Protector Role of Intraocular Lenses under Artificial Light Conditions.

INTRODUCTION: The aim of this work was to analyze, in an in vitro model, the possible protective effects of ultraviolet- (UV-) or UV/blue-filtering intraocular lens (IOL) under light-emitting diode (LED) lighting conditions. METHODS: Ten models of IOLs were evaluated. Light transmission spectrum was recorded from 300 to 800 nm, in steps of 1 nm. Photodamage in vitro model was induced in ARPE-19 cells by blue LED light (465-475 nm). Changes in cell viability and oxidative stress variables were studied to assess the protective effect of IOLs. RESULTS: UV/blue-filtering IOLs models block blue light spectrum in different proportion and UV-filtering IOLs blocking wavelength below 400 nm. However, in vitro study under blue LED light exposure does not show protective effects related with mitochondrial dysfunction and oxidative stress of UV/blue-filtering IOLs. CONCLUSIONS: The current in vitro study suggests that UV/blue filtering IOLs are not useful in terms of photoprotection in artificial light conditions. The results obtained indicate that it is needed to give attention to other IOL parameters besides the type of filter, as it seems they could have influence on the protective role.

Plasma Rich in Growth Factors Promotes Autophagy in ARPE19 Cells in Response to Oxidative Stress Induced by Blue Light.

Age-related macular degeneration (AMD) causes the degeneration of photoreceptors and retinal cells leading to vision loss in older subjects. Among possible exogenous risk factors, it has been recently proposed that long-term exposure to blue light could aggravate the course of AMD. In the search for therapeutic options, plasma rich in growth factors (PRGF) has been shown to enhance cell antioxidant pathways and protect photoreceptors against the harm produced by blue light, although its mechanism of action remains unknown. One possible mechanism, autophagy, is one of the most conservative cell renewal systems used in eukaryotes to destroy cellular components that have been damaged by some kind of insult. The oxidative stress of exposure to blue light is known to induce cell autophagy. In this study, we examined the combined effects on autophagy of blue light and PRGF in a retinal cell line, ARPE19. In response to treatment with both PRGF and blue light, we detected the modulated expression of autophagy markers such as NF-kB, p62/sqstm1, Atg5, LC3 and Beclin1, and inflammatory markers such as IL1B and IL18. Our findings suggest that PRGF promotes cell autophagy in response to exposure to blue light.

Plasma Rich in Growth Factors Enhances Cell Survival after in Situ Retinal Degeneration.

PURPOSE: The purpose of this study was to examine the effect of plasma rich in growth factors (PRGFs) under blue light conditions in an in vivo model of retinal degeneration. METHODS: Male Wistar rats were exposed to dark/blue light conditions for 9 days. On day 7, right eyes were injected with saline and left eyes with PRGF. Electroretinography (ERG) and intraocular pressure (IoP) measurements were performed before and after the experiment. After sacrifice, retinal samples were collected. Hematoxylin and eosin staining was performed to analyze the structure of retinal sections. Immunofluorescence for brain-specific homeobox/POU domain protein 3A (Brn3a), choline acetyltransferase (ChAT), rhodopsin, heme oxygenase-1 (HO-1), and glial fibrillary acidic protein (GFAP) was performed to study the retinal conditions. RESULTS: Retinal signaling measured by ERG was reduced by blue light and recovered with PRGF; however, IoP measurements did not show significant differences among treatments. Blue light reduced the expression for Brn3a, ChAT, and rhodopsin. Treatment with PRGF showed a recovery in their expressions. HO-1 and GFAP results showed that blue light increased their expression but the use of PRGF reduced the effect of light. CONCLUSIONS: Blue light causes retinal degeneration. PRGF mitigated the injury, restoring the functionality of these cells and maintaining the tissue integrity.

A new aggressive xenograft model of human colon cancer using cancer-associated fibroblasts.

