Pathogenic Role of Endoplasmic Reticulum Stress in Diabetic Corneal Endothelial Dysfunction.

PURPOSE: Progressive corneal edema and endothelial cell loss represent the major corneal complications observed in diabetic patients after intraocular surgery. However, the underlying pathogenesis and potential treatment remain incompletely understood. METHODS: We used streptozotocin-induced type 1 diabetic mice and db/db type 2 diabetic mice as diabetic animal models. These mice were treated with the endoplasmic reticulum (ER) stress agonist thapsigargin; 60-mmHg intraocular pressure (IOP) with the ER stress antagonist 4-phenylbutyric acid (4-PBA); mitochondria-targeted antioxidant SkQ1; or reactive oxygen species scavenger N-acetyl-l-cysteine (NAC). Corneal thickness and endothelial cell density were measured before and after treatment. Human corneal endothelial cells were treated with high glucose with or without 4-PBA. The expression of corneal endothelial- and ER stress-related genes was detected by western blot and immunofluorescence staining. Mitochondrial bioenergetics were measured with an Agilent Seahorse XFp Analyzer. RESULTS: In diabetic mice, the appearance of ER stress preceded morphological changes in the corneal endothelium. The persistent ER stress directly caused corneal edema and endothelial cell loss in normal mice. Pharmacological inhibition of ER stress was sufficient to mitigate corneal edema and endothelial cell loss in both diabetic mice after high IOP treatment. Mechanistically, inhibiting ER stress ameliorated the hyperglycemia-induced mitochondrial bioenergetic deficits and improved the barrier and pump functional recovery of the corneal endothelium. When compared with NAC, 4-PBA and SkQ1 exhibited better improvement of corneal edema and endothelial cell loss in diabetic mice. CONCLUSIONS: Hyperglycemia-induced ER stress contributes to the dysfunction of diabetic corneal endothelium, and inhibiting ER stress may offer therapeutic potential by improving mitochondrial bioenergetics.

The advanced glycation end-products (AGEs)/ROS/NLRP3 inflammasome axis contributes to delayed diabetic corneal wound healing and nerve regeneration.

Diabetic keratopathy (DK) is an important diabetic complication at the ocular surface. Chronic low-grade inflammation mediated by the NLRP3 inflammasome promotes pathogenesis of diabetes and its complications. However, the effect of the NLRP3 inflammasome on DK pathogenesis remains elusive. Wild-type (WT) and Nlrp3 knockout (KO) C57 mice were used to establish a type I diabetes model by intraperitoneal injection of streptozotocin. The effect of the NLRP3 inflammasome on diabetic corneal wound healing and never regeneration was examined by a corneal epithelial abrasion model. Western blot, immunofluorescence staining, enzyme-linked immunosorbent assay (ELISA) and pharmacological treatment were performed to investigate the regulatory mechanism of advanced glycation end products (AGEs) on NLRP3 inflammasome activation and corneal wound healing in vivo. The cultured mouse corneal epithelial cells (TKE2) were used to evaluate the effect and mechanism of AGEs on NLRP3 inflammasome activation in vitro. We revealed that NLRP3 inflammasome-mediated inflammation and pyroptosis contributed to DK pathogenesis. Under physiological conditions, the NLRP3 inflammasome was required for corneal wound healing and nerve regeneration. However, under a diabetic scenario, sustained activation of the NLRP3 inflammasome resulted in postponed corneal wound healing and impaired nerve regeneration. Mechanistically, the accumulated AGEs promoted hyperactivation of the NLRP3 inflammasome through ROS production. Moreover, genetically and pharmacologically blocking the AGEs/ROS/NLRP3 inflammasome axis significantly expedited diabetic corneal epithelial wound closure and nerve regeneration. Our results revealed that AGEs-induced hyperactivation of the NLRP3 inflammasome resulted in delayed diabetic corneal wound healing and impaired nerve regeneration, which further highlighted the NLRP3 inflammasome as a promising target for DK treatment.

