Activation of p38 and Erk Mitogen-Activated Protein Kinases Signaling in Ocular Rosacea.

Purpose: Rosacea-related cutaneous inflammation is a common cause of ocular surface disease. Currently, there are no specific pharmacologic therapies to treat ocular rosacea. Here, we aimed at determining the differences in intracellular signaling activity in eyelid skin from patients with and without ocular rosacea. Methods: This was an observational, comparative case series including 21 patients undergoing lower lid ectropion surgery at one practice during 2013 and 2014 (18 patients with rosacea, 13 control patients), and 24 paraffin-embedded archival samples from Albany Medical Center, selected randomly (12 patients with rosacea, 12 control patients). Cutaneous biopsies resulting from elective lower lid ectropion surgery were analyzed by Proteome Profiler Human Phospho-Kinase Array, Western blot, and/or immunohistochemistry. Results: Samples derived from ocular rosacea patients showed increased levels of phosphorylated (active) p38 and Erk kinases. Phosphoproteins were mainly localized to the epidermis of affected eyelids. Conclusions: This finding provides a novel potential therapeutic target for treatment of ocular rosacea and possibly other forms of rosacea. Further testing is required to determine if p38 and Erk activation have a causal role in ocular rosacea. The selective activation of keratinocytes in the affected skin suggests that topical pathway inhibition may be an effective treatment that will ultimately prevent ocular surface damage due to ocular rosacea.

Expression of heparanase in basal cell carcinoma and squamous cell carcinoma.

BACKGROUND:: Heparanase is an enzyme that cleaves heparan sulfate chains. Oligosaccharides generated by heparanase induce tumor progression. Basal cell carcinoma and squamous cell carcinoma comprise types of nonmelanoma skin cancer. OBJECTIVES:: Evaluate the glycosaminoglycans profile and expression of heparanase in two human cell lines established in culture, immortalized skin keratinocyte (HaCaT) and squamous cell carcinoma (A431) and also investigate the expression of heparanase in basal cell carcinoma, squamous cell carcinoma and eyelid skin of individuals not affected by the disease (control). METHODS:: Glycosaminoglycans were quantified by electrophoresis and indirect ELISA method. The heparanase expression was analyzed by quantitative RT-PCR (qRTPCR). RESULTS:: The A431 strain showed significant increase in the sulfated glycosaminoglycans, increased heparanase expression and decreased hyaluronic acid, comparing to the HaCaT lineage. The mRNA expression of heparanase was significantly higher in Basal cell carcinoma and squamous cell carcinoma compared with control skin samples. It was also observed increased heparanase expression in squamous cell carcinoma compared to the Basal cell carcinoma. CONCLUSION:: The glycosaminoglycans profile, as well as heparanase expression are different between HaCaT and A431 cell lines. The increased expression of heparanase in Basal cell carcinoma and squamous cell carcinoma suggests that this enzyme could be a marker for the diagnosis of such types of non-melanoma cancers, and may be useful as a target molecule for future alternative treatment.

Expression of heparanase in basal cell carcinoma and squamous cell carcinoma

Abstract: Background: Heparanase is an enzyme that cleaves heparan sulfate chains. Oligosaccharides generated by heparanase induce tumor progression. Basal cell carcinoma and squamous cell carcinoma comprise types of nonmelanoma skin cancer. Objectives: Evaluate the glycosaminoglycans profile and expression of heparanase in two human cell lines established in culture, immortalized skin keratinocyte (HaCaT) and squamous cell carcinoma (A431) and also investigate the expression of heparanase in basal cell carcinoma, squamous cell carcinoma and eyelid skin of individuals not affected by the disease (control). Methods: Glycosaminoglycans were quantified by electrophoresis and indirect ELISA method. The heparanase expression was analyzed by quantitative RT-PCR (qRTPCR). Results: The A431 strain showed significant increase in the sulfated glycosaminoglycans, increased heparanase expression and decreased hyaluronic acid, comparing to the HaCaT lineage. The mRNA expression of heparanase was significantly higher in Basal cell carcinoma and squamous cell carcinoma compared with control skin samples. It was also observed increased heparanase expression in squamous cell carcinoma compared to the Basal cell carcinoma. Conclusion: The glycosaminoglycans profile, as well as heparanase expression are different between HaCaT and A431 cell lines. The increased expression of heparanase in Basal cell carcinoma and squamous cell carcinoma suggests that this enzyme could be a marker for the diagnosis of such types of non-melanoma cancers, and may be useful as a target molecule for future alternative treatment.

Spry1 and Spry2 are necessary for eyelid closure.

