Immune modulatory and anti-angiogenic effects of cathepsin S knockdown in ARPE-19 cells.

We aimed to determine the role of cathepsin S (CTSS) in modulating oxidative stress-induced immune and inflammatory reactions and angiogenesis in age-related macular degeneration. Human retinal pigment epithelium cells line ARPE-19 (immature) were maintained and treated with H2O2. The expression of CTSS, inflammatory cytokines, and complement factors induced by oxidative stress was compared between cells incubated without (control) and with CTSS knockdown (using small interfering ribonucleic acid; siRNA). To evaluate the role of CTSS in angiogenesis, we assayed tube formation using human umbilical vein endothelial cells and conditioned medium from ARPE-19 cells. We also used a mouse model of laser-induced choroidal neovascularization. CTSS levels were higher in ARPE-19 cells treated with H2O2 than in control cells. Oxidative stress-induced CTSS resulted in significantly elevated transcription of nuclear factor kappa B-dependent inflammatory cytokines, complement factors C3a and C5a, membrane attack complex (C5b-9), and C3a and C5a receptors. siRNA-mediated knockdown of CTSS reduced the number of inflammatory signals. Furthermore, oxidative stress-induced CTSS regulated the expression of peroxisome proliferator-activated receptor γ and vascular endothelial growth factor A/ Akt serine/threonine kinase family signaling, which led to angiogenesis. Tube formation assays and mouse models of choroidal neovascularization revealed that CTSS knockdown ameliorated angiogenesis in vitro and in vivo. The present findings suggest that CTSS modulates the complement pathway, inflammatory reactions, and neovascularization, and that CTSS knockdown induces potent immunomodulatory effects. Hence, it could be a promising target for the prevention and treatment of early- and late-stage age-related macular degeneration.

Antimicrobial susceptibility pattern of aerobic bacteria responsible for post-surgical wound infection of the patients admitted into Khulna Medical College Hospital, Bangladesh.

Resistance against antimicrobial agents is dramatically increasing and gradually impacting treatment costs. Using existing drugs would have helped avoid bacterial infections in various circumstances. The primary objectives of this study were to determine the prevalence of pathogens responsible for postsurgical wound infections and their antimicrobial susceptibility and resistance pattern among the patients admitted to Khulna Medical College Hospital, Khulna Bangladesh. This cross-sectional study involved 250 patients suffering from postsurgical wound infection as respondents. The bacterial pathogens were isolated from pus samples obtained from those patients. The isolated bacterial pathogens were identified through several standard biochemical tests, and finally, the culture sensitivity tests of those bacterial isolates were performed. The study was conducted from August 2019 to June 2020. Data regarding the patient's age, gender, occupation, surgery performed, duration of hospital stay, and comorbidity were also documented using standard questionnaires. Five bacterial pathogens were identified with different frequencies, including Pseudomonas aeruginosa (36 %), Escherichia coli (21.2 %), Staphylococcus aureus (8.8 %), Klebsiella spp. (7.2 %) and Proteus spp. (4.8 %). These bacterial pathogens showed sensitivity to ciprofloxacin (75 %), piperacillin-tazobactam (56.7 %) and gentamicin (50 %). Besides, S. aureus showed sensitivity to linezolid and vancomycin and resistance to cefuroxime, ceftazidime and imipenem. Male patients (68.4 %) suffered more from postsurgical wound infection than female patients (31.6 %). Patients aged 31 to 40 years were more severely affected than patients from other age groups. Postsurgical wound infection was vigorously observed in the patients who underwent hand surgery. Intensive occurrence of this infection was found in the patients who stayed in the hospital from 31 to 40 days. Diabetic patients suffered more from postsurgical wound infection compared to the other patients. Throughout the study, ciprofloxacin has been the best performer against E. coli, Klebsiella spp., and Proteus spp., and gentamicin showed better performance against S. aureus. The antibiotic resistance pattern of these bacterial pathogens reflects the worldwide necessity of rational antibiotic management and proper steps to maintain hospital hygiene in Bangladesh.

Cathepsin S Knockdown Suppresses Endothelial Inflammation, Angiogenesis, and Complement Protein Activity under Hyperglycemic Conditions In Vitro by Inhibiting NF-κB Signaling.

Hyperglycemia plays a key role in the development of microvascular complications, endothelial dysfunction (ED), and inflammation. It has been demonstrated that cathepsin S (CTSS) is activated in hyperglycemia and is involved in inducing the release of inflammatory cytokines. We hypothesized that blocking CTSS might alleviate the inflammatory responses and reduce the microvascular complications and angiogenesis in hyperglycemic conditions. In this study, we treated human umbilical vein endothelial cells (HUVECs) with high glucose (HG; 30 mM) to induce hyperglycemia and measured the expression of inflammatory cytokines. When treated with glucose, hyperosmolarity could be linked to cathepsin S expression; however, many have mentioned the high expression of CTSS. Thus, we made an effort to concentrate on the immunomodulatory role of the CTSS knockdown in high glucose conditions. We validated that the HG treatment upregulated the expression of inflammatory cytokines and CTSS in HUVEC. Further, siRNA treatment significantly downregulated CTSS expression along with inflammatory marker levels by inhibiting the nuclear factor-kappa B (NF-κB) mediated signaling pathway. In addition, CTSS silencing led to the decreased expression of vascular endothelial markers and downregulated angiogenic activity in HUVECs, which was confirmed by a tube formation experiment. Concurrently, siRNA treatment reduced the activation of complement proteins C3a and C5a in HUVECs under hyperglycemic conditions. These findings show that CTSS silencing significantly reduces hyperglycemia-induced vascular inflammation. Hence, CTSS may be a novel target for preventing diabetes-induced microvascular complications.

