Analysis of prognostic and predictive factors in neovascular age-related macular degeneration Kuopio cohort.

PURPOSE: The aim of the study was to explore factors affecting the progression of neovascular age-related macular degeneration (nAMD) and identify predictive factors that can estimate the duration of intravitreal treatments. METHODS: This retrospective real-world study included 421 nAMD patients treated at the Kuopio University Hospital during years 2007-2021. The collected data included background demographics, treatment history, visual acuity and retinal biomarker analysis. Impact of baseline factors on age at diagnosis, treatment duration, received treatment intensity and visual acuity gains were analysed. RESULTS: Heavy smoking and high body mass index (BMI) were associated with an earlier onset, while the use of anticoagulation and anti-aggregation medication were associated with a later onset of nAMD. A low number of injections during the first year of treatment and the presence of intraretinal fluid (IRF) at baseline were associated with shorter treatment duration. Interestingly, when IRF only patients were compared to subretinal fluid (SRF) only patients, IRF patients showed higher occurrences of subretinal drusenoid deposits (43.5% vs. 15%, p = 0.04). In addition, when all patients with IRF were compared to SRF only patients, more hyperreflective foci (HRF) and complete RPE and outer retinal atrophy (cRORA; 20.7% vs. 5%, p = 0.02) were observed in patients with IRF. CONCLUSIONS: Our results reveal that heavy smoking and high BMI are accelerating factors for earlier emergence of nAMD, while the presence of IRF results in a fast-progressing disease. More intriguingly, the link between IRF and appearance of subretinal drusenoid deposits, HRF, and increased retinal atrophy was observed.

A comprehensive model for measuring real-life cost-effectiveness in eyecare: automation in care and evaluation of system (aces-rwm™).

This paper describes a holistic, yet simple and comprehensible, ecosystem model to deal with multiple and complex challenges in eyecare. It aims at producing the best possible wellbeing and eyesight with the available resources. When targeting to improve the real-world cost-effectiveness, what gets done in everyday practice needs be measured routinely, efficiently and unselectively. Collection of all real-world data of all patients will enable evaluation and comparison of eyecare systems and departments between themselves nationally and internationally. The concept advocates a strategy to optimize real-life effectiveness, sustainability and outcomes of the service delivery in ophthalmology. The model consists of three components: (1) resource-governing principles (i.e., to deal with increasing demand and limited resources), (2) real-world monitoring (i.e., to collect structured real-world data utilizing automation and visualization of clinical parameters, health-related quality of life and costs), and (3) digital innovation strategy (i.e., to evaluate and benchmark real-world outcomes and cost-effectiveness). The core value and strength of the model lies in the consensus and collaboration of all Finnish university eye clinics to collect and evaluate the uniformly structured real-world outcomes data. In addition to ophthalmology, the approach is adaptable to any medical discipline to efficiently generate real-world insights and resilience in health systems.

Long-term effects of intravitreal bevacizumab and aflibercept on intraocular pressure in wet age-related macular degeneration.

BACKGROUND: To evaluate the incidence of sustained elevation of intraocular pressure (SE-IOP) associated with intravitreal injections of anti-vascular endothelial growth factors (anti-VEGF) bevacizumab and aflibercept in patients with wet age-related macular degeneration (wAMD). METHODS: A retrospective cohort study consisting of 120 eyes from 120 patients with anti-VEGF treatment for wAMD. Three different anti-VEGF groups were considered: i) 71 cases receiving bevacizumab only, ii) 49 cases receiving bevacizumab before switch to aflibercept, iii) 49 cases after switch to aflibercept. 120 uninjected fellow eyes served as controls. SE-IOP was defined as an increase from baseline ≥5 mmHg on 2 consecutive follow-up visits. The incidence of SE-IOP was analysed using exact Poisson tests and survival analysis. The time course of IOP was evaluated with linear mixed effect modelling. RESULTS: In total, 6 treated eyes (2.38% incidence per eye-year) and 9 fellow eyes (3.58% incidence per eye-year) developed SE-IOP, and survival analysis showed no statistically significant difference (p = 0.43). Furthermore, the incidence of SE-IOP did not differ between the three anti-VEGF groups. Comparing the injected eyes of patients under 70 years to those of patients over 70 years, there was a statistically significant difference in survival without SE-IOP (incidence of 16.7% vs 0.7%, respectively, p < 0.0001). CONCLUSION: Intravitreal anti-VEGF injections were not associated with sustained elevation of IOP. These results do not support the claim that repeated anti-VEGF injections could elevate IOP.