Sproutys (Sprys) are downstream targets and negative feedback regulators of the FGF-Ras-ERK signaling pathway. Our previous studies have shown that Spry1 and Spry2, through negative modulation of FGF-ERK signaling, allow lens vesicle separation from the overlying ectoderm and regulate corneal epithelial proliferation. Here we show that Spry1 and Spry2 are necessary for eyelid closure. Murine palpebral conjunctival epithelial cells that differentiate as inner eyelids and adjacent mesenchymal cells express Spry1 and Spry2 prior to eyelid closure. Conditional deletion of both Spry1 and Spry2, but not either one alone, in the ocular surface epithelial cells result in the "EOB" (eyes open at birth) phenotype suggesting redundant roles for these proteins during eyelid closure. Spry mutant eyelids show increased proliferation of conjunctival epithelial cells with concomitant induction of FGF targets, Erm, Pea3 and Dusp6 and elevated ERK phosphorylation. Peridermal cells at the leading edge of Spry-mutant eyelids showed reduced c-Jun, but not ERK, phosphorylation, reduced F-actin polymerization and reduced motility in vitro. Spry mutant eyelids also showed disruptions in epithelial mesenchymal interactions reflected in the enhanced mesenchymal Spry1 and Spry4 expression, disaggregation of BMP4-positive mesenchymal cells and loss of Shh in the eyelid epithelium. Spry mutant eyelids also showed increased Wnt signaling and reduced expression of Foxc1 and Foxc2, two transcription factors previously shown to be necessary for eyelid closure. Collectively, our results show that conjunctival epithelial Spry1 and Spry2 redundantly promote eyelid closure by (a) stimulating ERK-independent, c-Jun-mediated peridermal migration, (b) suppressing conjunctival epithelial proliferation through FGF-ERK signaling, (c) mediating conjunctival epithelial-mesenchymal interactions and (d) maintaining expression of Foxc1 and Foxc2.

Differential transmission of MEKK1 morphogenetic signals by JNK1 and JNK2.

JNK1 and JNK2 are two ubiquitously expressed isoforms that exert redundant roles in many physiological processes, but the extent of their relative contributions to these processes has not been well characterized. We show that both JNK isoforms transmit MEK kinase 1 (MEKK1)-mediated morphogenetic signals during mouse embryonic eyelid closure. However, JNK1 and JNK2 are not synonymous, because MEKK1 is haploinsufficient for normal eyelid closure in Jnk1-null mice, but is haplosufficient in Jnk2-null mice. In the Mekk1 heterozygous background, a more efficient phosphorylation of JNK1 than JNK2 leads to differential downstream reactions, such as c-Jun phosphorylation and PAI1 expression in the developing eyelid epithelium. Differences in efficiency of phosphorylation are attributed to JNK1 Gly177 and Ser179 -- residues that are absent in JNK2 -- which promote a less ordered structural conformation. This leads to more favorable JNK phosphorylation by activin B morphogenetic signals mediated by the MEKK1-MKK4 pathway. Interestingly, Mekk1-Jnk1-Jnk2 triple hemizygotes display a partial eye-open phenotype at birth, suggesting that all three genes dose-dependently contribute to morphogenetic eyelid closure. We propose that a MEKK1-JNK1/2 axis governs the JNK activation levels to control downstream transcriptional events and eyelid morphogenesis and that reduction of upstream MEKK1 signals uncovers analogous but differential roles of JNK1 and JNK2 in a biological process.

Doxorubicin chemomyectomy is enhanced when performed two days following bupivacaine injections: the effect coincides with the peak of muscle satellite cell division.

PURPOSE: Doxorubicin is effective in permanently removing muscle after direct injection into the eyelid for treatment of blepharospasm and hemifacial spasm. However, patients often require two or more injection series before full abatement of their spasms is achieved. Local anesthetics cause muscle necrosis, followed by regeneration, a process that requires activation and division of muscle satellite cells. This study examined whether the muscle toxicity of doxorubicin could be amplified by injection of doxorubicin into the eyelid of rabbits 2 days after a local anesthetic injury, perhaps exploiting the toxic effects of doxoribicin on satellite cells at the peak time of their division after injury. METHODS: Rabbit eyelids received two series of injections of bupivacaine and hyaluronidase spaced 18 hours apart. Two days later, the eyelids were injected with either 0.5 or 1 mg doxorubicin. Animals were monitored daily for onset and duration of skin injury. After 1 month, the eyelids were assessed for muscle loss using histologic and morphometric techniques. RESULTS: Injection of doxorubicin during the peak of satellite cell activation and division 2 days after injury significantly increased muscle loss over doxorubicin alone. This treatment did not result in increased skin injury compared with doxorubicin alone. CONCLUSIONS: Permanent muscle loss was increased when doxorubicin was injected at the peak of satellite cell division 2 days after injury of the muscle with bupivacaine in rabbit eyelid, taking advantage of the antimitotic effects of doxorubicin on satellite cell division during the period of active regeneration. When local anesthetic injection immediately preceded the doxorubicin injection, increased myotoxicity was not seen. The injection of doxorubicin into muscle 2 days after a previous injury maximizes muscle loss. The increased muscle loss provided by this double treatment may decrease the number of injection visits required by blepharospasm and hemifacial spasm patients during their course of treatment, thus reducing the number of patients with side effects, which increases with repeated exposures of the eyelid to doxorubicin.

Sensory and autonomic innervation of the rat eyelid: neuronal origins and peptide phenotypes.

