anti-HCoV-OC43 activity. Antiviral activity had a moderately strong positive linear relationship (r = 0.7938) with andrographolide content. Although the methanol extract contained the highest andrographolide content (2.34 mg/ml), its anti-HCoV-OC43 activity was lower than that of the DES extracts containing lower andrographolide concentrations (0.92–1.46 mg/ml). ConclusionMethanol and the five acidic DES extracts of A. paniculata exhibited anti-HCoV-OC43 activity. However, the in vitro antiviral activity of A. paniculata extracts did not have a very strong positive linear relationship (r < 0.8) with andrographolide concentration in the extract. As a result, when comparing A. paniculata extracts, the anti-HCoV-OC43 test could provide a different result from the andrographolide concentration determination.

Cytoprotective Effects of Human Platelet Lysate during the Xeno-Free Culture of Human Donor Corneas.

We evaluated the suitability of 2% human platelet lysate medium (2%HPL) as a replacement for 2% fetal bovine serum medium (2%FBS) for the xeno-free organ culture of human donor corneas. A total of 32 corneas from 16 human donors were cultured in 2%FBS for 3 days (TP1), then evaluated using phase contrast microscopy (endothelial cell density (ECD) and cell morphology). Following an additional 25-day culture period (TP2) in either 2%FBS or 2%HPL, the pairs were again compared using microscopy; then stroma and Descemet membrane/endothelium (DmE) were processed for next generation sequencing (NGS). At TP2 the ECD was higher in the 2%HPL group (2179 ± 288 cells/mm2) compared to 2%FBS (2113 ± 331 cells/mm2; p = 0.03), and endothelial cell loss was lower (ECL HPL = -0.7% vs. FBS = -3.8%; p = 0.01). There were no significant differences in cell morphology between TP1 and 2, or between 2%HPL and 2%FBS. NGS showed the differential expression of 1644 genes in endothelial cells and 217 genes in stromal cells. It was found that 2%HPL led to the upregulation of cytoprotective, anti-inflammatory and anti-fibrotic genes (HMOX1, SERPINE1, ANGPTL4, LEFTY2, GADD45B, PLIN2, PTX3, GFRA1/2), and the downregulation of pro-inflammatory/apoptotic genes (e.g., CXCL14, SIK1B, PLK5, PPP2R3B, FABP5, MAL, GATA3). 2%HPL is a suitable xeno-free substitution for 2%FBS in human cornea organ culture, inducing less ECL and producing potentially beneficial alterations in gene expression.

A Human Corneal Organ Culture Model of Descemet's Stripping Only with Accelerated Healing Stimulated by Engineered Fibroblast Growth Factor 1.

Fuchs Endothelial Corneal Dystrophy (FECD) results from dysfunctional corneal endothelial cells (CECs) and is currently treated by transplantation of the whole cornea or Descemet's membrane. Recent developments in ocular surgery have established Descemet's Stripping Only (DSO), a surgical technique in which a central circle of guttae-dense Descemet's membrane is removed to allow for the migration of CECs onto the smooth stroma, restoring function and vision to the cornea. While this potential treatment option is of high interest in the field of ophthalmic research, no successful ex vivo models of DSO have been established and clinical data is limited. This work presents a novel wound-healing model simulating DSO in human donor corneas. Using this approach to evaluate the efficacy of the human engineered FGF1 (NM141), we found that treatment accelerated healing via stimulation of migration and proliferation of CECs. This finding was confirmed in 11 pairs of human corneas with signs of dystrophy reported by the eye banks in order to verify that these results can be replicated in patients with Fuchs' Dystrophy, as the target population of the DSO procedure.

A Human Corneal Organ Culture Model of Descemet's Stripping Only with Accelerated Healing Stimulated by Engineered Fibroblast Growth Factor 1

Fuchs Endothelial Corneal Dystrophy (FECD) results from dysfunctional corneal endothelial cells (CECs) and is currently treated by transplantation of the whole cornea or Descemet's membrane. Recent developments in ocular surgery have established Descemet's Stripping Only (DSO), a surgical technique in which a central circle of guttae-dense Descemet's membrane is removed to allow for the migration of CECs onto the smooth stroma, restoring function and vision to the cornea. While this potential treatment option is of high interest in the field of ophthalmic research, no successful ex vivo models of DSO have been established and clinical data is limited. This work presents a novel wound-healing model simulating DSO in human donor corneas. Using this approach to evaluate the efficacy of the human engineered FGF1 (NM141), we found that treatment accelerated healing via stimulation of migration and proliferation of CECs. This finding was confirmed in 11 pairs of human corneas with signs of dystrophy reported by the eye banks in order to verify that these results can be replicated in patients with Fuchs' Dystrophy, as the target population of the DSO procedure.

