Non-coding RNA-mediated endothelial-to-mesenchymal transition in human diabetic cardiomyopathy, potential regulation by DNA methylation.

AIMS: Diabetic cardiomyopathy (DCM) is a major complication of diabetes and a risk factor for cardiovascular disease. Endothelial dysfunction is central to DCM, and endothelial-to-mesenchymal transition (EndMT) is a key form of endothelial dysfunction in diabetes. EndMT in DCM has been well-studied in model systems and has been found to be epigenetically regulated by non-coding RNAs (ncRNAs). However, EndMT in DCM and its associated epigenetic changes need further characterization in human patients. It is also not known if ncRNAs are affected by changes in DNA methylation in DCM. This study aims to confirm in human hearts, the findings from animal and cell studies, and potentially provide novel insight into interactions between DNA methylation and ncRNAs in EndMT in DCM. METHODS AND RESULTS: Heart tissues were collected from autopsy patients, fixed in formalin, and embedded in paraffin. Thin sections from paraffin-embedded tissues were used for histology and immunofluorescence analyses, where we confirmed that diabetic patients showed increased cardiac fibrosis that EndMT had occurred. Tissue curls from the paraffin-embedded tissues were used for RT-qPCR and methylation analyses. RT-qPCR quantitatively showed that EndMT occurs in the hearts of diabetics, and that EndMT in human hearts corresponded to changes in key ncRNAs. Methylation analysis showed that some of the EndMT-related ncRNAs were regulated by DNA promoter methylation, while others may be regulated through different epigenetic mechanisms. CONCLUSIONS: We show that EndMT is a relevant pathological process in human hearts during DCM, and that its occurrence coincides with changes in relevant ncRNAs. We further find that interplay between DNA methylation and certain ncRNAs involved in the regulation of EndMT may contribute to the observed changes in ncRNA expression. These findings reinforce the role of EndMT in patients afflicted with DCM and underscore the complexities and importance of the interactions between different facets of epigenetic regulation.

Sustained over-expression of calpain-2 induces age-dependent dilated cardiomyopathy in mice through aberrant autophagy.

Calpains have been implicated in heart diseases. While calpain-1 has been detrimental to the heart, the role of calpain-2 in cardiac pathology remains controversial. In this study we investigated whether sustained over-expression of calpain-2 had any adverse effects on the heart and the underlying mechanisms. Double transgenic mice (Tg-Capn2/tTA) were generated, which express human CAPN2 restricted to cardiomyocytes. The mice were subjected to echocardiography at age 3, 6, 8 and 12 months, and their heart tissues and sera were collected for analyses. We showed that transgenic mice over-expressing calpain-2 restricted to cardiomyocytes had normal heart function with no evidence of cardiac pathological remodeling at age 3 months. However, they exhibited features of dilated cardiomyopathy including increased heart size, enlarged heart chambers and heart dysfunction from age 8 months; histological analysis revealed loss of cardiomyocytes replaced by myocardial fibrosis and cardiomyocyte hypertrophy in transgenic mice from age 8 months. These cardiac alterations closely correlated with aberrant autophagy evidenced by significantly increased LC3BII and p62 protein levels and accumulation of autophagosomes in the hearts of transgenic mice. Notably, injection of 3-methyladenine, a well-established inhibitor of autophagy (30 mg/kg, i.p. once every 3 days starting from age 6 months for 2 months) prevented aberrant autophagy, attenuated myocardial injury and improved heart function in the transgenic mice. In cultured cardiomyocytes, over-expression of calpain-2 blocked autophagic flux by impairing lysosomal function. Furthermore, over-expression of calpain-2 resulted in lower levels of junctophilin-2 protein in the heart of transgenic mice and in cultured cardiomyocytes, which was attenuated by 3-methyladenine. In addition, blockade of autophagic flux by bafilomycin A (100 nM) induced a reduction of junctophilin-2 protein in cardiomyocytes. In summary, transgenic over-expression of calpain-2 induces age-dependent dilated cardiomyopathy in mice, which may be mediated through aberrant autophagy and a reduction of junctophilin-2. Thus, a sustained increase in calpain-2 may be detrimental to the heart.

Fibroblast transdifferentiation promotes conversion of M1 macrophages and replenishment of cardiac resident macrophages following cardiac injury in mice.