Periplocin ameliorates mouse age-related meibomian gland dysfunction through up-regulation of Na/K-ATPase via SRC pathway.

Age-related meibomian gland dysfunction (MGD) is the main cause of evaporative dry eye disease in an aging population. Decreased meibocyte cell renewal and lipid synthesis are associated with age-related MGD. Here, we found an obvious decline of Ki67, ΔNp63, and Na+/K+ ATPase expression in aged meibomian glands. Potential Na+/K+ ATPase agonist periplocin, a naturally occurring compound extracted from the traditional herbal medicine cortex periplocae, could promote the proliferation and stem cell activity of meibocyte cells in vitro. Moreover, we observed that periplocin treatment effectively increased the expression of Na+ /K+ ATPase, accompanied with the enhanced expression of Ki67 and ΔNp63 in aged meibomian glands, indicating that periplocin may accelerate meibocyte cell renewal in aged mice. LipidTox staining showed increased lipid accumulation after periplocin treatment in cultured meibomian gland cells and aged meibomian glands. Furthermore, we demonstrated that the SRC pathway was inhibited in aged meibomian glands; however, it was activated by periplocin. Accordingly, the inhibition of the SRC signaling pathway by saracatinib blocked periplocin-induced proliferation and lipid accumulation in meibomian gland cells. In sum, we suggest periplocin-ameliorated meibocyte cell renewal and lipid synthesis in aged meibomian glands via the SRC pathway, which could be a promising candidate for age-related MGD.

Efficacy of rhNGF-loaded amniotic membrane transplantation for rabbit corneal epithelial and nerve regeneration.

AIM: To evaluate the efficacy of recombinant human nerve growth factor-loaded amniotic membrane (rhNGF-AM) on corneal epithelial and nerve regeneration in rabbit model. METHODS: Freshly prepared human amniotic membrane (AM) were immersed into PBS buffer containing 100 or 500 µg/mL rhNGF for 15, 30, and 60min at 4°C. The in vitro release kinetics of rhNGF was measured with ELISA. For in vivo evaluation, the AM were immersed with 500 µg/mL rhNGF for 30min. Fifty-seven rabbits were selected to establish corneal epithelial defect model. In addition to the 19 rabbits in control group, 38 rabbits received AM transplantation with or without rhNGF after the removal of central epithelium. Corneal epithelial defect area, sub-epithelial nerve fiber density, corneal sensitivity, rhNGF contents in resident AM and corneas were measured after the surgery. RESULTS: rhNGF was sustained release from the AM within 14d in vitro, with the positive correlation with initial immersion concentration. The immersion of AM in 500 µg/mL rhNGF for 30min achieved the most stable release within 14d. After transplantation in rabbit cornea, a high concentration of rhNGF in resident rhNGF-AM and cornea was maintained within 8d. Corneal epithelial healing, nerve fiber regeneration and the recovery of corneal sensitivity were significantly accelerated after the rhNGF-AM transplantation when compared to simple AM transplantation (all P<0.05). CONCLUSION: Simple immersion of AM achieves the sustained release of rhNGF, and promotes corneal epithelial wound healing and nerve regeneration, as well as the recovery of corneal sensitivity in rabbit.

Midterm outcomes of penetrating keratoplasty following allogeneic cultivated limbal epithelial transplantation in patients with bilateral limbal stem cell deficiency.