Lagerstroemia indica extract regulates human hair dermal papilla cell growth and degeneration via modulation of β-catenin, Stat6, and TGF-β signaling pathway.

Lagerstroemia indica (L.indica) is reported to have diverse biological activities including anti-inflammatory, anti-cancer, neuro-regulatory, antidiabetic, and antioxidant activity. The purpose of this study is to examine the potential of hair growth promotion and/or hair loss prevention by L.indica extract. The effects of L.indica on hair growth have been studied in human hair follicle dermal papillary (hHFDP) cells and follicular organ culture ex vivo by cell proliferation assay, PCR, western blot analysis, and reporter gene activity assay. Moreover, a clinical trial was conducted in healthy volunteers. Lagerstroemia indica significantly promoted the proliferation of hHFDP cells, which was associated with increased expression of TCF/LEF, VEGF, and Gli1 mRNA, and inhibition of STAT6 and Smad2 mRNA. Treatment with L.indica also increased the TCF/LEF reporter gene activity but downregulated the SBE- and STAT6-luciferase activities. The expression of total β-catenin, CDK4, and CDK2 were elevated, while that of STAT6 and SMAD2/3 was suppressed upon treatment with L.indica. In human hair follicles organ culture, L.indica significantly inhibited hair follicular degeneration. The clinical trial showed a statistically significant rise in total hair count in test group (n=24) after 24 weeks of applying the hair tonic enriched with L.indica (141.46 ± 21.27 number/cm2 , p < 0.05). We suggest that L.indica extract prevents hair loss as well as stimulate hair growth by regulating the Wnt-β-catenin, JAK3-STAT6, and TGF-β1-Smad signaling pathways, and may be further developed as a novel functional cosmetic for preventing hair loss.

OP37 Bacterial suppression of intestinal fungi via activation of human gut Vδ2+ T-cells

Abstract Background The human gut is colonized by diverse microbes including fungi such as Candida albicans, which has been implicated in the pathogenesis of Crohn’s disease (CD). Intestinal C. albicans typically exists as commensal yeast, but can also form tissue-invasive filaments that secrete Candidalysin toxin to disrupt epithelial barriers. Commensal gut bacteria produce molecules that induce host immunity, including phosphoantigen HMB-PP which stimulates Vδ2+ T-cell homing to intestine and mucosal expression of IL-22 to promote barrier integrity. We aimed to identify mechanisms of gut barrier defence against fungal invasion in health and CD. Methods Colonic biopsies / resected tissue were stimulated or not with HMB-PP in the presence or absence of anti-IL-22 blocking antibody and analysed by flow-cytometry / microscopy to determine extent of fungal outgrowth. Fungal isolates were identified using standard morphological and biochemical criteria before assessment of filamentation, cell wall composition, and NETosis induction. Results Mucosal Vδ2+ T-cell activation with bacterial phosphoantigen HMB-PP, in the presence of epithelial damage-associated cytokine IL-15, potently suppressed growth of endogenous fungi in human colonic organ cultures. Vδ2-mediated fungal suppression required IL-22 and was sufficient to restrict the growth of multiple fungal species, including a range of emerging pathogenic moulds as well as archetypal pathobiont C. albicans. In Vδ2-deficient CD patients, mucosal biopsy stimulation with HMB-PP failed to control fungal growth. CD-derived C. albicans strains also displayed rapid filamentation ex vivo and higher levels of NOD2 ligand chitin than were observed in isolates from healthy controls. Comparing hypoxic with oxygenated cultures that mimic inflamed intestine, pSILAC proteomic analysis of a representative CD-derived C. albicans strain confirmed features of invasive growth (e.g. DAO1, IHD1), whereas a healthy control isolate exhibited metabolic hallmarks of symbiosis (Jen1p, SCH9). These divergent characteristics directly impacted on host anti-fungal immune responses, since the CD-derived strain rapidly induced neutrophil extracellular traps in vitro, whereas the healthy control isolate did not. Conclusion These data suggest that commensal bacteria can activate host Vδ2 T-cells to suppress fungal invasion of the gut epithelium via an IL22-dependent mechanism that is deficient in CD patients.

Induction of hair growth in hair follicle cells and organ cultures upon treatment with 30 kHz frequency inaudible sound via cell proliferation and antiapoptotic effects.

Androgenic alopecia is a hair loss disease mediated by dihydrotestosterone (DHT) and is currently treated using minoxidil, finasteride, or low-level laser therapy. However, these treatments have side-effects, indicating the need for an alternative treatment. In the present study, it was demonstrated that inaudible sound at 30 kHz significantly induced proliferative and anti-apoptotic effects in human dermal papilla cells (hDPCs) and outer root sheath keratinocytes. Cell viability assay, ELISA, reverse transcription quantitative PCR and TUNEL assays were performed to evaluate the effect of inaudible sound. Inaudible sound was also demonstrated to significantly inhibit the hair loss signals induced by DHT treatment in hDPCs. Furthermore, inaudible sound significantly induced hair follicle (HF) elongation and hair matrix keratinocyte proliferation in human HF organ culture. Overall, the results suggested that inaudible sound may be effective in treating hair loss and could be used to develop a new hair loss treatment approach.