BACKGROUND: Colorectal cancer is the second leading cause of cancer death. Almost half of the patients present recurrence within 5 years after the treatment of the primary tumor, the majority, with metastasis. On the other hand, in the search for new animal models that simulate metastatic cancer, it has been suggested that fibroblasts immersed in the peritumoral stroma (cancer-associated fibroblasts (CAFs)), play a relevant role in the development of cancer. The objective of this study was to identify an adequate animal model to study metastatic colon cancer and the application of new treatments. METHODS: Human CAFs and normal fibroblasts (NF) for transplant and culture were obtained from surgical fresh samples of patients with adenocarcinoma of sigmoid colon. Stromal cell purity was evaluated by morphology and immunostaining with vimentin (VIM) as a fibroblast marker and anti-proColXIα1 as a specific human CAF marker. Phenotypic characterization of cultured stromal cells was performed by co-staining with mesenchymal and epithelial cell markers. For identification in mice, human CAFs were labeled with the PKH26 red fluorescence dye. Cell line HT-29 was used as tumor cells. Transplant in the head of the pancreas of 34 SCID mice was performed in four different groups, as follows: I. 150,000 CAFS (n = 12), IIa. 1.5 million HT29 cells (n = 7), IIb. 150,000 NF+1.5 million HT29 cells (n = 5), III. 150,000 CAFS+1.5 million HT29 cells (n = 10). After euthanasia performed one month later, histological analysis was made using hematoxylin-eosin and anti-proColXIα1. A histopathological score system based on three features (tumor volume, desmoplasia and number of metastasized organs) was established to compare the tumor severity. RESULTS: The CAFs and NF cultured were proColXIα1+/VIM+, proColXIα1/alphaSMA+ and proColXIα1+/CK19+ in different proportions without differences among them, but the CAFs growth curve was significantly larger than that of the NF (p < 0.05). No tumor developed in those animals that only received CAFs. When comparing group II (a + b) vs. group III, both groups showed 100% hepatic metastases. Median hepatic nodules, tumor burden, lung metastases and severity score were bigger in group III vs group II (a + b), although without being significant, except in the case of the median tumor volume, that was significantly higher in group III (154.8 (76.9-563.2) mm3) vs group II (46.7 (3.7-239.6) mm3), p = 0.04. A correlation was observed between the size of the tumor developed in the pancreas and the metastatic tumor burden in the liver and with the severity score. CONCLUSION: Our experiments demonstrate that cultured CAFs have a higher growth than NF and that when human CAFs are associated to human tumor cells, larger tumors with liver and lung metastases are generated than if only colon cancer cells with/without NF are transplanted. This emphasizes the importance of the tumor stroma, and especially the CAFs, in the development of cancer.

Antioxidant Role of PRGF on RPE Cells after Blue Light Insult as a Therapy for Neurodegenerative Diseases.

Oxidative stress has a strong impact on the development of retinal diseases such as age-related macular degeneration (AMD). Plasma rich in growth factors (PRGF) is a novel therapeutic approach in ophthalmological pathologies. The aim of this study was to analyze the antioxidant effect of PRGF in retinal epithelial cells (EPR) in in vitro and ex vivo retinal phototoxicity models. In vitro analyses were performed on ARPE19 human cell line. Viability and mitochondrial status were assessed in order to test the primary effects of PRGF. GSH level, and protein and gene expression of the main antioxidant pathway (Keap1, Nrf2, GCL, HO-1, and NQO1) were also studied. Ex vivo analyses were performed on rat RPE, and HO-1 and Nrf2 gene and protein expression were evaluated. The results show that PRGF reduces light insult by stimulating the cell response against oxidative damage and modulates the antioxidant pathway. We conclude that PRGF's protective effect could prove useful as a new therapy for treating neurodegenerative disorders such as AMD.

Transcriptomic Analysis of a Diabetic Skin-Humanized Mouse Model Dissects Molecular Pathways Underlying the Delayed Wound Healing Response.