Relationship Between Prolonged Intraocular Inflammation and Macular Edema After Cataract Surgery.

Purpose: To assess whether aqueous flare is related to an increased risk of pseudophakic cystoid macular edema (PCME) following uneventful cataract surgery in nondiabetic and diabetic patients. Methods: A post hoc analysis of five consecutive randomized clinical trials in the Department of Ophthalmology, Kymenlaakso Central Hospital, Finland. Aqueous flare levels were recorded in 448 eyes of 448 patients before surgery, and after the course of topical anti-inflammatory treatment 28 days and three months after cataract surgery. Results: Aqueous flare increase of <50%, ≥50%, ≥100%, and ≥200% associated in central subfield macular thickness (CSMT) increase across the groups at 28 days and three months after surgery. Increase of aqueous flare ≥100% compared to those with <100% was associated with increased CSMT (P = 0.022 at 28 days, and P = 0.027 at three months). At three months, macular thickening (at least 10% CSMT increase) was observed in 12.7% compared to 4.6% of eyes when using a cutoff value of 100% increase in aqueous flare (P = 0.033). Although diabetic patients presented higher aqueous flare levels at baseline compared to nondiabetic patients (12.9 ± 11.8 vs. 9.8 ± 8.2 photon units/ms P < 0.001), the postoperative levels illustrated a similar profile in aqueous flare increase between the two groups. Conclusions: At 28 days, aqueous flare increase was associated with macular thickening. A 100% cutoff value could potentially be used when studying anti-inflammatory efficacy of different treatment protocols. Flare values exceeding this cutoff value could be considered as an indication for extending anti-inflammatory therapy. Translational Relevance: A 100% increase in aqueous flare at 28 days after cataract surgery from baseline predicted macular thickening up to three months postoperatively. Identifying a correlation between increased aqueous flare levels and pseudophakic cystoid macular edema may allow recognition of the most vulnerable patients, development of prophylactic treatment strategies and reduction of the number and severity of postoperative complications.

Case report: bilateral Mooren ulcer in association with hepatitis C.

BACKGROUND: Mooren ulcer has been considered as an idiopathic autoimmune keratitis. However, it has been in some cases suggested to be associated with hepatitis C, although the evidence is very vague. CASE PRESENTATION: We present a case of a man who was diagnosed with a primary Mooren ulcer in his right eye. The eye became blind despite of intensive treatment with local medications and extensive surgical procedures. After 10 years, the patient was diagnosed with the same disease, now in his left, previously healthy eye. There was no history that would suggest a secondary Mooren ulcer, but a chronic hepatitis C infection was detected. Treatment was targeted against hepatitis C (ribavirin and interferon) in addition to immunosuppressive medical and surgical treatment which resulted in a full and more than 6 years lasting remission of the disease. CONCLUSIONS: Whether the immunomodulatory and immunosuppressive medication against hepatitis C was the key reason for the good results in the treatment of the second eye, remains elusive. The causality of hepatitis C with respect to the pathogenesis of Mooren ulcer on this patient remains open, but should be considered as one of the possible etiological factors of the disease.

The Finnish national guideline for diagnosis, treatment and follow-up of patients with wet age-related macular degeneration.

Age-related macular degeneration (AMD) is the main cause of visual impairment in developed countries. Several improvements in the visualization of posterior segment of the eye together with the introduction of intravitreal anti-VEGF treatment have revolutionized the prognosis of the wet form of AMD (wAMD). Increasing incidence of wAMD together with the limited resources of society and of the healthcare system poses challenges for the provision and development of care. In context of these current aspects, we aimed to set evidence-based medical guidelines for diagnosis, treatment and follow-up of patients with wAMD.

Hypoxia and inflammation in the release of VEGF and interleukins from human retinal pigment epithelial cells.