Hyper- and hypo-perfusion of choriocapillaris in the eyes with pachychoroid pigment epitheliopathy.

PURPOSE: To evaluate the changes in choriocapillaris vessel density (VD) in eyes with pachychoroid pigment epitheliopathy (PPE) using swept-source optical coherence tomography (OCT) angiography (OCTA). METHODS: This study included 83 eyes with PPE and 42 control eyes. We collected OCT and OCTA parameters, including central point thickness, subfoveal choroidal thickness (SFChT), and choriocapillaris VD of the fovea (CC fovea) and parafovea. The parafoveal area was divided into superior, nasal, inferior, and temporal choriocapillaris areas. Maximum (CC max) and minimum (CC min) choriocapillaris VD were defined as the highest and lowest values among the four parafoveal subfield VDs, respectively. We analyzed the average choriocapillaris VD, CC max, CC min, CC fovea, and the difference between CC max and CC min (CC delta) individually and compared all the parameters between PPE and control eyes. RESULTS: CC max (56.0% ± 1.7%) was significantly higher and CC min (50.9% ± 2.0%) significantly lower in eyes with PPE than in control eyes (CC max, 55.3% ± 1.0%, P = 0.006; CC min, 51.5% ± 1.3%, P = 0.046). The CC delta value (5.0% ± 2.1%) and SFChT (389.9 ± 129.9 µm) were also significantly higher in eyes with PPE than in the control group (3.7% ± 1.5%, P < 0.001; 268.2 ± 102.2 µm, P < 0.001; respectively). CONCLUSIONS: Choriocapillaris VD showed higher variability (hyperperfusion and hypoperfusion) in eyes with PPE than in control eyes. Choriocapillaris hypoperfusion may precede the development of PPE; however, choriocapillaris hyperperfusion is associated with projection artifacts.

A biphasic calcium phosphate ceramic scaffold loaded with oxidized cellulose nanofiber-gelatin hydrogel with immobilized simvastatin drug for osteogenic differentiation.

A novel bone scaffold containing bioceramic and biopolymer materials with an osteoinductive simvastatin molecule was developed to enhance bone regeneration. An oxidized cellulose nanofiber (OCNF)-Gelatin (Gel) hydrogel was loaded into a biphasic calcium phosphate (BCP) ceramic in which simvastatin was entrapped, resulting in a scaffold with both osteoconductive and osteoinductive properties. The fabricated scaffold showed interconnected porosity with micro- and macroporous orientation. After loading the OCNF-Gel (HG), the mechanical stability of the ceramic BCP scaffold was increased suitable for the application of hard tissue regeneration. Fourier-transform infrared spectroscopy showed that simvastatin was successfully coated on the BCPHG scaffolds. OCNF, with its slower degradation, may contribute to the sustained release of drug from the scaffold. Initially simvastatin was released from the scaffold at high levels, then was constantly and gradually released for up to 4 weeks. Pre-osteoblast MC3T3E1 cells were seeded on the scaffolds to investigate cell viability, morphology, and differentiation. The simvastatin-loaded BCPHG-S scaffolds showed better cell proliferation and spreading compared to other scaffolds. Immunostaining assays showed the expression of proteins responsible for osteogenic differentiation. Alkaline phosphatase and osteopontin were more highly expressed in the BCPHG-S scaffold than in other scaffolds. These results suggest that simvastatin-loaded BCPHG scaffolds provided physiological environments suitable for better osteogenic differentiation.

Thermal cycling effect on osteogenic differentiation of MC3T3-E1 cells loaded on 3D-porous Biphasic Calcium Phosphate (BCP) scaffolds for early osteogenesis.

The application of heat stress on a defect site during the healing process is a promising technique for early bone regeneration. The primary goal of this study was to investigate the effect of periodic heat shock on bone formation. MC3T3-E1 cells were seeded onto biphasic calcium phosphate (BCP) scaffolds, followed by periodic heating to evaluate osteogenic differentiation. Heat was applied to cells seeded onto scaffolds at 41 °C for 1 h once, twice, and four times a day for seven days and their viability, morphology, and differentiation were analyzed. BCP scaffolds with interconnected porous structures mimic bone biology for cellular studies. MTT and confocal studies have shown that heat shock significantly increased cell proliferation without any toxic effects. Compared to non-heated samples, heat shock enhanced calcium deposition and mineralization, which could be visualized by SEM observation and Alizarin red S staining. Immunostaining images showed the localization of osteogenic proteins ALP and OPN on heat-shocked cells. qRT-PCR analysis revealed the presence of more osteospecific markers, osteopontin (OPN), osteocalcin, collagen type X, and Runx2, in the heat-shocked samples than in the non-heated sample. Periodic heat shock significantly upregulated both heat shock proteins (HSP70 and HSP27) in differentiated MC3T3-E1 cells. The results of this study demonstrated that periodically heat applied especially two times a day was better approach for osteogenic differentiation. Hence, this work provides a define temperature and time schedule for the development of a clinical heating device in future for early bone regeneration during the postsurgical period.