AIM: To evaluate the midterm outcomes of penetrating keratoplasty (PK) following allogeneic cultivated limbal epithelial transplantation (CLET) for bilateral total limbal stem cell deficiency (LSCD). METHODS: Ten patients (10 eyes) with bilateral LSCD were enrolled in this prospective noncomparative case series study. Each participant underwent PK approximately 6mo after a CLET. Topical tacrolimus, topical and systemic steroids, and oral ciclosporin were administered postoperatively. Best-corrected visual acuity (BCVA), intraocular pressure (IOP), ocular surface grading scores (OSS), corneal graft epithelial rehabilitation, persistent epithelial defect (PED), immunological rejection, and graft survival rate were assessed. RESULTS: The time interval between PK and allogeneic CLET was 6.90±1.29 (6-10)mo. BCVA improved from 2.46±0.32 logMAR preoperatively to 0.77±0.55 logMAR post-PK (P<0.001). Kaplan-Meier analysis of mean graft survival revealed graft survival rates of 100% at 12 and 24mo and 80.0% at 36mo. PEDs appeared in 5 eyes at different periods post-PK, and graft rejection occurred in 4 eyes. The total OSS decreased from 12.4±4.4 before allogeneic CLET to 1.4±1.51 after PK. CONCLUSION: A sequential therapy design of PK following allogeneic CLET can maintain a stable ocular surface with improved BCVA despite the relatively high graft rejection rate.

Revealing the Angiopathy of Lacrimal Gland Lesion in Type 2 Diabetes.

For a better understanding of diabetic angiopathy (DA), the potential biomarkers in lacrimal DA and its potential mechanism, we evaluated the morphological and hemodynamic alterations of lacrimal glands (LGs) in patients with type 2 diabetes and healthy counterparts by color Doppler flow imaging (CDFI). We further established a type 2 diabetic mice model and performed hematoxylin-eosin (HE) staining, immunofluorescence staining of CD31, RNA-sequencing analysis, and connectivity map (CMap) analysis. We found atrophy and ischemia in patients with type 2 diabetes and mice models. Furthermore, we identified 846 differentially expressed genes (DEGs) between type 2 diabetes mellitus (T2DM) and vehicle mice by RNA-seq. The gene ontology (GO) analysis indicated significant enrichment of immune system process, regulation of blood circulation, apoptotic, regulation of secretion, regulation of blood vessel diameter, and so on. The molecular complex detection (MCODE) showed 17 genes were involved in the most significant module, and 6/17 genes were involved in vascular disorders. CytoHubba revealed the top 10 hub genes of DEGs, and four hub genes (App, F5, Fgg, and Gas6) related to vascular regulation were identified repeatedly by MCODE and cytoHubba. GeneMANIA analysis demonstrated functions of the four hub genes above and their associated molecules were primarily related to the regulation of circulation and coagulation. CMap analysis found several small molecular compounds to reverse the altered DEGs, including disulfiram, bumetanide, genistein, and so on. Our outputs could empower the novel potential targets to treat lacrimal angiopathy, diabetes dry eye, and other diabetes-related diseases.

Lipidomic analysis of meibomian glands from type-1 diabetes mouse model and preliminary studies of potential mechanism.

Diabetes is a significant risk factor for meibomian gland dysfunction (MGD), but its mechanism is poorly understood. The main function of the meibomian glands (MGs) is to synthesize, store, and secrete lipids. In this study, we found that the amount of lipids in the meibomian acini in STZ-induced type 1 diabetic mice decreased, and the lipid droplets became larger and irregular. In all, 31 lipid subclasses were identified in the mouse MGs, which contained 1378 lipid species in total through lipidomics analysis based on LC-MS/MS. Diabetes caused a significant increase in the content of ceramides (Cer) in the MGs but a significant decrease in the ration of sphingomyelin to ceramides (SM/Cer). The quantity of meibocytes in diabetic mice was dramatically decreased, and the proliferation activity was alleviated, which may be associated with cell cycle arrest caused by diabetes-induced abnormal Cer metabolism in MGs. We found an increase in macrophage and neutrophils infiltration in the diabetic MGs, which may be related to the significant reduction of AcCa in diabetic MGs. Taken together, the results of the present study demonstrated that diabetes induced disruption of lipid homeostasis in MGs, which may mediate the decreased cell proliferation and increased inflammation caused by diabetes in MGs.