Resident cardiac macrophages play important roles in homeostasis, maintenance of cardiac function, and tissue repair. After cardiac injury, monocytes infiltrate the tissue, undergo phenotypic and functional changes, and are involved in inflammatory injury and functional remodelling. However, the fate of cardiac infiltrating/polarized macrophages and the relationship between these cells and resident cardiac macrophage replenishment following injury remain unclear. Our results showed that angiotensin II induces cardiac fibroblast transdifferentiation into cardiac myofibroblasts (MFBs). In cocultures with MFBs and murine macrophages, the MFBs promoted macrophage polarization to M1 phenotype, followed by selective apoptosis, which was associated with TNF/TNFR1 axis and independent of NO production. Surprisingly, after 36 h of coculture, the surviving macrophages were converted to M2 phenotype and settled in heart, which was dependent on leptin produced by MFBs or polarized macrophages via the PI3K or Akt pathway. CCR2+ CD45.2+ cells adoptively transferred into CD45.1+ mice with viral myocarditis, differentiated into CD45.2+ CCR2+ CX3CR1+ M2 cells during the resolution of inflammation and settled within the heart. Our data highlight a novel mechanism related to the renewal or replenishment of cardiac resident macrophages following cardiac injury; and suggest that transdifferentiation of cardiac fibroblasts may promote the resolution of inflammation.

IgG4-related disease as a rare cause of gastric outlet obstruction: a case report and literature review.

BACKGROUND: IgG4-related disease involvement of the digestive tract is very rare. In few reported cases of isolated gastric/duodenal IgG4-related disease, none of which resulted in luminal obstruction. CASE PRESENTATION: A 59 years old female presented with longstanding gastrointestinal symptoms. CT showed mural thickening of the proximal duodenum. Gastroscopy showed antral ulcer extending into the duodenum with outlet obstruction and biopsy showed acute on chronic duodenitis. Whipple's procedure was performed and IgG4-related disease was diagnosed on final pathology. Symptoms were revolved on mycophenolate mofetil and prednisone with no recurrence. CONCLUSIONS: Our case is the only reported case with gastric outlet obstruction secondary to gastroduodenal IgG4-related disease. The diagnosis should be considered in the differential diagnosis of unexplained duodenal stricture, gastric outlet obstruction or gastrointestinal ulceration. IgG4-related disease usually responds to steroids but long-term response rates to steroid-sparing agents, especially in the subset of patients with luminal IgG4-related disease remains to be determined.

Glucose-induced, duration-dependent genome-wide DNA methylation changes in human endothelial cells.

DNA methylation, a critical epigenetic mechanism, plays an important role in governing gene expressions during biological processes such as aging, which is well known to be accelerated in hyperglycemia (diabetes). In the present study, we investigated the effects of glucose on whole genome DNA methylation in small [human retinal microvascular endothelial cells (HRECs)] and large [human umbilical vein endothelial cells (HUVECs)] vessel endothelial cell (EC) lines exposed to basal or high glucose-containing media for variable lengths of time. Using the Infinium EPIC array, we obtained 773,133 CpG sites (probes) for analysis. Unsupervised clustering of the top 5% probes identified four distinct clusters within EC groups, with significant methylation differences attributed to EC types and the duration of cell culture rather than glucose stimuli alone. When comparing the ECs incubated for 2 days versus 7 days, hierarchical clustering analyses [methylation change >10% and false discovery rate (FDR) <0.05] identified 17,354 and 128 differentially methylated CpGs for HUVECs and HRECs, respectively. Predominant DNA hypermethylation was associated with the length of culture and was enriched for gene enhancer elements and regions surrounding CpG shores and shelves. We identified 88 differentially methylated regions (DMRs) for HUVECs and 8 DMRs for HRECs (all FDR <0.05). Pathway enrichment analyses of DMRs highlighted involvement of regulators of embryonic development (i.e., HOX genes) and cellular differentiation [transforming growth factor-ß (TGF-ß) family members]. Collectively, our findings suggest that DNA methylation is a complex process that involves tightly coordinated, cell-specific mechanisms. Such changes in methylation overlap genes critical for cellular differentiation and embryonic development.

lncRNA H19 prevents endothelial-mesenchymal transition in diabetic retinopathy.

