Ilkka Immonen

Crosstalk between Hsp70 molecular chaperone, lysosomes and proteasomes in autophagy‐mediated proteolysis in human retinal pigment epithelial cells

The pathogenesis of age‐related macular degeneration involves chronic oxidative stress, impaired degradation of membranous discs shed from photoreceptor outer segments and accumulation of lysosomal lipofuscin in retinal pigment epithelial (RPE) cells. It has been estimated that a major part of cellular proteolysis occurs in proteasomes, but the importance of proteasomes and the other proteolytic pathways including autophagy in RPE cells is poorly understood. Prior to proteolysis, heat shock proteins (Hsps), agents that function as molecular chaperones, attempt to refold misfolded proteins and thus prevent the accumulation of cytoplasmic protein aggregates. In the present study, the roles of the Hsp70 molecular chaperone and proteasomal and lysosomal proteolytic pathways were evaluated in human RPE cells (ARPE‐19). The Hsp70 and ubiquitin protein levels and localization were analysed by Western blotting and immunofluorescense. Confocal and transmission electron microscopy were used to detect cellular organelles and to evaluate the morphological changes. Hsp70 levels were modulated using RNA interference and overexpression techniques. Cell viability was measured by colorimetric assay. The proteasome inhibitor MG‐132 evoked the accumulation of perinuclear aggregates positive for Hsp70, ubiquitin‐protein conjugates and the lysosomal membrane protein LAMP‐2. Interestingly, the hsp70 mRNA depletion significantly increased cell death in conjunction with proteasome inhibition. We found that the accumulation of lysosomes was reversible: a cessation of proteasome inhibition led to clearance of the deposits via a mechanism believed to include autophagy. The molecular chaperone Hsp70, proteasomes and autophagy have an important regulatory role in the protein turnover of human RPE cells and may thus open new avenues for understanding degenerative processes in retinal cells.

Radiation related complications after ruthenium plaque radiotherapy of uveal melanoma.

AIMS/BACKGROUND: To analyse radiation related complications and secondary enucleation after irradiation of malignant uveal melanoma with ruthenium-106 plaques. METHODS: A series of 100 consecutive eyes irradiated in 1981-91 was analysed using the life table method and the Cox proportional hazards model. The median apical and scleral tumour dose was 100 Gy (range 15-200 Gy) and 1000 Gy (range 200-1200 Gy), respectively. The median follow up time was 2.8 and 2.0 years (range 1 month to 10 years) for anterior and posterior segment complications, respectively. RESULTS: The 3 and 5 year probabilities of being without radiation cataract were 73% and 63%, without neovascular glaucoma 91% and 81%, without vitreous haemorrhage 83% and 74%, without radiation maculopathy 85% and 70%, and without radiation optic neuropathy 90% and 88%, respectively. The risk of radiation cataract was highest with large tumour size (T1 + T2 v T3, p = 0.0027; height < or = 5 v > 5 mm, p = 0.029; largest basal diameter (LBD) < or = 15 v > 15 mm, p < 0.0001) and location of anterior tumour margin anterior v posterior to the equator (p = 0.0003); the risk of neovascular glaucoma with large size (T1 + T2 v T3, p = 0.039; LBD < or = 15 mm v 15 mm, p = 0.021); and the risk of maculopathy and optic neuropathy with proximity of the posterior tumour margin to the fovea and the optic disc (< or = 1.5 v > 1.5 mm; p = 0.030 and p = 0.0004, respectively). In Coxs multivariate analysis the strongest risk indicator for radiation cataract (RR 1.5, 95% CI 1.4-1.6) and vitreous haemorrhage (RR 1.6, 95% CI 1.4-1.8) was the height of the tumour; for neovascular glaucoma the TNM class (RR 6.2, 95% CI 2.7-13.8); for radiation maculopathy location of posterior tumour margin within 2 mm from the fovea (RR 3.4, 95% CI 2.0-6.0); and for radiation optic neuropathy location of tumour margin within 1 DD of the optic disc (RR 6.1, 95% CI 3.0-12.4). The 3 and 5 year probabilities of avoiding enucleation were 92% and 85%, respectively. Ten eyes were enucleated--six because of recurrent tumour growth, three because of treatment complications, and one because of mistakenly suspected extraocular growth. CONCLUSION: The results suggest that the frequency of radiation related complications after ruthenium brachytherapy of uveal melanoma is acceptable, in particular as regard irradiation of small and medium sized tumours for which ruthenium therapy generally is recommended.