Neuronal origins, peptide phenotypes and target distributions were determined for sensory and autonomic nerves projecting to the eyelid. The retrograde tracer, Fluoro-Ruby, was injected into the superior tarsal muscle and meibomian gland of Sprague-Dawley rats. Labelled neurons were observed within the pterygopalatine (31 +/- 6 of a total of 8238 +/- 1610 ganglion neurons), trigeminal (173 +/- 43 of 62,082 +/- 5869) and superior cervical ganglia (184 +/- 35 of 21,900 +/- 1741). Immunostaining revealed vasoactive intestinal polypeptide immunoreactivity (VIP-ir) in nearly all Fluoro-Ruby-labelled pterygopalatine ganglion neurons (86 +/- 5%) but only rarely in trigeminal (0.3 +/- 0.3%) or superior cervical (1.4 +/- 1.4%) ganglion neurons. Calcitonin gene-related peptide (CGRP)-ir was not observed in pterygopalatine or superior cervical ganglion somata, but was present in 24 +/- 4% of trigeminal neurons. Bright dopamine beta-hydroxylase (DBH) immunofluorescence was observed in the majority of eyelid-projecting neurons within the superior cervical ganglia (65 +/- 5%) and lighter staining was detected in pterygopalatine neurons (63 +/- 3%), but no DBH-ir was observed in trigeminal neurons. Examination of eyelid sections revealed dense VIP-ir innervation of meibomian gland acini and vasculature and modest distribution within tarsal muscle. CGRP-ir fibers surrounded ductal and vascular elements of the meibomian gland and the perimeter of tarsal muscle. DBH-ir fibers were associated with meibomian gland blood vessels and acini, and were more densely distributed within tarsal muscle. This study provides evidence for prominent meibomian gland innervation by parasympathetic pterygopalatine ganglion VIP-ir neurons, with more restricted innervation by sensory trigeminal CGRP-ir and sympathetic neurons. Tarsal muscle receives abundant sympathetic innervation, as well as moderate parasympathetic and sensory CGRP-ir projections. The eyelid contains substantial non-CGRP-ir sensory innervation, the targets of which remain undetermined. The distribution of identified autonomic and sensory fibers is consistent with the idea that meibomian gland function, as well as that of the tarsal muscle, is regulated by peripheral innervation.

Orbicularis oculi muscle in children. Histologic and histochemical characteristics.

This is the first study devoted to the histologic and histochemical characteristics of the orbicularis oculi muscle in children to the authors' knowledge. The orbicularis muscle was compared with extraocular, facial, and limb striated muscle. Light microscopy showed the orbicularis oculi muscle to be much smaller and more loosely packed than skeletal limb muscles. It further showed these muscle fibers to have greater variation in fiber size and shape and more endomysial and perimysial connective tissue. Finally, analysis of the histochemical reactions showed the orbicularis oculi had a higher percentage of fast-contracting fibers (Type II). This study establishes the histologic and histochemical standard characteristics for the orbicularis oculi muscle in children. It was found that orbicularis oculi muscles have some histologic and histochemical features in common with other facial muscles and other features in common with extraocular muscles.

Fatty acid chain elongation by microsomal enzymes from the bovine meibomian gland.

The composition of meibomian gland lipids suggested that fatty acid chain elongation might play a major role in the synthesis of such lipids. A fatty acid synthase preparation from the bovine meibomian gland catalyzed the formation of C16 acid and the enzyme was immunologically quite similar to that in the mammary gland. The microsomal fraction from the gland, on the other hand, catalyzed elongation of endogenous fatty acids in the presence of ATP and Mg2+ and of exogenous C18-CoA using malonyl-CoA and NADPH as the preferred reductant. The elongated products, ranging up to C28 in chain length, were found mainly as CoA esters and products derived from them. With C18-CoA as the exogenous primer, the elongation rate was linear with incubation time up to 20 min but the rate changed in a sigmoidal manner with increasing protein concentration. The elongation rate was maximal at a pH around 7.0. Typical Michaelis-Menten-type substrate saturation patterns were observed with both malonyl-CoA and NADPH. From linear double-reciprocal plots, the Km values for the two substrates were calculated to be 52 and 11 microM, respectively, with a V of about 340 pmol min-1 mg protein-1 with respect to malonyl-CoA. Exogenous CoA esters of C16 to C22 fatty acids were elongated to give products up to C28 without exhibiting any preference for the primer. The present elongation system could account for the formation of most of the very long chains found in meibomian lipids.

Lactate dehydrogenase in tears.

Lactate dehydrogenase levels in tears were measured in normal subjects and in patients with retinoblastoma. When specimens were collected without trauma in normal subjects, there were usually no detectable levels of lactate dehydrogenase. When the eyelids were rubbed (probably liberating epithelial cells) lactate dehydrogenase levels were detectable and were five to ten times those of normal aqueous humor. When lactate dehydrogenase isoenzymes in such specimens were analyzed, the level of lactate dehydrogenase 5 was always higher than that of lactate dehydrogenase 1 (similar to normal aqueous humor). While occasional patients with retinoblastoma do have elevated levels of lactate dehydrogenase in tears, it is uncertain if this is due to epithelial destruction or retinoblastoma itself. Lactate dehydrogenase in tears does not appear to be a useful test for the diagnosis of retinoblastoma given present techniques for collection and analysis.