AIMS/HYPOTHESIS: The pathophysiology of diabetic retinopathy is linked to hyperglycaemia and its effect on retinal microvascular tissues. The resulting endothelial injury changes the endothelial cell phenotype to acquire mesenchymal properties (i.e. endothelial-mesenchymal transition [EndMT]). Such changes can be regulated by epigenetic mechanisms, including long non-coding RNAs (lncRNAs). lncRNA H19 may influence EndMT through TGF-ß. We investigated the role of H19 in regulating EndMT during diabetic retinopathy. METHODS: H19 was overexpressed or silenced in human retinal endothelial cells exposed to various glucose levels. The cells were examined for H19, endothelial and mesenchymal markers. We then expanded the study to retinal tissues in a mouse model of diabetic retinopathy and also examined vitreous humour samples from individuals with proliferative diabetic retinopathy. RESULTS: Expression of H19 was downregulated in high glucose conditions (25 mmol/l). H19 overexpression prevented glucose-induced EndMT. Such changes appear to involve TGF-ß through a Smad-independent mechanism. Diabetes caused downregulation of retinal H19. Using H19 knockout mice, we demonstrated similar EndMT in the retina. Examination of vitreous humour from individuals with proliferative diabetic retinopathy also reinforced the downregulation of H19 in diabetes. CONCLUSIONS/INTERPRETATION: We therefore concluded that H19 regulates EndMT in diabetic retinopathy through specific mechanisms. DATA AVAILABILITY: The results from our previous microarray can be found online using the GEO accession number GSE122189.

ANRIL regulates production of extracellular matrix proteins and vasoactive factors in diabetic complications.

noncoding RNAs (lncRNAs) have gained widespread interest due to their prevailing presence in various diseases. lncRNA ANRIL (a. k. a. CDKN2B-AS1) is located on human chromosome 9 (p21.3) and transcribed in opposite direction to the INK4b-ARF-INK4a gene cluster. It has been identified as a highly susceptible region for diseases such as coronary artery diseases and type 2 diabetes. Here, we explored its regulatory role in diabetic nephropathy (DN) and diabetic cardiomyopathy (DCM) in association with epigenetic modifiers p300 and polycomb repressive complex 2 (PRC2) complex. We used an ANRIL-knockout (ANRILKO) mouse model for this study. The wild-type and ANRILKO animals with or without streptozotocin-induced diabetes were monitored for 2 min. At the end of the time point, urine and tissues were collected. The tissues were measured for fibronectin (FN), type IV collagen (Col1α4), and VEGF mRNA and protein expressions. Renal function was determined by the measurement of 24-h urine volume and albumin/creatinine ratio at euthanasia. Renal and cardiac structures were investigated using periodic acid-Schiff stain and/or immunohistochemical analysis. Elevated expressions of extracellular matrix (ECM) proteins were prevented in ANRILKO diabetic animals. Furthermore, ANRILKO had a protective effect on diabetic mouse kidneys, as evidenced by lowering of urine volume and urine albumin levels in comparison with the wild-type diabetic animals. These alterations regulated by ANRIL may be mediated by p300 and enhancer of zeste 2 (EZH2) of the PRC2 complex. Our study concludes that ANRIL regulates functional and structural alterations in the kidneys and hearts in diabetes through controlling the expressions of ECM proteins and VEGF.

Mitotic chromosome condensation mediated by the retinoblastoma protein is tumor-suppressive.

Condensation and segregation of mitotic chromosomes is a critical process for cellular propagation, and, in mammals, mitotic errors can contribute to the pathogenesis of cancer. In this report, we demonstrate that the retinoblastoma protein (pRB), a well-known regulator of progression through the G1 phase of the cell cycle, plays a critical role in mitotic chromosome condensation that is independent of G1-to-S-phase regulation. Using gene targeted mutant mice, we studied this aspect of pRB function in isolation, and demonstrate that it is an essential part of pRB-mediated tumor suppression. Cancer-prone Trp53(-/-) mice succumb to more aggressive forms of cancer when pRB's ability to condense chromosomes is compromised. Furthermore, we demonstrate that defective mitotic chromosome structure caused by mutant pRB accelerates loss of heterozygosity, leading to earlier tumor formation in Trp53(+/-) mice. These data reveal a new mechanism of tumor suppression, facilitated by pRB, in which genome stability is maintained by proper condensation of mitotic chromosomes.

miR-146a mediates inflammatory changes and fibrosis in the heart in diabetes.