The Schlemm’s canal is a VEGF-C/VEGFR-3–responsive lymphatic-like vessel

In glaucoma, aqueous outflow into the Schlemms canal (SC) is obstructed. Despite striking structural and functional similarities with the lymphatic vascular system, it is unknown whether the SC is a blood or lymphatic vessel. Here, we demonstrated the expression of lymphatic endothelial cell markers by the SC in murine and zebrafish models as well as in human eye tissue. The initial stages of SC development involved induction of the transcription factor PROX1 and the lymphangiogenic receptor tyrosine kinase VEGFR-3 in venous endothelial cells in postnatal mice. Using gene deletion and function-blocking antibodies in mice, we determined that the lymphangiogenic growth factor VEGF-C and its receptor, VEGFR-3, are essential for SC development. Delivery of VEGF-C into the adult eye resulted in sprouting, proliferation, and growth of SC endothelial cells, whereas VEGF-A obliterated the aqueous outflow system. Furthermore, a single injection of recombinant VEGF-C induced SC growth and was associated with trend toward a sustained decrease in intraocular pressure in adult mice. These results reveal the evolutionary conservation of the lymphatic-like phenotype of the SC, implicate VEGF-C and VEGFR-3 as critical regulators of SC lymphangiogenesis, and provide a basis for further studies on therapeutic manipulation of the SC with VEGF-C in glaucoma treatment.

Photodynamic Ablation of Lymphatic Vessels and Intralymphatic Cancer Cells Prevents Metastasis

Destruction of lymphatic vessels by photodynamic therapy suppresses metastasis by eradicating both the in-transit tumor cells and their conduit to distant tissues. Shining a Light on Tumor Metastasis Tumor cells have several routes that enable them to move from the primary tumor to distant tissues, a process called metastasis. It is metastasis of the primary tumor that kills most cancer patients. One of the least studied routes of metastasis is the lymphatic system. Many tumors produce vascular endothelial growth factor–C (VEGF-C) or VEGF-D, factors that promote the formation of new lymphatic vessels (lymphangiogenesis). The newly formed lymphatic vessels enable tumor cells to travel from the primary tumor to the regional lymph nodes from whence they can spread throughout the body. In a new study, Tammela and colleagues investigate whether eradicating tumor-associated lymphatic vessels and the tumor cells they contain using photodynamic therapy (PDT) with a light-activated cytotoxic compound could reduce or eliminate tumor metastasis. They selected the mouse ear as their model system because it is easy to image both the lymphatic vessels and the in-transit tumor cells. The mouse ear is also amenable to PDT because it is very thin, thus enabling sufficient tissue penetration by the infrared light that activates the cytotoxic compound (verteporfin). The authors implanted mouse melanoma cells or human lung tumor cells into the mouse ear tip and waited 2 weeks for the primary tumors to become established and to induce lymphangiogenesis. Using immunohistochemistry, they observed that the newly formed lymphatic vessels contained in-transit tumor cells as well as small tumor nodules. Using pathology sections taken from a patient with recurrent melanoma, they confirmed that the tumor-associated lymphatic vessels of cancer patients contain in-transit tumor cells and tumor nodules. But could PDT eliminate both tumor-associated lymphatic vessels and the tumor cells inside them? The authors injected verteporfin in a liposomal preparation intradermally into the mouse ear and showed that this cytotoxic dye accumulated specifically in the lymphatic vessels. When they illuminated the mouse ear with infrared laser light, the lymphatic vessels started to shrink and fragment, and became leaky. Could this treatment prevent relapse when combined with surgery? The authors implanted mouse melanoma cells into the flanks of mice and waited 2 weeks until the tumor cells had become established and had metastasized via the lymphatic vessels to the axillary lymph nodes. They then injected verteporfin into the primary tumor and surrounding tissue and illuminated the whole flank with infrared laser light before surgically removing the primary tumor and lymph nodes. They found that the mice receiving PDT and surgery had a much lower relapse rate than those that underwent surgery alone. But will PDT be useful for eradicating tumor-associated lymphatic vessels deep within the tissues of human cancer patients? Using a pig model, Tammela and colleagues demonstrated that this should be feasible. They injected verteporfin into pig hoofs and then used a special laser catheter inserted at the knee to activate the dye. They showed that the lymphatic system ceased to drain as the vessels fragmented and became clogged, indicating that PDT can target lymphatic vessels deep within the body. PDT using verteporfin is currently used clinically to destroy overgrown blood vessels in the retina of patients with macular degeneration. So it should certainly be feasible to use PDT to destroy tumor-associated lymphatic vessels in cancer patients undergoing surgery. Shining a light deep within the body should ensure that an oft-overlooked conduit for tumor metastasis will remain illuminated. The dissemination of tumor cells to sites far from the primary tumor (metastasis) is the principal cause of death in cancer patients. Tumor-associated lymphatic vessels are a key conduit for metastatic tumor cells, which typically first colonize the lymph nodes. Although the primary tumor and affected lymph nodes can be removed during surgery, tumor cells inside lymphatic vessels are left behind. Here, we show that in-transit tumor cells inside lymphatic vessels in mice bearing mouse melanomas or human lung tumors give rise to metastases. Using photodynamic therapy with the benzoporphyrin derivative verteporfin, we selectively destroyed lymphatic vessels in mice and pigs. Destruction of tumor-associated lymphatic vessels also eradicated intralymphatic tumor cells and prevented metastasis of mouse melanoma cells and subsequent relapse. Photodynamic therapy, when combined with anti-lymphangiogenic therapy, prevented further tumor invasion of lymphatic vessels. These findings highlight the potential of targeting in-transit tumor cells in patients.