Defective healing leading to cutaneous ulcer formation is one of the most feared complications of diabetes due to its consequences on patients' quality of life and on the healthcare system. A more in-depth analysis of the underlying molecular pathophysiology is required to develop effective healing-promoting therapies for those patients. Major architectural and functional differences with human epidermis limit extrapolation of results coming from rodents and other small mammal-healing models. Therefore, the search for reliable humanized models has become mandatory. Previously, we developed a diabetes-induced delayed humanized wound healing model that faithfully recapitulated the major histological features of such skin repair-deficient condition. Herein, we present the results of a transcriptomic and functional enrichment analysis followed by a mechanistic analysis performed in such humanized wound healing model. The deregulation of genes implicated in functions such as angiogenesis, apoptosis, and inflammatory signaling processes were evidenced, confirming published data in diabetic patients that in fact might also underlie some of the histological features previously reported in the delayed skin-humanized healing model. Altogether, these molecular findings support the utility of such preclinical model as a valuable tool to gain insight into the molecular basis of the delayed diabetic healing with potential impact in the translational medicine field.

Human plasma gels: Their preparation and rheological characterization for cell culture applications in tissue engineering.

Tissue engineering is one of the fields of clinical medicine that has forged ahead in recent years, especially because of its role as a potential alternative to organ transplantation. The main aim of this study has been the development of biocompatible materials to form extracellular matrix (ECM) structures in order to provide the necessary conditions for the settlement, proliferation and differentiation of dermal cells such as fibroblasts. To this end, human plasma gels were synthesized with the addition of increasing concentrations of transglutaminase (TGase), which catalyses the formation of covalent bonds between Lys and Glu residues. These materials were structurally characterized using rheology and texturometry and were found to have good structural resistance and elasticity for fibroblast culture. A remarkable improvement in the mechanical properties of the human plasma gels was detected when the two highest TGase concentrations were tested, which may be interpreted as an increase in the number of covalent and non-covalent bonds formed between the plasma protein chains. Furthermore, a human fibroblast primary culture was seeded on human plasma scaffolds and satisfactorily proliferated at 37 °C. This was verified in the images obtained by optical microscopy (OM) and by scanning electron microscopy (SEM), which confirmed that the structure of this type of material is suitable for the growth and proliferation of dermal fibroblasts.

Ectopic bone formation during tissue-engineered cartilage repair using autologous chondrocytes and novel plasma-derived albumin scaffolds.

PURPOSE: The aims of this study were twofold: first, to evaluate the production of cartilaginous tissue in vitro and in vivo using a novel plasma-derived scaffold, and second, to test the repair of experimental defects made on ears of New Zealand rabbits (NZr) using this approach. MATERIALS AND METHODS: Scaffolds were seeded with chondrocytes and cultured in vitro for 3 months to check in vitro cartilage production. To evaluate in vivo cartilage production, a chondrocyte-seeded scaffold was transplanted subcutaneously to a nude mouse. To check in vivo repair, experimental defects made in the ears of five New Zealand rabbits (NZr) were filled with chondrocyte-seeded scaffolds. RESULTS: In vitro culture produced mature chondrocytes with no extracellular matrix (ECM). Histological examination of redifferentiated in vitro cultures showed differentiated chondrocytes adhered to scaffold pores. Subcutaneous transplantation of these constructs to a nude mouse produced cartilage, confirmed by histological study. Experimental cartilage repair in five NZr showed cartilaginous tissue repairing the defects, mixed with calcified areas of bone formation. CONCLUSION: It is possible to produce cartilaginous tissue in vivo and to repair experimental auricular defects by means of chondrocyte cultures and the novel plasma-derived scaffold. Further studies are needed to determine the significance of bone formation in the samples.

Feeder Layer Cell Actions and Applications.