An ex vivo model of human corneal rim perfusion organ culture

The human anterior segment perfusion culture model is a valuable tool for studying the trabecular meshwork (TM) and aqueous humor outflow in glaucoma. The traditional model relies on whole eye globes resulting in high cost and limited availability. Here, we developed a glue-based method which enabled us to use human corneal rims for perfusion culture experiments. Human corneal rim perfusion culture plates were 3D printed. Human corneal rims containing intact TM were attached and sealed to the plate using low viscosity and high viscosity glues, respectively. The human corneal rims were perfused using the constant flow mode, and the pressure changes were recorded using a computerized system. Outflow facility, TM stiffness, and TM morphology were evaluated. When perfused at rates from 1.2 to 3.6 μl/min, the outflow facility was 0.359 ± 0.216 μl/min/mmHg among 10 human corneal rims. The stiffness of the TM in naïve human corneal rim was similar to that of perfusion cultured human corneal rim. Also, the stiffness of TM of corneal rims perfused with dexamethasone was significantly higher than the control. Human corneal rims with glue contamination in the TM could be differentiated by high baseline intraocular pressure as well as high TM stiffness. Histology studies showed that the TM tissues perfused with plain medium appeared normal. We believed that our glued-based method is a useful tool and low-cost alternative to the traditional anterior segment perfusion culture model.

Human hair follicles operate an internal Cori cycle and modulate their growth via glycogen phosphorylase

Hair follicles (HFs) are unique, multi-compartment, mini-organs that cycle through phases of active hair growth and pigmentation (anagen), apoptosis-driven regression (catagen) and relative quiescence (telogen). Anagen HFs have high demands for energy and biosynthesis precursors mainly fulfilled by aerobic glycolysis. Histochemistry reports the outer root sheath (ORS) contains high levels of glycogen. To investigate a functional role for glycogen in the HF we quantified glycogen by Periodic-Acid Schiff (PAS) histomorphometry and colorimetric quantitative assay showing ORS of anagen VI HFs contained high levels of glycogen that decreased in catagen. qPCR and immunofluorescence microscopy showed the ORS expressed all enzymes for glycogen synthesis and metabolism. Using human ORS keratinocytes (ORS-KC) and ex vivo human HF organ culture we showed active glycogen metabolism by nutrient starvation and use of a specific glycogen phosphorylase (PYGL) inhibitor. Glycogen in ORS-KC was significantly increased by incubation with lactate demonstrating a functional Cori cycle. Inhibition of PYGL significantly stimulated the ex vivo growth of HFs and delayed onset of catagen. This study defines translationally relevant and therapeutically targetable new features of HF metabolism showing that human scalp HFs operate an internal Cori cycle, synthesize glycogen in the presence of lactate and modulate their growth via PYGL activity.

T cell-depleted cultured pediatric thymus tissue as a model for some aspects of human age-related thymus involution.

Human age-related thymus involution is characterized by loss of developing thymocytes and the thymic epithelial network that supports them, with replacement by adipose tissue. The mechanisms that drive these changes are difficult to study in vivo due to constant trafficking to and from the thymus. We hypothesized that the loss of thymocytes that occurs during human thymic organ cultures could model some aspects of thymus involution and begin to identify mechanisms that drive age-related changes in the thymic microenvironment. Potential mechanistically important candidate molecules were initially identified by screening conditioned media from human thymus organ cultures using antibody microarrays. These candidates were further validated using cultured tissue extracts and conditioned media. Results were compared with gene expression studies from a panel of well-characterized (non-cultured) human thymus tissues from human donors aged 5days to 78years. L-selectin released into conditioned media was identified as a biomarker for the content of viable thymocytes within the cultured thymus. Levels of the chemokines CCL21 and CXCL12, likely produced by surviving thymic epithelial cells, increased markedly in conditioned media as thymocytes were lost during culture. Native non-cultured thymus from adults older than 18years also showed a strong trend toward increased CCL21 expression, in conjunction with significant decreases in thymocyte-related mRNAs compared with thymus from subjects younger than 18years. Together, these findings demonstrate that use of postnatal human thymus organ cultures can model some aspects of human age-related thymic involution.