Visual function index (VF-14) in exudative age-related macular degeneration of long duration

PURPOSE To evaluate the Visual Function Index (VF-14) questionnaire for its effectiveness in assessing visual function in patients with longstanding exudative age-related macular degeneration (AMD). DESIGN Observational case series. METHODS The records of 167 consecutive patients with recent neovascularization related to AMD between June 1990 and December 1994 at the Helsinki University Eye Clinic were analyzed in 1999. Of 121 patients still living, 74 (61%) attended the reexamination. After exclusions, data from 62 patients were analyzed. The VF-14 score, plus global assessment scores of satisfaction with vision and quality of vision, in which patients graded the subjective level of difficulty with their vision, best-corrected visual acuity (BCVA), contrast sensitivity, the area of the AMD lesion, and the shortest distance and direction from the center of the fovea to the edge of the subfoveal lesion, were analyzed. RESULTS The VF-14 score correlated significantly with BCVA (P <.01), contrast sensitivity (P <.01), and global assessment scores (P <.01), showing stronger correlations with global assessment scores than did BCVA. In multivariate regression analysis, the global assessment scale of overall quality of vision and BCVA in the better eye were significant predictors (P <.001) of the variability in the VF-14 score. CONCLUSIONS The VF-14 reflects visual function of patients with late AMD more effectively than BCVA measurement alone. The VF-14 can thus be used to compare the visual handicap of late AMD patients with that of patients with other eye diseases.

Dislocated nuclear fragments after cataract surgery

Posterior nuclear dislocation is a serious complication of cataract surgery, especially when using the phacoemulsification technique. So far, there have been only a few reports concerning the indications and timing of vitrectomy with nuclear removal as well as long-term visual outcome of these eyes. We analysed 23 consecutive patients (follow-up at least 3 months) with intravitreal nuclear remnants after cataract surgery treated with vitrectomy and removal of posteriorly dislocated nuclei. All the eyes had increased intraocular pressure pre-vitrectomy, 63% had corneal oedema, 67% marked uveitis and 26% either retinal tear or detachment. Vitrectomy was performed within 1 week in 70% of eyes. In 14 eyes (61%) the final visual acuity was 20/40 or more. The main reason for poor visual outcome was retinal detachment (9%). These results indicate that with vitrectomy and removal of the nucleus good visual results can be achieved in a large proportion of eyes with posterior dislocation of nuclear remnants.

Elevation of tear fluid plasmin in corneal disease

Abstract. Plasmin concentration was determined in tear fluid from 76 eyes showing corneal epithelial disorders, such as corneal ulcers and erosions due to trauma or contact lens wear. Nearly 70% of the eyes with corneal disease had plasmin in their tear fluid, whereas plasmin was present in only 20% of the eyes in a control group of 50 eyes. Re‐examination of the plasmin positive control eyes revealed conjunctival bacterial growth or mild subclinical viral infection in most cases. We conclude that plasmin is released into the tear fluid in the presence of corneal or conjunctival lesions or infections, suggesting a pathogenic role of plasmin in these disorders. The significance of the occurrence of plasmin in tear fluid during corneal wound healing is discussed.

Ang-2 upregulation correlates with increased levels of MMP-9, VEGF, EPO and TGFβ1 in diabetic eyes undergoing vitrectomy.