Leucine-rich α-2-glycoprotein-1 promotes diabetic corneal epithelial wound healing and nerve regeneration via regulation of matrix metalloproteinases.

Leucine-rich α-2-glycoprotein-1 (LRG1) is involved in several pathophysiological processes, including angiogenesis, cutaneous wound repair and cancer metastasis. In this study, we investigated the potential role and mechanism of LRG1 in corneal re-epithelialisation and nerve regeneration in streptozotocin-induced diabetic mice. We found decreased levels of LRG1 in the corneal epithelium after wounding in diabetic mice compared to normal controls. Hyperglycaemia downregulated the LRG1 expression in the corneal epithelium in vivo, as well as in vitro in a cultured mouse corneal epithelial stem/progenitor cell line (TKE2 cells) exposed to high glucose (HG; 30 mM) in the culture medium. Exogenous application of LRG1 accelerated corneal re-epithelialisation and nerve regeneration in normal mice and diabetic mice. LRG1 also overcame the suppression of wound healing in TKE2 cells by HG conditions, and it activated repair-related signalling by JAK2/STAT3, AKT, epidermal growth factor receptor (EGFR) and transforming growth factor (TGF)-ß3. We also found that LRG1 treatment overcame the hyperglycaemia-suppressed expression of matrix metalloproteinase 3 (MMP3) and metalloproteinase 13 (MMP13) in the regenerated corneal epithelium. The promoted effects of LRG1 on corneal re-epithelialisation and nerve regeneration were blocked by inhibitors of MMP3 and MMP13. Subconjunctival injection of 0.5 µg MMP inhibitors did not cause any obvious toxic damage in corneal epithelial cells. Immunoprecipitation and proximity ligation assay experiments confirmed that endogenous LRG1 coprecipitated with MMP3 and MMP13 in TKE2 cells. These results indicate that LRG1 promoted wound repair and nerve regeneration in the diabetic corneal epithelium by regulation of MMPs. Our findings reveal a new function and mechanism for LRG1 in the cornea, and they provide new insights for a better understanding of diabetic keratopathy.

[Components of Urinary Nanocrystals and Their Influence on Formation of Calcium Oxalate Stones].

High-resolution transmission electron microscopy, X-ray diffraction, selected area electron diffraction (SAED), and energy dispersive spectroscopy (EDS) were accurately performed to analyze the components of nanocrystals in the urine of patients with calcium oxalate (CaOx) stones. XRD, SAED and FFT detected the presence of calcium oxalate monohydrate (COM), uric acid (UA), and calcium phosphate (CaP). EDS detected the elements of C, O, Ca, with a small amount of N and P. These results showed that the main components of urinary nanocrystals were COM, with a small amount UA and phosphate. HRTEM observation showed that the particle size of urinary nanocrystals was dozens of nanometers. The result was consistent with the calculation by Debye-Scherrer equation. When the urine was filtered through a microporous membrane of 0.45, 1.2, and 3 µm, respectively, the number of diffraction peaks of the obtained urine crystallites increased with the increased pore size, indicating the increase of urinary crystallite species. Crystal nucleation, growth, aggregation, and adhesion of crystals to the renal epithelial cells are important processes for CaOx stone formation. The presence of a large amount of COM crystals in patients' urine is a critical factor for CaOx stones formation. Nano UA and CaP crystallite can induce the CaOx stone formation as central nidus.

Component analyses of urinary nanocrystallites of uric acid stone formers by combination of high-resolution transmission electron microscopy, fast Fourier transformation, energy dispersive X-ray spectroscopy, X-ray diffraction and Fourier transform infrared spectroscopy.