Inhibitory activity of FDA-approved drugs cetilistat, abiraterone, diiodohydroxyquinoline, bexarotene, remdesivir, and hydroxychloroquine on COVID-19 main protease and human ACE2 receptor: A comparative in silico approach

By September 1, 2021, SARS-CoV-2, a respiratory virus that prompted Coronavirus Disease in 2019, had infected approximately 218,567,442 patients and claimed 4,534,151 lives. There are currently no specific treatments available for this lethal virus, although several drugs, including remdesivir and hydroxychloroquine, have been tested. The purpose of this study is to assess the activity of FDA-approved drugs cetilistat, abiraterone, diiodohydroxyquinoline, bexarotene, remdesivir, and hydroxychloroquine as potential SARS-CoV-2 main protease inhibitors. Additionally, this study aims to provide insight into the development of potential inhibitors that may inhibit ACE2, thereby preventing SARS-CoV-2 entry into the host cell and infection. To this end, remdesivir and hydroxychloroquine were used as comparator drugs. The calculations revealed that cetilistat, abiraterone, diiodohydroxyquinoline, and bexarotene inhibit main protease and ACE2 receptors more effectively than the well-known drug hydroxychloroquine when used against COVID-19. Meanwhile, bexarotene and cetilistat bind more tightly to the SARS-CoV-2 main protease and the ACE2 receptor, respectively, than remdesivir, a potential treatment for COVID-19 that is the first FDA-approved drug against this virus. As a result, the molecular dynamic simulations of these two drugs in the presence of proteins were investigated. The MD simulation results demonstrated that these drugs interact to stabilize the systems, allowing them to be used as effective inhibitors of these proteins. Meanwhile, bexarotene, abiraterone, cetilistat, and diiodohydroxyquinoline's systemic effects should be further investigated in suitable ex vivo human organ culture or organoids, animal models, or clinical trials.

Organ Cultures for Retinal Diseases.

The successful development of novel therapies is closely linked with understanding the underlying pathomechanisms of a disease. To do so, model systems that reflect human diseases and allow for the evaluation of new therapeutic approaches are needed. Yet, preclinical animal studies often have limited success in predicting human physiology, pathology, and therapeutic responses. Moreover, animal testing is facing increasing ethical and bureaucratic hurdles, while human cell cultures are limited in their ability to represent in vivo situations due to the lack of the tissue microenvironment, which may alter cellular responses. To overcome these struggles, organ cultures, especially those of complex organs such as the retina, can be used to study physiological reactions to substances or stressors. Human and animal organ cultures are now well established and recognized. This mini-review discusses how retinal organ cultures can be used to preserve tissue architecture more realistically and therefore better represent disease-related changes. It also shows how molecular biological, biochemical, and histological techniques can be combined to investigate how anatomical localization may alter cellular responses. Examples for the use of retinal organ cultures, including models to study age-related macular degeneration (AMD), retinitis pigmentosa (RP), central artery occlusion (CRAO), and glaucoma are presented, and their advantages and disadvantages are discussed. We conclude that organ cultures significantly improve our understanding of complex retinal diseases and may advance treatment testing without the need for animal testing.

Impaired arterial vitamin D signaling occurs in the development of vascular calcification.

Conflicting data exists as to whether vitamin D receptor agonists (VDRa) are protective of arterial calcification. Confounding this, is the inherent physiological differences between human and animal experimental models and our current fragmented understanding of arterial vitamin D metabolism, their alterations in disease states and responses to VDRa’s. Herein, the study aims to address these problems by leveraging frontiers in human arterial organ culture models. Human arteries were collected from a total of 24 patients (healthy controls, n = 12; end-stage CKD, n = 12). Cross-sectional and interventional studies were performed using arterial organ cultures treated with normal and calcifying (containing 5mmol/L CaCl2 and 5mmol/L β-glycerophosphate) medium, ex vivo. To assess the role of VDRa therapy, arteries were treated with either calcitriol or paricalcitol. We found that human arteries express a functionally active vitamin D system, including the VDR, 1α-hydroxylase and 24-hydroxylase (24-OHase) components and these were dysregulated in CKD arteries. VDRa therapy increased VDR expression in healthy arteries (p<0.01) but not in CKD arteries. Arterial 1α-OHase (p<0.05) and 24-OHase mRNA and protein expression were modulated differentially in healthy and CKD arteries by VDRa therapy. VDRa exposure suppressed Runx2 and MMP-9 expression in CKD arteries, however only paricalcitol suppressed MMP-2. VDRa exposure did not modulate arterial calcification in all organ culture models. However, VDRa reduced expression of senescence associated β-galactosidase (SAβG) staining in human aortic-smooth muscle cells under calcifying conditions, in vitro. In conclusion, maladaptation of arterial vitamin D signaling components occurs in CKD. VDRa exposure can exert vasculo-protective effects and seems critical for the regulation of arterial health in CKD.