Hyperglycemia induced endothelial injury is a key pathogenetic factor in diabetic cardiomyopathy. In diabetes, changes in pro-inflammatory cytokines are a key mechanism leading to cardiac fibrosis. We have previously demonstrated alteration of miR-146a in chronic diabetic complications. Here, we investigated the role of endothelial miR-146a in mediating inflammation and fibrosis in diabetic cardiomyopathy. To examine the effects of miR-146a on the inflammatory mediators, an endothelial specific miR-146a overexpressing transgenic mice (TG) using tie-2 promoter, was generated. We examined these mice and wild type littermate controls with or without STZ induced diabetes. Transthoracic echocardiography was performed. Cardiac tissues were examined for inflammatory cytokine mRNAs and proteins by real time RT-PCR or ELISA. Cardiac fibrosis was examined by histology staining. Human cardiac microvascular endothelial cells (HCMECs) and primary endothelial cells isolated from mice were used following incubation with various levels of glucose with or without miR-146a mimics or antagomir transfection. In hearts of wild type mice with diabetes, increased expression of inflammatory markers and extracellular matrix proteins (IL6, TNFα, IL-1ß, MCP-1, NF-κB, Col1α1, Col4α1) were seen compared to wild type controls. These changes were prevented in the diabetic TG mice. In addition, WT diabetic mice showed cardiac functional abnormalities, which were improved in the diabetic TG mice. In vitro studies showed glucose induced increase the expressions of the above inflammatory cytokines and specific NF-κB regulators (IRAK1 &TRAF6). Such changes were corrected in the HCMECs following miR-146a mimic transfection. These data indicate that in diabetes, increased inflammatory cytokine and extracellular matrix protein productions and associated cardiac functional alterations are regulated by endothelial miR-146a. Identification of such mechanisms may potentially lead to the development of novel RNA based therapeutics.

Fibroblast Growth Factor 9 Imparts Hierarchy and Vasoreactivity to the Microcirculation of Renal Tumors and Suppresses Metastases.

Tumor vessel normalization has been proposed as a therapeutic paradigm. However, normal microvessels are hierarchical and vasoreactive with single file transit of red blood cells through capillaries. Such a network has not been identified in malignant tumors. We tested whether the chaotic tumor microcirculation could be reconfigured by the mesenchyme-selective growth factor, FGF9. Delivery of FGF9 to renal tumors in mice yielded microvessels that were covered by pericytes, smooth muscle cells, and a collagen-fortified basement membrane. This was associated with reduced pulmonary metastases. Intravital microvascular imaging revealed a haphazard web of channels in control tumors but a network of arterioles, bona fide capillaries, and venules in FGF9-expressing tumors. Moreover, whereas vasoreactivity was absent in control tumors, arterioles in FGF9-expressing tumors could constrict and dilate in response to adrenergic and nitric oxide releasing agents, respectively. These changes were accompanied by reduced hypoxia in the tumor core and reduced expression of the angiogenic factor VEGF-A. FGF9 was found to selectively amplify a population of PDGFRß-positive stromal cells in the tumor and blocking PDGFRß prevented microvascular differentiation by FGF9 and also worsened metastases. We conclude that harnessing local mesenchymal stromal cells with FGF9 can differentiate the tumor microvasculature to an extent not observed previously.

Collectivization of Vascular Smooth Muscle Cells via TGF-ß-Cadherin-11-Dependent Adhesive Switching.

OBJECTIVE: Smooth muscle cells (SMCs) in healthy arteries are arranged as a collective. However, in diseased arteries, SMCs commonly exist as individual cells, unconnected to each other. The purpose of this study was to elucidate the events that enable individualized SMCs to enter into a stable and interacting cell collective. APPROACH AND RESULTS: Human SMCs stimulated to undergo programmed collectivization were tracked by time-lapse microscopy. We uncovered a switch in the behavior of contacting SMCs from semiautonomous motility to cell-cell adherence. Central to the cell-adherent phenotype was the formation of uniquely elongated adherens junctions, up to 60 µm in length, which appeared to strap adjacent SMCs to each other. Remarkably, these junctions contained both N-cadherin and cadherin-11. Ground-state depletion super-resolution microscopy revealed that these hybrid assemblies were comprised of 2 parallel nanotracks of each cadherin, separated by 50 nm. Blocking either N-cadherin or cadherin-11 inhibited collectivization. Cell-cell adhesion and adherens junction elongation were associated with reduced transforming growth factor-ß signaling, and exogenous transforming growth factor-ß1 suppressed junction elongation via the noncanonical p38 pathway. Imaging of fura-2-loaded SMCs revealed that SMC assemblies displayed coordinated calcium oscillations and cell-cell transmission of calcium waves which, together with increased connexin 43-containing junctions, depended on cadherin-11 and N-cadherin function. CONCLUSIONS: SMCs can self-organize, structurally and functionally, via transforming growth factor-ß-p38-dependent adhesive switching and a novel adherens junction architecture comprised of hybrid nanotracks of cadherin-11 and N-cadherin. The findings define a mechanism for the assembly of SMCs into networks, a process that may be relevant to the stability and function of blood vessels.