PURPOSE: Retinal diseases are closely associated with both decreased oxygenation and increased inflammation. It is not known if hypoxia-induced vascular endothelial growth factor (VEGF) expression in the retina itself evokes inflammation, or whether inflammation is a prerequisite for the development of neovascularization. METHODS: Human ARPE-19 cell line and primary human retinal pigment epithelium (RPE) cells were used. ARPE-19 cells were kept either under normoxic (24 h or 48 h) or hypoxic conditions (1% O2, 24 h). Part of the cells were re-oxygenated (24 h). Some ARPE-19 cells were additionally pre-treated with bacterial lipopolysaccharide (LPS). The levels of IL-6, IL-8, IL-1ß, and IL-18 were determined from medium samples by an enzyme-linked immunosorbent assay (ELISA) method. Primary human RPE cells were exposed to hypoxia for 24 h, and the subsequent release of IL-6 and IL-8 was measured with ELISA. VEGF secretion from ARPE-19 cells was determined up to 24 h. RESULTS: Hypoxia induced significant (P < 0.01) increases in the levels of both IL-6 and IL-8 in ARPE-19 cells, and LPS pre-treatment further enhanced these responses. Hypoxia exposure did not affect the IL-1ß or IL-18 release irrespective of LPS pre-treatment. If primary RPE cells were incubated for 4 h in hypoxic conditions, IL-6 and IL-8 concentrations were increased by 7 and 8-fold respectively. Hypoxia increased the VEGF secretion from ARPE-19 cells in a similar manner with or without pre-treatment with LPS. CONCLUSIONS: Hypoxia causes an inflammatory reaction in RPE cells that is potentiated by pre-treatment with the Toll-like receptor-activating agent, LPS. The secretion of VEGF from these cells is regulated directly by hypoxia and is not mediated by inflammation.

Hypoxic conditions stimulate the release of B-type natriuretic peptide from human retinal pigment epithelium cell culture.

PURPOSE: A-type peptide, a natriuretic peptide belonging to the natriuretic peptide family, has been shown to be increased in the vitreous of patients suffering from diabetic retinopathy and that human retina has a well-developed natriuretic peptide system. The stimulus to which the synthesis of natriuretic peptides responded in these patients has, however, remained unknown. As the natriuretic peptides have recently been shown to respond to hypoxic conditions, the genes of both A-type and B-type have a hypoxia-response element (HRE) in their promoter sequence, we therefore hypothesized that hypoxia in the human retinal pigment epithelium will increase the secretion of NT-proBNP, the most common natriuretic peptide monitored in clinical medicine. METHODS: We used cultured human retinal pigment epithelium cell line (ARPE-19) which was exposed either to normoxia or to hypoxia for 2 hr, 4 hr, 6 hr and 24 hr. NT-proBNP was measured with enzyme immunoassay, VEGF with ELISA and HIF-1α with Western blotting. RESULTS: Hypoxia induced VEGF 165 release in culture medium and HIF-1α expression in cultured ARPE-19 cells. Time-dependent NT-proBNP release was detected when the ARPE-19 cells were cultured under normoxia. When hypoxia was induced, a statistically significant increase in NT-proBNP release was demonstrated in the culture medium. CONCLUSIONS: Hypoxic conditions increase the release of a natriuretic peptide from retinal pigment epithelium (RPE) cells. The secretion of VEGF was also enhanced. The responses were associated with the up-regulation of the HIF-1α transcription factor. These results explain the previous findings from patients with diabetes, which also suggest that hypoxia is a ubiquitous stimulus for the secretion of natriuretic peptides in human body.

Impaired gap junction formation and intercellular calcium signaling in urinary bladder cancer cells can be improved by Gö6976.