Cultures of growth-arrested feeder cells have been used for years to promote cell proliferation, particularly with low-density inocula. Basically, feeder cells consist in a layer of cells unable to divide, which provides extracellular secretions to help another cell to proliferate. It differs from a coculture system because only one cell type is capable to proliferate. It is known that feeder cells support the growth of target cells by releasing growth factors to the culture media, but this is not the only way that feeder cells promote the growth of target cells. In this work, we discuss the different mechanisms of action of feeder cells, tackling questions as to why for some cell cultures the presence of feeder cell layers is mandatory, while in some other cases, the growth of target cells can be achieved with just a conditioned medium. Different treatments to avoid feeder cells to proliferate are revised, not only the classical treatments as mitomycin or γ-irradiation but also the not so common treatments as electric pulses or chemical fixation. Regenerative medicine has been gaining importance in recent years as a discipline that moves biomedical technology from the laboratory to the patients. In this context, human stem and pluripotent cells play an important role, but the presence of feeder cells is necessary for these progenitor cells to grow and differentiate. This review addresses recent specific applications, including those associated to the growth of embryonic and induced pluripotent stem cells. In addition, we have also dealt with safety issues, including feeder cell sources, as major factors of concern for clinical applications.

Overexpression of COL11A1 by cancer-associated fibroblasts: clinical relevance of a stromal marker in pancreatic cancer.

BACKGROUND: The collagen11A1 (COL11A1) gene is overexpressed in pancreatic cancer. The expression of COL11A1 protein could be involved in desmoplastic events in pancreatic cancer, but an antibody that specifically stains the COL11A1 protein is not currently available. METHODS AND FINDINGS: A total of 54 pancreatic ductal adenocarcinomas (PDAC), 23 chronic pancreatitis (CP) samples, and cultured peritumoral stromal cells of PDAC (passages 3-6) were studied. Normal human pancreas tissue samples were obtained through a cadaveric organ donation program. 1) Validation of COL11A1 gene overexpression by q-RT-PCR. FINDINGS: the expression of COL11A1 gene is significantly increased in PDAC samples vs. normal and CP samples. 2) Analysis of COL11A1 by immunohistochemistry using highly specific anti-proCOL11A1 antibodies. FINDINGS: anti-proCOL11A1 stains stromal cells/cancer-associated fibroblasts (CAFs) of PDAC but it does not stain chronic benign condition (chronic pancreatitis) stromal cells, epithelial cells, or normal fibroblasts. 3) Evaluation of the discrimination ability of the antibody. FINDINGS: anti-proCOL11A1 immunostaining accurately discriminates between PDAC and CP (AUC 0.936, 95% CI 0.851, 0.981). 4) Phenotypic characterization of proCOL11A1+ stromal cells co-staining with mesenchymal, epithelial and stellate cell markers on pancreatic tissue samples and cultured peritumoral pancreatic cancer stromal cells. FINDINGS: ProCOL11A1+ cells present co-staining with mesenchymal, stellate and epithelial markers (EMT phenotype) in different proportions. CONCLUSIONS/SIGNIFICANCE: Detection of proCOL11A1 through immunostaining with this newly-developed antibody allows for a highly accurate distinction between PDAC and CP. Unlike other available antibodies commonly used to detect CAFs, anti-proCOL11A1 is negative in stromal cells of the normal pancreas and almost absent in benign inflammation. These results strongly suggest that proCOL11A1 is a specific marker for CAFs, and thus, anti-proCOL11A1 is a powerful new tool for cancer research and clinical diagnostics.

A functional polymorphism in MMP1 could influence osteomyelitis development.