Lactodifucotetraose, a human milk oligosaccharide, attenuates platelet function and inflammatory cytokine release.

Human milk strongly quenches inflammatory processes in vitro, and breastfed infants have lower incidence of inflammatory diseases than those fed artificially. Platelets from neonates, in contrast to those from adults, are less responsive to platelet agonists such as collagen, thrombin, ADP, and epinephrine. Breastfed infants absorb oligosaccharides intact from the human milk in their gut to the circulation. This study was to determine whether these oligosaccharides can attenuate platelet function and platelet secretion of pro-inflammatory proteins, and to identify the active component. The natural mixture of oligosaccharides from human milk and pure individual human milk oligosaccharides were tested for their ability to modulate responses of platelets isolated from human blood following exposure to thrombin, ADP, and collagen. Human milk and the natural mixture of human milk oligosaccharides inhibited platelet release of inflammatory proteins. Of the purified human milk oligosaccharides tested, only lactodifucotetraose (LDFT) significantly inhibited thrombin induced release of the pro-inflammatory proteins RANTES and sCD40L. LDFT also inhibited platelet adhesion to a collagen-coated surface, as well as platelet aggregation induced by ADP or collagen. These data indicate that LDFT may help modulate hemostasis by suppressing platelet-induced inflammatory processes in breastfed infants. This activity suggests further study of LDFT for its potential as a therapeutic agent in infants and adults.

Curcumin protects hearts from FFA-induced injury by activating Nrf2 and inactivating NF-κB both in vitro and in vivo.

Obesity and increased free fatty acid (FFA) level are tightly linked, leading to the development of cardiovascular disorders. Curcumin is a natural product from Curcuma longa with multiple bioactivities and is known to have cardioprotective effects in several cellular and animal models. The current study was designed to evaluate the cardioprotective effects of curcumin and demonstrate the underlying mechanism in FFA-induced cardiac injury. Using cell culture studies and high fat in vivo model, we explored the mechanistic basis of anti-inflammatory and antioxidant activities of curcumin. We observed that palmitate (PA) treatment in cardiac derived H9C2 cells induced a marked increase in reactive oxygen species, inflammation, apoptosis and hypertrophy. All of these changes were effectively suppressed by curcumin treatment. In addition, oral administration of curcumin at 50mg/kg completely suppressed high fat diet-induced oxidative stress, inflammation, apoptosis, fibrosis, hypertrophy and tissue remodeling in mice. The beneficial actions of curcumin are closely associated with its ability to increase Nrf2 expression and inhibit NF-κB activation. Thus, both in vitro and in vivo studies showed a promising role of curcumin as a cardioprotective agent against palmitate and high fat diet mediated cardiac dysfunction. We indicated the regulatory roles of Nrf2 and NF-κB in obesity-induced heart injury, and suggested that they may be important therapeutic targets in the treatment of obesity-related disorders.

SIRT1 reduction causes renal and retinal injury in diabetes through endothelin 1 and transforming growth factor ß1.