This study aimed to analyse the components of nanocrystallites in urines of patients with uric acid (UA) stones. X-ray diffraction (XRD), Fourier transform infrared spectroscopy, high-resolution transmission electron microscopy (HRTEM), fast Fourier transformation (FFT) of HRTEM, and energy dispersive X-ray spectroscopy (EDS) were performed to analyse the components of these nanocrystallites. XRD and FFT showed that the main component of urinary nanocrystallites was UA, which contains a small amount of calcium oxalate monohydrate and phosphates. EDS showed the characteristic absorption peaks of C, O, Ca and P. The formation of UA stones was closely related to a large number of UA nanocrystallites in urine. A combination of HRTEM, FFT, EDS and XRD analyses could be performed accurately to analyse the components of urinary nanocrystallites.

Inhibition of urinary macromolecule heparin on aggregation of nano-COM and nano-COD crystals.

PURPOSE: This research aims to study the influences of heparin (HP) on the aggregation of nano calcium oxalate monohydrate (COM) and nano calcium oxalate dihydrate (COD) with mean diameter of about 50 nm. METHOD: The influences of different concentrations of HP on the mean diameter and Zeta potential of nano COM and nano COD were investigated using a nanoparticle size Zeta potential analyzer. RESULTS: HP could be adsorbed on the surface of nano COM and nano COD crystals, leading to an increase in the absolute value of Zeta potential on the crystals and an increase in the electrostatic repulsion force between crystals. Consequently, the aggregation of the crystals is reduced and the stability of the system is improved. The strong adsorption ability of HP was closely related to the -OSO3- and -COO- groups contained in the HP molecules. X-ray photoelectron spectroscopy confirmed the coordination of HP with Ca2+ ions of COM and COD crystals. CONCLUSION: HP could inhibit the aggregation of nano COM and nano COD crystals and increase their stability in aqueous solution, which is conducive in inhibiting the formation of calcium oxalate stones.

Nanouric acid or nanocalcium phosphate as central nidus to induce calcium oxalate stone formation: a high-resolution transmission electron microscopy study on urinary nanocrystallites.

PURPOSE: This study aimed to accurately analyze the relationship between calcium oxalate (CaOx) stone formation and the components of urinary nanocrystallites. METHOD: High-resolution transmission electron microscopy (HRTEM), selected area electron diffraction, fast Fourier transformation of HRTEM, and energy dispersive X-ray spectroscopy were performed to analyze the components of these nanocrystallites. RESULTS: The main components of CaOx stones are calcium oxalate monohydrate and a small amount of dehydrate, while those of urinary nanocrystallites are calcium oxalate monohydrate, uric acid, and calcium phosphate. The mechanism of formation of CaOx stones was discussed based on the components of urinary nanocrystallites. CONCLUSION: The formation of CaOx stones is closely related both to the properties of urinary nanocrystallites and to the urinary components. The combination of HRTEM, fast Fourier transformation, selected area electron diffraction, and energy dispersive X-ray spectroscopy could be accurately performed to analyze the components of single urinary nanocrystallites. This result provides evidence for nanouric acid and/or nanocalcium phosphate crystallites as the central nidus to induce CaOx stone formation.

Property changes of urinary nanocrystallites and urine of uric acid stone formers after taking potassium citrate.

The property changes of urinary nanocrystallites in 20 cases of uric acid (UA) stone formers after 1 week of potassium citrate (K3cit) intake were comparatively studied by X-ray diffraction analysis, Fourier transform infrared spectroscopy, nanoparticle size analysis, and transmission electron microscopy. Before K3cit intake, the urinary crystallites mainly contained UA and calcium oxalate. After K3cit intake, the components changed to urate and UA; the qualities, species, and amounts of aggregated crystallites decreased; urine pH, citrate, and glycosaminoglycan excretions increased; and UA excretion, Zeta potential, and crystallite size decreased. The stability of crystallites followed the order: controls>patients after taking K3cit>patients before taking K3cit. Therefore, the components of urinary stones were closely related to the components of urinary crystallites.