Purpose:  Angiogenesis in diabetic retinopathy (DR) is a multifactorial process regulated by hypoxia‐induced growth factors and inflammatory cytokines. In addition to the angiogenic switch, the proteolytic processing and altered synthesis of the extracellular matrix are critical steps in this disease. This study was performed to evaluate the levels of matrix metalloproteinase‐2 and matrix metalloproteinase‐9 (MMP‐2 and MMP‐9), angiopoietin‐1 and angiopoietin‐2 (Ang‐1 and Ang‐2), vascular endothelial growth factor (VEGF), erythropoietin (EPO) and transforming growth factor‐β1 (totalTGFβ1) in the vitreous of diabetic eyes undergoing vitrectomy compared with control eyes operated because of macular hole or pucker.

Vascular Endothelial Growth Factor Gene Variation and the Response to Photodynamic Therapy in Age-Related Macular Degeneration

PURPOSE To evaluate the role of vascular endothelial growth factor (VEGF) gene polymorphisms in exudative age-related macular degeneration (AMD). DESIGN Retrospective, comparative case series. PARTICIPANTS Patients with recent exudative AMD (n = 162) and age-matched subjects without AMD (n = 85). METHODS Fluorescein angiography (FA), clinical examination, and single nucleotide polymorphism (SNP) genotyping. MAIN OUTCOME MEASURES The frequencies of 3 VEGF gene SNPs were analyzed, 1 at the promoter site (rs699947, A-->C) and 2 intronic SNPs (rs2146323, A-->C, and rs3025033, A-->G), in relation to the risk of AMD, to choroidal neovascular (CNV) lesion size and configuration, and to the anatomic response to photodynamic therapy (PDT). These SNPs were chosen to cover all the haploblocks of the VEGF gene. The 86 patients who had undergone PDT were classified as either PDT responders or PDT nonresponders based on the outcome of PDT after the last treatment session. For the PDT responders, the treating physician had deemed the lesion to be clinically dry and without leakage from CNV in FA at a visit scheduled at least 12 weeks after the last PDT treatment. For the PDT nonresponders, the PDT sessions had been discontinued by the treating retina specialist because of an apparently poor response and a still exudative lesion after several PDT sessions. RESULTS The presence of exudative AMD or lesion size or configuration was not associated with the SNPs studied here. The frequencies of the rs699947 were significantly different in PDT nonresponders and PDT responders. The AA, AC, and CC genotypes were 14%, 39%, and 46%, respectively, in PDT nonresponders, compared with 40%, 48%, and 12%, respectively, in the PDT responders (P = 0.0008). The corresponding frequencies for the rs2146323 AA, AC, and CC genotypes were 4%, 32%, and 64%, respectively, in nonresponders and 24%, 38%, and 38%, respectively, in responders (P = 0.0369). The genotypes of the rs3025033 SNP were distributed evenly between the responders and nonresponders. CONCLUSIONS The VEGF gene polymorphic SNPs at rs699947 and rs2146323 are strong determinants of the anatomic outcome after PDT, but the SNPs studied were not associated with the presence of exudative AMD or with the CNV lesion size or configuration. FINANCIAL DISCLOSURE(S) The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Strontium plaque irradiation of subfoveal neovascular membranes in age-related macular degeneration

Abstract• Purpose: To determine the effect of focal strontium-90 plaque radiotherapy on subfoveal choroidal neovascularization in patients with age-related macular degeneration. • Methods: Twenty patients with age-related macular degeneration, presenting with a recent subfoveal neovascular membrane, were treated with local episcleral radiotherapy using a strontium-90 applicator. The applicator was applied to the sciera under the macular region and held there tightly for 54 min to obtain a dose of 15 Gy at a depth of 1.75 mm. The main parameters evaluated at follow-up after 3, 6 and 12 months were visual acuity and changes in fluorescein angiography. Twelve untreated patients with the same criteria were followed as controls. • Results: Early effects of radiation could be seen at 3 months, but became more obvious after follow-up for 6 and 12 months. At 6 months, regression of the choroidal neovascularization was detected in 14/19 patients (74%) as a decrease in the size of the lesion or as diminished leakage in late-phase fluorescein angiography. Likewise, in 14/19 patients (74%) at 12 months the neovascular membrane was partially or totally occluded. In all patients showing regression of the choroidal neovascular membrane, the neurosensory detachment had also dried. Visual acuity was unchanged (within 1 line) or improved in 11/20 (55%) and 9/20 (45%) treated patients after 6 and 12 months, respectively. In the controls, the choroidal neovascularization had increased in size in 9/12 patients (75%) at the last follow-up (mean 12.0 months). Visual acuity was unchanged in 3/12 patients (25%). • Conclusion: Local low-energy beta irradiation with a strontium-90 applicator can induce regression of choroidal neovascularization. The effect of irradiation is seen as a decrease in the size of the choroidal neovascular membrane and disappearance of the neurosensory detachment and exudates.