In diabetes, hyperglycaemia causes up-regulation of endothelin 1 (ET-1) and transforming growth factor beta 1 (TGF-ß1). Previously we showed glucose reduces sirtuin1 (SIRT1), a class III histone deacetylase. Here, we investigated the regulatory role of SIRT1 on ET-1 and TGF-ß1 expression. Human microvascular endothelial cells were examined following incubation with 25 mmol/l glucose (HG) and 5 mmol/l glucose (NG) with or without SIRT1 or histone acetylase p300 overexpression or knockdown. mRNA expressions of ET-1, TGF-ß1, SIRT1, p300 and collagen 1α(I) were examined. SIRT1 enzyme activity, ET-1 and TGF-ß1 protein levels were measured. Histone acetylation and endothelial permeability were further investigated. Similar analyses were performed in the kidneys and retinas of SIRT1 overexpressing transgenic mice with or without streptozotocin induced diabetes. Renal functions were evaluated. In the endothelial cells (ECs), HG caused increased permeability and escalated production of ET-1, TGF-ß1, collagen Iα(I). These cells also showed increased p300 expression, histone acetylation and reduced SIRT1 levels. These changes were rectified in the ECs following p300 silencing or by SIRT1 overexpression, whereas SIRT1 knockdown or p300 overexpression in NG mimicked the effects of HG. High ET-1 and TGF-ß1 levels were seen in the kidneys and retinas of diabetic mice along with micro-albuminuria and increased fibronectin protein (marker of glucose-induced cell injury) levels. Interestingly, these detrimental changes were blunted in SIRT1 overexpressing transgenic mice with diabetes. This study showed a novel SIRT1 mediated protection against renal and retinal injury in diabetes, regulated through p300, ET-1 and TGF-ß1.

Long non-coding RNA MALAT1 regulates hyperglycaemia induced inflammatory process in the endothelial cells.

To examine whether the long non-coding RNA (lncRNA) metastasis associated lung adenocarcinoma transcript 1 (MALAT1) is altered in the endothelial cells in response to glucose and the significance of such alteration. We incubated human umbilical vein endothelial cells with media containing various glucose levels. We found an increase in MALAT1 expression peaking after 12 hrs of incubation in high glucose. This increase was associated with parallel increase in serum amyloid antigen 3 (SAA3), an inflammatory ligand and target of MALAT1 and was further accompanied by increase in mRNAs and proteins of inflammatory mediators, tumour necrosis factor alpha (TNF-α) and interleukin 6 (IL-6). Renal tissue from the diabetic animals showed similar changes. Such cellular alterations were prevented following MALAT1 specific siRNA transfection. Results of this study indicate that LncRNA MALAT1 regulates glucose-induced up-regulation of inflammatory mediators IL-6 and TNF-α through activation of SAA3. Identification of such novel mechanism may lead to the development of RNA-based therapeutics targeting MALAT1 for diabetes-induced micro and macro vascular complications.

Modulation of ERK5 is a novel mechanism by which Cdc42 regulates migration of breast cancer cells.

Members of Rho family GTPases including Cdc42 are known to play pivotal roles in cell migration. Cell migration is also known to be regulated by many protein kinases. Kinetworks KPSS 11.0 phospho-site screening of Cdc42-silenced Hs578T breast cancer cells revealed most dramatic change in ERK5 MAP kinase. In the present study, we set out to determine the relationship between Cdc42 and ERK5 and its significance in breast cancer cell migration and invasion. Specific siRNAs were used for knocking down Cdc42 or ERK5 in breast cancer cells. Increased ERK5 phosphorylation in breast cancer cells was achieved by infection of constitutively active MEK5 adenovirus. The cells were then subjected to cell migration or invasion assay without the presence of serum or any growth factor. We found that Cdc42 negatively regulated phosphorylation of ERK5, which in turn exhibited an inverse relationship with migration and invasiveness of breast cancer cells. To find out some in vivo relevance of the results of our in vitro experiments we also examined the expression of ERK5 in the breast cancer tissues and their adjacent normal control tissues by real-time RT-PCR and immunocytochemistry. ERK5 expression was found to be reduced in breast cancer tissues as compared with their adjacent uninvolved mammary tissues. Therefore, Cdc42 may promote breast cancer cell migration and invasion by inhibiting ERK5 phosphorylation and ERK5 expression may be inversely correlated with the progression of some breast tumors.

P63 Positive Mucoepidermoid Tumor of the Lacrimal Sac with Associated Papilloma.

We report a case of a 44-year-old man who presented with a left medial canthal mass and epiphora. Imaging was suggestive of a mass continuous with the nasolacrimal sac. Subsequent surgical exploration revealed a mass adherent to bone with invasion of the lacrimal system. Histological examination revealed a squamous/transitional cell papilloma overlying a low-grade mucoepidermoid carcinoma (MEC). Complete surgical resection was completed and pathology confirmed the diagnosis. This is the first case in which a MEC has been reported concurrently with an overlying papilloma, providing support for the hypothesis that MECs arise from papillomas in the lacrimal sac. Additionally, the tissue stained positive for p63, which is congruent with MEC immunoreactivity in the salivary gland. The description of these unique histopathological findings may assist in definitive diagnosis and improve our understanding of the pathophysiology underlying lacrimal sac MEC tumors.

miR-195 regulates SIRT1-mediated changes in diabetic retinopathy.