Changes in urinary nanocrystallites in calcium oxalate stone formers before and after potassium citrate intake.

The property changes of urinary nanocrystallites in 13 patients with calcium oxalate (CaOx) stones were studied before and after ingestion of potassium citrate (K3cit), a therapeutic drug for stones. The analytical techniques included nanoparticle size analysis, transmission electron microscopy, X-ray diffraction, and Fourier-transform infrared spectroscopy. The studied properties included the components, morphologies, zeta potentials, particle size distributions, light intensity autocorrelation curves, and polydispersity indices (PDIs) of the nanocrystallites. The main components of the urinary nanocrystallites before K3cit intake included uric acid, ß-calcium phosphate, and calcium oxalate monohydrate. After K3cit intake, the quantities, species, and percentages of aggregated crystals decreased, whereas the percentages of monosodium urate and calcium oxalate dehydrate increased, and some crystallites became blunt. Moreover, the urinary pH increased from 5.96 ± 0.43 to 6.46 ± 0.50, the crystallite size decreased from 524 ± 320 nm to 354 ± 173 nm, and the zeta potential decreased from -4.85 ± 2.87 mV to -8.77 ± 3.03 mV. The autocorrelation curves became smooth, the decay time decreased from 11.4 ± 3.2 ms to 4.3 ± 1.7 ms, and the PDI decreased from 0.67 ± 0.14 to 0.53 ± 0.19. These changes helped inhibit CaOx calculus formation.

Coordination dynamics and coordination mechanism of a new type of anticoagulant diethyl citrate with ca(2+) ions.

Diethyl citrate (Et2Cit) is a new potential anticoagulant. The coordination dynamics and coordination mechanism of Et2Cit with Ca(2+) ions and the effect of pH on the complex were examined. The result was compared with that for the conventional anticoagulant sodium citrate (Na3Cit). The reaction order (n) of Et2Cit and Na3Cit with Ca(2+) was 2.46 and 2.44, respectively. The reaction rate constant (k) was 120 and 289 L·mol(-1) ·s(-1). The reverse reaction rate constant (k re) was 0.52 and 0.15 L·mol(-1) ·s(-1), respectively. It is indicated that the coordination ability of Et2Cit with Ca(2+) was weaker than that of Na3Cit. However, the dissociation rate of the calcium complex of Et2Cit was faster than that of Na3Cit. Increased pH accelerated the dissociation rate of the complex and improved its anticoagulant effect. The Et2Cit complex with calcium was synthesized and characterized by elemental analysis, XRD, FT-IR, (1)H NMR, and ICP. These characteristics indicated that O in -COOH and C-O-C of Et2Cit was coordinated with Ca(2+) in a bidentate manner with 1 : 1 coordination proportion; that is, complex CaEt2Cit was formed. Given that CaEt2Cit released Ca(2+) more easily than Na3Cit, a calcium solution was not needed in intravenous infusions using Et2Cit as anticoagulant unlike using Na3Cit. Consequently, hypocalcemia and hypercalcemia were avoided.

Stabilization of submicron calcium oxalate suspension by chondroitin sulfate C may be an efficient protection from stone formation.

The influences of chondroitin sulfate C (C6S) on size, aggregation, sedimentation, and Zeta potential of sub-micron calcium oxalate monohydrate (COM) and calcium oxalate dihydrate (COD) crystallites with mean sizes of about 330 nm were investigated using an X-ray diffractometer, nanoparticle size Zeta potential analyzer, ultraviolet spectrophotometer, and scanning electron microscope, after which the results were compared with those of micron-grade crystals. C6S inhibited the conversion of COD to COM and the aggregation of COM and COD crystallitesis; it also decreased their sedimentation rate, thus increasing their stability in aqueous solution. The smaller the size of the COD crystallites, the easier they can be converted to COM. The stability of sub-micron COD was worse than that of micron-grade crystals. C6S can inhibit the formation of calcium oxalate stones.