Osteomyelitis (OM) is a bone infection characterized by necrosis and new formation of bone. Because matrix metalloproteases (MMPs) play an important role in bone extracellular matrix remodeling, we investigated the role of some MMP polymorphisms in OM patients. A total of 118 OM patients and 300 blood donors were genotyped for the polymorphisms of MMP1 (-1607 1G/2G) and MMP13 (-77A/G). Levels of MMPs (-1, -2, -3, -8, -9, -10, and -13) and tissue inhibitors of metaloproteases (TIMP-1, -2, and -4) in serum and in human osteoblasts obtained from OM biopsies also were determined. The MMP1 (-1607 2G/2G) genotype was significantly more frequent among OM patients compared with controls [65.3% versus 33.7%, chi(2) = 26.85, odds ratio (OR) = 3.24, 95% confidence interval (CI) 2.03-5.2, p < .0001]. The MMP1 2G allele also was more frequent in OM patients (73.3% versus 57.2%, chi(2) = 37.76, OR = 2.75, 95% CI 1.96-3.85, p < .0001). Carriers of the 2G allele had significantly higher osteoblast MMP1 mRNA and MMP-1 serum levels than noncarriers (p < .04). Interleukin 1alpha (IL-1alpha) increased MMP-1 and -13 protein secretion and Ets1 mRNA expression by OM patients' osteoblasts. No association of the MMP13 (-77 A/G) polymorphism with OM was observed. The MMP1 (-1607 1G/2G) polymorphism might contribute to OM pathogenesis. This could be due to increased expression of MMP-1 by osteoblasts and is regulated by IL-1alpha.

Plasma-fibroblast gel as scaffold for islet transplantation.

The transplant of pancreatic islets into the liver can restore normal blood glucose levels in patients with type I diabetes. However, long-term results have indicated that the site and method of transplantation still need to be optimized to improve islet engraftment. This study was designed to assess the efficiency of the use of clotted blood plasma containing fibroblasts ("plasma-fibroblast gel") as a scaffold for subcutaneous islet transplantation in diabetic athymic mice. Islets embedded in the plasma-fibroblast gel were able to resolve hyperglycemia in transplanted mice, restoring normoglycemia over a 60-day period and allowing gradual body weight recovery. Glucose clearances were significantly improved when compared to those recorded in diabetic animals and similar to those observed in the control group (free islets transplanted beneath the kidney capsule). Histological evaluation revealed functional islets within a subcutaneous tissue rich in collagen fibers that was well vascularized, with blood vessels observed around and inside the islets. These findings suggest that this approach could be used as an alternative option for the treatment of type I diabetes in human clinical practice.

Local loperamide inhibits thermal hyperalgesia but not mechanical allodynia induced by intratibial inoculation of melanoma cells in mice.

The stimulation of peripheral opioid receptors counteracts thermal hyperalgesia produced by the intratibial inoculation of NCTC 2472 cells in mice, through the activation of the nitric oxide/cGMP/ATP-sensitive K+-channels (NO/cGMP/K(+) (ATP)) cascade (Menéndez et al. 2007, Neuropharmacology 53:71-80). We aimed to elucidate whether this peripheral opioid antihyperalgesic effect is exclusive to this model or might also occur in other types of bone neoplastic processes. In C57BL/6 mice intratibially inoculated with B16-F10 melanoma cells, the progressive tumoral damage was accompanied by the establishment of thermal hyperalgesia (unilateral hot plate test) and mechanical allodynia (von Frey test). Intraplantar administration of loperamide (15 microg, 30 min before) inhibited thermal hyperalgesia, but did not modify the intense mechanical allodynia. The fact that the coadministration of naloxone-methiodide (5 microg) completely suppressed the thermal antihyperalgesic effect induced by loperamide indicates its production through the stimulation of peripheral opioid receptors. Furthermore, its prevention by the coadministration of the non-selective inhibitor of the NO synthase, N(G)-monomethyl-L-arginine (L-NMMA, 10 microg), the selective inhibitor of neural NOS, N-omega-propyl-L-arginine (1-10 microg), or the K+ (ATP) channel blocker, glibenclamide (10 microg) demonstrated the involvement of the NO/cGMP/K(+) (ATP) pathway in the antihyperalgesic effect induced by loperamide. Overall, the present results show that the intratibial inoculation of B16-F10 cells to C57BL/6 mice evokes thermal hyperalgesia and mechanical allodynia and that, as occurred in the osteosarcoma model, the stimulation of peripheral opioid receptors is not effective in modifying neoplastic allodynia but completely inhibits thermal hyperalgesia through the activation of the NO/cGMP/K+ (ATP) cascade.