AIMS/HYPOTHESIS: Endothelial cell (EC) damage is a key mechanism causing retinal microvascular injury in diabetes. Several microRNAs (miRNAs) have been found to regulate sirtuin 1 (SIRT1, which is involved in regulation of the cell cycle, survival and metabolism) in various tissues and disease states, but no studies have been conducted on the role of miRNA in regulation of SIRT1 in diabetic retinopathy. Here we investigated the effect of miRNA-195 (miR-195), a SIRT1-targeting miRNA, on the development of diabetes-induced changes in ECs and retina. METHODS: The level of miR-195 was measured in human retinal and dermal microvascular ECs (HRECs, HMECs) following exposure to 25 mmol/l glucose (high glucose, HG) and 5 mmol/l glucose (normal glucose, NG). SIRT1 and fibronectin levels were examined following transfection with miR-195 mimic or antagomir or forced expression of SIRT1. Retinal tissues from diabetic rats were similarly studied following intravitreal injection of an miR-195 antagomir or mimic. In situ hybridisation was used to localise retinal miR-195. RESULTS: HG caused increased miR-195 levels and decreased SIRT1 expression (compared with NG) in both HRECs and HMECs. Transfection with miR-195 antagomir and forced expression of SIRT1 prevented such changes, whereas transfection with miR-195 mimic produced HG-like effects. A luciferase assay confirmed the binding of miR-195 to the 3' untranslated region of SIRT1. miR-195 expression was upregulated in retinas of diabetic rats and intravitreal injection of miR-195 antagomir ameliorated levels of SIRT1. CONCLUSIONS/INTERPRETATION: These studies identified a novel mechanism whereby miR-195 regulates SIRT1-mediated tissue damage in diabetic retinopathy.

Cardiac miR-133a overexpression prevents early cardiac fibrosis in diabetes.

Diabetic cardiomyopathy is a cascade of complex events leading to eventual failure of the heart and cardiac fibrosis being considered as one of its major causes. miR-133a is one of the most abundantly expressed microRNAs in the heart. We investigated the role of miR-133a during severe hyperglycaemia. And, our aim was to find out what role miR-133a plays during diabetes-induced cardiac fibrosis. We saw a drastic decrease in miR-133a expression in the hearts of streptozotocin-induced diabetic animals, as measured by RT-qPCR. This decrease was accompanied by an increase in the transcriptional co-activator EP300 mRNA and major markers of fibrosis [transforming growth factor-ß1, connective tissue growth factor, fibronectin (FN1) and COL4A1]; in addition, focal cardiac fibrosis assessed by Masson's trichome stain was increased. Interestingly, in diabetic mice with cardiac-specific miR-133aa overexpression, cardiac fibrosis was significantly decreased, as observed by RT-qPCR and immunoblotting of COL4A1, ELISA for FN1 and microscopic examination. Furthermore, Cardiac miR-133a overexpression prevented ERK1/2 and SMAD-2 phosphorylation. These findings show that miR-133a could be a potential therapeutic target for diabetes-induced cardiac fibrosis and related cardiac dysfunction.

Glucose-induced cell signaling in the pathogenesis of diabetic cardiomyopathy.

Chronic diabetic complications affect multiple organ systems and lead to significant morbidity and mortality in the diabetic population. Diabetic cardiomyopathy is a major etiologic factor causing heart failure. Dysfunction of both vascular endothelial cells and cardiomyocytes contributes in the pathogenesis of diabetic cardiomyopathy. Hyperglycemia has been identified as the key determinant in the development of several chronic diabetic complications. Hyperglycemia leads to oxidative stress and several other abnormalities causing changes in cellular signaling. These diabetes-mediated biochemical anomalies show cross-interaction and complex interplay. Such changes also cause alteration of transcriptional and post-transcriptional machinery causing altered production of vasoactive and cardioactive factors. In this review, we will highlight some of the important signaling changes leading to diabetic cardiomyopathy and discuss possible potential therapeutic remedies.