PURPOSE: Calcium wave propagation and connexin 26, 32 and 43 expression were studied in normal and malignant urothelial cells. MATERIALS AND METHODS: Human urothelial cell cultures were established from tissue biopsies obtained from three healthy control persons and compared to human transitional cell carcinoma (TCC) cell line 5637. Fluo-3 was used to study intercellular calcium signaling in urothelial cells. The cells were stimulated mechanically in the presence of inhibitors of gap-junctional or ATP-mediated communication to determine which pathways are operative in intercellular calcium signaling. In addition, Gö6976 was used to determine the effects of PKC alpha and betaI inhibition on intercellular calcium signaling. RESULTS: In normal urothelial cells, the primary pathway for intercellular calcium mediated cell signaling was gap junctional intercellular communication (GJIC), but the paracrine ATP-mediated signaling was also operative. In 5637 TCC cells, GJIC and ATP-mediated signaling routes were altered when compared to normal urothelial cells. More specifically, inhibition of GJIC resulted in a complete block of intercellular calcium signaling, while inhibition of ATP-mediated signaling decreased signal transduction in 5637 TCC cells. The results of the present study also demonstrated that connexin 26 was the most abundant gap junction plaque protein in cultured normal human urothelial cells and that it did not form gap junction plaques in 5637 TCC cell culture. Treatment with Gö6976 induced gap junction plaque formation by connexin 26 in 5637 TCC cells. In addition, the exposure to Gö6976 enhanced intercellular calcium mediated signaling in 5637 TCC cells, but not in normal cells. CONCLUSIONS: The results of the present study suggest that gap junctions play a major role in intercellular calcium signaling in urothelial cells. In addition, intercellular calcium signaling is altered in urinary bladder carcinoma cells, and it can be improved by PKC alpha and betaI inhibition. (Supplementary materials are available for this article. Go to the publisher's online edition of Cell Communication and Adhesion for the following free supplemental resources; Movie files of Fig. 2normal Gö6976-, normal Gö6976+, TCC Gö6976-, TCC Gö6976+ and image of Supplementary Figure 1).

PKC inhibitor Go6976 induces mitosis and enhances doxorubicin-paclitaxel cytotoxicity in urinary bladder carcinoma cells.

Protein kinase C (PKC) alpha/betaI isoenzyme inhibitor Go6976 has been suggested to be a G2 checkpoint abrogator by direct Chk1 inhibition. In the present study, we demonstrate that Go6976 induces mitosis in doxorubicin treated G2-arrested 5637 urinary bladder transitional cell carcinoma cells and interestingly also in non-synchronized 5637 cells. Importantly, the results demonstrated that both doxorubicin treated and non-synchronized cancer cells are forced to mitosis by Go6976. However, part of the cells avoid the death in mitosis and continue in the cell cycle which may increase the probability of genomic instability. Cytotoxicity of Go6976 alone and in combination with chemotherapeutic agents was further studied. Go6976 treatment alone induced apoptotic cell death. Cytostatic doxorubicin pre-treatment induced G2 arrest and inhibited the cytotoxic effects of mitosis specific drug paclitaxel. Cytotoxicities of doxorubicin-paclitaxel and doxorubicin-Go6976 sequences could be markedly enhanced by combining Go6976 with paclitaxel after doxorubicin pre-treatment. In doxorubicin-Go6976+paclitaxel sequence, paclitaxel arrested the cells to mitosis and unfavourable progression of the cell cycle was inhibited. Analyzes of the molecular mechanisms underlying Go6976 induced mitosis showed that PKC inhibiting concentrations of Go6976 induced cdc2 activation concentration-dependently in non-synchronized and in DNA damaged cells. Simultaneously, Chk1/2 became deactivated and cdc25C activated in DNA damaged cells, indicating regulatory events upstream. In non-synchronized cells, activation of cdc25C, but not Chk1/2, was observed, suggesting inactivation of c-TAK1. The results of the current study suggest that Go6976 has a synergistic cytotoxic effect when combined with doxorubicin and paclitaxel.

Heterogeneity of cellular proliferation within transitional cell carcinoma: correlation of protein kinase C alpha/betaI expression and activity.

A total of 18 histological samples containing both transitional cell carcinoma (TCC) and normal urothelial epithelium were analyzed for protein kinase C (PKC)-alpha and -betaI expression, and for their phosphorylated substrates. The results showed an increased expression of PKC-alpha in 13 out of 18 samples and -betaI in 11 out of 18 TCC samples when compared with normal urothelium. In addition, 11 out of 18 of the TCC tumors displayed heterogeneous expression of the PKC isoenzymes, with different levels of immunosignal in different areas of the tumor. Within the same sample, areas of highest PKC isoenzyme expression also showed highest classical PKC activity, as estimated by immunodetection of phosphorylated forms of PKC substrates. The areas of highest expression of PKC-alpha and/or -betaI isoenzymes showed also the highest number of cells positive for Ki67, an indicator of proliferation. Immunofluorescence and Western blotting demonstrated that in cultured TCC cells, PKC-alpha was located in the cytoplasm, whereas PKC-betaI was located primarily in the nucleus as a 65-kDa fragment and in the cytoplasm as a full-size 79-kDa protein. Our results indicate that increased expression of PKC-alpha and -betaI leads to increased total classical PKC kinase activity and suggest that increased activity of the isoenzymes plays a role in accelerated growth of TCC. Furthermore, these results suggest that even in carcinoma tissue, PKC expression and activity are under strict control.

Protein kinase C (PKC) family in cancer progression.

PKC family consist of a number of serine-threonine kinases which are divided into three groups based on their activating factors. PKCs have been linked to carcinogenesis since PKC activators can act as tumor promoters. Furthermore, functional studies have suggested that PKCs play a role in the carcinogenesis and maintenance of malignant phenotype. Potentiation of malignant phenotype may be mediated by activation of selective PKC isoenzymes or through altered isoenzyme expression profile compared to the originating tissue. Activation of PKCalpha and beta isoenzymes have often been linked to malignant phenotype while PKCdelta is thought to mediate anti-cancer effects. This review will focus on the regulation and significance of PKC isoenzymes to cancer progression.

NF1 tumor suppressor in epidermal wound healing with special focus on wound healing in patients with type 1 neurofibromatosis.

Type 1 neurofibromatosis syndrome (NF1) has been linked with mutations of the NF1 gene which encodes tumor suppressor neurofibromin, a regulator of Ras-MAPK signaling. In human epidermis, keratinocytes express NF1 tumor suppressor and it may have a distinctive function in these cells during wound healing, such as regulating Ras activity. NF1 expression was first studied during the epidermal wound healing using suction blister method. NF1 gene expression increased both in hypertrophic and migrating zones of the healing epidermis, and also in dermal fibroblasts underneath the injury. This prompted us to study epidermal wound healing in NF1 patients. Wound healing efficiency was evaluated 4 days after blister induction by clinical, physiological and histological methods. Epidermal wound healing was equally effective in NF1 patients and healthy controls. In addition, dermal wound healing appears to function normally in NF1 patients based on retrospective and follow-up study of biopsy scars. Furthermore, the healing wounds were analyzed immunohistochemically for cell proliferation rate and Ras-MAPK activity. Neither epidermal keratinocytes nor dermal fibroblasts showed difference in the cell proliferation rate or Ras-MAPK activity between NF1 patients and controls. Interestingly, NF1 patients displayed increased cell proliferation rate and Ras-MAPK activity in periarteriolar tissue underneath the wound. The results of the study suggest that epidermal wound healing is not markedly altered in NF1 patients. Furthermore, NF1 protein seems not to have an important function as a Ras-MAPK regulator in epidermal keratinocytes or dermal fibroblasts but instead appears to be regulator of Ras-MAPK signaling in vascular tissues.

Protein kinase C alpha/beta inhibitor Go6976 promotes formation of cell junctions and inhibits invasion of urinary bladder carcinoma cells.

Changes in activation balance of different protein kinase C (PKC) isoenzymes have been linked to cancer development. The current study investigated the effect of different PKC inhibitors on cellular contacts in cultured high-grade urinary bladder carcinoma cells (5637 and T24). Exposure of the cells to isoenzyme-specific PKC inhibitors yielded variable results: Go6976, an inhibitor of PKCalpha and PKCbeta isoenzymes, induced rapid clustering of cultured carcinoma cells and formation of an increased number of desmosomes and adherens junctions. Safingol, a PKCalpha inhibitor, had similar but less pronounced effects. In contrast, a PKCdelta inhibitor, rottlerin, had an opposite effect on cell clustering and caused dissociation of cell junctions. A broad-spectrum PKC inhibitor bisindolylmaleimide I did not have any apparent effect on the morphology of the cultures or on the number of cell junctions. Additional studies with Go6976 demonstrated that inhibition of PKCalpha and beta isoenzymes induced translocation of beta1-integrin from the cell-matrix junctions and that beta4-integrin was translocated to face the culture substratum. Go6976 was also highly effective in inhibiting migration of carcinoma cells and inhibited invasion through artificial basement membrane. Our results on urinary bladder carcinoma cells emphasize that Go6976 is a potential anticancer drug due to its effects on cell-cell and cell-matrix junctions, migration, and invasion. Furthermore, the results may be explained by changes in PKC activation balance promoted by inhibition of PKCalpha/beta.